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Os06g0594600

2014-06-08T14:46:35Z

Xiaoyanjie2012: /* Evolution */

Please input one-sentence summary here.

==Annotated Information==
OsAt10 is a gene in rice(Oryza sativa).Its RAP ID is Os06g0594600,and its MSU ID LOC_Os06g39390. Overexpression of this gene can alters rice cell wall hydroxycinnamic acid Content and sacchariﬁcation.
===Function===
The overexpression of the rice gene -- OsAt10(LOC_Os06g39390) -- can effect the hydroxycinnamic acid content and saccharification in the rice cell wall.rice mutants with altered expression of four of these genes have altered cell wall hydroxycinnamate content. In-depth characterization of ectopic expression lines for one gene, OsAt10, revealed that this modification increases matrix polysaccharideassociated ester-linkedp-CA while simultaneously decreasing matrix polysaccharide-associated FA.OsAt10 overexpression plants exhibit increased in vitro saccharification, with no discernible effects on vegetative development. Thus, this gene is a useful target for improving biofuel and feed production.
Grass cell wall properties influence food, feed, and biofuel feedstock usage efficiency. The glucuronoarabinoxylan of grass cell walls is esterified with the phenylpropanoid-derived hydroxycinnamic acids ferulic acid (FA) and para-coumaric acid (p-CA). Feruloyl esters undergo oxidative coupling with neighboring phenylpropanoids on glucuronoarabinoxylan and lignin. Examination of rice (Oryza sativa) mutants in a grass-expanded and -diverged clade of BAHD acyl coenzyme A-utilizing transferases identified four mutants with altered cell wall FA or p-CA contents.Some researchers reported on the effects of overexpressing one of these genes, OsAt10 (LOC_Os06g39390), in rice. An activation-tagged line, OsAT10-D1, shows a 60% reduction in matrix polysaccharide-bound FA and an approximately 300% increase in p-CA in young leaf tissue but no discernible phenotypic alterations in vegetative development, lignin content, or lignin composition. Two additional independent OsAt10 overexpression lines show similar changes in FA and p-CA content. Cell wall fractionation and liquid chromatography-mass spectrometry experiments isolate the cell wall alterations in the mutant to ester conjugates of a five-carbon sugar with p-CA and FA. These results suggest that OsAT10 is a p-coumaroyl coenzyme A transferase involved in glucuronoarabinoxylan modification. Biomass from OsAT10-D1 exhibits a 20% to 40% increase in saccharification yield depending on the assay. Thus, OsAt10 is an attractive target for improving grass cell wall quality for fuel and animal feed.<ref name="ref1" />
====1.The Mitchell Clade of BAHD Acyltransferases Is Expanded and Diverged in Grasses====
Mitchell et al. (2007) identified what we term the Mitchell clade of BAHD acyl-CoA-dependent acyltransferases on the basis of high gene expression in grasses relative to dicots. To refine the hypothesis that these enzymes might be involved in grass-diverged cell wall synthesis, we systematically characterized the distribution of this clade in selected plant species and compared the clade with other characterized BAHD proteins. We identified BAHD proteins from the genomes of a diverse set of sequenced plant species available at the time of the analysis and examined the phylogenetic relationships among them and a reference set of BAHDs (Table I). To gain higher sensitivity relative to local sequence alignment (i.e. BLAST) for recognizing sequences with low, but potentially still significant, homology, we used a hidden Markov model to identify putative BAHD proteins (Finn et al., 2011)
The researchers then inferred an initial model of the phylo-genetic relationships among the putative BAHD proteins from each genome and the set of biochemically characterized BAHD proteins cataloged by D’Auria (2006). While we are aware that recent analyses have included the presence of a strict HXXXD motif as indicative of whether the protein is an active BAHD (Banks et al., 2011; Tuominen et al., 2011), we have included proteins with single amino acid alterations to this motif, since one of the known biochemically active proteins for the family involved in taxol biosynthesis, BAPT (National Center for Biotechnology Information identifier AAL92459; Walker et al., 2002), possesses a variation of this motif in which the His is replaced by a Ser.
As observed by Tuominen et al. (2011), the distribution of BAHD proteins varies among species (Table I). The Mitchell clade is embedded within clade V, or clade Va of Tuominen et al. (2011). Furthermore, we find that the Mitchell clade includes a biochemically characterized banana (Musa spp.) alcohol CoA acyltransferase, BanAAT (Beekwilder et al., 2004), and is related to a group of BAHD proteins that participate in taxol biosynthesis (Fig. 1B).

[[File:Table 1xxy.png]][[File:figure 1xxy.png]]
====2.Screen of Rice Mutants for Altered Cell Wall Hydroxycinnamic Acid Content====

[[File:Figure 2xxy.png]]

====3.The OsAT10-D1 Line Has an Increase in Cell Wall Glc Content====
Compensatory changes are often seen among the components of the cell wall (Humphrey et al., 2007). Quantification of sugars released by acid treatment of
destarched AIR preparations from mature straw suggests that the Glc content is increased by approximately 20% (w/w) for the mutant relative to the wild type (Fig. 9A). We observed the difference both with TFA treatment, which liberates monosaccharides derived from matrix polysaccharides and amorphous cellulose, and when the TFA residue was further treated with sulfuric acid, which breaks down crystalline cellulose (Fig. 9A). The difference in the mutant compared with the wild type is most apparent when the products of both treatments are summed together, which gives an increase in Glc in the mutant compared with the wild type of 19% 6 11%. By mass, we did not observe any other significant changes in sugar amounts in the mutant compared with the wild type. We also observed no change in the total mass percentage of sugars in AIR. When the TFA-solubilized sugars are expressed in terms of mol%, the data also indicate an increase in Glc content of 11% 6 5% (Fig. 9B). The sum of the mol % of other measured sugars (i.e. Xyl, Ara, and the sum of minor sugars) decreases proportionally to the Glc increase (7% 6 7%). This balance in mol % change suggests that the change in polysaccharide content in the mutant is restricted to the Glc-containing polymers.

[[File:Figure 9xxy.png]]

====4.The OsAT10-D1 Line Shows No Alterations in Lignin Content or Composition====
OsAT10-D1 mature straw samples show no significant differences in the content of acetylbromide-soluble lignin after saponification relative to the wild type
(Table II). We obtained a similar result via py-MBMS for mature straw and separate, young leaf and sheath samples. The py-MBMS also revealed no difference in the syringyl-guaiacyl (S:G) lignin ratio in the mutant compared with the wild type after saponification (Table II). We also collected py-MBMS data for unprocessed straw and AIR of OsAT10-D1. Separate analyses of the saponified and unsaponified samples reveals distinctions between the wild type and mutant in the unsaponified samples (Fig. 10A). Principal component 1 explains the alcohol extraction (30% of the variation), and principal component 2 explains the differences between wild-type and mutant samples (19% of the variation). The loadings for principal component 2 show that the major ions that distinguish wild-type and mutant samples are phenolics (Fig. 10B). The mass spectrometry fragmentation pattern is consistent with an interpretation in which there is an increase of p-CA, as reflected by peaks 120, 94, and 91, and a decrease in FA, as reflected in the reduction in the coniferyl ion, peak 150 (Evans and Milne, 1987). Because principal component analysis no longer distinguishes the samples after saponification (Fig. 10C), the observed differences in phenylpropanoids between OsAT10-D1 and the wild type are likely associated with ester-linked hydroxycinnamates and not lignin, consistent with the other results.
A limitation of the pyrolysis method for determining lignin composition is that it inaccurately measures H-lignin, which volatilizes poorly and instead turns to char upon heating. Because of the increase in p-CA, a precursor of H-lignin, in OsAT10-D1 cell walls relative to the wild type, we sought to determine whether there is a change in the char content of OsAT10-D1 using a thermogravimetric pyrolysis instrument. Duplicate runs per genotype of the thermogravimetric instrument did not detect a difference in the mass remaining from mature straw after pyrolysis, again consistent with there being no difference in core lignin composition or content between OsAT10-D1 and the wild type.

[[File:Table 2xxy.png]]
[[File:Figure 10xxy.png]]

====The OsAT10-D1 Line Shows an Increase in Saccharification====
Ferulate in grass biomass is inversely correlated with digestibility across diverse grass accessions (Lam et al., 2003; Casler and Jung, 2006). The phenotype of the OsAT10-D1 line provided the opportunity to determine whether there is also an increase in enzymatic digestibility with reduced FA content when comparing two near-isogenic plant lines. We found that destarched AIR after mild pretreatment followed by incubation with a cellulase cocktail resulted in the release of approximately 20% more reducing sugar from the mutant compared with the wild type at each time point examined (Fig. 11A)
Acid-pretreated rice straw of the wild type and OsAT10-D1 to the mesophilic fungus, Penicillium sp. YT02. This recently characterized fungus shows significantly higher xylanase and b-glucosidase activities with various insoluble lignocellulosic substrates compared with the commonly used fungal strain, Trichoderma reesei (ATCC 24449; Kovacs et al., 2009; L. Gao and J. Zhou, unpublished data). In the fungal treatments, the biomass-derived sugars initially accumulate but are gradually depleted via incorporation into fungal biomass. Qualitatively consistent with the enzymatic deconstruction results, Penicillium sp. YT02 incubation released 46% more Glc, 82% more Xyl, and 25% more Ara into the medium from OsAT10-D1 straw than from wild-type straw (Fig. 11B). Averaged over the entire time course (12–120 h), the improvement in yield is more dramatic with the fungus than with the simple enzymatic treatment, with a total sugar yield increase of approximately 40%. Cellulase and b-glucosidase enzymatic activities in the slurry are unchanged on the mutant straw (Fig. 11C), suggesting that the fungus grew similarly on both. In contrast, and of relevance to the nature of the change caused by the increased expression of OsAt10, xylanase activity is dramatically enhanced, especially at later time points (Fig. 11C).

[[File:Figure 11xxy.png]]

===Expression===
For the remaining 11 lines, we characterized the alkali-labile hydroxycinnamoyl ester content of cell wall alcohol-insoluble residue (AIR) from leaf blades and sheaths of side tillers. We compared homozygous, mutant, and wild-type segregant plants 7 or 10 weeks after planting. The screen revealed four mutants with possible cell wall hydroxycinnamic acid phenotypes . All four lines showed changes in the expression of the nearest acyltransferase
gene to the T-DNA insertion site via quantitative reverse transcription (qRT)-PCR. Homozygous mutant progeny of4A-03423(here afterreferred to asOsAT10-D1), which has increased expression of OsAt10, exhibited reduced FA (approximately 60% less) and an increase in p-CA (approximately 300% more) in sheaths and leaves.TheT-DNA insertions it efort helinewerefertoas OsAT10-D1(PFG_4A-03423) is approximately 8.5 kb downstream of the transcriptional start site forOsAt10. The insert is oriented with the activating sequences proximate toOsAt10and in the range observed to activate expression (Jeong et al., 2006). As mentioned above, qRT-PCR indicated that, indeed,the expression ofOsAt10was increased by more than 100-fold in the leaves of homozygous OsAT10-D1 plants (Fig. 3B). InOsAT10-D1, besides OsAt10the expression of other genes proximate to the site of the T-DNA insertion does not vary significantly relative to the wild type (Fig. 3B). Similarly, the expression of related OsAtgenesdoesnotvarysignificantly in OsAT10-D1(Supplemental Fig. S2), reducing the possibility that the observed phenotype is due to compensation at the level of gene expression of a related acyltransferase. Of the acyltransferase transcripts examined in this survey, OsAt6appears to vary the most, although not significantly. However, OsAt6(LOC_Os01g08380) is expressed near the lower limitofourdetectionand,infact,wasreportedas undetectable in a previous qRT-PCR study (Piston et al., 2010). OsAT10-D1lines show no change in size and dry mass at maturity (Fig. 4, A and B). However, we did measure an approximately 20% to 30% decrease in total seed mass per plant for the mutant compared with the wild type.

[[File:Figure 3xxy.png]][[File:Figure 4xxy.png]]

===Evolution===
Altered expression of members of a grass-diverged and -expanded clade of BAHD acyl-CoA acyltransferases alters the amounts of hydroxycinnamic acids in grass cell walls. In particular, increased expression of OsAt10 increases p-CA content but decreases FA content of rice matrix polysaccharide, consistent with our tentative assignment of this enzyme as a p-coumaroyl-CoA transferase. Together with the recent report that OsAT4 has p-CA monolignol transferase activity, this suggests that other members of the Mitchell clade of acyl-CoA acyltransferases likely possess feruloyl transferase activity(ies). This insight opens the possibility of a detailed examination of the biological functions of and selective basis for acylation of the different grass cell wall polymers with hydroxycinnamates. Of practical importance toward improving the efficiency of biofuel production from grass biomass and the nutritional value of forage
grasses, we have found that the increased OsAt10 expression increases straw Glc content and improves in vitro digestibility. The fact that this is an over -expression effect will facilitate the rapid testing of this gene in other grass species.

==Labs working on this gene==
Department of Microbiology and Plant Biology (L.E.B., M.L.P., L.G., F.L., J.Z.), Department of Chemistry and Biochemistry (W.L.S., S.B.F.), and Department of Chemical, Biological, and Materials Engineering (X.Z., R.E.J.),University of Oklahoma, Norman, Oklahoma 73019; Department of Plant Pathology and The Genome Center, University of California, Davis, California 95616 (L.E.B., D.M.C., M.E.V.-S., P.E.C., P.C., S.B., P.C.R.); Joint BioEnergy Institute, Emeryville, California 94608 (L.E.B., B.E., C.M., D.M.C., C.R., M.E.V.-S., P.I.B., P.C., J.D.K., H.V.S., P.C.R.); Crop Biotech Institute and Department of Genetic Engineering, Kyung Hee University, Yongin 446–701, Republic of Korea (S.-R.K., G.A., P.C.R.); Physical Biosciences Division, Lawrence Berkeley National
Laboratory, Berkeley, California 94720 (B.E., C.R., M.E.V.-S., P.I.B., J.D.K., H.V.S., P.C.R.); BioEnergy Sciences Center, National Renewable Energy Laboratory, Golden, Colorado 80401 (R.S., A.Z.); China Tobacco Gene Research Center, Zhengzhou Tobacco Research Institute, Zhengzhou, Henan 450001, China (P.C.); and Department of Chemical and Biomolecular Engineering, Department of Bioengineering, University of California, Berkeley, California 94720 (J.D.K.).

==References==
<references>
<ref name="ref1"> Bartley, L.E., et al., Overexpression of a BAHD acyltransferase, OsAt10, alters rice cell wall hydroxycinnamic acid content and saccharification. Plant physiology, 2013. 161(4): p. 1615-1633.

==Structured Information==
{{JaponicaGene|
GeneName = Os06g0594600|
Description = The start codon is not identified.|
Version = NM_001064516.2 GI:297606110 GeneID:4341427|
Length = 1126 bp|
Definition = Oryza sativa Japonica Group Os06g0594600, complete gene.|
Source = Oryza sativa Japonica Group

ORGANISM Oryza sativa Japonica Group
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP
clade; Ehrhartoideae; Oryzeae; Oryza.
|
Chromosome = [[:category:Japonica Chromosome 6|Chromosome 6]]|
AP = Chromosome 6:24256206..24257331|
CDS = 24256486..24257331|
GCID = <gbrowseImage1>
name=NC_008399:24256206..24257331
source=RiceChromosome06
preset=GeneLocation
</gbrowseImage1>|
GSID = <gbrowseImage2>
name=NC_008399:24256206..24257331
source=RiceChromosome06
preset=GeneLocation
</gbrowseImage2>|
CDNA = <cdnaseq>gatttgcaggtgacgacgttcacctgcggcggcttcgtgatcgggctgcgcaccaaccacgcggtggcggacggcaccggcgccgcccagttcatgaacgccgtcggcgacctcgcccgcggcctcccggagccgcgggtgaagccgatctgggcgcgcgaccgcttcccggacccggacatcaagcccggcccgctgccggagctccccgtgctgccgctccagtacatcgccttcgacttccccgccgcctacctcggcaagctcaaggcgcagtacgccgccaccgccggcgccagcaagatctgctccgccttcgacatcgtcatcgccaagctctggcagtgccggacgcgcgccatcgccgccgaccccgccgcggccgtcaagctctgcttcttcgccagcgcccgccaggtgctcggcctggagaccggctactggggcaacgccatcttcccggtgaaggtgtccgcggcggcgggggaggtggcggcgtcgtcggtgatcgagctcgtcggcgtggtccgggaggcgaagcggcggatggccggcgagtgcctgcgctgggcggaggggcgcaccggcggcgccgacccgttccagatgacgttcgactacgagtccgtgtacgtgtcggactggagcaagctcgggttcaacgacgtcgactacgggtacggcgcgccgtcggcggcggggccgctggtgaactgcgacctcatctcgtcggtgatcgtcatgcgggcgccggcgccgctcgccggcacgcggctgctggcgagctgcgtcaccaaggagcacgccgacgacttcgccgccaggatgagggaggatctcgtctaa</cdnaseq>|
AA = <aaseq>DLQVTTFTCGGFVIGLRTNHAVADGTGAAQFMNAVGDLARGLPE PRVKPIWARDRFPDPDIKPGPLPELPVLPLQYIAFDFPAAYLGKLKAQYAATAGASKI CSAFDIVIAKLWQCRTRAIAADPAAAVKLCFFASARQVLGLETGYWGNAIFPVKVSAA AGEVAASSVIELVGVVREAKRRMAGECLRWAEGRTGGADPFQMTFDYESVYVSDWSKL GFNDVDYGYGAPSAAGPLVNCDLISSVIVMRAPAPLAGTRLLASCVTKEHADDFAARM REDLV</aaseq>|
DNA = <dnaseqindica>1..846#gatttgcaggtgacgacgttcacctgcggcggcttcgtgatcgggctgcgcaccaaccacgcggtggcggacggcaccggcgccgcccagttcatgaacgccgtcggcgacctcgcccgcggcctcccggagccgcgggtgaagccgatctgggcgcgcgaccgcttcccggacccggacatcaagcccggcccgctgccggagctccccgtgctgccgctccagtacatcgccttcgacttccccgccgcctacctcggcaagctcaaggcgcagtacgccgccaccgccggcgccagcaagatctgctccgccttcgacatcgtcatcgccaagctctggcagtgccggacgcgcgccatcgccgccgaccccgccgcggccgtcaagctctgcttcttcgccagcgcccgccaggtgctcggcctggagaccggctactggggcaacgccatcttcccggtgaaggtgtccgcggcggcgggggaggtggcggcgtcgtcggtgatcgagctcgtcggcgtggtccgggaggcgaagcggcggatggccggcgagtgcctgcgctgggcggaggggcgcaccggcggcgccgacccgttccagatgacgttcgactacgagtccgtgtacgtgtcggactggagcaagctcgggttcaacgacgtcgactacgggtacggcgcgccgtcggcggcggggccgctggtgaactgcgacctcatctcgtcggtgatcgtcatgcgggcgccggcgccgctcgccggcacgcggctgctggcgagctgcgtcaccaaggagcacgccgacgacttcgccgccaggatgagggaggatctcgtctaatataccatggccgcccccaataacattattagtcacgtacaatattgtcactgaatattaatttgttcgtgtatatctattgtggtatgtattttttttttcataggaaaaaagtagtagtacgacaataggtagctgggagctacctaatatcgacctctggtttgagagtaagtgagcgagcgagagatgtaaaccacctcagtttttaccgtgctttgtgacatgcgtggtacagtactggtactattaatcattggccgtgaaaaattatctaccc</dnaseqindica>|
Link = [http://www.ncbi.nlm.nih.gov/nuccore/NM_001064516.2 RefSeq:Os06g0594600]|
}}
[[Category:Genes]]
[[Category:Japonica mRNA]]
[[Category:Oryza Sativa Japonica Group]]
[[Category:Japonica Genes]]
[[Category:Japonica Chromosome 6]]
[[Category:Chromosome 6]]

Os06g0594600

2014-06-08T14:42:18Z

Xiaoyanjie2012: /* References */

Please input one-sentence summary here.

==Annotated Information==
OsAt10 is a gene in rice(Oryza sativa).Its RAP ID is Os06g0594600,and its MSU ID LOC_Os06g39390. Overexpression of this gene can alters rice cell wall hydroxycinnamic acid Content and sacchariﬁcation.
===Function===
The overexpression of the rice gene -- OsAt10(LOC_Os06g39390) -- can effect the hydroxycinnamic acid content and saccharification in the rice cell wall.rice mutants with altered expression of four of these genes have altered cell wall hydroxycinnamate content. In-depth characterization of ectopic expression lines for one gene, OsAt10, revealed that this modification increases matrix polysaccharideassociated ester-linkedp-CA while simultaneously decreasing matrix polysaccharide-associated FA.OsAt10 overexpression plants exhibit increased in vitro saccharification, with no discernible effects on vegetative development. Thus, this gene is a useful target for improving biofuel and feed production.
Grass cell wall properties influence food, feed, and biofuel feedstock usage efficiency. The glucuronoarabinoxylan of grass cell walls is esterified with the phenylpropanoid-derived hydroxycinnamic acids ferulic acid (FA) and para-coumaric acid (p-CA). Feruloyl esters undergo oxidative coupling with neighboring phenylpropanoids on glucuronoarabinoxylan and lignin. Examination of rice (Oryza sativa) mutants in a grass-expanded and -diverged clade of BAHD acyl coenzyme A-utilizing transferases identified four mutants with altered cell wall FA or p-CA contents.Some researchers reported on the effects of overexpressing one of these genes, OsAt10 (LOC_Os06g39390), in rice. An activation-tagged line, OsAT10-D1, shows a 60% reduction in matrix polysaccharide-bound FA and an approximately 300% increase in p-CA in young leaf tissue but no discernible phenotypic alterations in vegetative development, lignin content, or lignin composition. Two additional independent OsAt10 overexpression lines show similar changes in FA and p-CA content. Cell wall fractionation and liquid chromatography-mass spectrometry experiments isolate the cell wall alterations in the mutant to ester conjugates of a five-carbon sugar with p-CA and FA. These results suggest that OsAT10 is a p-coumaroyl coenzyme A transferase involved in glucuronoarabinoxylan modification. Biomass from OsAT10-D1 exhibits a 20% to 40% increase in saccharification yield depending on the assay. Thus, OsAt10 is an attractive target for improving grass cell wall quality for fuel and animal feed.<ref name="ref1" />
====1.The Mitchell Clade of BAHD Acyltransferases Is Expanded and Diverged in Grasses====
Mitchell et al. (2007) identified what we term the Mitchell clade of BAHD acyl-CoA-dependent acyltransferases on the basis of high gene expression in grasses relative to dicots. To refine the hypothesis that these enzymes might be involved in grass-diverged cell wall synthesis, we systematically characterized the distribution of this clade in selected plant species and compared the clade with other characterized BAHD proteins. We identified BAHD proteins from the genomes of a diverse set of sequenced plant species available at the time of the analysis and examined the phylogenetic relationships among them and a reference set of BAHDs (Table I). To gain higher sensitivity relative to local sequence alignment (i.e. BLAST) for recognizing sequences with low, but potentially still significant, homology, we used a hidden Markov model to identify putative BAHD proteins (Finn et al., 2011)
The researchers then inferred an initial model of the phylo-genetic relationships among the putative BAHD proteins from each genome and the set of biochemically characterized BAHD proteins cataloged by D’Auria (2006). While we are aware that recent analyses have included the presence of a strict HXXXD motif as indicative of whether the protein is an active BAHD (Banks et al., 2011; Tuominen et al., 2011), we have included proteins with single amino acid alterations to this motif, since one of the known biochemically active proteins for the family involved in taxol biosynthesis, BAPT (National Center for Biotechnology Information identifier AAL92459; Walker et al., 2002), possesses a variation of this motif in which the His is replaced by a Ser.
As observed by Tuominen et al. (2011), the distribution of BAHD proteins varies among species (Table I). The Mitchell clade is embedded within clade V, or clade Va of Tuominen et al. (2011). Furthermore, we find that the Mitchell clade includes a biochemically characterized banana (Musa spp.) alcohol CoA acyltransferase, BanAAT (Beekwilder et al., 2004), and is related to a group of BAHD proteins that participate in taxol biosynthesis (Fig. 1B).

[[File:Table 1xxy.png]][[File:figure 1xxy.png]]
====2.Screen of Rice Mutants for Altered Cell Wall Hydroxycinnamic Acid Content====

[[File:Figure 2xxy.png]]

====3.The OsAT10-D1 Line Has an Increase in Cell Wall Glc Content====
Compensatory changes are often seen among the components of the cell wall (Humphrey et al., 2007). Quantification of sugars released by acid treatment of
destarched AIR preparations from mature straw suggests that the Glc content is increased by approximately 20% (w/w) for the mutant relative to the wild type (Fig. 9A). We observed the difference both with TFA treatment, which liberates monosaccharides derived from matrix polysaccharides and amorphous cellulose, and when the TFA residue was further treated with sulfuric acid, which breaks down crystalline cellulose (Fig. 9A). The difference in the mutant compared with the wild type is most apparent when the products of both treatments are summed together, which gives an increase in Glc in the mutant compared with the wild type of 19% 6 11%. By mass, we did not observe any other significant changes in sugar amounts in the mutant compared with the wild type. We also observed no change in the total mass percentage of sugars in AIR. When the TFA-solubilized sugars are expressed in terms of mol%, the data also indicate an increase in Glc content of 11% 6 5% (Fig. 9B). The sum of the mol % of other measured sugars (i.e. Xyl, Ara, and the sum of minor sugars) decreases proportionally to the Glc increase (7% 6 7%). This balance in mol % change suggests that the change in polysaccharide content in the mutant is restricted to the Glc-containing polymers.

[[File:Figure 9xxy.png]]

====4.The OsAT10-D1 Line Shows No Alterations in Lignin Content or Composition====
OsAT10-D1 mature straw samples show no significant differences in the content of acetylbromide-soluble lignin after saponification relative to the wild type
(Table II). We obtained a similar result via py-MBMS for mature straw and separate, young leaf and sheath samples. The py-MBMS also revealed no difference in the syringyl-guaiacyl (S:G) lignin ratio in the mutant compared with the wild type after saponification (Table II). We also collected py-MBMS data for unprocessed straw and AIR of OsAT10-D1. Separate analyses of the saponified and unsaponified samples reveals distinctions between the wild type and mutant in the unsaponified samples (Fig. 10A). Principal component 1 explains the alcohol extraction (30% of the variation), and principal component 2 explains the differences between wild-type and mutant samples (19% of the variation). The loadings for principal component 2 show that the major ions that distinguish wild-type and mutant samples are phenolics (Fig. 10B). The mass spectrometry fragmentation pattern is consistent with an interpretation in which there is an increase of p-CA, as reflected by peaks 120, 94, and 91, and a decrease in FA, as reflected in the reduction in the coniferyl ion, peak 150 (Evans and Milne, 1987). Because principal component analysis no longer distinguishes the samples after saponification (Fig. 10C), the observed differences in phenylpropanoids between OsAT10-D1 and the wild type are likely associated with ester-linked hydroxycinnamates and not lignin, consistent with the other results.
A limitation of the pyrolysis method for determining lignin composition is that it inaccurately measures H-lignin, which volatilizes poorly and instead turns to char upon heating. Because of the increase in p-CA, a precursor of H-lignin, in OsAT10-D1 cell walls relative to the wild type, we sought to determine whether there is a change in the char content of OsAT10-D1 using a thermogravimetric pyrolysis instrument. Duplicate runs per genotype of the thermogravimetric instrument did not detect a difference in the mass remaining from mature straw after pyrolysis, again consistent with there being no difference in core lignin composition or content between OsAT10-D1 and the wild type.

[[File:Table 2xxy.png]]
[[File:Figure 10xxy.png]]

====The OsAT10-D1 Line Shows an Increase in Saccharification====
Ferulate in grass biomass is inversely correlated with digestibility across diverse grass accessions (Lam et al., 2003; Casler and Jung, 2006). The phenotype of the OsAT10-D1 line provided the opportunity to determine whether there is also an increase in enzymatic digestibility with reduced FA content when comparing two near-isogenic plant lines. We found that destarched AIR after mild pretreatment followed by incubation with a cellulase cocktail resulted in the release of approximately 20% more reducing sugar from the mutant compared with the wild type at each time point examined (Fig. 11A)
Acid-pretreated rice straw of the wild type and OsAT10-D1 to the mesophilic fungus, Penicillium sp. YT02. This recently characterized fungus shows significantly higher xylanase and b-glucosidase activities with various insoluble lignocellulosic substrates compared with the commonly used fungal strain, Trichoderma reesei (ATCC 24449; Kovacs et al., 2009; L. Gao and J. Zhou, unpublished data). In the fungal treatments, the biomass-derived sugars initially accumulate but are gradually depleted via incorporation into fungal biomass. Qualitatively consistent with the enzymatic deconstruction results, Penicillium sp. YT02 incubation released 46% more Glc, 82% more Xyl, and 25% more Ara into the medium from OsAT10-D1 straw than from wild-type straw (Fig. 11B). Averaged over the entire time course (12–120 h), the improvement in yield is more dramatic with the fungus than with the simple enzymatic treatment, with a total sugar yield increase of approximately 40%. Cellulase and b-glucosidase enzymatic activities in the slurry are unchanged on the mutant straw (Fig. 11C), suggesting that the fungus grew similarly on both. In contrast, and of relevance to the nature of the change caused by the increased expression of OsAt10, xylanase activity is dramatically enhanced, especially at later time points (Fig. 11C).

[[File:Figure 11xxy.png]]

===Expression===
For the remaining 11 lines, we characterized the alkali-labile hydroxycinnamoyl ester content of cell wall alcohol-insoluble residue (AIR) from leaf blades and sheaths of side tillers. We compared homozygous, mutant, and wild-type segregant plants 7 or 10 weeks after planting. The screen revealed four mutants with possible cell wall hydroxycinnamic acid phenotypes . All four lines showed changes in the expression of the nearest acyltransferase
gene to the T-DNA insertion site via quantitative reverse transcription (qRT)-PCR. Homozygous mutant progeny of4A-03423(here afterreferred to asOsAT10-D1), which has increased expression of OsAt10, exhibited reduced FA (approximately 60% less) and an increase in p-CA (approximately 300% more) in sheaths and leaves.TheT-DNA insertions it efort helinewerefertoas OsAT10-D1(PFG_4A-03423) is approximately 8.5 kb downstream of the transcriptional start site forOsAt10. The insert is oriented with the activating sequences proximate toOsAt10and in the range observed to activate expression (Jeong et al., 2006). As mentioned above, qRT-PCR indicated that, indeed,the expression ofOsAt10was increased by more than 100-fold in the leaves of homozygous OsAT10-D1 plants (Fig. 3B). InOsAT10-D1, besides OsAt10the expression of other genes proximate to the site of the T-DNA insertion does not vary significantly relative to the wild type (Fig. 3B). Similarly, the expression of related OsAtgenesdoesnotvarysignificantly in OsAT10-D1(Supplemental Fig. S2), reducing the possibility that the observed phenotype is due to compensation at the level of gene expression of a related acyltransferase. Of the acyltransferase transcripts examined in this survey, OsAt6appears to vary the most, although not significantly. However, OsAt6(LOC_Os01g08380) is expressed near the lower limitofourdetectionand,infact,wasreportedas undetectable in a previous qRT-PCR study (Piston et al., 2010). OsAT10-D1lines show no change in size and dry mass at maturity (Fig. 4, A and B). However, we did measure an approximately 20% to 30% decrease in total seed mass per plant for the mutant compared with the wild type.

[[File:Figure 3xxy.png]][[File:Figure 4xxy.png]]

===Evolution===
Please input evolution information here.

You can also add sub-section(s) at will.

==Labs working on this gene==
Department of Microbiology and Plant Biology (L.E.B., M.L.P., L.G., F.L., J.Z.), Department of Chemistry and Biochemistry (W.L.S., S.B.F.), and Department of Chemical, Biological, and Materials Engineering (X.Z., R.E.J.),University of Oklahoma, Norman, Oklahoma 73019; Department of Plant Pathology and The Genome Center, University of California, Davis, California 95616 (L.E.B., D.M.C., M.E.V.-S., P.E.C., P.C., S.B., P.C.R.); Joint BioEnergy Institute, Emeryville, California 94608 (L.E.B., B.E., C.M., D.M.C., C.R., M.E.V.-S., P.I.B., P.C., J.D.K., H.V.S., P.C.R.); Crop Biotech Institute and Department of Genetic Engineering, Kyung Hee University, Yongin 446–701, Republic of Korea (S.-R.K., G.A., P.C.R.); Physical Biosciences Division, Lawrence Berkeley National
Laboratory, Berkeley, California 94720 (B.E., C.R., M.E.V.-S., P.I.B., J.D.K., H.V.S., P.C.R.); BioEnergy Sciences Center, National Renewable Energy Laboratory, Golden, Colorado 80401 (R.S., A.Z.); China Tobacco Gene Research Center, Zhengzhou Tobacco Research Institute, Zhengzhou, Henan 450001, China (P.C.); and Department of Chemical and Biomolecular Engineering, Department of Bioengineering, University of California, Berkeley, California 94720 (J.D.K.).

==References==
<references>
<ref name="ref1"> Bartley, L.E., et al., Overexpression of a BAHD acyltransferase, OsAt10, alters rice cell wall hydroxycinnamic acid content and saccharification. Plant physiology, 2013. 161(4): p. 1615-1633.

==Structured Information==
{{JaponicaGene|
GeneName = Os06g0594600|
Description = The start codon is not identified.|
Version = NM_001064516.2 GI:297606110 GeneID:4341427|
Length = 1126 bp|
Definition = Oryza sativa Japonica Group Os06g0594600, complete gene.|
Source = Oryza sativa Japonica Group

ORGANISM Oryza sativa Japonica Group
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP
clade; Ehrhartoideae; Oryzeae; Oryza.
|
Chromosome = [[:category:Japonica Chromosome 6|Chromosome 6]]|
AP = Chromosome 6:24256206..24257331|
CDS = 24256486..24257331|
GCID = <gbrowseImage1>
name=NC_008399:24256206..24257331
source=RiceChromosome06
preset=GeneLocation
</gbrowseImage1>|
GSID = <gbrowseImage2>
name=NC_008399:24256206..24257331
source=RiceChromosome06
preset=GeneLocation
</gbrowseImage2>|
CDNA = <cdnaseq>gatttgcaggtgacgacgttcacctgcggcggcttcgtgatcgggctgcgcaccaaccacgcggtggcggacggcaccggcgccgcccagttcatgaacgccgtcggcgacctcgcccgcggcctcccggagccgcgggtgaagccgatctgggcgcgcgaccgcttcccggacccggacatcaagcccggcccgctgccggagctccccgtgctgccgctccagtacatcgccttcgacttccccgccgcctacctcggcaagctcaaggcgcagtacgccgccaccgccggcgccagcaagatctgctccgccttcgacatcgtcatcgccaagctctggcagtgccggacgcgcgccatcgccgccgaccccgccgcggccgtcaagctctgcttcttcgccagcgcccgccaggtgctcggcctggagaccggctactggggcaacgccatcttcccggtgaaggtgtccgcggcggcgggggaggtggcggcgtcgtcggtgatcgagctcgtcggcgtggtccgggaggcgaagcggcggatggccggcgagtgcctgcgctgggcggaggggcgcaccggcggcgccgacccgttccagatgacgttcgactacgagtccgtgtacgtgtcggactggagcaagctcgggttcaacgacgtcgactacgggtacggcgcgccgtcggcggcggggccgctggtgaactgcgacctcatctcgtcggtgatcgtcatgcgggcgccggcgccgctcgccggcacgcggctgctggcgagctgcgtcaccaaggagcacgccgacgacttcgccgccaggatgagggaggatctcgtctaa</cdnaseq>|
AA = <aaseq>DLQVTTFTCGGFVIGLRTNHAVADGTGAAQFMNAVGDLARGLPE PRVKPIWARDRFPDPDIKPGPLPELPVLPLQYIAFDFPAAYLGKLKAQYAATAGASKI CSAFDIVIAKLWQCRTRAIAADPAAAVKLCFFASARQVLGLETGYWGNAIFPVKVSAA AGEVAASSVIELVGVVREAKRRMAGECLRWAEGRTGGADPFQMTFDYESVYVSDWSKL GFNDVDYGYGAPSAAGPLVNCDLISSVIVMRAPAPLAGTRLLASCVTKEHADDFAARM REDLV</aaseq>|
DNA = <dnaseqindica>1..846#gatttgcaggtgacgacgttcacctgcggcggcttcgtgatcgggctgcgcaccaaccacgcggtggcggacggcaccggcgccgcccagttcatgaacgccgtcggcgacctcgcccgcggcctcccggagccgcgggtgaagccgatctgggcgcgcgaccgcttcccggacccggacatcaagcccggcccgctgccggagctccccgtgctgccgctccagtacatcgccttcgacttccccgccgcctacctcggcaagctcaaggcgcagtacgccgccaccgccggcgccagcaagatctgctccgccttcgacatcgtcatcgccaagctctggcagtgccggacgcgcgccatcgccgccgaccccgccgcggccgtcaagctctgcttcttcgccagcgcccgccaggtgctcggcctggagaccggctactggggcaacgccatcttcccggtgaaggtgtccgcggcggcgggggaggtggcggcgtcgtcggtgatcgagctcgtcggcgtggtccgggaggcgaagcggcggatggccggcgagtgcctgcgctgggcggaggggcgcaccggcggcgccgacccgttccagatgacgttcgactacgagtccgtgtacgtgtcggactggagcaagctcgggttcaacgacgtcgactacgggtacggcgcgccgtcggcggcggggccgctggtgaactgcgacctcatctcgtcggtgatcgtcatgcgggcgccggcgccgctcgccggcacgcggctgctggcgagctgcgtcaccaaggagcacgccgacgacttcgccgccaggatgagggaggatctcgtctaatataccatggccgcccccaataacattattagtcacgtacaatattgtcactgaatattaatttgttcgtgtatatctattgtggtatgtattttttttttcataggaaaaaagtagtagtacgacaataggtagctgggagctacctaatatcgacctctggtttgagagtaagtgagcgagcgagagatgtaaaccacctcagtttttaccgtgctttgtgacatgcgtggtacagtactggtactattaatcattggccgtgaaaaattatctaccc</dnaseqindica>|
Link = [http://www.ncbi.nlm.nih.gov/nuccore/NM_001064516.2 RefSeq:Os06g0594600]|
}}
[[Category:Genes]]
[[Category:Japonica mRNA]]
[[Category:Oryza Sativa Japonica Group]]
[[Category:Japonica Genes]]
[[Category:Japonica Chromosome 6]]
[[Category:Chromosome 6]]

Os06g0594600

2014-06-08T14:38:37Z

Xiaoyanjie2012: /* References */

Please input one-sentence summary here.

==Annotated Information==
OsAt10 is a gene in rice(Oryza sativa).Its RAP ID is Os06g0594600,and its MSU ID LOC_Os06g39390. Overexpression of this gene can alters rice cell wall hydroxycinnamic acid Content and sacchariﬁcation.
===Function===
The overexpression of the rice gene -- OsAt10(LOC_Os06g39390) -- can effect the hydroxycinnamic acid content and saccharification in the rice cell wall.rice mutants with altered expression of four of these genes have altered cell wall hydroxycinnamate content. In-depth characterization of ectopic expression lines for one gene, OsAt10, revealed that this modification increases matrix polysaccharideassociated ester-linkedp-CA while simultaneously decreasing matrix polysaccharide-associated FA.OsAt10 overexpression plants exhibit increased in vitro saccharification, with no discernible effects on vegetative development. Thus, this gene is a useful target for improving biofuel and feed production.
Grass cell wall properties influence food, feed, and biofuel feedstock usage efficiency. The glucuronoarabinoxylan of grass cell walls is esterified with the phenylpropanoid-derived hydroxycinnamic acids ferulic acid (FA) and para-coumaric acid (p-CA). Feruloyl esters undergo oxidative coupling with neighboring phenylpropanoids on glucuronoarabinoxylan and lignin. Examination of rice (Oryza sativa) mutants in a grass-expanded and -diverged clade of BAHD acyl coenzyme A-utilizing transferases identified four mutants with altered cell wall FA or p-CA contents.Some researchers reported on the effects of overexpressing one of these genes, OsAt10 (LOC_Os06g39390), in rice. An activation-tagged line, OsAT10-D1, shows a 60% reduction in matrix polysaccharide-bound FA and an approximately 300% increase in p-CA in young leaf tissue but no discernible phenotypic alterations in vegetative development, lignin content, or lignin composition. Two additional independent OsAt10 overexpression lines show similar changes in FA and p-CA content. Cell wall fractionation and liquid chromatography-mass spectrometry experiments isolate the cell wall alterations in the mutant to ester conjugates of a five-carbon sugar with p-CA and FA. These results suggest that OsAT10 is a p-coumaroyl coenzyme A transferase involved in glucuronoarabinoxylan modification. Biomass from OsAT10-D1 exhibits a 20% to 40% increase in saccharification yield depending on the assay. Thus, OsAt10 is an attractive target for improving grass cell wall quality for fuel and animal feed.<ref name="ref1" />
====1.The Mitchell Clade of BAHD Acyltransferases Is Expanded and Diverged in Grasses====
Mitchell et al. (2007) identified what we term the Mitchell clade of BAHD acyl-CoA-dependent acyltransferases on the basis of high gene expression in grasses relative to dicots. To refine the hypothesis that these enzymes might be involved in grass-diverged cell wall synthesis, we systematically characterized the distribution of this clade in selected plant species and compared the clade with other characterized BAHD proteins. We identified BAHD proteins from the genomes of a diverse set of sequenced plant species available at the time of the analysis and examined the phylogenetic relationships among them and a reference set of BAHDs (Table I). To gain higher sensitivity relative to local sequence alignment (i.e. BLAST) for recognizing sequences with low, but potentially still significant, homology, we used a hidden Markov model to identify putative BAHD proteins (Finn et al., 2011)
The researchers then inferred an initial model of the phylo-genetic relationships among the putative BAHD proteins from each genome and the set of biochemically characterized BAHD proteins cataloged by D’Auria (2006). While we are aware that recent analyses have included the presence of a strict HXXXD motif as indicative of whether the protein is an active BAHD (Banks et al., 2011; Tuominen et al., 2011), we have included proteins with single amino acid alterations to this motif, since one of the known biochemically active proteins for the family involved in taxol biosynthesis, BAPT (National Center for Biotechnology Information identifier AAL92459; Walker et al., 2002), possesses a variation of this motif in which the His is replaced by a Ser.
As observed by Tuominen et al. (2011), the distribution of BAHD proteins varies among species (Table I). The Mitchell clade is embedded within clade V, or clade Va of Tuominen et al. (2011). Furthermore, we find that the Mitchell clade includes a biochemically characterized banana (Musa spp.) alcohol CoA acyltransferase, BanAAT (Beekwilder et al., 2004), and is related to a group of BAHD proteins that participate in taxol biosynthesis (Fig. 1B).

[[File:Table 1xxy.png]][[File:figure 1xxy.png]]
====2.Screen of Rice Mutants for Altered Cell Wall Hydroxycinnamic Acid Content====

[[File:Figure 2xxy.png]]

====3.The OsAT10-D1 Line Has an Increase in Cell Wall Glc Content====
Compensatory changes are often seen among the components of the cell wall (Humphrey et al., 2007). Quantification of sugars released by acid treatment of
destarched AIR preparations from mature straw suggests that the Glc content is increased by approximately 20% (w/w) for the mutant relative to the wild type (Fig. 9A). We observed the difference both with TFA treatment, which liberates monosaccharides derived from matrix polysaccharides and amorphous cellulose, and when the TFA residue was further treated with sulfuric acid, which breaks down crystalline cellulose (Fig. 9A). The difference in the mutant compared with the wild type is most apparent when the products of both treatments are summed together, which gives an increase in Glc in the mutant compared with the wild type of 19% 6 11%. By mass, we did not observe any other significant changes in sugar amounts in the mutant compared with the wild type. We also observed no change in the total mass percentage of sugars in AIR. When the TFA-solubilized sugars are expressed in terms of mol%, the data also indicate an increase in Glc content of 11% 6 5% (Fig. 9B). The sum of the mol % of other measured sugars (i.e. Xyl, Ara, and the sum of minor sugars) decreases proportionally to the Glc increase (7% 6 7%). This balance in mol % change suggests that the change in polysaccharide content in the mutant is restricted to the Glc-containing polymers.

[[File:Figure 9xxy.png]]

====4.The OsAT10-D1 Line Shows No Alterations in Lignin Content or Composition====
OsAT10-D1 mature straw samples show no significant differences in the content of acetylbromide-soluble lignin after saponification relative to the wild type
(Table II). We obtained a similar result via py-MBMS for mature straw and separate, young leaf and sheath samples. The py-MBMS also revealed no difference in the syringyl-guaiacyl (S:G) lignin ratio in the mutant compared with the wild type after saponification (Table II). We also collected py-MBMS data for unprocessed straw and AIR of OsAT10-D1. Separate analyses of the saponified and unsaponified samples reveals distinctions between the wild type and mutant in the unsaponified samples (Fig. 10A). Principal component 1 explains the alcohol extraction (30% of the variation), and principal component 2 explains the differences between wild-type and mutant samples (19% of the variation). The loadings for principal component 2 show that the major ions that distinguish wild-type and mutant samples are phenolics (Fig. 10B). The mass spectrometry fragmentation pattern is consistent with an interpretation in which there is an increase of p-CA, as reflected by peaks 120, 94, and 91, and a decrease in FA, as reflected in the reduction in the coniferyl ion, peak 150 (Evans and Milne, 1987). Because principal component analysis no longer distinguishes the samples after saponification (Fig. 10C), the observed differences in phenylpropanoids between OsAT10-D1 and the wild type are likely associated with ester-linked hydroxycinnamates and not lignin, consistent with the other results.
A limitation of the pyrolysis method for determining lignin composition is that it inaccurately measures H-lignin, which volatilizes poorly and instead turns to char upon heating. Because of the increase in p-CA, a precursor of H-lignin, in OsAT10-D1 cell walls relative to the wild type, we sought to determine whether there is a change in the char content of OsAT10-D1 using a thermogravimetric pyrolysis instrument. Duplicate runs per genotype of the thermogravimetric instrument did not detect a difference in the mass remaining from mature straw after pyrolysis, again consistent with there being no difference in core lignin composition or content between OsAT10-D1 and the wild type.

[[File:Table 2xxy.png]]
[[File:Figure 10xxy.png]]

====The OsAT10-D1 Line Shows an Increase in Saccharification====
Ferulate in grass biomass is inversely correlated with digestibility across diverse grass accessions (Lam et al., 2003; Casler and Jung, 2006). The phenotype of the OsAT10-D1 line provided the opportunity to determine whether there is also an increase in enzymatic digestibility with reduced FA content when comparing two near-isogenic plant lines. We found that destarched AIR after mild pretreatment followed by incubation with a cellulase cocktail resulted in the release of approximately 20% more reducing sugar from the mutant compared with the wild type at each time point examined (Fig. 11A)
Acid-pretreated rice straw of the wild type and OsAT10-D1 to the mesophilic fungus, Penicillium sp. YT02. This recently characterized fungus shows significantly higher xylanase and b-glucosidase activities with various insoluble lignocellulosic substrates compared with the commonly used fungal strain, Trichoderma reesei (ATCC 24449; Kovacs et al., 2009; L. Gao and J. Zhou, unpublished data). In the fungal treatments, the biomass-derived sugars initially accumulate but are gradually depleted via incorporation into fungal biomass. Qualitatively consistent with the enzymatic deconstruction results, Penicillium sp. YT02 incubation released 46% more Glc, 82% more Xyl, and 25% more Ara into the medium from OsAT10-D1 straw than from wild-type straw (Fig. 11B). Averaged over the entire time course (12–120 h), the improvement in yield is more dramatic with the fungus than with the simple enzymatic treatment, with a total sugar yield increase of approximately 40%. Cellulase and b-glucosidase enzymatic activities in the slurry are unchanged on the mutant straw (Fig. 11C), suggesting that the fungus grew similarly on both. In contrast, and of relevance to the nature of the change caused by the increased expression of OsAt10, xylanase activity is dramatically enhanced, especially at later time points (Fig. 11C).

[[File:Figure 11xxy.png]]

===Expression===
For the remaining 11 lines, we characterized the alkali-labile hydroxycinnamoyl ester content of cell wall alcohol-insoluble residue (AIR) from leaf blades and sheaths of side tillers. We compared homozygous, mutant, and wild-type segregant plants 7 or 10 weeks after planting. The screen revealed four mutants with possible cell wall hydroxycinnamic acid phenotypes . All four lines showed changes in the expression of the nearest acyltransferase
gene to the T-DNA insertion site via quantitative reverse transcription (qRT)-PCR. Homozygous mutant progeny of4A-03423(here afterreferred to asOsAT10-D1), which has increased expression of OsAt10, exhibited reduced FA (approximately 60% less) and an increase in p-CA (approximately 300% more) in sheaths and leaves.TheT-DNA insertions it efort helinewerefertoas OsAT10-D1(PFG_4A-03423) is approximately 8.5 kb downstream of the transcriptional start site forOsAt10. The insert is oriented with the activating sequences proximate toOsAt10and in the range observed to activate expression (Jeong et al., 2006). As mentioned above, qRT-PCR indicated that, indeed,the expression ofOsAt10was increased by more than 100-fold in the leaves of homozygous OsAT10-D1 plants (Fig. 3B). InOsAT10-D1, besides OsAt10the expression of other genes proximate to the site of the T-DNA insertion does not vary significantly relative to the wild type (Fig. 3B). Similarly, the expression of related OsAtgenesdoesnotvarysignificantly in OsAT10-D1(Supplemental Fig. S2), reducing the possibility that the observed phenotype is due to compensation at the level of gene expression of a related acyltransferase. Of the acyltransferase transcripts examined in this survey, OsAt6appears to vary the most, although not significantly. However, OsAt6(LOC_Os01g08380) is expressed near the lower limitofourdetectionand,infact,wasreportedas undetectable in a previous qRT-PCR study (Piston et al., 2010). OsAT10-D1lines show no change in size and dry mass at maturity (Fig. 4, A and B). However, we did measure an approximately 20% to 30% decrease in total seed mass per plant for the mutant compared with the wild type.

[[File:Figure 3xxy.png]][[File:Figure 4xxy.png]]

===Evolution===
Please input evolution information here.

You can also add sub-section(s) at will.

==Labs working on this gene==
Department of Microbiology and Plant Biology (L.E.B., M.L.P., L.G., F.L., J.Z.), Department of Chemistry and Biochemistry (W.L.S., S.B.F.), and Department of Chemical, Biological, and Materials Engineering (X.Z., R.E.J.),University of Oklahoma, Norman, Oklahoma 73019; Department of Plant Pathology and The Genome Center, University of California, Davis, California 95616 (L.E.B., D.M.C., M.E.V.-S., P.E.C., P.C., S.B., P.C.R.); Joint BioEnergy Institute, Emeryville, California 94608 (L.E.B., B.E., C.M., D.M.C., C.R., M.E.V.-S., P.I.B., P.C., J.D.K., H.V.S., P.C.R.); Crop Biotech Institute and Department of Genetic Engineering, Kyung Hee University, Yongin 446–701, Republic of Korea (S.-R.K., G.A., P.C.R.); Physical Biosciences Division, Lawrence Berkeley National
Laboratory, Berkeley, California 94720 (B.E., C.R., M.E.V.-S., P.I.B., J.D.K., H.V.S., P.C.R.); BioEnergy Sciences Center, National Renewable Energy Laboratory, Golden, Colorado 80401 (R.S., A.Z.); China Tobacco Gene Research Center, Zhengzhou Tobacco Research Institute, Zhengzhou, Henan 450001, China (P.C.); and Department of Chemical and Biomolecular Engineering, Department of Bioengineering, University of California, Berkeley, California 94720 (J.D.K.).

==References==
[[1]]. Bartley, L.E., et al., Overexpression of a BAHD acyltransferase, OsAt10, alters rice cell wall hydroxycinnamic acid content and saccharification. Plant physiology, 2013. 161(4): p. 1615-1633.

==Structured Information==
{{JaponicaGene|
GeneName = Os06g0594600|
Description = The start codon is not identified.|
Version = NM_001064516.2 GI:297606110 GeneID:4341427|
Length = 1126 bp|
Definition = Oryza sativa Japonica Group Os06g0594600, complete gene.|
Source = Oryza sativa Japonica Group

ORGANISM Oryza sativa Japonica Group
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP
clade; Ehrhartoideae; Oryzeae; Oryza.
|
Chromosome = [[:category:Japonica Chromosome 6|Chromosome 6]]|
AP = Chromosome 6:24256206..24257331|
CDS = 24256486..24257331|
GCID = <gbrowseImage1>
name=NC_008399:24256206..24257331
source=RiceChromosome06
preset=GeneLocation
</gbrowseImage1>|
GSID = <gbrowseImage2>
name=NC_008399:24256206..24257331
source=RiceChromosome06
preset=GeneLocation
</gbrowseImage2>|
CDNA = <cdnaseq>gatttgcaggtgacgacgttcacctgcggcggcttcgtgatcgggctgcgcaccaaccacgcggtggcggacggcaccggcgccgcccagttcatgaacgccgtcggcgacctcgcccgcggcctcccggagccgcgggtgaagccgatctgggcgcgcgaccgcttcccggacccggacatcaagcccggcccgctgccggagctccccgtgctgccgctccagtacatcgccttcgacttccccgccgcctacctcggcaagctcaaggcgcagtacgccgccaccgccggcgccagcaagatctgctccgccttcgacatcgtcatcgccaagctctggcagtgccggacgcgcgccatcgccgccgaccccgccgcggccgtcaagctctgcttcttcgccagcgcccgccaggtgctcggcctggagaccggctactggggcaacgccatcttcccggtgaaggtgtccgcggcggcgggggaggtggcggcgtcgtcggtgatcgagctcgtcggcgtggtccgggaggcgaagcggcggatggccggcgagtgcctgcgctgggcggaggggcgcaccggcggcgccgacccgttccagatgacgttcgactacgagtccgtgtacgtgtcggactggagcaagctcgggttcaacgacgtcgactacgggtacggcgcgccgtcggcggcggggccgctggtgaactgcgacctcatctcgtcggtgatcgtcatgcgggcgccggcgccgctcgccggcacgcggctgctggcgagctgcgtcaccaaggagcacgccgacgacttcgccgccaggatgagggaggatctcgtctaa</cdnaseq>|
AA = <aaseq>DLQVTTFTCGGFVIGLRTNHAVADGTGAAQFMNAVGDLARGLPE PRVKPIWARDRFPDPDIKPGPLPELPVLPLQYIAFDFPAAYLGKLKAQYAATAGASKI CSAFDIVIAKLWQCRTRAIAADPAAAVKLCFFASARQVLGLETGYWGNAIFPVKVSAA AGEVAASSVIELVGVVREAKRRMAGECLRWAEGRTGGADPFQMTFDYESVYVSDWSKL GFNDVDYGYGAPSAAGPLVNCDLISSVIVMRAPAPLAGTRLLASCVTKEHADDFAARM REDLV</aaseq>|
DNA = <dnaseqindica>1..846#gatttgcaggtgacgacgttcacctgcggcggcttcgtgatcgggctgcgcaccaaccacgcggtggcggacggcaccggcgccgcccagttcatgaacgccgtcggcgacctcgcccgcggcctcccggagccgcgggtgaagccgatctgggcgcgcgaccgcttcccggacccggacatcaagcccggcccgctgccggagctccccgtgctgccgctccagtacatcgccttcgacttccccgccgcctacctcggcaagctcaaggcgcagtacgccgccaccgccggcgccagcaagatctgctccgccttcgacatcgtcatcgccaagctctggcagtgccggacgcgcgccatcgccgccgaccccgccgcggccgtcaagctctgcttcttcgccagcgcccgccaggtgctcggcctggagaccggctactggggcaacgccatcttcccggtgaaggtgtccgcggcggcgggggaggtggcggcgtcgtcggtgatcgagctcgtcggcgtggtccgggaggcgaagcggcggatggccggcgagtgcctgcgctgggcggaggggcgcaccggcggcgccgacccgttccagatgacgttcgactacgagtccgtgtacgtgtcggactggagcaagctcgggttcaacgacgtcgactacgggtacggcgcgccgtcggcggcggggccgctggtgaactgcgacctcatctcgtcggtgatcgtcatgcgggcgccggcgccgctcgccggcacgcggctgctggcgagctgcgtcaccaaggagcacgccgacgacttcgccgccaggatgagggaggatctcgtctaatataccatggccgcccccaataacattattagtcacgtacaatattgtcactgaatattaatttgttcgtgtatatctattgtggtatgtattttttttttcataggaaaaaagtagtagtacgacaataggtagctgggagctacctaatatcgacctctggtttgagagtaagtgagcgagcgagagatgtaaaccacctcagtttttaccgtgctttgtgacatgcgtggtacagtactggtactattaatcattggccgtgaaaaattatctaccc</dnaseqindica>|
Link = [http://www.ncbi.nlm.nih.gov/nuccore/NM_001064516.2 RefSeq:Os06g0594600]|
}}
[[Category:Genes]]
[[Category:Japonica mRNA]]
[[Category:Oryza Sativa Japonica Group]]
[[Category:Japonica Genes]]
[[Category:Japonica Chromosome 6]]
[[Category:Chromosome 6]]

Os06g0594600

2014-06-08T14:37:20Z

Xiaoyanjie2012: /* Labs working on this gene */

Please input one-sentence summary here.

==Annotated Information==
OsAt10 is a gene in rice(Oryza sativa).Its RAP ID is Os06g0594600,and its MSU ID LOC_Os06g39390. Overexpression of this gene can alters rice cell wall hydroxycinnamic acid Content and sacchariﬁcation.
===Function===
The overexpression of the rice gene -- OsAt10(LOC_Os06g39390) -- can effect the hydroxycinnamic acid content and saccharification in the rice cell wall.rice mutants with altered expression of four of these genes have altered cell wall hydroxycinnamate content. In-depth characterization of ectopic expression lines for one gene, OsAt10, revealed that this modification increases matrix polysaccharideassociated ester-linkedp-CA while simultaneously decreasing matrix polysaccharide-associated FA.OsAt10 overexpression plants exhibit increased in vitro saccharification, with no discernible effects on vegetative development. Thus, this gene is a useful target for improving biofuel and feed production.
Grass cell wall properties influence food, feed, and biofuel feedstock usage efficiency. The glucuronoarabinoxylan of grass cell walls is esterified with the phenylpropanoid-derived hydroxycinnamic acids ferulic acid (FA) and para-coumaric acid (p-CA). Feruloyl esters undergo oxidative coupling with neighboring phenylpropanoids on glucuronoarabinoxylan and lignin. Examination of rice (Oryza sativa) mutants in a grass-expanded and -diverged clade of BAHD acyl coenzyme A-utilizing transferases identified four mutants with altered cell wall FA or p-CA contents.Some researchers reported on the effects of overexpressing one of these genes, OsAt10 (LOC_Os06g39390), in rice. An activation-tagged line, OsAT10-D1, shows a 60% reduction in matrix polysaccharide-bound FA and an approximately 300% increase in p-CA in young leaf tissue but no discernible phenotypic alterations in vegetative development, lignin content, or lignin composition. Two additional independent OsAt10 overexpression lines show similar changes in FA and p-CA content. Cell wall fractionation and liquid chromatography-mass spectrometry experiments isolate the cell wall alterations in the mutant to ester conjugates of a five-carbon sugar with p-CA and FA. These results suggest that OsAT10 is a p-coumaroyl coenzyme A transferase involved in glucuronoarabinoxylan modification. Biomass from OsAT10-D1 exhibits a 20% to 40% increase in saccharification yield depending on the assay. Thus, OsAt10 is an attractive target for improving grass cell wall quality for fuel and animal feed.<ref name="ref1" />
====1.The Mitchell Clade of BAHD Acyltransferases Is Expanded and Diverged in Grasses====
Mitchell et al. (2007) identified what we term the Mitchell clade of BAHD acyl-CoA-dependent acyltransferases on the basis of high gene expression in grasses relative to dicots. To refine the hypothesis that these enzymes might be involved in grass-diverged cell wall synthesis, we systematically characterized the distribution of this clade in selected plant species and compared the clade with other characterized BAHD proteins. We identified BAHD proteins from the genomes of a diverse set of sequenced plant species available at the time of the analysis and examined the phylogenetic relationships among them and a reference set of BAHDs (Table I). To gain higher sensitivity relative to local sequence alignment (i.e. BLAST) for recognizing sequences with low, but potentially still significant, homology, we used a hidden Markov model to identify putative BAHD proteins (Finn et al., 2011)
The researchers then inferred an initial model of the phylo-genetic relationships among the putative BAHD proteins from each genome and the set of biochemically characterized BAHD proteins cataloged by D’Auria (2006). While we are aware that recent analyses have included the presence of a strict HXXXD motif as indicative of whether the protein is an active BAHD (Banks et al., 2011; Tuominen et al., 2011), we have included proteins with single amino acid alterations to this motif, since one of the known biochemically active proteins for the family involved in taxol biosynthesis, BAPT (National Center for Biotechnology Information identifier AAL92459; Walker et al., 2002), possesses a variation of this motif in which the His is replaced by a Ser.
As observed by Tuominen et al. (2011), the distribution of BAHD proteins varies among species (Table I). The Mitchell clade is embedded within clade V, or clade Va of Tuominen et al. (2011). Furthermore, we find that the Mitchell clade includes a biochemically characterized banana (Musa spp.) alcohol CoA acyltransferase, BanAAT (Beekwilder et al., 2004), and is related to a group of BAHD proteins that participate in taxol biosynthesis (Fig. 1B).

[[File:Table 1xxy.png]][[File:figure 1xxy.png]]
====2.Screen of Rice Mutants for Altered Cell Wall Hydroxycinnamic Acid Content====

[[File:Figure 2xxy.png]]

====3.The OsAT10-D1 Line Has an Increase in Cell Wall Glc Content====
Compensatory changes are often seen among the components of the cell wall (Humphrey et al., 2007). Quantification of sugars released by acid treatment of
destarched AIR preparations from mature straw suggests that the Glc content is increased by approximately 20% (w/w) for the mutant relative to the wild type (Fig. 9A). We observed the difference both with TFA treatment, which liberates monosaccharides derived from matrix polysaccharides and amorphous cellulose, and when the TFA residue was further treated with sulfuric acid, which breaks down crystalline cellulose (Fig. 9A). The difference in the mutant compared with the wild type is most apparent when the products of both treatments are summed together, which gives an increase in Glc in the mutant compared with the wild type of 19% 6 11%. By mass, we did not observe any other significant changes in sugar amounts in the mutant compared with the wild type. We also observed no change in the total mass percentage of sugars in AIR. When the TFA-solubilized sugars are expressed in terms of mol%, the data also indicate an increase in Glc content of 11% 6 5% (Fig. 9B). The sum of the mol % of other measured sugars (i.e. Xyl, Ara, and the sum of minor sugars) decreases proportionally to the Glc increase (7% 6 7%). This balance in mol % change suggests that the change in polysaccharide content in the mutant is restricted to the Glc-containing polymers.

[[File:Figure 9xxy.png]]

====4.The OsAT10-D1 Line Shows No Alterations in Lignin Content or Composition====
OsAT10-D1 mature straw samples show no significant differences in the content of acetylbromide-soluble lignin after saponification relative to the wild type
(Table II). We obtained a similar result via py-MBMS for mature straw and separate, young leaf and sheath samples. The py-MBMS also revealed no difference in the syringyl-guaiacyl (S:G) lignin ratio in the mutant compared with the wild type after saponification (Table II). We also collected py-MBMS data for unprocessed straw and AIR of OsAT10-D1. Separate analyses of the saponified and unsaponified samples reveals distinctions between the wild type and mutant in the unsaponified samples (Fig. 10A). Principal component 1 explains the alcohol extraction (30% of the variation), and principal component 2 explains the differences between wild-type and mutant samples (19% of the variation). The loadings for principal component 2 show that the major ions that distinguish wild-type and mutant samples are phenolics (Fig. 10B). The mass spectrometry fragmentation pattern is consistent with an interpretation in which there is an increase of p-CA, as reflected by peaks 120, 94, and 91, and a decrease in FA, as reflected in the reduction in the coniferyl ion, peak 150 (Evans and Milne, 1987). Because principal component analysis no longer distinguishes the samples after saponification (Fig. 10C), the observed differences in phenylpropanoids between OsAT10-D1 and the wild type are likely associated with ester-linked hydroxycinnamates and not lignin, consistent with the other results.
A limitation of the pyrolysis method for determining lignin composition is that it inaccurately measures H-lignin, which volatilizes poorly and instead turns to char upon heating. Because of the increase in p-CA, a precursor of H-lignin, in OsAT10-D1 cell walls relative to the wild type, we sought to determine whether there is a change in the char content of OsAT10-D1 using a thermogravimetric pyrolysis instrument. Duplicate runs per genotype of the thermogravimetric instrument did not detect a difference in the mass remaining from mature straw after pyrolysis, again consistent with there being no difference in core lignin composition or content between OsAT10-D1 and the wild type.

[[File:Table 2xxy.png]]
[[File:Figure 10xxy.png]]

====The OsAT10-D1 Line Shows an Increase in Saccharification====
Ferulate in grass biomass is inversely correlated with digestibility across diverse grass accessions (Lam et al., 2003; Casler and Jung, 2006). The phenotype of the OsAT10-D1 line provided the opportunity to determine whether there is also an increase in enzymatic digestibility with reduced FA content when comparing two near-isogenic plant lines. We found that destarched AIR after mild pretreatment followed by incubation with a cellulase cocktail resulted in the release of approximately 20% more reducing sugar from the mutant compared with the wild type at each time point examined (Fig. 11A)
Acid-pretreated rice straw of the wild type and OsAT10-D1 to the mesophilic fungus, Penicillium sp. YT02. This recently characterized fungus shows significantly higher xylanase and b-glucosidase activities with various insoluble lignocellulosic substrates compared with the commonly used fungal strain, Trichoderma reesei (ATCC 24449; Kovacs et al., 2009; L. Gao and J. Zhou, unpublished data). In the fungal treatments, the biomass-derived sugars initially accumulate but are gradually depleted via incorporation into fungal biomass. Qualitatively consistent with the enzymatic deconstruction results, Penicillium sp. YT02 incubation released 46% more Glc, 82% more Xyl, and 25% more Ara into the medium from OsAT10-D1 straw than from wild-type straw (Fig. 11B). Averaged over the entire time course (12–120 h), the improvement in yield is more dramatic with the fungus than with the simple enzymatic treatment, with a total sugar yield increase of approximately 40%. Cellulase and b-glucosidase enzymatic activities in the slurry are unchanged on the mutant straw (Fig. 11C), suggesting that the fungus grew similarly on both. In contrast, and of relevance to the nature of the change caused by the increased expression of OsAt10, xylanase activity is dramatically enhanced, especially at later time points (Fig. 11C).

[[File:Figure 11xxy.png]]

===Expression===
For the remaining 11 lines, we characterized the alkali-labile hydroxycinnamoyl ester content of cell wall alcohol-insoluble residue (AIR) from leaf blades and sheaths of side tillers. We compared homozygous, mutant, and wild-type segregant plants 7 or 10 weeks after planting. The screen revealed four mutants with possible cell wall hydroxycinnamic acid phenotypes . All four lines showed changes in the expression of the nearest acyltransferase
gene to the T-DNA insertion site via quantitative reverse transcription (qRT)-PCR. Homozygous mutant progeny of4A-03423(here afterreferred to asOsAT10-D1), which has increased expression of OsAt10, exhibited reduced FA (approximately 60% less) and an increase in p-CA (approximately 300% more) in sheaths and leaves.TheT-DNA insertions it efort helinewerefertoas OsAT10-D1(PFG_4A-03423) is approximately 8.5 kb downstream of the transcriptional start site forOsAt10. The insert is oriented with the activating sequences proximate toOsAt10and in the range observed to activate expression (Jeong et al., 2006). As mentioned above, qRT-PCR indicated that, indeed,the expression ofOsAt10was increased by more than 100-fold in the leaves of homozygous OsAT10-D1 plants (Fig. 3B). InOsAT10-D1, besides OsAt10the expression of other genes proximate to the site of the T-DNA insertion does not vary significantly relative to the wild type (Fig. 3B). Similarly, the expression of related OsAtgenesdoesnotvarysignificantly in OsAT10-D1(Supplemental Fig. S2), reducing the possibility that the observed phenotype is due to compensation at the level of gene expression of a related acyltransferase. Of the acyltransferase transcripts examined in this survey, OsAt6appears to vary the most, although not significantly. However, OsAt6(LOC_Os01g08380) is expressed near the lower limitofourdetectionand,infact,wasreportedas undetectable in a previous qRT-PCR study (Piston et al., 2010). OsAT10-D1lines show no change in size and dry mass at maturity (Fig. 4, A and B). However, we did measure an approximately 20% to 30% decrease in total seed mass per plant for the mutant compared with the wild type.

[[File:Figure 3xxy.png]][[File:Figure 4xxy.png]]

===Evolution===
Please input evolution information here.

You can also add sub-section(s) at will.

==Labs working on this gene==
Department of Microbiology and Plant Biology (L.E.B., M.L.P., L.G., F.L., J.Z.), Department of Chemistry and Biochemistry (W.L.S., S.B.F.), and Department of Chemical, Biological, and Materials Engineering (X.Z., R.E.J.),University of Oklahoma, Norman, Oklahoma 73019; Department of Plant Pathology and The Genome Center, University of California, Davis, California 95616 (L.E.B., D.M.C., M.E.V.-S., P.E.C., P.C., S.B., P.C.R.); Joint BioEnergy Institute, Emeryville, California 94608 (L.E.B., B.E., C.M., D.M.C., C.R., M.E.V.-S., P.I.B., P.C., J.D.K., H.V.S., P.C.R.); Crop Biotech Institute and Department of Genetic Engineering, Kyung Hee University, Yongin 446–701, Republic of Korea (S.-R.K., G.A., P.C.R.); Physical Biosciences Division, Lawrence Berkeley National
Laboratory, Berkeley, California 94720 (B.E., C.R., M.E.V.-S., P.I.B., J.D.K., H.V.S., P.C.R.); BioEnergy Sciences Center, National Renewable Energy Laboratory, Golden, Colorado 80401 (R.S., A.Z.); China Tobacco Gene Research Center, Zhengzhou Tobacco Research Institute, Zhengzhou, Henan 450001, China (P.C.); and Department of Chemical and Biomolecular Engineering, Department of Bioengineering, University of California, Berkeley, California 94720 (J.D.K.).

==References==
1. Bartley, L.E., et al., Overexpression of a BAHD acyltransferase, OsAt10, alters rice cell wall hydroxycinnamic acid content and saccharification. Plant physiology, 2013. 161(4): p. 1615-1633.

==Structured Information==
{{JaponicaGene|
GeneName = Os06g0594600|
Description = The start codon is not identified.|
Version = NM_001064516.2 GI:297606110 GeneID:4341427|
Length = 1126 bp|
Definition = Oryza sativa Japonica Group Os06g0594600, complete gene.|
Source = Oryza sativa Japonica Group

ORGANISM Oryza sativa Japonica Group
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP
clade; Ehrhartoideae; Oryzeae; Oryza.
|
Chromosome = [[:category:Japonica Chromosome 6|Chromosome 6]]|
AP = Chromosome 6:24256206..24257331|
CDS = 24256486..24257331|
GCID = <gbrowseImage1>
name=NC_008399:24256206..24257331
source=RiceChromosome06
preset=GeneLocation
</gbrowseImage1>|
GSID = <gbrowseImage2>
name=NC_008399:24256206..24257331
source=RiceChromosome06
preset=GeneLocation
</gbrowseImage2>|
CDNA = <cdnaseq>gatttgcaggtgacgacgttcacctgcggcggcttcgtgatcgggctgcgcaccaaccacgcggtggcggacggcaccggcgccgcccagttcatgaacgccgtcggcgacctcgcccgcggcctcccggagccgcgggtgaagccgatctgggcgcgcgaccgcttcccggacccggacatcaagcccggcccgctgccggagctccccgtgctgccgctccagtacatcgccttcgacttccccgccgcctacctcggcaagctcaaggcgcagtacgccgccaccgccggcgccagcaagatctgctccgccttcgacatcgtcatcgccaagctctggcagtgccggacgcgcgccatcgccgccgaccccgccgcggccgtcaagctctgcttcttcgccagcgcccgccaggtgctcggcctggagaccggctactggggcaacgccatcttcccggtgaaggtgtccgcggcggcgggggaggtggcggcgtcgtcggtgatcgagctcgtcggcgtggtccgggaggcgaagcggcggatggccggcgagtgcctgcgctgggcggaggggcgcaccggcggcgccgacccgttccagatgacgttcgactacgagtccgtgtacgtgtcggactggagcaagctcgggttcaacgacgtcgactacgggtacggcgcgccgtcggcggcggggccgctggtgaactgcgacctcatctcgtcggtgatcgtcatgcgggcgccggcgccgctcgccggcacgcggctgctggcgagctgcgtcaccaaggagcacgccgacgacttcgccgccaggatgagggaggatctcgtctaa</cdnaseq>|
AA = <aaseq>DLQVTTFTCGGFVIGLRTNHAVADGTGAAQFMNAVGDLARGLPE PRVKPIWARDRFPDPDIKPGPLPELPVLPLQYIAFDFPAAYLGKLKAQYAATAGASKI CSAFDIVIAKLWQCRTRAIAADPAAAVKLCFFASARQVLGLETGYWGNAIFPVKVSAA AGEVAASSVIELVGVVREAKRRMAGECLRWAEGRTGGADPFQMTFDYESVYVSDWSKL GFNDVDYGYGAPSAAGPLVNCDLISSVIVMRAPAPLAGTRLLASCVTKEHADDFAARM REDLV</aaseq>|
DNA = <dnaseqindica>1..846#gatttgcaggtgacgacgttcacctgcggcggcttcgtgatcgggctgcgcaccaaccacgcggtggcggacggcaccggcgccgcccagttcatgaacgccgtcggcgacctcgcccgcggcctcccggagccgcgggtgaagccgatctgggcgcgcgaccgcttcccggacccggacatcaagcccggcccgctgccggagctccccgtgctgccgctccagtacatcgccttcgacttccccgccgcctacctcggcaagctcaaggcgcagtacgccgccaccgccggcgccagcaagatctgctccgccttcgacatcgtcatcgccaagctctggcagtgccggacgcgcgccatcgccgccgaccccgccgcggccgtcaagctctgcttcttcgccagcgcccgccaggtgctcggcctggagaccggctactggggcaacgccatcttcccggtgaaggtgtccgcggcggcgggggaggtggcggcgtcgtcggtgatcgagctcgtcggcgtggtccgggaggcgaagcggcggatggccggcgagtgcctgcgctgggcggaggggcgcaccggcggcgccgacccgttccagatgacgttcgactacgagtccgtgtacgtgtcggactggagcaagctcgggttcaacgacgtcgactacgggtacggcgcgccgtcggcggcggggccgctggtgaactgcgacctcatctcgtcggtgatcgtcatgcgggcgccggcgccgctcgccggcacgcggctgctggcgagctgcgtcaccaaggagcacgccgacgacttcgccgccaggatgagggaggatctcgtctaatataccatggccgcccccaataacattattagtcacgtacaatattgtcactgaatattaatttgttcgtgtatatctattgtggtatgtattttttttttcataggaaaaaagtagtagtacgacaataggtagctgggagctacctaatatcgacctctggtttgagagtaagtgagcgagcgagagatgtaaaccacctcagtttttaccgtgctttgtgacatgcgtggtacagtactggtactattaatcattggccgtgaaaaattatctaccc</dnaseqindica>|
Link = [http://www.ncbi.nlm.nih.gov/nuccore/NM_001064516.2 RefSeq:Os06g0594600]|
}}
[[Category:Genes]]
[[Category:Japonica mRNA]]
[[Category:Oryza Sativa Japonica Group]]
[[Category:Japonica Genes]]
[[Category:Japonica Chromosome 6]]
[[Category:Chromosome 6]]

Os06g0594600

2014-06-08T14:35:17Z

Xiaoyanjie2012: /* 4.The OsAT10-D1 Line Shows No Alterations in Lignin Content or Composition */

Please input one-sentence summary here.

==Annotated Information==
OsAt10 is a gene in rice(Oryza sativa).Its RAP ID is Os06g0594600,and its MSU ID LOC_Os06g39390. Overexpression of this gene can alters rice cell wall hydroxycinnamic acid Content and sacchariﬁcation.
===Function===
The overexpression of the rice gene -- OsAt10(LOC_Os06g39390) -- can effect the hydroxycinnamic acid content and saccharification in the rice cell wall.rice mutants with altered expression of four of these genes have altered cell wall hydroxycinnamate content. In-depth characterization of ectopic expression lines for one gene, OsAt10, revealed that this modification increases matrix polysaccharideassociated ester-linkedp-CA while simultaneously decreasing matrix polysaccharide-associated FA.OsAt10 overexpression plants exhibit increased in vitro saccharification, with no discernible effects on vegetative development. Thus, this gene is a useful target for improving biofuel and feed production.
Grass cell wall properties influence food, feed, and biofuel feedstock usage efficiency. The glucuronoarabinoxylan of grass cell walls is esterified with the phenylpropanoid-derived hydroxycinnamic acids ferulic acid (FA) and para-coumaric acid (p-CA). Feruloyl esters undergo oxidative coupling with neighboring phenylpropanoids on glucuronoarabinoxylan and lignin. Examination of rice (Oryza sativa) mutants in a grass-expanded and -diverged clade of BAHD acyl coenzyme A-utilizing transferases identified four mutants with altered cell wall FA or p-CA contents.Some researchers reported on the effects of overexpressing one of these genes, OsAt10 (LOC_Os06g39390), in rice. An activation-tagged line, OsAT10-D1, shows a 60% reduction in matrix polysaccharide-bound FA and an approximately 300% increase in p-CA in young leaf tissue but no discernible phenotypic alterations in vegetative development, lignin content, or lignin composition. Two additional independent OsAt10 overexpression lines show similar changes in FA and p-CA content. Cell wall fractionation and liquid chromatography-mass spectrometry experiments isolate the cell wall alterations in the mutant to ester conjugates of a five-carbon sugar with p-CA and FA. These results suggest that OsAT10 is a p-coumaroyl coenzyme A transferase involved in glucuronoarabinoxylan modification. Biomass from OsAT10-D1 exhibits a 20% to 40% increase in saccharification yield depending on the assay. Thus, OsAt10 is an attractive target for improving grass cell wall quality for fuel and animal feed.<ref name="ref1" />
====1.The Mitchell Clade of BAHD Acyltransferases Is Expanded and Diverged in Grasses====
Mitchell et al. (2007) identified what we term the Mitchell clade of BAHD acyl-CoA-dependent acyltransferases on the basis of high gene expression in grasses relative to dicots. To refine the hypothesis that these enzymes might be involved in grass-diverged cell wall synthesis, we systematically characterized the distribution of this clade in selected plant species and compared the clade with other characterized BAHD proteins. We identified BAHD proteins from the genomes of a diverse set of sequenced plant species available at the time of the analysis and examined the phylogenetic relationships among them and a reference set of BAHDs (Table I). To gain higher sensitivity relative to local sequence alignment (i.e. BLAST) for recognizing sequences with low, but potentially still significant, homology, we used a hidden Markov model to identify putative BAHD proteins (Finn et al., 2011)
The researchers then inferred an initial model of the phylo-genetic relationships among the putative BAHD proteins from each genome and the set of biochemically characterized BAHD proteins cataloged by D’Auria (2006). While we are aware that recent analyses have included the presence of a strict HXXXD motif as indicative of whether the protein is an active BAHD (Banks et al., 2011; Tuominen et al., 2011), we have included proteins with single amino acid alterations to this motif, since one of the known biochemically active proteins for the family involved in taxol biosynthesis, BAPT (National Center for Biotechnology Information identifier AAL92459; Walker et al., 2002), possesses a variation of this motif in which the His is replaced by a Ser.
As observed by Tuominen et al. (2011), the distribution of BAHD proteins varies among species (Table I). The Mitchell clade is embedded within clade V, or clade Va of Tuominen et al. (2011). Furthermore, we find that the Mitchell clade includes a biochemically characterized banana (Musa spp.) alcohol CoA acyltransferase, BanAAT (Beekwilder et al., 2004), and is related to a group of BAHD proteins that participate in taxol biosynthesis (Fig. 1B).

[[File:Table 1xxy.png]][[File:figure 1xxy.png]]
====2.Screen of Rice Mutants for Altered Cell Wall Hydroxycinnamic Acid Content====

[[File:Figure 2xxy.png]]

====3.The OsAT10-D1 Line Has an Increase in Cell Wall Glc Content====
Compensatory changes are often seen among the components of the cell wall (Humphrey et al., 2007). Quantification of sugars released by acid treatment of
destarched AIR preparations from mature straw suggests that the Glc content is increased by approximately 20% (w/w) for the mutant relative to the wild type (Fig. 9A). We observed the difference both with TFA treatment, which liberates monosaccharides derived from matrix polysaccharides and amorphous cellulose, and when the TFA residue was further treated with sulfuric acid, which breaks down crystalline cellulose (Fig. 9A). The difference in the mutant compared with the wild type is most apparent when the products of both treatments are summed together, which gives an increase in Glc in the mutant compared with the wild type of 19% 6 11%. By mass, we did not observe any other significant changes in sugar amounts in the mutant compared with the wild type. We also observed no change in the total mass percentage of sugars in AIR. When the TFA-solubilized sugars are expressed in terms of mol%, the data also indicate an increase in Glc content of 11% 6 5% (Fig. 9B). The sum of the mol % of other measured sugars (i.e. Xyl, Ara, and the sum of minor sugars) decreases proportionally to the Glc increase (7% 6 7%). This balance in mol % change suggests that the change in polysaccharide content in the mutant is restricted to the Glc-containing polymers.

[[File:Figure 9xxy.png]]

====4.The OsAT10-D1 Line Shows No Alterations in Lignin Content or Composition====
OsAT10-D1 mature straw samples show no significant differences in the content of acetylbromide-soluble lignin after saponification relative to the wild type
(Table II). We obtained a similar result via py-MBMS for mature straw and separate, young leaf and sheath samples. The py-MBMS also revealed no difference in the syringyl-guaiacyl (S:G) lignin ratio in the mutant compared with the wild type after saponification (Table II). We also collected py-MBMS data for unprocessed straw and AIR of OsAT10-D1. Separate analyses of the saponified and unsaponified samples reveals distinctions between the wild type and mutant in the unsaponified samples (Fig. 10A). Principal component 1 explains the alcohol extraction (30% of the variation), and principal component 2 explains the differences between wild-type and mutant samples (19% of the variation). The loadings for principal component 2 show that the major ions that distinguish wild-type and mutant samples are phenolics (Fig. 10B). The mass spectrometry fragmentation pattern is consistent with an interpretation in which there is an increase of p-CA, as reflected by peaks 120, 94, and 91, and a decrease in FA, as reflected in the reduction in the coniferyl ion, peak 150 (Evans and Milne, 1987). Because principal component analysis no longer distinguishes the samples after saponification (Fig. 10C), the observed differences in phenylpropanoids between OsAT10-D1 and the wild type are likely associated with ester-linked hydroxycinnamates and not lignin, consistent with the other results.
A limitation of the pyrolysis method for determining lignin composition is that it inaccurately measures H-lignin, which volatilizes poorly and instead turns to char upon heating. Because of the increase in p-CA, a precursor of H-lignin, in OsAT10-D1 cell walls relative to the wild type, we sought to determine whether there is a change in the char content of OsAT10-D1 using a thermogravimetric pyrolysis instrument. Duplicate runs per genotype of the thermogravimetric instrument did not detect a difference in the mass remaining from mature straw after pyrolysis, again consistent with there being no difference in core lignin composition or content between OsAT10-D1 and the wild type.

[[File:Table 2xxy.png]]
[[File:Figure 10xxy.png]]

====The OsAT10-D1 Line Shows an Increase in Saccharification====
Ferulate in grass biomass is inversely correlated with digestibility across diverse grass accessions (Lam et al., 2003; Casler and Jung, 2006). The phenotype of the OsAT10-D1 line provided the opportunity to determine whether there is also an increase in enzymatic digestibility with reduced FA content when comparing two near-isogenic plant lines. We found that destarched AIR after mild pretreatment followed by incubation with a cellulase cocktail resulted in the release of approximately 20% more reducing sugar from the mutant compared with the wild type at each time point examined (Fig. 11A)
Acid-pretreated rice straw of the wild type and OsAT10-D1 to the mesophilic fungus, Penicillium sp. YT02. This recently characterized fungus shows significantly higher xylanase and b-glucosidase activities with various insoluble lignocellulosic substrates compared with the commonly used fungal strain, Trichoderma reesei (ATCC 24449; Kovacs et al., 2009; L. Gao and J. Zhou, unpublished data). In the fungal treatments, the biomass-derived sugars initially accumulate but are gradually depleted via incorporation into fungal biomass. Qualitatively consistent with the enzymatic deconstruction results, Penicillium sp. YT02 incubation released 46% more Glc, 82% more Xyl, and 25% more Ara into the medium from OsAT10-D1 straw than from wild-type straw (Fig. 11B). Averaged over the entire time course (12–120 h), the improvement in yield is more dramatic with the fungus than with the simple enzymatic treatment, with a total sugar yield increase of approximately 40%. Cellulase and b-glucosidase enzymatic activities in the slurry are unchanged on the mutant straw (Fig. 11C), suggesting that the fungus grew similarly on both. In contrast, and of relevance to the nature of the change caused by the increased expression of OsAt10, xylanase activity is dramatically enhanced, especially at later time points (Fig. 11C).

[[File:Figure 11xxy.png]]

===Expression===
For the remaining 11 lines, we characterized the alkali-labile hydroxycinnamoyl ester content of cell wall alcohol-insoluble residue (AIR) from leaf blades and sheaths of side tillers. We compared homozygous, mutant, and wild-type segregant plants 7 or 10 weeks after planting. The screen revealed four mutants with possible cell wall hydroxycinnamic acid phenotypes . All four lines showed changes in the expression of the nearest acyltransferase
gene to the T-DNA insertion site via quantitative reverse transcription (qRT)-PCR. Homozygous mutant progeny of4A-03423(here afterreferred to asOsAT10-D1), which has increased expression of OsAt10, exhibited reduced FA (approximately 60% less) and an increase in p-CA (approximately 300% more) in sheaths and leaves.TheT-DNA insertions it efort helinewerefertoas OsAT10-D1(PFG_4A-03423) is approximately 8.5 kb downstream of the transcriptional start site forOsAt10. The insert is oriented with the activating sequences proximate toOsAt10and in the range observed to activate expression (Jeong et al., 2006). As mentioned above, qRT-PCR indicated that, indeed,the expression ofOsAt10was increased by more than 100-fold in the leaves of homozygous OsAT10-D1 plants (Fig. 3B). InOsAT10-D1, besides OsAt10the expression of other genes proximate to the site of the T-DNA insertion does not vary significantly relative to the wild type (Fig. 3B). Similarly, the expression of related OsAtgenesdoesnotvarysignificantly in OsAT10-D1(Supplemental Fig. S2), reducing the possibility that the observed phenotype is due to compensation at the level of gene expression of a related acyltransferase. Of the acyltransferase transcripts examined in this survey, OsAt6appears to vary the most, although not significantly. However, OsAt6(LOC_Os01g08380) is expressed near the lower limitofourdetectionand,infact,wasreportedas undetectable in a previous qRT-PCR study (Piston et al., 2010). OsAT10-D1lines show no change in size and dry mass at maturity (Fig. 4, A and B). However, we did measure an approximately 20% to 30% decrease in total seed mass per plant for the mutant compared with the wild type.

[[File:Figure 3xxy.png]][[File:Figure 4xxy.png]]

===Evolution===
Please input evolution information here.

You can also add sub-section(s) at will.

==Labs working on this gene==
Please input related labs here.

==References==
1. Bartley, L.E., et al., Overexpression of a BAHD acyltransferase, OsAt10, alters rice cell wall hydroxycinnamic acid content and saccharification. Plant physiology, 2013. 161(4): p. 1615-1633.

==Structured Information==
{{JaponicaGene|
GeneName = Os06g0594600|
Description = The start codon is not identified.|
Version = NM_001064516.2 GI:297606110 GeneID:4341427|
Length = 1126 bp|
Definition = Oryza sativa Japonica Group Os06g0594600, complete gene.|
Source = Oryza sativa Japonica Group

ORGANISM Oryza sativa Japonica Group
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP
clade; Ehrhartoideae; Oryzeae; Oryza.
|
Chromosome = [[:category:Japonica Chromosome 6|Chromosome 6]]|
AP = Chromosome 6:24256206..24257331|
CDS = 24256486..24257331|
GCID = <gbrowseImage1>
name=NC_008399:24256206..24257331
source=RiceChromosome06
preset=GeneLocation
</gbrowseImage1>|
GSID = <gbrowseImage2>
name=NC_008399:24256206..24257331
source=RiceChromosome06
preset=GeneLocation
</gbrowseImage2>|
CDNA = <cdnaseq>gatttgcaggtgacgacgttcacctgcggcggcttcgtgatcgggctgcgcaccaaccacgcggtggcggacggcaccggcgccgcccagttcatgaacgccgtcggcgacctcgcccgcggcctcccggagccgcgggtgaagccgatctgggcgcgcgaccgcttcccggacccggacatcaagcccggcccgctgccggagctccccgtgctgccgctccagtacatcgccttcgacttccccgccgcctacctcggcaagctcaaggcgcagtacgccgccaccgccggcgccagcaagatctgctccgccttcgacatcgtcatcgccaagctctggcagtgccggacgcgcgccatcgccgccgaccccgccgcggccgtcaagctctgcttcttcgccagcgcccgccaggtgctcggcctggagaccggctactggggcaacgccatcttcccggtgaaggtgtccgcggcggcgggggaggtggcggcgtcgtcggtgatcgagctcgtcggcgtggtccgggaggcgaagcggcggatggccggcgagtgcctgcgctgggcggaggggcgcaccggcggcgccgacccgttccagatgacgttcgactacgagtccgtgtacgtgtcggactggagcaagctcgggttcaacgacgtcgactacgggtacggcgcgccgtcggcggcggggccgctggtgaactgcgacctcatctcgtcggtgatcgtcatgcgggcgccggcgccgctcgccggcacgcggctgctggcgagctgcgtcaccaaggagcacgccgacgacttcgccgccaggatgagggaggatctcgtctaa</cdnaseq>|
AA = <aaseq>DLQVTTFTCGGFVIGLRTNHAVADGTGAAQFMNAVGDLARGLPE PRVKPIWARDRFPDPDIKPGPLPELPVLPLQYIAFDFPAAYLGKLKAQYAATAGASKI CSAFDIVIAKLWQCRTRAIAADPAAAVKLCFFASARQVLGLETGYWGNAIFPVKVSAA AGEVAASSVIELVGVVREAKRRMAGECLRWAEGRTGGADPFQMTFDYESVYVSDWSKL GFNDVDYGYGAPSAAGPLVNCDLISSVIVMRAPAPLAGTRLLASCVTKEHADDFAARM REDLV</aaseq>|
DNA = <dnaseqindica>1..846#gatttgcaggtgacgacgttcacctgcggcggcttcgtgatcgggctgcgcaccaaccacgcggtggcggacggcaccggcgccgcccagttcatgaacgccgtcggcgacctcgcccgcggcctcccggagccgcgggtgaagccgatctgggcgcgcgaccgcttcccggacccggacatcaagcccggcccgctgccggagctccccgtgctgccgctccagtacatcgccttcgacttccccgccgcctacctcggcaagctcaaggcgcagtacgccgccaccgccggcgccagcaagatctgctccgccttcgacatcgtcatcgccaagctctggcagtgccggacgcgcgccatcgccgccgaccccgccgcggccgtcaagctctgcttcttcgccagcgcccgccaggtgctcggcctggagaccggctactggggcaacgccatcttcccggtgaaggtgtccgcggcggcgggggaggtggcggcgtcgtcggtgatcgagctcgtcggcgtggtccgggaggcgaagcggcggatggccggcgagtgcctgcgctgggcggaggggcgcaccggcggcgccgacccgttccagatgacgttcgactacgagtccgtgtacgtgtcggactggagcaagctcgggttcaacgacgtcgactacgggtacggcgcgccgtcggcggcggggccgctggtgaactgcgacctcatctcgtcggtgatcgtcatgcgggcgccggcgccgctcgccggcacgcggctgctggcgagctgcgtcaccaaggagcacgccgacgacttcgccgccaggatgagggaggatctcgtctaatataccatggccgcccccaataacattattagtcacgtacaatattgtcactgaatattaatttgttcgtgtatatctattgtggtatgtattttttttttcataggaaaaaagtagtagtacgacaataggtagctgggagctacctaatatcgacctctggtttgagagtaagtgagcgagcgagagatgtaaaccacctcagtttttaccgtgctttgtgacatgcgtggtacagtactggtactattaatcattggccgtgaaaaattatctaccc</dnaseqindica>|
Link = [http://www.ncbi.nlm.nih.gov/nuccore/NM_001064516.2 RefSeq:Os06g0594600]|
}}
[[Category:Genes]]
[[Category:Japonica mRNA]]
[[Category:Oryza Sativa Japonica Group]]
[[Category:Japonica Genes]]
[[Category:Japonica Chromosome 6]]
[[Category:Chromosome 6]]

File:Figure 11xxy.png

2014-06-08T14:34:49Z

Xiaoyanjie2012:

Os06g0594600

2014-06-08T14:28:37Z

Xiaoyanjie2012: /* 2.Screen of Rice Mutants for Altered Cell Wall Hydroxycinnamic Acid Content */

Please input one-sentence summary here.

==Annotated Information==
OsAt10 is a gene in rice(Oryza sativa).Its RAP ID is Os06g0594600,and its MSU ID LOC_Os06g39390. Overexpression of this gene can alters rice cell wall hydroxycinnamic acid Content and sacchariﬁcation.
===Function===
The overexpression of the rice gene -- OsAt10(LOC_Os06g39390) -- can effect the hydroxycinnamic acid content and saccharification in the rice cell wall.rice mutants with altered expression of four of these genes have altered cell wall hydroxycinnamate content. In-depth characterization of ectopic expression lines for one gene, OsAt10, revealed that this modification increases matrix polysaccharideassociated ester-linkedp-CA while simultaneously decreasing matrix polysaccharide-associated FA.OsAt10 overexpression plants exhibit increased in vitro saccharification, with no discernible effects on vegetative development. Thus, this gene is a useful target for improving biofuel and feed production.
Grass cell wall properties influence food, feed, and biofuel feedstock usage efficiency. The glucuronoarabinoxylan of grass cell walls is esterified with the phenylpropanoid-derived hydroxycinnamic acids ferulic acid (FA) and para-coumaric acid (p-CA). Feruloyl esters undergo oxidative coupling with neighboring phenylpropanoids on glucuronoarabinoxylan and lignin. Examination of rice (Oryza sativa) mutants in a grass-expanded and -diverged clade of BAHD acyl coenzyme A-utilizing transferases identified four mutants with altered cell wall FA or p-CA contents.Some researchers reported on the effects of overexpressing one of these genes, OsAt10 (LOC_Os06g39390), in rice. An activation-tagged line, OsAT10-D1, shows a 60% reduction in matrix polysaccharide-bound FA and an approximately 300% increase in p-CA in young leaf tissue but no discernible phenotypic alterations in vegetative development, lignin content, or lignin composition. Two additional independent OsAt10 overexpression lines show similar changes in FA and p-CA content. Cell wall fractionation and liquid chromatography-mass spectrometry experiments isolate the cell wall alterations in the mutant to ester conjugates of a five-carbon sugar with p-CA and FA. These results suggest that OsAT10 is a p-coumaroyl coenzyme A transferase involved in glucuronoarabinoxylan modification. Biomass from OsAT10-D1 exhibits a 20% to 40% increase in saccharification yield depending on the assay. Thus, OsAt10 is an attractive target for improving grass cell wall quality for fuel and animal feed.<ref name="ref1" />
====1.The Mitchell Clade of BAHD Acyltransferases Is Expanded and Diverged in Grasses====
Mitchell et al. (2007) identified what we term the Mitchell clade of BAHD acyl-CoA-dependent acyltransferases on the basis of high gene expression in grasses relative to dicots. To refine the hypothesis that these enzymes might be involved in grass-diverged cell wall synthesis, we systematically characterized the distribution of this clade in selected plant species and compared the clade with other characterized BAHD proteins. We identified BAHD proteins from the genomes of a diverse set of sequenced plant species available at the time of the analysis and examined the phylogenetic relationships among them and a reference set of BAHDs (Table I). To gain higher sensitivity relative to local sequence alignment (i.e. BLAST) for recognizing sequences with low, but potentially still significant, homology, we used a hidden Markov model to identify putative BAHD proteins (Finn et al., 2011)
The researchers then inferred an initial model of the phylo-genetic relationships among the putative BAHD proteins from each genome and the set of biochemically characterized BAHD proteins cataloged by D’Auria (2006). While we are aware that recent analyses have included the presence of a strict HXXXD motif as indicative of whether the protein is an active BAHD (Banks et al., 2011; Tuominen et al., 2011), we have included proteins with single amino acid alterations to this motif, since one of the known biochemically active proteins for the family involved in taxol biosynthesis, BAPT (National Center for Biotechnology Information identifier AAL92459; Walker et al., 2002), possesses a variation of this motif in which the His is replaced by a Ser.
As observed by Tuominen et al. (2011), the distribution of BAHD proteins varies among species (Table I). The Mitchell clade is embedded within clade V, or clade Va of Tuominen et al. (2011). Furthermore, we find that the Mitchell clade includes a biochemically characterized banana (Musa spp.) alcohol CoA acyltransferase, BanAAT (Beekwilder et al., 2004), and is related to a group of BAHD proteins that participate in taxol biosynthesis (Fig. 1B).

[[File:Table 1xxy.png]][[File:figure 1xxy.png]]
====2.Screen of Rice Mutants for Altered Cell Wall Hydroxycinnamic Acid Content====

[[File:Figure 2xxy.png]]

====3.The OsAT10-D1 Line Has an Increase in Cell Wall Glc Content====
Compensatory changes are often seen among the components of the cell wall (Humphrey et al., 2007). Quantification of sugars released by acid treatment of
destarched AIR preparations from mature straw suggests that the Glc content is increased by approximately 20% (w/w) for the mutant relative to the wild type (Fig. 9A). We observed the difference both with TFA treatment, which liberates monosaccharides derived from matrix polysaccharides and amorphous cellulose, and when the TFA residue was further treated with sulfuric acid, which breaks down crystalline cellulose (Fig. 9A). The difference in the mutant compared with the wild type is most apparent when the products of both treatments are summed together, which gives an increase in Glc in the mutant compared with the wild type of 19% 6 11%. By mass, we did not observe any other significant changes in sugar amounts in the mutant compared with the wild type. We also observed no change in the total mass percentage of sugars in AIR. When the TFA-solubilized sugars are expressed in terms of mol%, the data also indicate an increase in Glc content of 11% 6 5% (Fig. 9B). The sum of the mol % of other measured sugars (i.e. Xyl, Ara, and the sum of minor sugars) decreases proportionally to the Glc increase (7% 6 7%). This balance in mol % change suggests that the change in polysaccharide content in the mutant is restricted to the Glc-containing polymers.

[[File:Figure 9xxy.png]]

====4.The OsAT10-D1 Line Shows No Alterations in Lignin Content or Composition====
OsAT10-D1 mature straw samples show no significant differences in the content of acetylbromide-soluble lignin after saponification relative to the wild type
(Table II). We obtained a similar result via py-MBMS for mature straw and separate, young leaf and sheath samples. The py-MBMS also revealed no difference in the syringyl-guaiacyl (S:G) lignin ratio in the mutant compared with the wild type after saponification (Table II). We also collected py-MBMS data for unprocessed straw and AIR of OsAT10-D1. Separate analyses of the saponified and unsaponified samples reveals distinctions between the wild type and mutant in the unsaponified samples (Fig. 10A). Principal component 1 explains the alcohol extraction (30% of the variation), and principal component 2 explains the differences between wild-type and mutant samples (19% of the variation). The loadings for principal component 2 show that the major ions that distinguish wild-type and mutant samples are phenolics (Fig. 10B). The mass spectrometry fragmentation pattern is consistent with an interpretation in which there is an increase of p-CA, as reflected by peaks 120, 94, and 91, and a decrease in FA, as reflected in the reduction in the coniferyl ion, peak 150 (Evans and Milne, 1987). Because principal component analysis no longer distinguishes the samples after saponification (Fig. 10C), the observed differences in phenylpropanoids between OsAT10-D1 and the wild type are likely associated with ester-linked hydroxycinnamates and not lignin, consistent with the other results.
A limitation of the pyrolysis method for determining lignin composition is that it inaccurately measures H-lignin, which volatilizes poorly and instead turns to char upon heating. Because of the increase in p-CA, a precursor of H-lignin, in OsAT10-D1 cell walls relative to the wild type, we sought to determine whether there is a change in the char content of OsAT10-D1 using a thermogravimetric pyrolysis instrument. Duplicate runs per genotype of the thermogravimetric instrument did not detect a difference in the mass remaining from mature straw after pyrolysis, again consistent with there being no difference in core lignin composition or content between OsAT10-D1 and the wild type.

[[File:Table 2xxy.png]]
[[File:Figure 10xxy.png]]

===Expression===
For the remaining 11 lines, we characterized the alkali-labile hydroxycinnamoyl ester content of cell wall alcohol-insoluble residue (AIR) from leaf blades and sheaths of side tillers. We compared homozygous, mutant, and wild-type segregant plants 7 or 10 weeks after planting. The screen revealed four mutants with possible cell wall hydroxycinnamic acid phenotypes . All four lines showed changes in the expression of the nearest acyltransferase
gene to the T-DNA insertion site via quantitative reverse transcription (qRT)-PCR. Homozygous mutant progeny of4A-03423(here afterreferred to asOsAT10-D1), which has increased expression of OsAt10, exhibited reduced FA (approximately 60% less) and an increase in p-CA (approximately 300% more) in sheaths and leaves.TheT-DNA insertions it efort helinewerefertoas OsAT10-D1(PFG_4A-03423) is approximately 8.5 kb downstream of the transcriptional start site forOsAt10. The insert is oriented with the activating sequences proximate toOsAt10and in the range observed to activate expression (Jeong et al., 2006). As mentioned above, qRT-PCR indicated that, indeed,the expression ofOsAt10was increased by more than 100-fold in the leaves of homozygous OsAT10-D1 plants (Fig. 3B). InOsAT10-D1, besides OsAt10the expression of other genes proximate to the site of the T-DNA insertion does not vary significantly relative to the wild type (Fig. 3B). Similarly, the expression of related OsAtgenesdoesnotvarysignificantly in OsAT10-D1(Supplemental Fig. S2), reducing the possibility that the observed phenotype is due to compensation at the level of gene expression of a related acyltransferase. Of the acyltransferase transcripts examined in this survey, OsAt6appears to vary the most, although not significantly. However, OsAt6(LOC_Os01g08380) is expressed near the lower limitofourdetectionand,infact,wasreportedas undetectable in a previous qRT-PCR study (Piston et al., 2010). OsAT10-D1lines show no change in size and dry mass at maturity (Fig. 4, A and B). However, we did measure an approximately 20% to 30% decrease in total seed mass per plant for the mutant compared with the wild type.

[[File:Figure 3xxy.png]][[File:Figure 4xxy.png]]

===Evolution===
Please input evolution information here.

You can also add sub-section(s) at will.

==Labs working on this gene==
Please input related labs here.

==References==
1. Bartley, L.E., et al., Overexpression of a BAHD acyltransferase, OsAt10, alters rice cell wall hydroxycinnamic acid content and saccharification. Plant physiology, 2013. 161(4): p. 1615-1633.

==Structured Information==
{{JaponicaGene|
GeneName = Os06g0594600|
Description = The start codon is not identified.|
Version = NM_001064516.2 GI:297606110 GeneID:4341427|
Length = 1126 bp|
Definition = Oryza sativa Japonica Group Os06g0594600, complete gene.|
Source = Oryza sativa Japonica Group

ORGANISM Oryza sativa Japonica Group
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP
clade; Ehrhartoideae; Oryzeae; Oryza.
|
Chromosome = [[:category:Japonica Chromosome 6|Chromosome 6]]|
AP = Chromosome 6:24256206..24257331|
CDS = 24256486..24257331|
GCID = <gbrowseImage1>
name=NC_008399:24256206..24257331
source=RiceChromosome06
preset=GeneLocation
</gbrowseImage1>|
GSID = <gbrowseImage2>
name=NC_008399:24256206..24257331
source=RiceChromosome06
preset=GeneLocation
</gbrowseImage2>|
CDNA = <cdnaseq>gatttgcaggtgacgacgttcacctgcggcggcttcgtgatcgggctgcgcaccaaccacgcggtggcggacggcaccggcgccgcccagttcatgaacgccgtcggcgacctcgcccgcggcctcccggagccgcgggtgaagccgatctgggcgcgcgaccgcttcccggacccggacatcaagcccggcccgctgccggagctccccgtgctgccgctccagtacatcgccttcgacttccccgccgcctacctcggcaagctcaaggcgcagtacgccgccaccgccggcgccagcaagatctgctccgccttcgacatcgtcatcgccaagctctggcagtgccggacgcgcgccatcgccgccgaccccgccgcggccgtcaagctctgcttcttcgccagcgcccgccaggtgctcggcctggagaccggctactggggcaacgccatcttcccggtgaaggtgtccgcggcggcgggggaggtggcggcgtcgtcggtgatcgagctcgtcggcgtggtccgggaggcgaagcggcggatggccggcgagtgcctgcgctgggcggaggggcgcaccggcggcgccgacccgttccagatgacgttcgactacgagtccgtgtacgtgtcggactggagcaagctcgggttcaacgacgtcgactacgggtacggcgcgccgtcggcggcggggccgctggtgaactgcgacctcatctcgtcggtgatcgtcatgcgggcgccggcgccgctcgccggcacgcggctgctggcgagctgcgtcaccaaggagcacgccgacgacttcgccgccaggatgagggaggatctcgtctaa</cdnaseq>|
AA = <aaseq>DLQVTTFTCGGFVIGLRTNHAVADGTGAAQFMNAVGDLARGLPE PRVKPIWARDRFPDPDIKPGPLPELPVLPLQYIAFDFPAAYLGKLKAQYAATAGASKI CSAFDIVIAKLWQCRTRAIAADPAAAVKLCFFASARQVLGLETGYWGNAIFPVKVSAA AGEVAASSVIELVGVVREAKRRMAGECLRWAEGRTGGADPFQMTFDYESVYVSDWSKL GFNDVDYGYGAPSAAGPLVNCDLISSVIVMRAPAPLAGTRLLASCVTKEHADDFAARM REDLV</aaseq>|
DNA = <dnaseqindica>1..846#gatttgcaggtgacgacgttcacctgcggcggcttcgtgatcgggctgcgcaccaaccacgcggtggcggacggcaccggcgccgcccagttcatgaacgccgtcggcgacctcgcccgcggcctcccggagccgcgggtgaagccgatctgggcgcgcgaccgcttcccggacccggacatcaagcccggcccgctgccggagctccccgtgctgccgctccagtacatcgccttcgacttccccgccgcctacctcggcaagctcaaggcgcagtacgccgccaccgccggcgccagcaagatctgctccgccttcgacatcgtcatcgccaagctctggcagtgccggacgcgcgccatcgccgccgaccccgccgcggccgtcaagctctgcttcttcgccagcgcccgccaggtgctcggcctggagaccggctactggggcaacgccatcttcccggtgaaggtgtccgcggcggcgggggaggtggcggcgtcgtcggtgatcgagctcgtcggcgtggtccgggaggcgaagcggcggatggccggcgagtgcctgcgctgggcggaggggcgcaccggcggcgccgacccgttccagatgacgttcgactacgagtccgtgtacgtgtcggactggagcaagctcgggttcaacgacgtcgactacgggtacggcgcgccgtcggcggcggggccgctggtgaactgcgacctcatctcgtcggtgatcgtcatgcgggcgccggcgccgctcgccggcacgcggctgctggcgagctgcgtcaccaaggagcacgccgacgacttcgccgccaggatgagggaggatctcgtctaatataccatggccgcccccaataacattattagtcacgtacaatattgtcactgaatattaatttgttcgtgtatatctattgtggtatgtattttttttttcataggaaaaaagtagtagtacgacaataggtagctgggagctacctaatatcgacctctggtttgagagtaagtgagcgagcgagagatgtaaaccacctcagtttttaccgtgctttgtgacatgcgtggtacagtactggtactattaatcattggccgtgaaaaattatctaccc</dnaseqindica>|
Link = [http://www.ncbi.nlm.nih.gov/nuccore/NM_001064516.2 RefSeq:Os06g0594600]|
}}
[[Category:Genes]]
[[Category:Japonica mRNA]]
[[Category:Oryza Sativa Japonica Group]]
[[Category:Japonica Genes]]
[[Category:Japonica Chromosome 6]]
[[Category:Chromosome 6]]

Os06g0594600

2014-06-08T14:27:45Z

Xiaoyanjie2012: /* 2.Screen of Rice Mutants for Altered Cell Wall Hydroxycinnamic Acid Content */

Please input one-sentence summary here.

==Annotated Information==
OsAt10 is a gene in rice(Oryza sativa).Its RAP ID is Os06g0594600,and its MSU ID LOC_Os06g39390. Overexpression of this gene can alters rice cell wall hydroxycinnamic acid Content and sacchariﬁcation.
===Function===
The overexpression of the rice gene -- OsAt10(LOC_Os06g39390) -- can effect the hydroxycinnamic acid content and saccharification in the rice cell wall.rice mutants with altered expression of four of these genes have altered cell wall hydroxycinnamate content. In-depth characterization of ectopic expression lines for one gene, OsAt10, revealed that this modification increases matrix polysaccharideassociated ester-linkedp-CA while simultaneously decreasing matrix polysaccharide-associated FA.OsAt10 overexpression plants exhibit increased in vitro saccharification, with no discernible effects on vegetative development. Thus, this gene is a useful target for improving biofuel and feed production.
Grass cell wall properties influence food, feed, and biofuel feedstock usage efficiency. The glucuronoarabinoxylan of grass cell walls is esterified with the phenylpropanoid-derived hydroxycinnamic acids ferulic acid (FA) and para-coumaric acid (p-CA). Feruloyl esters undergo oxidative coupling with neighboring phenylpropanoids on glucuronoarabinoxylan and lignin. Examination of rice (Oryza sativa) mutants in a grass-expanded and -diverged clade of BAHD acyl coenzyme A-utilizing transferases identified four mutants with altered cell wall FA or p-CA contents.Some researchers reported on the effects of overexpressing one of these genes, OsAt10 (LOC_Os06g39390), in rice. An activation-tagged line, OsAT10-D1, shows a 60% reduction in matrix polysaccharide-bound FA and an approximately 300% increase in p-CA in young leaf tissue but no discernible phenotypic alterations in vegetative development, lignin content, or lignin composition. Two additional independent OsAt10 overexpression lines show similar changes in FA and p-CA content. Cell wall fractionation and liquid chromatography-mass spectrometry experiments isolate the cell wall alterations in the mutant to ester conjugates of a five-carbon sugar with p-CA and FA. These results suggest that OsAT10 is a p-coumaroyl coenzyme A transferase involved in glucuronoarabinoxylan modification. Biomass from OsAT10-D1 exhibits a 20% to 40% increase in saccharification yield depending on the assay. Thus, OsAt10 is an attractive target for improving grass cell wall quality for fuel and animal feed.<ref name="ref1" />
====1.The Mitchell Clade of BAHD Acyltransferases Is Expanded and Diverged in Grasses====
Mitchell et al. (2007) identified what we term the Mitchell clade of BAHD acyl-CoA-dependent acyltransferases on the basis of high gene expression in grasses relative to dicots. To refine the hypothesis that these enzymes might be involved in grass-diverged cell wall synthesis, we systematically characterized the distribution of this clade in selected plant species and compared the clade with other characterized BAHD proteins. We identified BAHD proteins from the genomes of a diverse set of sequenced plant species available at the time of the analysis and examined the phylogenetic relationships among them and a reference set of BAHDs (Table I). To gain higher sensitivity relative to local sequence alignment (i.e. BLAST) for recognizing sequences with low, but potentially still significant, homology, we used a hidden Markov model to identify putative BAHD proteins (Finn et al., 2011)
The researchers then inferred an initial model of the phylo-genetic relationships among the putative BAHD proteins from each genome and the set of biochemically characterized BAHD proteins cataloged by D’Auria (2006). While we are aware that recent analyses have included the presence of a strict HXXXD motif as indicative of whether the protein is an active BAHD (Banks et al., 2011; Tuominen et al., 2011), we have included proteins with single amino acid alterations to this motif, since one of the known biochemically active proteins for the family involved in taxol biosynthesis, BAPT (National Center for Biotechnology Information identifier AAL92459; Walker et al., 2002), possesses a variation of this motif in which the His is replaced by a Ser.
As observed by Tuominen et al. (2011), the distribution of BAHD proteins varies among species (Table I). The Mitchell clade is embedded within clade V, or clade Va of Tuominen et al. (2011). Furthermore, we find that the Mitchell clade includes a biochemically characterized banana (Musa spp.) alcohol CoA acyltransferase, BanAAT (Beekwilder et al., 2004), and is related to a group of BAHD proteins that participate in taxol biosynthesis (Fig. 1B).

[[File:Table 1xxy.png]][[File:figure 1xxy.png]]
====2.Screen of Rice Mutants for Altered Cell Wall Hydroxycinnamic Acid Content====
Data are for homozygous wild-type segregant plants (gray bars) and homozygous mutant plants (hatched bars). Each plant line is designated by the repository identifier and the putative target gene. A and D, Average FA content from an AIR preparation. B and E, p-CA content from AIR. C and F, The ratio of FA to p-CA. Side tillers from lines 1B-00523 and 2D-40243 were harvested 10 weeks after germination. All other lines were harvested 7 weeks after germination. Averages from samples from two to three plants for each genotype were measured independently.

[[File:Figure 2xxy.png]]

====3.The OsAT10-D1 Line Has an Increase in Cell Wall Glc Content====
Compensatory changes are often seen among the components of the cell wall (Humphrey et al., 2007). Quantification of sugars released by acid treatment of
destarched AIR preparations from mature straw suggests that the Glc content is increased by approximately 20% (w/w) for the mutant relative to the wild type (Fig. 9A). We observed the difference both with TFA treatment, which liberates monosaccharides derived from matrix polysaccharides and amorphous cellulose, and when the TFA residue was further treated with sulfuric acid, which breaks down crystalline cellulose (Fig. 9A). The difference in the mutant compared with the wild type is most apparent when the products of both treatments are summed together, which gives an increase in Glc in the mutant compared with the wild type of 19% 6 11%. By mass, we did not observe any other significant changes in sugar amounts in the mutant compared with the wild type. We also observed no change in the total mass percentage of sugars in AIR. When the TFA-solubilized sugars are expressed in terms of mol%, the data also indicate an increase in Glc content of 11% 6 5% (Fig. 9B). The sum of the mol % of other measured sugars (i.e. Xyl, Ara, and the sum of minor sugars) decreases proportionally to the Glc increase (7% 6 7%). This balance in mol % change suggests that the change in polysaccharide content in the mutant is restricted to the Glc-containing polymers.

[[File:Figure 9xxy.png]]

====4.The OsAT10-D1 Line Shows No Alterations in Lignin Content or Composition====
OsAT10-D1 mature straw samples show no significant differences in the content of acetylbromide-soluble lignin after saponification relative to the wild type
(Table II). We obtained a similar result via py-MBMS for mature straw and separate, young leaf and sheath samples. The py-MBMS also revealed no difference in the syringyl-guaiacyl (S:G) lignin ratio in the mutant compared with the wild type after saponification (Table II). We also collected py-MBMS data for unprocessed straw and AIR of OsAT10-D1. Separate analyses of the saponified and unsaponified samples reveals distinctions between the wild type and mutant in the unsaponified samples (Fig. 10A). Principal component 1 explains the alcohol extraction (30% of the variation), and principal component 2 explains the differences between wild-type and mutant samples (19% of the variation). The loadings for principal component 2 show that the major ions that distinguish wild-type and mutant samples are phenolics (Fig. 10B). The mass spectrometry fragmentation pattern is consistent with an interpretation in which there is an increase of p-CA, as reflected by peaks 120, 94, and 91, and a decrease in FA, as reflected in the reduction in the coniferyl ion, peak 150 (Evans and Milne, 1987). Because principal component analysis no longer distinguishes the samples after saponification (Fig. 10C), the observed differences in phenylpropanoids between OsAT10-D1 and the wild type are likely associated with ester-linked hydroxycinnamates and not lignin, consistent with the other results.
A limitation of the pyrolysis method for determining lignin composition is that it inaccurately measures H-lignin, which volatilizes poorly and instead turns to char upon heating. Because of the increase in p-CA, a precursor of H-lignin, in OsAT10-D1 cell walls relative to the wild type, we sought to determine whether there is a change in the char content of OsAT10-D1 using a thermogravimetric pyrolysis instrument. Duplicate runs per genotype of the thermogravimetric instrument did not detect a difference in the mass remaining from mature straw after pyrolysis, again consistent with there being no difference in core lignin composition or content between OsAT10-D1 and the wild type.

[[File:Table 2xxy.png]]
[[File:Figure 10xxy.png]]

===Expression===
For the remaining 11 lines, we characterized the alkali-labile hydroxycinnamoyl ester content of cell wall alcohol-insoluble residue (AIR) from leaf blades and sheaths of side tillers. We compared homozygous, mutant, and wild-type segregant plants 7 or 10 weeks after planting. The screen revealed four mutants with possible cell wall hydroxycinnamic acid phenotypes . All four lines showed changes in the expression of the nearest acyltransferase
gene to the T-DNA insertion site via quantitative reverse transcription (qRT)-PCR. Homozygous mutant progeny of4A-03423(here afterreferred to asOsAT10-D1), which has increased expression of OsAt10, exhibited reduced FA (approximately 60% less) and an increase in p-CA (approximately 300% more) in sheaths and leaves.TheT-DNA insertions it efort helinewerefertoas OsAT10-D1(PFG_4A-03423) is approximately 8.5 kb downstream of the transcriptional start site forOsAt10. The insert is oriented with the activating sequences proximate toOsAt10and in the range observed to activate expression (Jeong et al., 2006). As mentioned above, qRT-PCR indicated that, indeed,the expression ofOsAt10was increased by more than 100-fold in the leaves of homozygous OsAT10-D1 plants (Fig. 3B). InOsAT10-D1, besides OsAt10the expression of other genes proximate to the site of the T-DNA insertion does not vary significantly relative to the wild type (Fig. 3B). Similarly, the expression of related OsAtgenesdoesnotvarysignificantly in OsAT10-D1(Supplemental Fig. S2), reducing the possibility that the observed phenotype is due to compensation at the level of gene expression of a related acyltransferase. Of the acyltransferase transcripts examined in this survey, OsAt6appears to vary the most, although not significantly. However, OsAt6(LOC_Os01g08380) is expressed near the lower limitofourdetectionand,infact,wasreportedas undetectable in a previous qRT-PCR study (Piston et al., 2010). OsAT10-D1lines show no change in size and dry mass at maturity (Fig. 4, A and B). However, we did measure an approximately 20% to 30% decrease in total seed mass per plant for the mutant compared with the wild type.

[[File:Figure 3xxy.png]][[File:Figure 4xxy.png]]

===Evolution===
Please input evolution information here.

You can also add sub-section(s) at will.

==Labs working on this gene==
Please input related labs here.

==References==
1. Bartley, L.E., et al., Overexpression of a BAHD acyltransferase, OsAt10, alters rice cell wall hydroxycinnamic acid content and saccharification. Plant physiology, 2013. 161(4): p. 1615-1633.

==Structured Information==
{{JaponicaGene|
GeneName = Os06g0594600|
Description = The start codon is not identified.|
Version = NM_001064516.2 GI:297606110 GeneID:4341427|
Length = 1126 bp|
Definition = Oryza sativa Japonica Group Os06g0594600, complete gene.|
Source = Oryza sativa Japonica Group

ORGANISM Oryza sativa Japonica Group
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP
clade; Ehrhartoideae; Oryzeae; Oryza.
|
Chromosome = [[:category:Japonica Chromosome 6|Chromosome 6]]|
AP = Chromosome 6:24256206..24257331|
CDS = 24256486..24257331|
GCID = <gbrowseImage1>
name=NC_008399:24256206..24257331
source=RiceChromosome06
preset=GeneLocation
</gbrowseImage1>|
GSID = <gbrowseImage2>
name=NC_008399:24256206..24257331
source=RiceChromosome06
preset=GeneLocation
</gbrowseImage2>|
CDNA = <cdnaseq>gatttgcaggtgacgacgttcacctgcggcggcttcgtgatcgggctgcgcaccaaccacgcggtggcggacggcaccggcgccgcccagttcatgaacgccgtcggcgacctcgcccgcggcctcccggagccgcgggtgaagccgatctgggcgcgcgaccgcttcccggacccggacatcaagcccggcccgctgccggagctccccgtgctgccgctccagtacatcgccttcgacttccccgccgcctacctcggcaagctcaaggcgcagtacgccgccaccgccggcgccagcaagatctgctccgccttcgacatcgtcatcgccaagctctggcagtgccggacgcgcgccatcgccgccgaccccgccgcggccgtcaagctctgcttcttcgccagcgcccgccaggtgctcggcctggagaccggctactggggcaacgccatcttcccggtgaaggtgtccgcggcggcgggggaggtggcggcgtcgtcggtgatcgagctcgtcggcgtggtccgggaggcgaagcggcggatggccggcgagtgcctgcgctgggcggaggggcgcaccggcggcgccgacccgttccagatgacgttcgactacgagtccgtgtacgtgtcggactggagcaagctcgggttcaacgacgtcgactacgggtacggcgcgccgtcggcggcggggccgctggtgaactgcgacctcatctcgtcggtgatcgtcatgcgggcgccggcgccgctcgccggcacgcggctgctggcgagctgcgtcaccaaggagcacgccgacgacttcgccgccaggatgagggaggatctcgtctaa</cdnaseq>|
AA = <aaseq>DLQVTTFTCGGFVIGLRTNHAVADGTGAAQFMNAVGDLARGLPE PRVKPIWARDRFPDPDIKPGPLPELPVLPLQYIAFDFPAAYLGKLKAQYAATAGASKI CSAFDIVIAKLWQCRTRAIAADPAAAVKLCFFASARQVLGLETGYWGNAIFPVKVSAA AGEVAASSVIELVGVVREAKRRMAGECLRWAEGRTGGADPFQMTFDYESVYVSDWSKL GFNDVDYGYGAPSAAGPLVNCDLISSVIVMRAPAPLAGTRLLASCVTKEHADDFAARM REDLV</aaseq>|
DNA = <dnaseqindica>1..846#gatttgcaggtgacgacgttcacctgcggcggcttcgtgatcgggctgcgcaccaaccacgcggtggcggacggcaccggcgccgcccagttcatgaacgccgtcggcgacctcgcccgcggcctcccggagccgcgggtgaagccgatctgggcgcgcgaccgcttcccggacccggacatcaagcccggcccgctgccggagctccccgtgctgccgctccagtacatcgccttcgacttccccgccgcctacctcggcaagctcaaggcgcagtacgccgccaccgccggcgccagcaagatctgctccgccttcgacatcgtcatcgccaagctctggcagtgccggacgcgcgccatcgccgccgaccccgccgcggccgtcaagctctgcttcttcgccagcgcccgccaggtgctcggcctggagaccggctactggggcaacgccatcttcccggtgaaggtgtccgcggcggcgggggaggtggcggcgtcgtcggtgatcgagctcgtcggcgtggtccgggaggcgaagcggcggatggccggcgagtgcctgcgctgggcggaggggcgcaccggcggcgccgacccgttccagatgacgttcgactacgagtccgtgtacgtgtcggactggagcaagctcgggttcaacgacgtcgactacgggtacggcgcgccgtcggcggcggggccgctggtgaactgcgacctcatctcgtcggtgatcgtcatgcgggcgccggcgccgctcgccggcacgcggctgctggcgagctgcgtcaccaaggagcacgccgacgacttcgccgccaggatgagggaggatctcgtctaatataccatggccgcccccaataacattattagtcacgtacaatattgtcactgaatattaatttgttcgtgtatatctattgtggtatgtattttttttttcataggaaaaaagtagtagtacgacaataggtagctgggagctacctaatatcgacctctggtttgagagtaagtgagcgagcgagagatgtaaaccacctcagtttttaccgtgctttgtgacatgcgtggtacagtactggtactattaatcattggccgtgaaaaattatctaccc</dnaseqindica>|
Link = [http://www.ncbi.nlm.nih.gov/nuccore/NM_001064516.2 RefSeq:Os06g0594600]|
}}
[[Category:Genes]]
[[Category:Japonica mRNA]]
[[Category:Oryza Sativa Japonica Group]]
[[Category:Japonica Genes]]
[[Category:Japonica Chromosome 6]]
[[Category:Chromosome 6]]

Os06g0594600

2014-06-08T14:27:05Z

Xiaoyanjie2012: /* 2.Screen of Rice Mutants for Altered Cell Wall Hydroxycinnamic Acid Content */

Please input one-sentence summary here.

==Annotated Information==
OsAt10 is a gene in rice(Oryza sativa).Its RAP ID is Os06g0594600,and its MSU ID LOC_Os06g39390. Overexpression of this gene can alters rice cell wall hydroxycinnamic acid Content and sacchariﬁcation.
===Function===
The overexpression of the rice gene -- OsAt10(LOC_Os06g39390) -- can effect the hydroxycinnamic acid content and saccharification in the rice cell wall.rice mutants with altered expression of four of these genes have altered cell wall hydroxycinnamate content. In-depth characterization of ectopic expression lines for one gene, OsAt10, revealed that this modification increases matrix polysaccharideassociated ester-linkedp-CA while simultaneously decreasing matrix polysaccharide-associated FA.OsAt10 overexpression plants exhibit increased in vitro saccharification, with no discernible effects on vegetative development. Thus, this gene is a useful target for improving biofuel and feed production.
Grass cell wall properties influence food, feed, and biofuel feedstock usage efficiency. The glucuronoarabinoxylan of grass cell walls is esterified with the phenylpropanoid-derived hydroxycinnamic acids ferulic acid (FA) and para-coumaric acid (p-CA). Feruloyl esters undergo oxidative coupling with neighboring phenylpropanoids on glucuronoarabinoxylan and lignin. Examination of rice (Oryza sativa) mutants in a grass-expanded and -diverged clade of BAHD acyl coenzyme A-utilizing transferases identified four mutants with altered cell wall FA or p-CA contents.Some researchers reported on the effects of overexpressing one of these genes, OsAt10 (LOC_Os06g39390), in rice. An activation-tagged line, OsAT10-D1, shows a 60% reduction in matrix polysaccharide-bound FA and an approximately 300% increase in p-CA in young leaf tissue but no discernible phenotypic alterations in vegetative development, lignin content, or lignin composition. Two additional independent OsAt10 overexpression lines show similar changes in FA and p-CA content. Cell wall fractionation and liquid chromatography-mass spectrometry experiments isolate the cell wall alterations in the mutant to ester conjugates of a five-carbon sugar with p-CA and FA. These results suggest that OsAT10 is a p-coumaroyl coenzyme A transferase involved in glucuronoarabinoxylan modification. Biomass from OsAT10-D1 exhibits a 20% to 40% increase in saccharification yield depending on the assay. Thus, OsAt10 is an attractive target for improving grass cell wall quality for fuel and animal feed.<ref name="ref1" />
====1.The Mitchell Clade of BAHD Acyltransferases Is Expanded and Diverged in Grasses====
Mitchell et al. (2007) identified what we term the Mitchell clade of BAHD acyl-CoA-dependent acyltransferases on the basis of high gene expression in grasses relative to dicots. To refine the hypothesis that these enzymes might be involved in grass-diverged cell wall synthesis, we systematically characterized the distribution of this clade in selected plant species and compared the clade with other characterized BAHD proteins. We identified BAHD proteins from the genomes of a diverse set of sequenced plant species available at the time of the analysis and examined the phylogenetic relationships among them and a reference set of BAHDs (Table I). To gain higher sensitivity relative to local sequence alignment (i.e. BLAST) for recognizing sequences with low, but potentially still significant, homology, we used a hidden Markov model to identify putative BAHD proteins (Finn et al., 2011)
The researchers then inferred an initial model of the phylo-genetic relationships among the putative BAHD proteins from each genome and the set of biochemically characterized BAHD proteins cataloged by D’Auria (2006). While we are aware that recent analyses have included the presence of a strict HXXXD motif as indicative of whether the protein is an active BAHD (Banks et al., 2011; Tuominen et al., 2011), we have included proteins with single amino acid alterations to this motif, since one of the known biochemically active proteins for the family involved in taxol biosynthesis, BAPT (National Center for Biotechnology Information identifier AAL92459; Walker et al., 2002), possesses a variation of this motif in which the His is replaced by a Ser.
As observed by Tuominen et al. (2011), the distribution of BAHD proteins varies among species (Table I). The Mitchell clade is embedded within clade V, or clade Va of Tuominen et al. (2011). Furthermore, we find that the Mitchell clade includes a biochemically characterized banana (Musa spp.) alcohol CoA acyltransferase, BanAAT (Beekwilder et al., 2004), and is related to a group of BAHD proteins that participate in taxol biosynthesis (Fig. 1B).

[[File:Table 1xxy.png]][[File:figure 1xxy.png]]
====2.Screen of Rice Mutants for Altered Cell Wall Hydroxycinnamic Acid Content====
Data are for homozygous wild-type segregant plants (gray bars) and homozygous mutant plants (hatched bars). Each plant line is designated by the repository identifier and the putative target gene. A and D, Average FA content from an AIR preparation. B and E, p-CA content from AIR. C and F, The ratio of FA to p-CA. Side tillers from lines 1B-00523 and 2D-40243 were harvested 10 weeks after germination. All other lines were harvested 7 weeks after germination. Averages from samples from two to three plants for each genotype were measured independently. Error bars indicate SD . *Significant difference at P , 0.05,**significant difference at P , 0.01 (Student’s t test).

[[File:Figure 2xxy.png]]
====3.The OsAT10-D1 Line Has an Increase in Cell Wall Glc Content====
Compensatory changes are often seen among the components of the cell wall (Humphrey et al., 2007). Quantification of sugars released by acid treatment of
destarched AIR preparations from mature straw suggests that the Glc content is increased by approximately 20% (w/w) for the mutant relative to the wild type (Fig. 9A). We observed the difference both with TFA treatment, which liberates monosaccharides derived from matrix polysaccharides and amorphous cellulose, and when the TFA residue was further treated with sulfuric acid, which breaks down crystalline cellulose (Fig. 9A). The difference in the mutant compared with the wild type is most apparent when the products of both treatments are summed together, which gives an increase in Glc in the mutant compared with the wild type of 19% 6 11%. By mass, we did not observe any other significant changes in sugar amounts in the mutant compared with the wild type. We also observed no change in the total mass percentage of sugars in AIR. When the TFA-solubilized sugars are expressed in terms of mol%, the data also indicate an increase in Glc content of 11% 6 5% (Fig. 9B). The sum of the mol % of other measured sugars (i.e. Xyl, Ara, and the sum of minor sugars) decreases proportionally to the Glc increase (7% 6 7%). This balance in mol % change suggests that the change in polysaccharide content in the mutant is restricted to the Glc-containing polymers.

[[File:Figure 9xxy.png]]

====4.The OsAT10-D1 Line Shows No Alterations in Lignin Content or Composition====
OsAT10-D1 mature straw samples show no significant differences in the content of acetylbromide-soluble lignin after saponification relative to the wild type
(Table II). We obtained a similar result via py-MBMS for mature straw and separate, young leaf and sheath samples. The py-MBMS also revealed no difference in the syringyl-guaiacyl (S:G) lignin ratio in the mutant compared with the wild type after saponification (Table II). We also collected py-MBMS data for unprocessed straw and AIR of OsAT10-D1. Separate analyses of the saponified and unsaponified samples reveals distinctions between the wild type and mutant in the unsaponified samples (Fig. 10A). Principal component 1 explains the alcohol extraction (30% of the variation), and principal component 2 explains the differences between wild-type and mutant samples (19% of the variation). The loadings for principal component 2 show that the major ions that distinguish wild-type and mutant samples are phenolics (Fig. 10B). The mass spectrometry fragmentation pattern is consistent with an interpretation in which there is an increase of p-CA, as reflected by peaks 120, 94, and 91, and a decrease in FA, as reflected in the reduction in the coniferyl ion, peak 150 (Evans and Milne, 1987). Because principal component analysis no longer distinguishes the samples after saponification (Fig. 10C), the observed differences in phenylpropanoids between OsAT10-D1 and the wild type are likely associated with ester-linked hydroxycinnamates and not lignin, consistent with the other results.
A limitation of the pyrolysis method for determining lignin composition is that it inaccurately measures H-lignin, which volatilizes poorly and instead turns to char upon heating. Because of the increase in p-CA, a precursor of H-lignin, in OsAT10-D1 cell walls relative to the wild type, we sought to determine whether there is a change in the char content of OsAT10-D1 using a thermogravimetric pyrolysis instrument. Duplicate runs per genotype of the thermogravimetric instrument did not detect a difference in the mass remaining from mature straw after pyrolysis, again consistent with there being no difference in core lignin composition or content between OsAT10-D1 and the wild type.

[[File:Table 2xxy.png]]
[[File:Figure 10xxy.png]]

===Expression===
For the remaining 11 lines, we characterized the alkali-labile hydroxycinnamoyl ester content of cell wall alcohol-insoluble residue (AIR) from leaf blades and sheaths of side tillers. We compared homozygous, mutant, and wild-type segregant plants 7 or 10 weeks after planting. The screen revealed four mutants with possible cell wall hydroxycinnamic acid phenotypes . All four lines showed changes in the expression of the nearest acyltransferase
gene to the T-DNA insertion site via quantitative reverse transcription (qRT)-PCR. Homozygous mutant progeny of4A-03423(here afterreferred to asOsAT10-D1), which has increased expression of OsAt10, exhibited reduced FA (approximately 60% less) and an increase in p-CA (approximately 300% more) in sheaths and leaves.TheT-DNA insertions it efort helinewerefertoas OsAT10-D1(PFG_4A-03423) is approximately 8.5 kb downstream of the transcriptional start site forOsAt10. The insert is oriented with the activating sequences proximate toOsAt10and in the range observed to activate expression (Jeong et al., 2006). As mentioned above, qRT-PCR indicated that, indeed,the expression ofOsAt10was increased by more than 100-fold in the leaves of homozygous OsAT10-D1 plants (Fig. 3B). InOsAT10-D1, besides OsAt10the expression of other genes proximate to the site of the T-DNA insertion does not vary significantly relative to the wild type (Fig. 3B). Similarly, the expression of related OsAtgenesdoesnotvarysignificantly in OsAT10-D1(Supplemental Fig. S2), reducing the possibility that the observed phenotype is due to compensation at the level of gene expression of a related acyltransferase. Of the acyltransferase transcripts examined in this survey, OsAt6appears to vary the most, although not significantly. However, OsAt6(LOC_Os01g08380) is expressed near the lower limitofourdetectionand,infact,wasreportedas undetectable in a previous qRT-PCR study (Piston et al., 2010). OsAT10-D1lines show no change in size and dry mass at maturity (Fig. 4, A and B). However, we did measure an approximately 20% to 30% decrease in total seed mass per plant for the mutant compared with the wild type.

[[File:Figure 3xxy.png]][[File:Figure 4xxy.png]]

===Evolution===
Please input evolution information here.

You can also add sub-section(s) at will.

==Labs working on this gene==
Please input related labs here.

==References==
1. Bartley, L.E., et al., Overexpression of a BAHD acyltransferase, OsAt10, alters rice cell wall hydroxycinnamic acid content and saccharification. Plant physiology, 2013. 161(4): p. 1615-1633.

==Structured Information==
{{JaponicaGene|
GeneName = Os06g0594600|
Description = The start codon is not identified.|
Version = NM_001064516.2 GI:297606110 GeneID:4341427|
Length = 1126 bp|
Definition = Oryza sativa Japonica Group Os06g0594600, complete gene.|
Source = Oryza sativa Japonica Group

ORGANISM Oryza sativa Japonica Group
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP
clade; Ehrhartoideae; Oryzeae; Oryza.
|
Chromosome = [[:category:Japonica Chromosome 6|Chromosome 6]]|
AP = Chromosome 6:24256206..24257331|
CDS = 24256486..24257331|
GCID = <gbrowseImage1>
name=NC_008399:24256206..24257331
source=RiceChromosome06
preset=GeneLocation
</gbrowseImage1>|
GSID = <gbrowseImage2>
name=NC_008399:24256206..24257331
source=RiceChromosome06
preset=GeneLocation
</gbrowseImage2>|
CDNA = <cdnaseq>gatttgcaggtgacgacgttcacctgcggcggcttcgtgatcgggctgcgcaccaaccacgcggtggcggacggcaccggcgccgcccagttcatgaacgccgtcggcgacctcgcccgcggcctcccggagccgcgggtgaagccgatctgggcgcgcgaccgcttcccggacccggacatcaagcccggcccgctgccggagctccccgtgctgccgctccagtacatcgccttcgacttccccgccgcctacctcggcaagctcaaggcgcagtacgccgccaccgccggcgccagcaagatctgctccgccttcgacatcgtcatcgccaagctctggcagtgccggacgcgcgccatcgccgccgaccccgccgcggccgtcaagctctgcttcttcgccagcgcccgccaggtgctcggcctggagaccggctactggggcaacgccatcttcccggtgaaggtgtccgcggcggcgggggaggtggcggcgtcgtcggtgatcgagctcgtcggcgtggtccgggaggcgaagcggcggatggccggcgagtgcctgcgctgggcggaggggcgcaccggcggcgccgacccgttccagatgacgttcgactacgagtccgtgtacgtgtcggactggagcaagctcgggttcaacgacgtcgactacgggtacggcgcgccgtcggcggcggggccgctggtgaactgcgacctcatctcgtcggtgatcgtcatgcgggcgccggcgccgctcgccggcacgcggctgctggcgagctgcgtcaccaaggagcacgccgacgacttcgccgccaggatgagggaggatctcgtctaa</cdnaseq>|
AA = <aaseq>DLQVTTFTCGGFVIGLRTNHAVADGTGAAQFMNAVGDLARGLPE PRVKPIWARDRFPDPDIKPGPLPELPVLPLQYIAFDFPAAYLGKLKAQYAATAGASKI CSAFDIVIAKLWQCRTRAIAADPAAAVKLCFFASARQVLGLETGYWGNAIFPVKVSAA AGEVAASSVIELVGVVREAKRRMAGECLRWAEGRTGGADPFQMTFDYESVYVSDWSKL GFNDVDYGYGAPSAAGPLVNCDLISSVIVMRAPAPLAGTRLLASCVTKEHADDFAARM REDLV</aaseq>|
DNA = <dnaseqindica>1..846#gatttgcaggtgacgacgttcacctgcggcggcttcgtgatcgggctgcgcaccaaccacgcggtggcggacggcaccggcgccgcccagttcatgaacgccgtcggcgacctcgcccgcggcctcccggagccgcgggtgaagccgatctgggcgcgcgaccgcttcccggacccggacatcaagcccggcccgctgccggagctccccgtgctgccgctccagtacatcgccttcgacttccccgccgcctacctcggcaagctcaaggcgcagtacgccgccaccgccggcgccagcaagatctgctccgccttcgacatcgtcatcgccaagctctggcagtgccggacgcgcgccatcgccgccgaccccgccgcggccgtcaagctctgcttcttcgccagcgcccgccaggtgctcggcctggagaccggctactggggcaacgccatcttcccggtgaaggtgtccgcggcggcgggggaggtggcggcgtcgtcggtgatcgagctcgtcggcgtggtccgggaggcgaagcggcggatggccggcgagtgcctgcgctgggcggaggggcgcaccggcggcgccgacccgttccagatgacgttcgactacgagtccgtgtacgtgtcggactggagcaagctcgggttcaacgacgtcgactacgggtacggcgcgccgtcggcggcggggccgctggtgaactgcgacctcatctcgtcggtgatcgtcatgcgggcgccggcgccgctcgccggcacgcggctgctggcgagctgcgtcaccaaggagcacgccgacgacttcgccgccaggatgagggaggatctcgtctaatataccatggccgcccccaataacattattagtcacgtacaatattgtcactgaatattaatttgttcgtgtatatctattgtggtatgtattttttttttcataggaaaaaagtagtagtacgacaataggtagctgggagctacctaatatcgacctctggtttgagagtaagtgagcgagcgagagatgtaaaccacctcagtttttaccgtgctttgtgacatgcgtggtacagtactggtactattaatcattggccgtgaaaaattatctaccc</dnaseqindica>|
Link = [http://www.ncbi.nlm.nih.gov/nuccore/NM_001064516.2 RefSeq:Os06g0594600]|
}}
[[Category:Genes]]
[[Category:Japonica mRNA]]
[[Category:Oryza Sativa Japonica Group]]
[[Category:Japonica Genes]]
[[Category:Japonica Chromosome 6]]
[[Category:Chromosome 6]]

File:Figure 10xxy.png

2014-06-08T14:26:49Z

Xiaoyanjie2012:

File:Table 2xxy.png

2014-06-08T14:26:13Z

Xiaoyanjie2012:

File:Figure 9xxy.png

2014-06-08T14:17:29Z

Xiaoyanjie2012:

Os06g0594600

2014-06-08T14:13:02Z

Xiaoyanjie2012: /* Expression */

File:Figure 4xxy.png

2014-06-08T14:12:57Z

Xiaoyanjie2012:

File:Figure 3xxy.png

2014-06-08T14:12:13Z

Xiaoyanjie2012:

Os06g0594600

2014-06-08T14:09:42Z

Xiaoyanjie2012: /* 1.The Mitchell Clade of BAHD Acyltransferases Is Expanded and Diverged in Grasses */

File:Figure 2xxy.png

2014-06-08T14:09:23Z

Xiaoyanjie2012:

Os06g0594600

2014-06-08T14:05:57Z

Xiaoyanjie2012: /* Annotated Information */

File:Table 1xxy.png

2014-06-08T14:05:00Z

Xiaoyanjie2012:

File:Figure 1xxy.png

2014-06-08T14:04:37Z

Xiaoyanjie2012:

Os03g0411500

2014-06-08T08:39:00Z

Xiaoyanjie2012: /* Expression */

A nuclear gene coding caseinolyse positioned in plastid or mitochondrion regulating early morphogenesis.
VYL is a gene in rice.Its RAP ID is Os03g0411500 and its MSU ID is LOC_Os03g29810. The mutant of this gene produces chlorotic leaves throughout the entire growth period.
==Annotated Information==
===Function===
The protein encoded by this gene belongs to the peptidase family S14 and hydrolyzes proteins into small peptides in the presence of ATP and magnesium. The protein is transported into mitochondrial matrix and is associated with the inner mitochondrial membrane<ref name="ref2" />, or plastid inner membrane in plants<ref name="ref4" />.
The plastidic caseinolytic protease (Clp) of higher plants is an evolutionarily conserved protein degradation apparatus composed of a proteolytic core complex (the P and R rings) and a set of accessory proteins (ClpT, ClpC, and ClpS).<ref name="ref4" />
Rice yellow leaf mutant vyl, the performance of the entire growth period, new leaves chlorotic phenotype, then gradually turn green from the top down.<ref name="ref4" /> VYL Arabidopsis Clp protease subunit ClpP6 homologous protein in rice, is one of the subunits of the chloroplast Clp protease, with the Clp protease subunit interactions OsClpP3 and OsClpP4 respectively, play an important role in the biosynthesis of rice chloroplast.<ref name="ref4" />
What's more, the gene D53 product shares predicted features with the class I Clp ATPase proteins and can form a complex with the a/b hydrolase protein DWARF 14 (D14) and the F-box protein DWARF 3 (D3), two previously identified signalling components potentially responsible for SL(Strigolactones) perception, which means, in a D14- and D3-dependentmanner, SLs induce D53 degradation by the proteasome and abrogate its activity in promoting axillary bud outgrowth.<ref name="ref5" />
The role and molecular composition of Clps in higher plants has just begun to be unraveled, mostly from studies with the model dicotyledonous plant Arabidopsis (Arabidopsis thaliana).Some researchers isolated a virescent yellow leaf (vyl) mutant in rice (Oryza sativa), which produces chlorotic leaves throughout the entire growth period.<ref name="ref4" />
====1.The vyl Mutant Displays Reduced Chlorophyll Accumulation====
The vyl mutant was derived by transforming tissue cultures of the japonica rice variety Kita-ake. When grown under an alternating light/dark cycle (12 h of light at 30°C/12 h of darkness at 20°C) in a growth chamber, vyl mutant plants displayed a virescent yellow leaf pheno-type, and during development, leaves gradually turned green from their tips (more developed) to their bases (less developed; Fig. 1, A and B). At maturity, the vyl mutant plants also had reduced height and smaller seeds (Fig. 1, C and D). Mutant leaves also contained less chlorophyll than the wild type at various growth stages (Fig. 1E).
Additionally, vyl mutants developed chlorotic leaves under different temperature conditions and light/dark cycles, suggesting that the virescent yellow phenotype of the vyl mutant was developmentally regulated but independent of external cues (such as temperature and light)
[[File:figure 1.png]]

====2.The vyl Mutant Has Impaired Chloroplast Development====
Next, researchers investigated whether the virescent yellow phenotype of the vyl mutant was associated with ultra-structural changes in the chloroplasts. Leaf samples of L3U (upper half of the third leaf), L3L (basal half of the third leaf), and L4 (fourth leaf above the shoot base) were collected from wild-type and vyl mutant seedlings and compared (Fig. 2A). Normally, when the third leaf has fully emerged from the shoot base of a rice plant, the shoot also contains the fourth to the seventh immature leaves. The leaf cells in the L3L and L3U samples contain mature chloroplasts, whereas those in the shoot base and L4 samples contain proplastids and early developing immature chloroplasts (Sugimoto et al., 2004). Similar to the wild type, the chloroplasts from the L3U green leaf sample (already turned green) of vyl mutant seedlings displayed well-developed lamellar structures and were equipped with normally stacked grana and thylakoid membranes (Fig. 2, B and C). By contrast, the chloroplasts from the L3L and L4 pale leaves (still wrapped in leaf sheath) of the vyl mutant had much reduced thylakoid membrane networks compared with wild-type plants (Fig. 2, D–G). This developmental defect is similar to the phenotype reported in the Arabidopsis CLPP6 antisense transgenic plants (Sjögren et al., 2006).

[[File:Figure 2 .png]]

====3.The vyl Mutant Has Impaired Photosynthesis====
Chloroplasts are the organelles in plant cells that perform photosynthesis; therefore, they play an essential role in plant growth. To test whether the photosynthetic apparatus was affected in vyl mutants, we compared some key parameters of PSI and PSII between vyl and wild-type plants. Distinct differences in photochemical efficiency of PSII (FPSII), electron transport rate (ETR), nonphotochemical quenching (NPQ), and photochemical
quenching (Qp) were detected between vyl mutants and the wild type. In contrast, the maximal efficiency of PSII photochemistry (F v /F m ) values was almost comparable between vyl and wild-type plants (Table I). These observations indicate that vyl mutants absorbed much less light energy, as shown by the lower NPQ values. PSII photochemistry was reduced at both the donor and acceptor sites, as indicated by the greatly decreased Qp and ETR in vyl mutants. Notably, the PSII structure appeared intact in the mutant plants (indicated by the equivalent F v /F m values), but the actual FPSII was much lower in the mutants. These differences may underlie the defects in chloroplast biogenesis and retarded growth in the vyl mutant (Fig. 1, C and D)
[[File:Table_1.png‎]]

====4.Altered Expression of Genes Associated with Chloroplast Biogenesis and Photosynthesis in vyl Mutants====
Chloroplast biogenesis and physiological changes are tightly regulated by the coordinated expression of plastid and nuclear genes during leaf development and facilitated by protein quality control (Kusumi et al., 2010; Clarke, 2012). To examine whether the expression of genes associated with chloroplast biogenesis and photosynthesis was altered in vyl mutants, quantitative real-time reverse transcription (qRT)-PCR was performed on total RNAs extracted from the L4 and
L3U leaf sections of wild-type and vyl plants. Compared with the wild type, the transcript levels of Virescent1 (V1), V2, and V3 genes and some other genes encoding components of the plastid and nuclear transcription apparatus Sigma factor 2A (OsSig2A), Ribosomal Protein S15 (rps15) and the subunit RNA polymerase (RpoA and RpoTp) that are highly expressed in early stages of chloroplast development were significantly increased in vyl mutants. In contrast, several genes encoding components of the photosynthesis apparatus RuBisCO large subunit (RbcL), the subunit of photosystem I (PsaA), a core component of Photosystem II (PsbA), light-harvesting complex protein (Lhcp2) and Chlorophyll A/B binding protein1 (Cab1) that are highly expressed in later stages of chloroplast development were significantly reduced in vyl mutants (Fig. 3). These results suggested that VYL plays an important role in regulating chloroplast biogenesis.

[[File:Figure 3 .png]]

====5.The vyl Locus Maps to a Putative Gene Encoding OsClpP6====
Genetic analysis showed that the virescent yellow leaf phenotype in vyl mutants is controlled by a single recessive nuclear locus, VYL. Using a BC 1 F 2 mapping population of the vyl mutant and 93-11 (an indica variety), we mapped the vyl gene to a 137-kb genomic region on chromosome 3 between the insertion/deletion polymorphism (Indel) markers F17 and I53. Within this region, 17 open reading frames (ORFs) were predicted from published data (http://
www.gramene.org/; Fig. 4A). Genomic sequence analysis revealed that only the 13th ORF (LOC_Os03g29810) carries a single-nucleotide transition (G→T) at the position 1,509 bp from the ATG start codon (Fig. 4B). We obtained the full-length complementary DNA (cDNA) of LOC_Os03g29810 from both the wild type and the vyl mutant by reverse transcription (RT)-PCR. Sequence analysis showed that the full-length VYL cDNA is 780 bp long in the wild type but 842 bp in the mutant.
Comparison of the genomic and cDNA sequences revealed that the single-nucleotide substitution in the vyl mutant genome created a new splicing site and the addition of a partial fourth intron sequence in the cDNA (Fig. 4, C and D). The mutant cDNA is predicted to encode a truncated vyl mutant protein (DVYL) lacking the classic Ser protease triad (Ser-His-Asp) in the active site and the polypeptide-binding site (Fig. 4, C and E). Phylogenetic analysis and protein sequence alignment showed that VYL is most closely related to the Arabidopsis ClpP6 protein with a conserved S14_ClpP_2 domain and more remotely related to several other Arabidopsis ClpP subunits, and it apparently represents a single-copy gene in the rice genome (Fig. 4F).
To verify the identity of VYL, the plasmid pGVYL, containing a 6-kb genomic DNA fragment consisting of a 2.5-kb upstream sequence, the entire VYL coding region, including nine exons and eight introns, and a 0.6-kb downstream sequence, was constructed and introduced into the vyl mutant. All five transgenic lines containing pGVYL complemented the virescent phenotype of the vyl mutant (Fig. 5). To further confirm that disruption of the VYL gene was responsible for the vyl mutant phenotype, we generated RNA interference transgenic plants in the wild-type Kita-ake background and obtained more than eight independent transgenic
lines. qRT-PCR analysis revealed that the expression of VYL was reduced in three tested transgenic lines compared with the wild-type plants. These VYL knockdown transgenic plants showed reduced accumulation of chlorophyll a and chlorophyll b. Together, these results confirmed that LOC_Os03g29810 indeed corresponds to the VYL gene.
[[File:Figure 4 .png]]

====6.Interactions between VYL, OsClpP3, OsClpP4, OsClpP5, and OsClpT====
To gain an insight into the role of VYL in the rice Clp complex assembly, researchers sought to identify the proteins that directly interact with VYL. We first used a mass spectrometry-based tandem affinity purification proteomics approach. We generated a VYL-HBH construct in
which the C terminus of VYL was fused with a Hisbiotin tag (Tagwerker et al., 2006), and the fusion gene was driven by the Ubiquitin promoter. The VYL-HBH
fusion construct was introduced into the vyl mutant via Agrobacterium tumefaciens-mediated transformation. The VYL-HBH fusion protein transgene recovered the chlorotic phenotype of the vyl mutant, indicating that the fusion protein is biologically functiona SDS-PAGE and immunoblot analyses showed that the VYL-HBH fusion protein and several additional proteins could be effectively purified from the pUbi:: VYL-HBH transgenic plants using Ni 2+ -Sepharose and streptavidin beads (Fig. 8A). The copurified protein bands were excised from the SDS-PAGE gel and analyzed by matrix-assisted laser-desorption ionization time of flight (MALDI-TOF). Three bands were identified with confidence (P , 0.05), including two putative components of the Clp complex (LOC_Os03g29810/ VYL/OsClpP6 and LOC_Os10g43050/OsClpP4) and a vacuolar ATP synthase subunit E (Loc_Oso1g46980/ v-ATP-E; Fig. 8B; Table II). This result suggested that VYL is a bona fide component of the OsClp complex in vivo.
To further investigate the role of VYL in the OsClp core proteolytic complex assembly, we cloned the rice homologs of Arabidopsis ClpR1, ClpP3, ClpP5, and ClpT and named them OsClpR1, OsClpP3, OsClpP5, and OsClpT, respectively. It is noticeable that there is only one copy of the OsClpT gene in the rice genome.
Using yeast two-hybrid assays, we found that VYL protein directly interacted with OsClpP3 and OsClpP4 but not with vacuolar ATP synthase subunit E (v-ATP-E;
Fig. 8C). Furthermore, we found that the yeast strain (Gold Saccharomyces cerevisiae) carrying BD-VYL+AD-OsClpP4 grew and developed the blue color faster than the yeast strain (Gold Saccharomyces cerevisiae) carrying BD-DVYL+AD-OsClpP4 or BD-VYL+AD-OsClpP3 on yeast growth medium containing 5-Bromo-4-chloro-3-
indoylla-galactoside (X-a-Gal). Furthermore, the yeast strain(GoldSaccharomycescerevisiae)carryingBD-DVYL+AD-OsClpP3 did not grow even after incubation for 5 d in a growth chamber (Fig. 8D). This result suggests that the C-terminal polypeptide-binding site of VYL likely plays a role in mediating the interaction between VYL with OsClpP3 and OsClpP4. In addition, they also found that OsClpP3 interacted with OsClpT, OsClpP4 interacted with OsClpP5 and OsClpT, and that both OsClpP4 and OsClpT interacted with themselves but OsClpP5 and OsClpP3 did not (Fig. 8E). Due to the self-activation activity of VYL-AD, we were not able to test whether VYL can homodimerize.

[[File:Figure 5 .png]][[File:Figure 6 .png]]

===Expression===
Expression is constitutive in most tissues examined (roots, stems, leaves, leaf sheath, panicle)but most abundant in leaf sections containing
chloroplasts in early stages of development,which can be light-mediated.<ref name="ref4" />
The young chlorotic leaves turn green in later developmental stages, accompanied by alterations in chlorophyll accumulation, chloroplast ultrastructure, and the expression of chloroplast development- and photosynthesis-related genes. Positional cloning revealed that the VYL gene encodes a protein homologous to the Arabidopsis ClpP6 subunit and that it is targeted to the chloroplast. VYL expression is constitutive in most tissues examined but most abundant in leaf sections containing chloroplasts in early stages of development. The mutation in vyl causes premature termination of the predicted gene product and loss of the conserved catalytic triad (serine-histidine-aspartate) and the polypeptide-binding site of VYL. Using a tandem affinity purification approach and mass spectrometry analysis, we identified OsClpP4 as a VYL-associated protein in vivo. In addition, yeast two-hybrid assays demonstrated that VYL directly interacts with OsClpP3 and OsClpP4. Furthermore, we found that OsClpP3 directly interacts with OsClpT, that OsClpP4 directly interacts with OsClpP5 and OsClpT, and that both OsClpP4 and OsClpT can homodimerize. Together, our data provide new insights into the function, assembly, and regulation of Clps in higher plants.<ref name="ref4" />
To investigate the expression patterns of VYL during chloroplast and leaf development, researchers analyzed VYL expression in different sections of leaves at various leaf developmental stages by qRT-PCR (Fig. 2A). They found that in wild-type plants, VYL was most highly expressed in the L4 section at the early chloroplast and leaf development stage (Fig. 6A). Furthermore, qRT-PCR analysis and histochemical staining of the pVYL::GUS reporter gene transgenic plants showed that VYL was constitutively expressed in young buds, young roots, stems, leaves, leaf shoots, and panicles (Fig. 6, B and C). To test whether VYL expression is regulated by light, They analyzed VYL expression during greening of etiolated seedlings. Wild-type rice plants were grown in continuous darkness for 10 d and subsequently exposed to light for 3, 6, 9, 12, 15, 18, 21, or 24 h. The expression of VYL was highly induced after 3 h of illumination and peaked after 6 h of illumination, then its expression gradually decreased over time, and by 15 h after illumination, its expression returned to the preillumination basal level (Fig. 6D). These observations suggested that VYL likely plays a role in the light regulation of chloroplast development. We next conducted qRT-PCR analysis to examine a possible effect of the vyl mutation on the expression of other genes encoding various components of the rice Clp. We
found that OsClpPs, OsClpT, and OsClpR4 all had similar expression patterns to VYL, with a peak accumulation at the early stage of chloroplast development, but the expression levels of these genes were higher in the vyl mutants compared with the wild type (Fig. 7). This observation suggested that there may be a compensatory mechanism to increase the expression of OsClpPs, OsClpT, and OsClpR4 in vyl mutants.

[[File:Figure 7 .png]][[File:Figure 8 .png]]

==Evolution==
ATP-dependent Clp protease proteolytic subunit is an enzyme that in humans is encoded by the CLPP gene.<ref name="ref1" /><ref name="ref2" /> It is found in mitochondria and is widely distributed in bacterial species.
In several bacteria, such as E. coli, proteins tagged with the SsrA peptide (ANDENYALAA) encoded by tmRNA are digested by Clp proteases.<ref name="ref3" />

[[File:612px-Protein_CLPP_PDB_1tg6.png]][[File:1867.png]]

==Labs working on this gene==
National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China

==References==
<references>
<ref name="ref1">
Bross P, Andresen BS, Knudsen I, Kruse TA, Gregersen N (Feb 1996). "Human ClpP protease: cDNA sequence, tissue-specific expression and chromosomal assignment of the gene". FEBS Lett 377 (2): 249–52. doi:10.1016/0014-5793(95)01353-9. PMID 8543061.
</ref>
<ref name="ref2">
a b "Entrez Gene: CLPP ClpP caseinolytic peptidase, ATP-dependent, proteolytic subunit homolog (E. coli)".
</ref>
<ref name="ref3">
Gottesman S, Roche E, Zhou Y, Sauer RT (1998). "The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system". Genes Dev 12 (9): 1338–47. doi:10.1101/gad.12.9.1338. PMC 316764. PMID 9573050
</ref>
<ref name="ref4">
Hui Dong et al. A Rice Virescent-Yellow Leaf Mutant Reveals New Insights into the Role and Assembly of Plastid Caseinolytic Protease in Higher Plants. Plant Physiology, 2013, 162(4): 1867-1880
</ref>
<ref name="ref5">
Zhou Feng et al.D14–SCFD3-dependent degradation of D53 regulates strigolactone signaling. Nature,2013. doi:10.1038/nature12878
</ref>
</references>
==Structured Information==
{{JaponicaGene|
GeneName = Os03g0411500|
Description = Peptidase S14, ClpP family protein|
Version = NM_001056884.1 GI:115453496 GeneID:4333096|
Length = 3448 bp|
Definition = Oryza sativa Japonica Group Os03g0411500, complete gene.|
Source = Oryza sativa Japonica Group

ORGANISM Oryza sativa Japonica Group
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP
clade; Ehrhartoideae; Oryzeae; Oryza.
|
Chromosome = [[:category:Japonica Chromosome 3|Chromosome 3]]|
AP = Chromosome 3:17630235..17633682|
CDS = 17630290..17630357,17630478..17630512,17631200..17631269,17631410..17631614,17631776..17631841 ,17632451..17632591,17632803..17632856,17632992..17633084,17633183..17633230 |
GCID = <gbrowseImage1>
name=NC_008396:17630235..17633682
source=RiceChromosome03
preset=GeneLocation
</gbrowseImage1>|
GSID = <gbrowseImage2>
name=NC_008396:17630235..17633682
source=RiceChromosome03
preset=GeneLocation
</gbrowseImage2>|
CDNA = <cdnaseq>atggcgcctatggccatctccaccccgctcgccctccgcgcctccccgacccgcctcctctcccgcaggcggagcggagccaaatcaggcgtggctctcccaggtccacaatttgtaccacctggtatttcttcaaagttggacgagaggatacattgtcattcttctctgaggaaaaatacaattgtagcatcagagaatgaaaatccacctttaatgcctgccataatgactcctgctggtgctcttgatctggcaactgtattgttggggaaccgcattatcttcattggtcaatatattaactcgcaagtagcacagcgtgtaatatcacagcttgtcacacttgctgctgttgatgaagaggctgatattctgatctacctgaactgccccggcggaagtctctactccatcttagcaatttatgattgcatgtcctggatcaagcccaaagttggaacagtgtgctttggtgttgttgctagccaggcagcaattatacttgctggcggtgagaagggaatgcgttatgccatgccaaatgctagagtaatgattcatcaacctcaaggtgtatcagagggtaatgtggaggaggtgaggcgacaggttggggaaaccatttatgctcgtgataaagttgataagatgtttgctgcttttactgggcaaaccttggatatggtacaacagtggacagagagggatcgtttcatgtcttcatctgaagccatggactttggactagttgatgccctgctggaaacaagatactaa</cdnaseq>|
AA = <aaseq>MAPMAISTPLALRASPTRLLSRRRSGAKSGVALPGPQFVPPGIS SKLDERIHCHSSLRKNTIVASENENPPLMPAIMTPAGALDLATVLLGNRIIFIGQYIN SQVAQRVISQLVTLAAVDEEADILIYLNCPGGSLYSILAIYDCMSWIKPKVGTVCFGV VASQAAIILAGGEKGMRYAMPNARVMIHQPQGVSEGNVEEVRRQVGETIYARDKVDKM FAAFTGQTLDMVQQWTERDRFMSSSEAMDFGLVDALLETRY</aaseq>|
DNA = <dnaseqindica>56..123#244..278#966..1035#1176..1380#1542..1607#2217..2357#2569..2622#2758..2850#2949..2996#actcctcagtcctcgcctcggctcggctccctcccacgctccagctccgcctccaatggcgcctatggccatctccaccccgctcgccctccgcgcctccccgacccgcctcctctcccgcaggtgagctccacggaactacaacttccacctccttcgctcgctcgctcgccccgcgcttctctcttcatggattcccccgttcttgtcccctcaccctctgtctggtccttctttcttcaggcggagcggagccaaatcaggcgtggctctcccaggtgagatttcctaacccttggtttagcaaaccatttcccttggtcagctcgttaggccaggactgtttggtggaatttgatcgttgatttgataagctttactgagatgttgtccatggtggtgcacatattagaacatgaccatttgggcagccgtcccatcagctcctagttgtctttctctgtgccaattttttatggagtcgtcattggtaggttttgcaaagcatatagaacctctgaatgtcggcattatccaagagtatgccgacctggggttatctgaaccagtgtcaactccttcctgggccaatgtctggtatgcatcagcattagctcactaagtacacgaaaaatggatgtgcttggttgaacggatatgtataaaaacgataacctgcatataacatatcacctcagtttggtctgattctgaaattagtttagggccttttagcaaactgctgaatgagatttccagactgtatatgtgttattgtgtttgtcagtaactcagtatggtgtattagcacaactcaacatgccataatgacaggatatgcggagcacaaacttttctttggacatgttttttggattcctttactgtttagtccatctgtctttcacatgatatattgctgcaaatgtgctgagttgcattctcactcaaatttccacacctaggtccacaatttgtaccacctggtatttcttcaaagttggacgagaggatacattgtcattcttctctgaggtgatatatttgaaactgtcatgcctgatatacaatgaggatacattgcctgatttgaaactgctcatctaggttttatttgtgctgtgtatcagaatcctgttttattcattcgtaatattgtaaatattttttcacaggaaaaatacaattgtagcatcagagaatgaaaatccacctttaatgcctgccataatgactcctgctggtgctcttgatctggcaactgtattgttggggaaccgcattatcttcattggtcaatatattaactcgcaagtagcacagcgtgtaatatcacagcttgtcacacttgctgctgttgatgaagaggctgatattctggttagtgtttatttttgtgtttttcagatcataacagttaccctattgttcactgcagcagctcttgattgctcaacttcactcccttggcttgctcctttagctcacaggtgtgttgctctatatcttataactccttttgtataattctgttttgccagatctacctgaactgccccggcggaagtctctactccatcttagcaatttatgattgcatgtcctgggtatgccatctatgttgagctactttttccatgtccttcatgcattcaaatttcagagattgtattcacctatatttattcttgtggatgctttctgagttattctcatctaatttaatatttacattgttggatgagaacacattatagatgcatctcaacattttgtatcttccacattatgcatgcccctagctagtgaatttatatttataatatagcacagatatagcatttacaggaaagcctaatgtaatttaggcaaaaattatatctcatatcaatggtagtgcttgcaacatttgtattcttatatttttattgtagtacatactagacatgagcatttgccatgctgagactgtgctaattgggtttggtctggtactgcagacaacagatctcatttcctgaaatcatgcctgtctctaaaactggctttgagctggaccagccttgttagttgttagatttggctgtgtttttacttgttatgccagttttccataaccaaatactttatctacaatttcgcctactgataaataaatcccagaatattaatctttttttgttgttctgcactaacacatgaaccatttattgcagatcaagcccaaagttggaacagtgtgctttggtgttgttgctagccaggcagcaattatacttgctggcggtgagaagggaatgcgttatgccatgccaaatgctagagtaatgattcatcaacctcaaggtgtatcagaggtatgattctggggctttctgcctttctgagttactgcagcgggtgcattatgattttctaacattgtgactacagtaaataataatcatcatcattttagctggccacatgaaacttacaatatacagtcttgctaggacatacttgcttgcctttgtgtttgttgtacttgaagtttgttttttattctaaaaattggatgatttgcagggtaatgtggaggaggtgaggcgacaggttggggaaaccatttatgctcgtgatgtaagtgttttgtgatagataacaagttctatattttcttcagtgtacatttgaattagatgtttgatgagggtaaggaatttctcttgattctgctctctaagcgattaaagcttctgaaaaatctggatgcagaaagttgataagatgtttgctgcttttactgggcaaaccttggatatggtacaacagtggacagagagggatcgtttcatgtcttcatctgaagtaactttcatctcttaaatgtatcagaagaaagtaaatcatcatttgccatgtgaattataacttatttcccccttctttttttggttttaccctaggccatggactttggactagttgatgccctgctggaaacaagatactaacaaacaaacacttaggcacagtttgattagcagaggctggtacaaggattgtgaaactggagctgaagttgagattttcgccgtccttctagttcaaggactccaatgacaggaggctggatctgcgagacttgaatgcatcgccatcgctcctatgagcaaaacatctctgcgttgagtgtgattttttgctcttcttttttggatctggttttacatgccgccagcctatggtctcaatgcattggtcctgctaatgtttagtgtagaccagatgatcctttagacagcaaaacatcagttattactccgtagattttcccatgagctgattccagactgtgtgcaacttttgtgttccaattgcaaagtttgcaacccaacccaacccaacacatgggctgatgggctgttgacaaaggagagattttacacttcacatatgtcaaact</dnaseqindica>|
Link = [http://www.ncbi.nlm.nih.gov/nuccore/NM_001056884.1 RefSeq:Os03g0411500]|
}}
[[Category:Genes]]
[[Category:Japonica mRNA]]
[[Category:Oryza Sativa Japonica Group]]
[[Category:Japonica Genes]]
[[Category:Japonica Chromosome 3]]
[[Category:Chromosome 3]]

Os03g0411500

2014-06-08T08:38:46Z

Xiaoyanjie2012: /* 6.Interactions between VYL, OsClpP3, OsClpP4, OsClpP5, and OsClpT */

A nuclear gene coding caseinolyse positioned in plastid or mitochondrion regulating early morphogenesis.
VYL is a gene in rice.Its RAP ID is Os03g0411500 and its MSU ID is LOC_Os03g29810. The mutant of this gene produces chlorotic leaves throughout the entire growth period.
==Annotated Information==
===Function===
The protein encoded by this gene belongs to the peptidase family S14 and hydrolyzes proteins into small peptides in the presence of ATP and magnesium. The protein is transported into mitochondrial matrix and is associated with the inner mitochondrial membrane<ref name="ref2" />, or plastid inner membrane in plants<ref name="ref4" />.
The plastidic caseinolytic protease (Clp) of higher plants is an evolutionarily conserved protein degradation apparatus composed of a proteolytic core complex (the P and R rings) and a set of accessory proteins (ClpT, ClpC, and ClpS).<ref name="ref4" />
Rice yellow leaf mutant vyl, the performance of the entire growth period, new leaves chlorotic phenotype, then gradually turn green from the top down.<ref name="ref4" /> VYL Arabidopsis Clp protease subunit ClpP6 homologous protein in rice, is one of the subunits of the chloroplast Clp protease, with the Clp protease subunit interactions OsClpP3 and OsClpP4 respectively, play an important role in the biosynthesis of rice chloroplast.<ref name="ref4" />
What's more, the gene D53 product shares predicted features with the class I Clp ATPase proteins and can form a complex with the a/b hydrolase protein DWARF 14 (D14) and the F-box protein DWARF 3 (D3), two previously identified signalling components potentially responsible for SL(Strigolactones) perception, which means, in a D14- and D3-dependentmanner, SLs induce D53 degradation by the proteasome and abrogate its activity in promoting axillary bud outgrowth.<ref name="ref5" />
The role and molecular composition of Clps in higher plants has just begun to be unraveled, mostly from studies with the model dicotyledonous plant Arabidopsis (Arabidopsis thaliana).Some researchers isolated a virescent yellow leaf (vyl) mutant in rice (Oryza sativa), which produces chlorotic leaves throughout the entire growth period.<ref name="ref4" />
====1.The vyl Mutant Displays Reduced Chlorophyll Accumulation====
The vyl mutant was derived by transforming tissue cultures of the japonica rice variety Kita-ake. When grown under an alternating light/dark cycle (12 h of light at 30°C/12 h of darkness at 20°C) in a growth chamber, vyl mutant plants displayed a virescent yellow leaf pheno-type, and during development, leaves gradually turned green from their tips (more developed) to their bases (less developed; Fig. 1, A and B). At maturity, the vyl mutant plants also had reduced height and smaller seeds (Fig. 1, C and D). Mutant leaves also contained less chlorophyll than the wild type at various growth stages (Fig. 1E).
Additionally, vyl mutants developed chlorotic leaves under different temperature conditions and light/dark cycles, suggesting that the virescent yellow phenotype of the vyl mutant was developmentally regulated but independent of external cues (such as temperature and light)
[[File:figure 1.png]]

====2.The vyl Mutant Has Impaired Chloroplast Development====
Next, researchers investigated whether the virescent yellow phenotype of the vyl mutant was associated with ultra-structural changes in the chloroplasts. Leaf samples of L3U (upper half of the third leaf), L3L (basal half of the third leaf), and L4 (fourth leaf above the shoot base) were collected from wild-type and vyl mutant seedlings and compared (Fig. 2A). Normally, when the third leaf has fully emerged from the shoot base of a rice plant, the shoot also contains the fourth to the seventh immature leaves. The leaf cells in the L3L and L3U samples contain mature chloroplasts, whereas those in the shoot base and L4 samples contain proplastids and early developing immature chloroplasts (Sugimoto et al., 2004). Similar to the wild type, the chloroplasts from the L3U green leaf sample (already turned green) of vyl mutant seedlings displayed well-developed lamellar structures and were equipped with normally stacked grana and thylakoid membranes (Fig. 2, B and C). By contrast, the chloroplasts from the L3L and L4 pale leaves (still wrapped in leaf sheath) of the vyl mutant had much reduced thylakoid membrane networks compared with wild-type plants (Fig. 2, D–G). This developmental defect is similar to the phenotype reported in the Arabidopsis CLPP6 antisense transgenic plants (Sjögren et al., 2006).

[[File:Figure 2 .png]]

====3.The vyl Mutant Has Impaired Photosynthesis====
Chloroplasts are the organelles in plant cells that perform photosynthesis; therefore, they play an essential role in plant growth. To test whether the photosynthetic apparatus was affected in vyl mutants, we compared some key parameters of PSI and PSII between vyl and wild-type plants. Distinct differences in photochemical efficiency of PSII (FPSII), electron transport rate (ETR), nonphotochemical quenching (NPQ), and photochemical
quenching (Qp) were detected between vyl mutants and the wild type. In contrast, the maximal efficiency of PSII photochemistry (F v /F m ) values was almost comparable between vyl and wild-type plants (Table I). These observations indicate that vyl mutants absorbed much less light energy, as shown by the lower NPQ values. PSII photochemistry was reduced at both the donor and acceptor sites, as indicated by the greatly decreased Qp and ETR in vyl mutants. Notably, the PSII structure appeared intact in the mutant plants (indicated by the equivalent F v /F m values), but the actual FPSII was much lower in the mutants. These differences may underlie the defects in chloroplast biogenesis and retarded growth in the vyl mutant (Fig. 1, C and D)
[[File:Table_1.png‎]]

====4.Altered Expression of Genes Associated with Chloroplast Biogenesis and Photosynthesis in vyl Mutants====
Chloroplast biogenesis and physiological changes are tightly regulated by the coordinated expression of plastid and nuclear genes during leaf development and facilitated by protein quality control (Kusumi et al., 2010; Clarke, 2012). To examine whether the expression of genes associated with chloroplast biogenesis and photosynthesis was altered in vyl mutants, quantitative real-time reverse transcription (qRT)-PCR was performed on total RNAs extracted from the L4 and
L3U leaf sections of wild-type and vyl plants. Compared with the wild type, the transcript levels of Virescent1 (V1), V2, and V3 genes and some other genes encoding components of the plastid and nuclear transcription apparatus Sigma factor 2A (OsSig2A), Ribosomal Protein S15 (rps15) and the subunit RNA polymerase (RpoA and RpoTp) that are highly expressed in early stages of chloroplast development were significantly increased in vyl mutants. In contrast, several genes encoding components of the photosynthesis apparatus RuBisCO large subunit (RbcL), the subunit of photosystem I (PsaA), a core component of Photosystem II (PsbA), light-harvesting complex protein (Lhcp2) and Chlorophyll A/B binding protein1 (Cab1) that are highly expressed in later stages of chloroplast development were significantly reduced in vyl mutants (Fig. 3). These results suggested that VYL plays an important role in regulating chloroplast biogenesis.

[[File:Figure 3 .png]]

====5.The vyl Locus Maps to a Putative Gene Encoding OsClpP6====
Genetic analysis showed that the virescent yellow leaf phenotype in vyl mutants is controlled by a single recessive nuclear locus, VYL. Using a BC 1 F 2 mapping population of the vyl mutant and 93-11 (an indica variety), we mapped the vyl gene to a 137-kb genomic region on chromosome 3 between the insertion/deletion polymorphism (Indel) markers F17 and I53. Within this region, 17 open reading frames (ORFs) were predicted from published data (http://
www.gramene.org/; Fig. 4A). Genomic sequence analysis revealed that only the 13th ORF (LOC_Os03g29810) carries a single-nucleotide transition (G→T) at the position 1,509 bp from the ATG start codon (Fig. 4B). We obtained the full-length complementary DNA (cDNA) of LOC_Os03g29810 from both the wild type and the vyl mutant by reverse transcription (RT)-PCR. Sequence analysis showed that the full-length VYL cDNA is 780 bp long in the wild type but 842 bp in the mutant.
Comparison of the genomic and cDNA sequences revealed that the single-nucleotide substitution in the vyl mutant genome created a new splicing site and the addition of a partial fourth intron sequence in the cDNA (Fig. 4, C and D). The mutant cDNA is predicted to encode a truncated vyl mutant protein (DVYL) lacking the classic Ser protease triad (Ser-His-Asp) in the active site and the polypeptide-binding site (Fig. 4, C and E). Phylogenetic analysis and protein sequence alignment showed that VYL is most closely related to the Arabidopsis ClpP6 protein with a conserved S14_ClpP_2 domain and more remotely related to several other Arabidopsis ClpP subunits, and it apparently represents a single-copy gene in the rice genome (Fig. 4F).
To verify the identity of VYL, the plasmid pGVYL, containing a 6-kb genomic DNA fragment consisting of a 2.5-kb upstream sequence, the entire VYL coding region, including nine exons and eight introns, and a 0.6-kb downstream sequence, was constructed and introduced into the vyl mutant. All five transgenic lines containing pGVYL complemented the virescent phenotype of the vyl mutant (Fig. 5). To further confirm that disruption of the VYL gene was responsible for the vyl mutant phenotype, we generated RNA interference transgenic plants in the wild-type Kita-ake background and obtained more than eight independent transgenic
lines. qRT-PCR analysis revealed that the expression of VYL was reduced in three tested transgenic lines compared with the wild-type plants. These VYL knockdown transgenic plants showed reduced accumulation of chlorophyll a and chlorophyll b. Together, these results confirmed that LOC_Os03g29810 indeed corresponds to the VYL gene.
[[File:Figure 4 .png]]

====6.Interactions between VYL, OsClpP3, OsClpP4, OsClpP5, and OsClpT====
To gain an insight into the role of VYL in the rice Clp complex assembly, researchers sought to identify the proteins that directly interact with VYL. We first used a mass spectrometry-based tandem affinity purification proteomics approach. We generated a VYL-HBH construct in
which the C terminus of VYL was fused with a Hisbiotin tag (Tagwerker et al., 2006), and the fusion gene was driven by the Ubiquitin promoter. The VYL-HBH
fusion construct was introduced into the vyl mutant via Agrobacterium tumefaciens-mediated transformation. The VYL-HBH fusion protein transgene recovered the chlorotic phenotype of the vyl mutant, indicating that the fusion protein is biologically functiona SDS-PAGE and immunoblot analyses showed that the VYL-HBH fusion protein and several additional proteins could be effectively purified from the pUbi:: VYL-HBH transgenic plants using Ni 2+ -Sepharose and streptavidin beads (Fig. 8A). The copurified protein bands were excised from the SDS-PAGE gel and analyzed by matrix-assisted laser-desorption ionization time of flight (MALDI-TOF). Three bands were identified with confidence (P , 0.05), including two putative components of the Clp complex (LOC_Os03g29810/ VYL/OsClpP6 and LOC_Os10g43050/OsClpP4) and a vacuolar ATP synthase subunit E (Loc_Oso1g46980/ v-ATP-E; Fig. 8B; Table II). This result suggested that VYL is a bona fide component of the OsClp complex in vivo.
To further investigate the role of VYL in the OsClp core proteolytic complex assembly, we cloned the rice homologs of Arabidopsis ClpR1, ClpP3, ClpP5, and ClpT and named them OsClpR1, OsClpP3, OsClpP5, and OsClpT, respectively. It is noticeable that there is only one copy of the OsClpT gene in the rice genome.
Using yeast two-hybrid assays, we found that VYL protein directly interacted with OsClpP3 and OsClpP4 but not with vacuolar ATP synthase subunit E (v-ATP-E;
Fig. 8C). Furthermore, we found that the yeast strain (Gold Saccharomyces cerevisiae) carrying BD-VYL+AD-OsClpP4 grew and developed the blue color faster than the yeast strain (Gold Saccharomyces cerevisiae) carrying BD-DVYL+AD-OsClpP4 or BD-VYL+AD-OsClpP3 on yeast growth medium containing 5-Bromo-4-chloro-3-
indoylla-galactoside (X-a-Gal). Furthermore, the yeast strain(GoldSaccharomycescerevisiae)carryingBD-DVYL+AD-OsClpP3 did not grow even after incubation for 5 d in a growth chamber (Fig. 8D). This result suggests that the C-terminal polypeptide-binding site of VYL likely plays a role in mediating the interaction between VYL with OsClpP3 and OsClpP4. In addition, they also found that OsClpP3 interacted with OsClpT, OsClpP4 interacted with OsClpP5 and OsClpT, and that both OsClpP4 and OsClpT interacted with themselves but OsClpP5 and OsClpP3 did not (Fig. 8E). Due to the self-activation activity of VYL-AD, we were not able to test whether VYL can homodimerize.

[[File:Figure 5 .png]][[File:Figure 6 .png]]

===Expression===
Expression is constitutive in most tissues examined (roots, stems, leaves, leaf sheath, panicle)but most abundant in leaf sections containing
chloroplasts in early stages of development,which can be light-mediated.<ref name="ref4" />
The young chlorotic leaves turn green in later developmental stages, accompanied by alterations in chlorophyll accumulation, chloroplast ultrastructure, and the expression of chloroplast development- and photosynthesis-related genes. Positional cloning revealed that the VYL gene encodes a protein homologous to the Arabidopsis ClpP6 subunit and that it is targeted to the chloroplast. VYL expression is constitutive in most tissues examined but most abundant in leaf sections containing chloroplasts in early stages of development. The mutation in vyl causes premature termination of the predicted gene product and loss of the conserved catalytic triad (serine-histidine-aspartate) and the polypeptide-binding site of VYL. Using a tandem affinity purification approach and mass spectrometry analysis, we identified OsClpP4 as a VYL-associated protein in vivo. In addition, yeast two-hybrid assays demonstrated that VYL directly interacts with OsClpP3 and OsClpP4. Furthermore, we found that OsClpP3 directly interacts with OsClpT, that OsClpP4 directly interacts with OsClpP5 and OsClpT, and that both OsClpP4 and OsClpT can homodimerize. Together, our data provide new insights into the function, assembly, and regulation of Clps in higher plants.<ref name="ref4" />
To investigate the expression patterns of VYL during chloroplast and leaf development, researchers analyzed VYL expression in different sections of leaves at various leaf developmental stages by qRT-PCR (Fig. 2A). They found that in wild-type plants, VYL was most highly expressed in the L4 section at the early chloroplast and leaf development stage (Fig. 6A). Furthermore, qRT-PCR analysis and histochemical staining of the pVYL::GUS reporter gene transgenic plants showed that VYL was constitutively expressed in young buds, young roots, stems, leaves, leaf shoots, and panicles (Fig. 6, B and C). To test whether VYL expression is regulated by light, They analyzed VYL expression during greening of etiolated seedlings. Wild-type rice plants were grown in continuous darkness for 10 d and subsequently exposed to light for 3, 6, 9, 12, 15, 18, 21, or 24 h. The expression of VYL was highly induced after 3 h of illumination and peaked after 6 h of illumination, then its expression gradually decreased over time, and by 15 h after illumination, its expression returned to the preillumination basal level (Fig. 6D). These observations suggested that VYL likely plays a role in the light regulation of chloroplast development. We next conducted qRT-PCR analysis to examine a possible effect of the vyl mutation on the expression of other genes encoding various components of the rice Clp. We
found that OsClpPs, OsClpT, and OsClpR4 all had similar expression patterns to VYL, with a peak accumulation at the early stage of chloroplast development, but the expression levels of these genes were higher in the vyl mutants compared with the wild type (Fig. 7). This observation suggested that there may be a compensatory mechanism to increase the expression of OsClpPs, OsClpT, and OsClpR4 in vyl mutants.

==Evolution==
ATP-dependent Clp protease proteolytic subunit is an enzyme that in humans is encoded by the CLPP gene.<ref name="ref1" /><ref name="ref2" /> It is found in mitochondria and is widely distributed in bacterial species.
In several bacteria, such as E. coli, proteins tagged with the SsrA peptide (ANDENYALAA) encoded by tmRNA are digested by Clp proteases.<ref name="ref3" />

[[File:612px-Protein_CLPP_PDB_1tg6.png]][[File:1867.png]]

==Labs working on this gene==
National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China

==References==
<references>
<ref name="ref1">
Bross P, Andresen BS, Knudsen I, Kruse TA, Gregersen N (Feb 1996). "Human ClpP protease: cDNA sequence, tissue-specific expression and chromosomal assignment of the gene". FEBS Lett 377 (2): 249–52. doi:10.1016/0014-5793(95)01353-9. PMID 8543061.
</ref>
<ref name="ref2">
a b "Entrez Gene: CLPP ClpP caseinolytic peptidase, ATP-dependent, proteolytic subunit homolog (E. coli)".
</ref>
<ref name="ref3">
Gottesman S, Roche E, Zhou Y, Sauer RT (1998). "The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system". Genes Dev 12 (9): 1338–47. doi:10.1101/gad.12.9.1338. PMC 316764. PMID 9573050
</ref>
<ref name="ref4">
Hui Dong et al. A Rice Virescent-Yellow Leaf Mutant Reveals New Insights into the Role and Assembly of Plastid Caseinolytic Protease in Higher Plants. Plant Physiology, 2013, 162(4): 1867-1880
</ref>
<ref name="ref5">
Zhou Feng et al.D14–SCFD3-dependent degradation of D53 regulates strigolactone signaling. Nature,2013. doi:10.1038/nature12878
</ref>
</references>
==Structured Information==
{{JaponicaGene|
GeneName = Os03g0411500|
Description = Peptidase S14, ClpP family protein|
Version = NM_001056884.1 GI:115453496 GeneID:4333096|
Length = 3448 bp|
Definition = Oryza sativa Japonica Group Os03g0411500, complete gene.|
Source = Oryza sativa Japonica Group

ORGANISM Oryza sativa Japonica Group
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP
clade; Ehrhartoideae; Oryzeae; Oryza.
|
Chromosome = [[:category:Japonica Chromosome 3|Chromosome 3]]|
AP = Chromosome 3:17630235..17633682|
CDS = 17630290..17630357,17630478..17630512,17631200..17631269,17631410..17631614,17631776..17631841 ,17632451..17632591,17632803..17632856,17632992..17633084,17633183..17633230 |
GCID = <gbrowseImage1>
name=NC_008396:17630235..17633682
source=RiceChromosome03
preset=GeneLocation
</gbrowseImage1>|
GSID = <gbrowseImage2>
name=NC_008396:17630235..17633682
source=RiceChromosome03
preset=GeneLocation
</gbrowseImage2>|
CDNA = <cdnaseq>atggcgcctatggccatctccaccccgctcgccctccgcgcctccccgacccgcctcctctcccgcaggcggagcggagccaaatcaggcgtggctctcccaggtccacaatttgtaccacctggtatttcttcaaagttggacgagaggatacattgtcattcttctctgaggaaaaatacaattgtagcatcagagaatgaaaatccacctttaatgcctgccataatgactcctgctggtgctcttgatctggcaactgtattgttggggaaccgcattatcttcattggtcaatatattaactcgcaagtagcacagcgtgtaatatcacagcttgtcacacttgctgctgttgatgaagaggctgatattctgatctacctgaactgccccggcggaagtctctactccatcttagcaatttatgattgcatgtcctggatcaagcccaaagttggaacagtgtgctttggtgttgttgctagccaggcagcaattatacttgctggcggtgagaagggaatgcgttatgccatgccaaatgctagagtaatgattcatcaacctcaaggtgtatcagagggtaatgtggaggaggtgaggcgacaggttggggaaaccatttatgctcgtgataaagttgataagatgtttgctgcttttactgggcaaaccttggatatggtacaacagtggacagagagggatcgtttcatgtcttcatctgaagccatggactttggactagttgatgccctgctggaaacaagatactaa</cdnaseq>|
AA = <aaseq>MAPMAISTPLALRASPTRLLSRRRSGAKSGVALPGPQFVPPGIS SKLDERIHCHSSLRKNTIVASENENPPLMPAIMTPAGALDLATVLLGNRIIFIGQYIN SQVAQRVISQLVTLAAVDEEADILIYLNCPGGSLYSILAIYDCMSWIKPKVGTVCFGV VASQAAIILAGGEKGMRYAMPNARVMIHQPQGVSEGNVEEVRRQVGETIYARDKVDKM FAAFTGQTLDMVQQWTERDRFMSSSEAMDFGLVDALLETRY</aaseq>|
DNA = <dnaseqindica>56..123#244..278#966..1035#1176..1380#1542..1607#2217..2357#2569..2622#2758..2850#2949..2996#actcctcagtcctcgcctcggctcggctccctcccacgctccagctccgcctccaatggcgcctatggccatctccaccccgctcgccctccgcgcctccccgacccgcctcctctcccgcaggtgagctccacggaactacaacttccacctccttcgctcgctcgctcgccccgcgcttctctcttcatggattcccccgttcttgtcccctcaccctctgtctggtccttctttcttcaggcggagcggagccaaatcaggcgtggctctcccaggtgagatttcctaacccttggtttagcaaaccatttcccttggtcagctcgttaggccaggactgtttggtggaatttgatcgttgatttgataagctttactgagatgttgtccatggtggtgcacatattagaacatgaccatttgggcagccgtcccatcagctcctagttgtctttctctgtgccaattttttatggagtcgtcattggtaggttttgcaaagcatatagaacctctgaatgtcggcattatccaagagtatgccgacctggggttatctgaaccagtgtcaactccttcctgggccaatgtctggtatgcatcagcattagctcactaagtacacgaaaaatggatgtgcttggttgaacggatatgtataaaaacgataacctgcatataacatatcacctcagtttggtctgattctgaaattagtttagggccttttagcaaactgctgaatgagatttccagactgtatatgtgttattgtgtttgtcagtaactcagtatggtgtattagcacaactcaacatgccataatgacaggatatgcggagcacaaacttttctttggacatgttttttggattcctttactgtttagtccatctgtctttcacatgatatattgctgcaaatgtgctgagttgcattctcactcaaatttccacacctaggtccacaatttgtaccacctggtatttcttcaaagttggacgagaggatacattgtcattcttctctgaggtgatatatttgaaactgtcatgcctgatatacaatgaggatacattgcctgatttgaaactgctcatctaggttttatttgtgctgtgtatcagaatcctgttttattcattcgtaatattgtaaatattttttcacaggaaaaatacaattgtagcatcagagaatgaaaatccacctttaatgcctgccataatgactcctgctggtgctcttgatctggcaactgtattgttggggaaccgcattatcttcattggtcaatatattaactcgcaagtagcacagcgtgtaatatcacagcttgtcacacttgctgctgttgatgaagaggctgatattctggttagtgtttatttttgtgtttttcagatcataacagttaccctattgttcactgcagcagctcttgattgctcaacttcactcccttggcttgctcctttagctcacaggtgtgttgctctatatcttataactccttttgtataattctgttttgccagatctacctgaactgccccggcggaagtctctactccatcttagcaatttatgattgcatgtcctgggtatgccatctatgttgagctactttttccatgtccttcatgcattcaaatttcagagattgtattcacctatatttattcttgtggatgctttctgagttattctcatctaatttaatatttacattgttggatgagaacacattatagatgcatctcaacattttgtatcttccacattatgcatgcccctagctagtgaatttatatttataatatagcacagatatagcatttacaggaaagcctaatgtaatttaggcaaaaattatatctcatatcaatggtagtgcttgcaacatttgtattcttatatttttattgtagtacatactagacatgagcatttgccatgctgagactgtgctaattgggtttggtctggtactgcagacaacagatctcatttcctgaaatcatgcctgtctctaaaactggctttgagctggaccagccttgttagttgttagatttggctgtgtttttacttgttatgccagttttccataaccaaatactttatctacaatttcgcctactgataaataaatcccagaatattaatctttttttgttgttctgcactaacacatgaaccatttattgcagatcaagcccaaagttggaacagtgtgctttggtgttgttgctagccaggcagcaattatacttgctggcggtgagaagggaatgcgttatgccatgccaaatgctagagtaatgattcatcaacctcaaggtgtatcagaggtatgattctggggctttctgcctttctgagttactgcagcgggtgcattatgattttctaacattgtgactacagtaaataataatcatcatcattttagctggccacatgaaacttacaatatacagtcttgctaggacatacttgcttgcctttgtgtttgttgtacttgaagtttgttttttattctaaaaattggatgatttgcagggtaatgtggaggaggtgaggcgacaggttggggaaaccatttatgctcgtgatgtaagtgttttgtgatagataacaagttctatattttcttcagtgtacatttgaattagatgtttgatgagggtaaggaatttctcttgattctgctctctaagcgattaaagcttctgaaaaatctggatgcagaaagttgataagatgtttgctgcttttactgggcaaaccttggatatggtacaacagtggacagagagggatcgtttcatgtcttcatctgaagtaactttcatctcttaaatgtatcagaagaaagtaaatcatcatttgccatgtgaattataacttatttcccccttctttttttggttttaccctaggccatggactttggactagttgatgccctgctggaaacaagatactaacaaacaaacacttaggcacagtttgattagcagaggctggtacaaggattgtgaaactggagctgaagttgagattttcgccgtccttctagttcaaggactccaatgacaggaggctggatctgcgagacttgaatgcatcgccatcgctcctatgagcaaaacatctctgcgttgagtgtgattttttgctcttcttttttggatctggttttacatgccgccagcctatggtctcaatgcattggtcctgctaatgtttagtgtagaccagatgatcctttagacagcaaaacatcagttattactccgtagattttcccatgagctgattccagactgtgtgcaacttttgtgttccaattgcaaagtttgcaacccaacccaacccaacacatgggctgatgggctgttgacaaaggagagattttacacttcacatatgtcaaact</dnaseqindica>|
Link = [http://www.ncbi.nlm.nih.gov/nuccore/NM_001056884.1 RefSeq:Os03g0411500]|
}}
[[Category:Genes]]
[[Category:Japonica mRNA]]
[[Category:Oryza Sativa Japonica Group]]
[[Category:Japonica Genes]]
[[Category:Japonica Chromosome 3]]
[[Category:Chromosome 3]]

Os03g0411500

2014-06-08T08:38:17Z

Xiaoyanjie2012: /* 6.Interactions between VYL, OsClpP3, OsClpP4, OsClpP5, and OsClpT */

A nuclear gene coding caseinolyse positioned in plastid or mitochondrion regulating early morphogenesis.
VYL is a gene in rice.Its RAP ID is Os03g0411500 and its MSU ID is LOC_Os03g29810. The mutant of this gene produces chlorotic leaves throughout the entire growth period.
==Annotated Information==
===Function===
The protein encoded by this gene belongs to the peptidase family S14 and hydrolyzes proteins into small peptides in the presence of ATP and magnesium. The protein is transported into mitochondrial matrix and is associated with the inner mitochondrial membrane<ref name="ref2" />, or plastid inner membrane in plants<ref name="ref4" />.
The plastidic caseinolytic protease (Clp) of higher plants is an evolutionarily conserved protein degradation apparatus composed of a proteolytic core complex (the P and R rings) and a set of accessory proteins (ClpT, ClpC, and ClpS).<ref name="ref4" />
Rice yellow leaf mutant vyl, the performance of the entire growth period, new leaves chlorotic phenotype, then gradually turn green from the top down.<ref name="ref4" /> VYL Arabidopsis Clp protease subunit ClpP6 homologous protein in rice, is one of the subunits of the chloroplast Clp protease, with the Clp protease subunit interactions OsClpP3 and OsClpP4 respectively, play an important role in the biosynthesis of rice chloroplast.<ref name="ref4" />
What's more, the gene D53 product shares predicted features with the class I Clp ATPase proteins and can form a complex with the a/b hydrolase protein DWARF 14 (D14) and the F-box protein DWARF 3 (D3), two previously identified signalling components potentially responsible for SL(Strigolactones) perception, which means, in a D14- and D3-dependentmanner, SLs induce D53 degradation by the proteasome and abrogate its activity in promoting axillary bud outgrowth.<ref name="ref5" />
The role and molecular composition of Clps in higher plants has just begun to be unraveled, mostly from studies with the model dicotyledonous plant Arabidopsis (Arabidopsis thaliana).Some researchers isolated a virescent yellow leaf (vyl) mutant in rice (Oryza sativa), which produces chlorotic leaves throughout the entire growth period.<ref name="ref4" />
====1.The vyl Mutant Displays Reduced Chlorophyll Accumulation====
The vyl mutant was derived by transforming tissue cultures of the japonica rice variety Kita-ake. When grown under an alternating light/dark cycle (12 h of light at 30°C/12 h of darkness at 20°C) in a growth chamber, vyl mutant plants displayed a virescent yellow leaf pheno-type, and during development, leaves gradually turned green from their tips (more developed) to their bases (less developed; Fig. 1, A and B). At maturity, the vyl mutant plants also had reduced height and smaller seeds (Fig. 1, C and D). Mutant leaves also contained less chlorophyll than the wild type at various growth stages (Fig. 1E).
Additionally, vyl mutants developed chlorotic leaves under different temperature conditions and light/dark cycles, suggesting that the virescent yellow phenotype of the vyl mutant was developmentally regulated but independent of external cues (such as temperature and light)
[[File:figure 1.png]]

====2.The vyl Mutant Has Impaired Chloroplast Development====
Next, researchers investigated whether the virescent yellow phenotype of the vyl mutant was associated with ultra-structural changes in the chloroplasts. Leaf samples of L3U (upper half of the third leaf), L3L (basal half of the third leaf), and L4 (fourth leaf above the shoot base) were collected from wild-type and vyl mutant seedlings and compared (Fig. 2A). Normally, when the third leaf has fully emerged from the shoot base of a rice plant, the shoot also contains the fourth to the seventh immature leaves. The leaf cells in the L3L and L3U samples contain mature chloroplasts, whereas those in the shoot base and L4 samples contain proplastids and early developing immature chloroplasts (Sugimoto et al., 2004). Similar to the wild type, the chloroplasts from the L3U green leaf sample (already turned green) of vyl mutant seedlings displayed well-developed lamellar structures and were equipped with normally stacked grana and thylakoid membranes (Fig. 2, B and C). By contrast, the chloroplasts from the L3L and L4 pale leaves (still wrapped in leaf sheath) of the vyl mutant had much reduced thylakoid membrane networks compared with wild-type plants (Fig. 2, D–G). This developmental defect is similar to the phenotype reported in the Arabidopsis CLPP6 antisense transgenic plants (Sjögren et al., 2006).

[[File:Figure 2 .png]]

====3.The vyl Mutant Has Impaired Photosynthesis====
Chloroplasts are the organelles in plant cells that perform photosynthesis; therefore, they play an essential role in plant growth. To test whether the photosynthetic apparatus was affected in vyl mutants, we compared some key parameters of PSI and PSII between vyl and wild-type plants. Distinct differences in photochemical efficiency of PSII (FPSII), electron transport rate (ETR), nonphotochemical quenching (NPQ), and photochemical
quenching (Qp) were detected between vyl mutants and the wild type. In contrast, the maximal efficiency of PSII photochemistry (F v /F m ) values was almost comparable between vyl and wild-type plants (Table I). These observations indicate that vyl mutants absorbed much less light energy, as shown by the lower NPQ values. PSII photochemistry was reduced at both the donor and acceptor sites, as indicated by the greatly decreased Qp and ETR in vyl mutants. Notably, the PSII structure appeared intact in the mutant plants (indicated by the equivalent F v /F m values), but the actual FPSII was much lower in the mutants. These differences may underlie the defects in chloroplast biogenesis and retarded growth in the vyl mutant (Fig. 1, C and D)
[[File:Table_1.png‎]]

====4.Altered Expression of Genes Associated with Chloroplast Biogenesis and Photosynthesis in vyl Mutants====
Chloroplast biogenesis and physiological changes are tightly regulated by the coordinated expression of plastid and nuclear genes during leaf development and facilitated by protein quality control (Kusumi et al., 2010; Clarke, 2012). To examine whether the expression of genes associated with chloroplast biogenesis and photosynthesis was altered in vyl mutants, quantitative real-time reverse transcription (qRT)-PCR was performed on total RNAs extracted from the L4 and
L3U leaf sections of wild-type and vyl plants. Compared with the wild type, the transcript levels of Virescent1 (V1), V2, and V3 genes and some other genes encoding components of the plastid and nuclear transcription apparatus Sigma factor 2A (OsSig2A), Ribosomal Protein S15 (rps15) and the subunit RNA polymerase (RpoA and RpoTp) that are highly expressed in early stages of chloroplast development were significantly increased in vyl mutants. In contrast, several genes encoding components of the photosynthesis apparatus RuBisCO large subunit (RbcL), the subunit of photosystem I (PsaA), a core component of Photosystem II (PsbA), light-harvesting complex protein (Lhcp2) and Chlorophyll A/B binding protein1 (Cab1) that are highly expressed in later stages of chloroplast development were significantly reduced in vyl mutants (Fig. 3). These results suggested that VYL plays an important role in regulating chloroplast biogenesis.

[[File:Figure 3 .png]]

====5.The vyl Locus Maps to a Putative Gene Encoding OsClpP6====
Genetic analysis showed that the virescent yellow leaf phenotype in vyl mutants is controlled by a single recessive nuclear locus, VYL. Using a BC 1 F 2 mapping population of the vyl mutant and 93-11 (an indica variety), we mapped the vyl gene to a 137-kb genomic region on chromosome 3 between the insertion/deletion polymorphism (Indel) markers F17 and I53. Within this region, 17 open reading frames (ORFs) were predicted from published data (http://
www.gramene.org/; Fig. 4A). Genomic sequence analysis revealed that only the 13th ORF (LOC_Os03g29810) carries a single-nucleotide transition (G→T) at the position 1,509 bp from the ATG start codon (Fig. 4B). We obtained the full-length complementary DNA (cDNA) of LOC_Os03g29810 from both the wild type and the vyl mutant by reverse transcription (RT)-PCR. Sequence analysis showed that the full-length VYL cDNA is 780 bp long in the wild type but 842 bp in the mutant.
Comparison of the genomic and cDNA sequences revealed that the single-nucleotide substitution in the vyl mutant genome created a new splicing site and the addition of a partial fourth intron sequence in the cDNA (Fig. 4, C and D). The mutant cDNA is predicted to encode a truncated vyl mutant protein (DVYL) lacking the classic Ser protease triad (Ser-His-Asp) in the active site and the polypeptide-binding site (Fig. 4, C and E). Phylogenetic analysis and protein sequence alignment showed that VYL is most closely related to the Arabidopsis ClpP6 protein with a conserved S14_ClpP_2 domain and more remotely related to several other Arabidopsis ClpP subunits, and it apparently represents a single-copy gene in the rice genome (Fig. 4F).
To verify the identity of VYL, the plasmid pGVYL, containing a 6-kb genomic DNA fragment consisting of a 2.5-kb upstream sequence, the entire VYL coding region, including nine exons and eight introns, and a 0.6-kb downstream sequence, was constructed and introduced into the vyl mutant. All five transgenic lines containing pGVYL complemented the virescent phenotype of the vyl mutant (Fig. 5). To further confirm that disruption of the VYL gene was responsible for the vyl mutant phenotype, we generated RNA interference transgenic plants in the wild-type Kita-ake background and obtained more than eight independent transgenic
lines. qRT-PCR analysis revealed that the expression of VYL was reduced in three tested transgenic lines compared with the wild-type plants. These VYL knockdown transgenic plants showed reduced accumulation of chlorophyll a and chlorophyll b. Together, these results confirmed that LOC_Os03g29810 indeed corresponds to the VYL gene.
[[File:Figure 4 .png]]

====6.Interactions between VYL, OsClpP3, OsClpP4, OsClpP5, and OsClpT====
To gain an insight into the role of VYL in the rice Clp complex assembly, researchers sought to identify the proteins that directly interact with VYL. We first used a mass spectrometry-based tandem affinity purification proteomics approach. We generated a VYL-HBH construct in
which the C terminus of VYL was fused with a Hisbiotin tag (Tagwerker et al., 2006), and the fusion gene was driven by the Ubiquitin promoter. The VYL-HBH
fusion construct was introduced into the vyl mutant via Agrobacterium tumefaciens-mediated transformation. The VYL-HBH fusion protein transgene recovered the chlorotic phenotype of the vyl mutant, indicating that the fusion protein is biologically functiona SDS-PAGE and immunoblot analyses showed that the VYL-HBH fusion protein and several additional proteins could be effectively purified from the pUbi:: VYL-HBH transgenic plants using Ni 2+ -Sepharose and streptavidin beads (Fig. 8A). The copurified protein bands were excised from the SDS-PAGE gel and analyzed by matrix-assisted laser-desorption ionization time of flight (MALDI-TOF). Three bands were identified with confidence (P , 0.05), including two putative components of the Clp complex (LOC_Os03g29810/ VYL/OsClpP6 and LOC_Os10g43050/OsClpP4) and a vacuolar ATP synthase subunit E (Loc_Oso1g46980/ v-ATP-E; Fig. 8B; Table II). This result suggested that VYL is a bona fide component of the OsClp complex in vivo.
To further investigate the role of VYL in the OsClp core proteolytic complex assembly, we cloned the rice homologs of Arabidopsis ClpR1, ClpP3, ClpP5, and ClpT and named them OsClpR1, OsClpP3, OsClpP5, and OsClpT, respectively. It is noticeable that there is only one copy of the OsClpT gene in the rice genome.
Using yeast two-hybrid assays, we found that VYL protein directly interacted with OsClpP3 and OsClpP4 but not with vacuolar ATP synthase subunit E (v-ATP-E;
Fig. 8C). Furthermore, we found that the yeast strain (Gold Saccharomyces cerevisiae) carrying BD-VYL+AD-OsClpP4 grew and developed the blue color faster than the yeast strain (Gold Saccharomyces cerevisiae) carrying BD-DVYL+AD-OsClpP4 or BD-VYL+AD-OsClpP3 on yeast growth medium containing 5-Bromo-4-chloro-3-
indoylla-galactoside (X-a-Gal). Furthermore, the yeast strain(GoldSaccharomycescerevisiae)carryingBD-DVYL+AD-OsClpP3 did not grow even after incubation for 5 d in a growth chamber (Fig. 8D). This result suggests that the C-terminal polypeptide-binding site of VYL likely plays a role in mediating the interaction between VYL with OsClpP3 and OsClpP4. In addition, they also found that OsClpP3 interacted with OsClpT, OsClpP4 interacted with OsClpP5 and OsClpT, and that both OsClpP4 and OsClpT interacted with themselves but OsClpP5 and OsClpP3 did not (Fig. 8E). Due to the self-activation activity of VYL-AD, we were not able to test whether VYL can homodimerize.

[[File:Figure 5 .png]][[File:Figure 6 .png]][[File:Figure 7 .png]][[File:Figure 8 .png]]

===Expression===
Expression is constitutive in most tissues examined (roots, stems, leaves, leaf sheath, panicle)but most abundant in leaf sections containing
chloroplasts in early stages of development,which can be light-mediated.<ref name="ref4" />
The young chlorotic leaves turn green in later developmental stages, accompanied by alterations in chlorophyll accumulation, chloroplast ultrastructure, and the expression of chloroplast development- and photosynthesis-related genes. Positional cloning revealed that the VYL gene encodes a protein homologous to the Arabidopsis ClpP6 subunit and that it is targeted to the chloroplast. VYL expression is constitutive in most tissues examined but most abundant in leaf sections containing chloroplasts in early stages of development. The mutation in vyl causes premature termination of the predicted gene product and loss of the conserved catalytic triad (serine-histidine-aspartate) and the polypeptide-binding site of VYL. Using a tandem affinity purification approach and mass spectrometry analysis, we identified OsClpP4 as a VYL-associated protein in vivo. In addition, yeast two-hybrid assays demonstrated that VYL directly interacts with OsClpP3 and OsClpP4. Furthermore, we found that OsClpP3 directly interacts with OsClpT, that OsClpP4 directly interacts with OsClpP5 and OsClpT, and that both OsClpP4 and OsClpT can homodimerize. Together, our data provide new insights into the function, assembly, and regulation of Clps in higher plants.<ref name="ref4" />
To investigate the expression patterns of VYL during chloroplast and leaf development, researchers analyzed VYL expression in different sections of leaves at various leaf developmental stages by qRT-PCR (Fig. 2A). They found that in wild-type plants, VYL was most highly expressed in the L4 section at the early chloroplast and leaf development stage (Fig. 6A). Furthermore, qRT-PCR analysis and histochemical staining of the pVYL::GUS reporter gene transgenic plants showed that VYL was constitutively expressed in young buds, young roots, stems, leaves, leaf shoots, and panicles (Fig. 6, B and C). To test whether VYL expression is regulated by light, They analyzed VYL expression during greening of etiolated seedlings. Wild-type rice plants were grown in continuous darkness for 10 d and subsequently exposed to light for 3, 6, 9, 12, 15, 18, 21, or 24 h. The expression of VYL was highly induced after 3 h of illumination and peaked after 6 h of illumination, then its expression gradually decreased over time, and by 15 h after illumination, its expression returned to the preillumination basal level (Fig. 6D). These observations suggested that VYL likely plays a role in the light regulation of chloroplast development. We next conducted qRT-PCR analysis to examine a possible effect of the vyl mutation on the expression of other genes encoding various components of the rice Clp. We
found that OsClpPs, OsClpT, and OsClpR4 all had similar expression patterns to VYL, with a peak accumulation at the early stage of chloroplast development, but the expression levels of these genes were higher in the vyl mutants compared with the wild type (Fig. 7). This observation suggested that there may be a compensatory mechanism to increase the expression of OsClpPs, OsClpT, and OsClpR4 in vyl mutants.

==Evolution==
ATP-dependent Clp protease proteolytic subunit is an enzyme that in humans is encoded by the CLPP gene.<ref name="ref1" /><ref name="ref2" /> It is found in mitochondria and is widely distributed in bacterial species.
In several bacteria, such as E. coli, proteins tagged with the SsrA peptide (ANDENYALAA) encoded by tmRNA are digested by Clp proteases.<ref name="ref3" />

[[File:612px-Protein_CLPP_PDB_1tg6.png]][[File:1867.png]]

==Labs working on this gene==
National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China

==References==
<references>
<ref name="ref1">
Bross P, Andresen BS, Knudsen I, Kruse TA, Gregersen N (Feb 1996). "Human ClpP protease: cDNA sequence, tissue-specific expression and chromosomal assignment of the gene". FEBS Lett 377 (2): 249–52. doi:10.1016/0014-5793(95)01353-9. PMID 8543061.
</ref>
<ref name="ref2">
a b "Entrez Gene: CLPP ClpP caseinolytic peptidase, ATP-dependent, proteolytic subunit homolog (E. coli)".
</ref>
<ref name="ref3">
Gottesman S, Roche E, Zhou Y, Sauer RT (1998). "The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system". Genes Dev 12 (9): 1338–47. doi:10.1101/gad.12.9.1338. PMC 316764. PMID 9573050
</ref>
<ref name="ref4">
Hui Dong et al. A Rice Virescent-Yellow Leaf Mutant Reveals New Insights into the Role and Assembly of Plastid Caseinolytic Protease in Higher Plants. Plant Physiology, 2013, 162(4): 1867-1880
</ref>
<ref name="ref5">
Zhou Feng et al.D14–SCFD3-dependent degradation of D53 regulates strigolactone signaling. Nature,2013. doi:10.1038/nature12878
</ref>
</references>
==Structured Information==
{{JaponicaGene|
GeneName = Os03g0411500|
Description = Peptidase S14, ClpP family protein|
Version = NM_001056884.1 GI:115453496 GeneID:4333096|
Length = 3448 bp|
Definition = Oryza sativa Japonica Group Os03g0411500, complete gene.|
Source = Oryza sativa Japonica Group

ORGANISM Oryza sativa Japonica Group
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP
clade; Ehrhartoideae; Oryzeae; Oryza.
|
Chromosome = [[:category:Japonica Chromosome 3|Chromosome 3]]|
AP = Chromosome 3:17630235..17633682|
CDS = 17630290..17630357,17630478..17630512,17631200..17631269,17631410..17631614,17631776..17631841 ,17632451..17632591,17632803..17632856,17632992..17633084,17633183..17633230 |
GCID = <gbrowseImage1>
name=NC_008396:17630235..17633682
source=RiceChromosome03
preset=GeneLocation
</gbrowseImage1>|
GSID = <gbrowseImage2>
name=NC_008396:17630235..17633682
source=RiceChromosome03
preset=GeneLocation
</gbrowseImage2>|
CDNA = <cdnaseq>atggcgcctatggccatctccaccccgctcgccctccgcgcctccccgacccgcctcctctcccgcaggcggagcggagccaaatcaggcgtggctctcccaggtccacaatttgtaccacctggtatttcttcaaagttggacgagaggatacattgtcattcttctctgaggaaaaatacaattgtagcatcagagaatgaaaatccacctttaatgcctgccataatgactcctgctggtgctcttgatctggcaactgtattgttggggaaccgcattatcttcattggtcaatatattaactcgcaagtagcacagcgtgtaatatcacagcttgtcacacttgctgctgttgatgaagaggctgatattctgatctacctgaactgccccggcggaagtctctactccatcttagcaatttatgattgcatgtcctggatcaagcccaaagttggaacagtgtgctttggtgttgttgctagccaggcagcaattatacttgctggcggtgagaagggaatgcgttatgccatgccaaatgctagagtaatgattcatcaacctcaaggtgtatcagagggtaatgtggaggaggtgaggcgacaggttggggaaaccatttatgctcgtgataaagttgataagatgtttgctgcttttactgggcaaaccttggatatggtacaacagtggacagagagggatcgtttcatgtcttcatctgaagccatggactttggactagttgatgccctgctggaaacaagatactaa</cdnaseq>|
AA = <aaseq>MAPMAISTPLALRASPTRLLSRRRSGAKSGVALPGPQFVPPGIS SKLDERIHCHSSLRKNTIVASENENPPLMPAIMTPAGALDLATVLLGNRIIFIGQYIN SQVAQRVISQLVTLAAVDEEADILIYLNCPGGSLYSILAIYDCMSWIKPKVGTVCFGV VASQAAIILAGGEKGMRYAMPNARVMIHQPQGVSEGNVEEVRRQVGETIYARDKVDKM FAAFTGQTLDMVQQWTERDRFMSSSEAMDFGLVDALLETRY</aaseq>|
DNA = <dnaseqindica>56..123#244..278#966..1035#1176..1380#1542..1607#2217..2357#2569..2622#2758..2850#2949..2996#actcctcagtcctcgcctcggctcggctccctcccacgctccagctccgcctccaatggcgcctatggccatctccaccccgctcgccctccgcgcctccccgacccgcctcctctcccgcaggtgagctccacggaactacaacttccacctccttcgctcgctcgctcgccccgcgcttctctcttcatggattcccccgttcttgtcccctcaccctctgtctggtccttctttcttcaggcggagcggagccaaatcaggcgtggctctcccaggtgagatttcctaacccttggtttagcaaaccatttcccttggtcagctcgttaggccaggactgtttggtggaatttgatcgttgatttgataagctttactgagatgttgtccatggtggtgcacatattagaacatgaccatttgggcagccgtcccatcagctcctagttgtctttctctgtgccaattttttatggagtcgtcattggtaggttttgcaaagcatatagaacctctgaatgtcggcattatccaagagtatgccgacctggggttatctgaaccagtgtcaactccttcctgggccaatgtctggtatgcatcagcattagctcactaagtacacgaaaaatggatgtgcttggttgaacggatatgtataaaaacgataacctgcatataacatatcacctcagtttggtctgattctgaaattagtttagggccttttagcaaactgctgaatgagatttccagactgtatatgtgttattgtgtttgtcagtaactcagtatggtgtattagcacaactcaacatgccataatgacaggatatgcggagcacaaacttttctttggacatgttttttggattcctttactgtttagtccatctgtctttcacatgatatattgctgcaaatgtgctgagttgcattctcactcaaatttccacacctaggtccacaatttgtaccacctggtatttcttcaaagttggacgagaggatacattgtcattcttctctgaggtgatatatttgaaactgtcatgcctgatatacaatgaggatacattgcctgatttgaaactgctcatctaggttttatttgtgctgtgtatcagaatcctgttttattcattcgtaatattgtaaatattttttcacaggaaaaatacaattgtagcatcagagaatgaaaatccacctttaatgcctgccataatgactcctgctggtgctcttgatctggcaactgtattgttggggaaccgcattatcttcattggtcaatatattaactcgcaagtagcacagcgtgtaatatcacagcttgtcacacttgctgctgttgatgaagaggctgatattctggttagtgtttatttttgtgtttttcagatcataacagttaccctattgttcactgcagcagctcttgattgctcaacttcactcccttggcttgctcctttagctcacaggtgtgttgctctatatcttataactccttttgtataattctgttttgccagatctacctgaactgccccggcggaagtctctactccatcttagcaatttatgattgcatgtcctgggtatgccatctatgttgagctactttttccatgtccttcatgcattcaaatttcagagattgtattcacctatatttattcttgtggatgctttctgagttattctcatctaatttaatatttacattgttggatgagaacacattatagatgcatctcaacattttgtatcttccacattatgcatgcccctagctagtgaatttatatttataatatagcacagatatagcatttacaggaaagcctaatgtaatttaggcaaaaattatatctcatatcaatggtagtgcttgcaacatttgtattcttatatttttattgtagtacatactagacatgagcatttgccatgctgagactgtgctaattgggtttggtctggtactgcagacaacagatctcatttcctgaaatcatgcctgtctctaaaactggctttgagctggaccagccttgttagttgttagatttggctgtgtttttacttgttatgccagttttccataaccaaatactttatctacaatttcgcctactgataaataaatcccagaatattaatctttttttgttgttctgcactaacacatgaaccatttattgcagatcaagcccaaagttggaacagtgtgctttggtgttgttgctagccaggcagcaattatacttgctggcggtgagaagggaatgcgttatgccatgccaaatgctagagtaatgattcatcaacctcaaggtgtatcagaggtatgattctggggctttctgcctttctgagttactgcagcgggtgcattatgattttctaacattgtgactacagtaaataataatcatcatcattttagctggccacatgaaacttacaatatacagtcttgctaggacatacttgcttgcctttgtgtttgttgtacttgaagtttgttttttattctaaaaattggatgatttgcagggtaatgtggaggaggtgaggcgacaggttggggaaaccatttatgctcgtgatgtaagtgttttgtgatagataacaagttctatattttcttcagtgtacatttgaattagatgtttgatgagggtaaggaatttctcttgattctgctctctaagcgattaaagcttctgaaaaatctggatgcagaaagttgataagatgtttgctgcttttactgggcaaaccttggatatggtacaacagtggacagagagggatcgtttcatgtcttcatctgaagtaactttcatctcttaaatgtatcagaagaaagtaaatcatcatttgccatgtgaattataacttatttcccccttctttttttggttttaccctaggccatggactttggactagttgatgccctgctggaaacaagatactaacaaacaaacacttaggcacagtttgattagcagaggctggtacaaggattgtgaaactggagctgaagttgagattttcgccgtccttctagttcaaggactccaatgacaggaggctggatctgcgagacttgaatgcatcgccatcgctcctatgagcaaaacatctctgcgttgagtgtgattttttgctcttcttttttggatctggttttacatgccgccagcctatggtctcaatgcattggtcctgctaatgtttagtgtagaccagatgatcctttagacagcaaaacatcagttattactccgtagattttcccatgagctgattccagactgtgtgcaacttttgtgttccaattgcaaagtttgcaacccaacccaacccaacacatgggctgatgggctgttgacaaaggagagattttacacttcacatatgtcaaact</dnaseqindica>|
Link = [http://www.ncbi.nlm.nih.gov/nuccore/NM_001056884.1 RefSeq:Os03g0411500]|
}}
[[Category:Genes]]
[[Category:Japonica mRNA]]
[[Category:Oryza Sativa Japonica Group]]
[[Category:Japonica Genes]]
[[Category:Japonica Chromosome 3]]
[[Category:Chromosome 3]]

Os03g0411500

2014-06-08T08:37:56Z

Xiaoyanjie2012: /* 6.Interactions between VYL, OsClpP3, OsClpP4, OsClpP5, and OsClpT */

A nuclear gene coding caseinolyse positioned in plastid or mitochondrion regulating early morphogenesis.
VYL is a gene in rice.Its RAP ID is Os03g0411500 and its MSU ID is LOC_Os03g29810. The mutant of this gene produces chlorotic leaves throughout the entire growth period.
==Annotated Information==
===Function===
The protein encoded by this gene belongs to the peptidase family S14 and hydrolyzes proteins into small peptides in the presence of ATP and magnesium. The protein is transported into mitochondrial matrix and is associated with the inner mitochondrial membrane<ref name="ref2" />, or plastid inner membrane in plants<ref name="ref4" />.
The plastidic caseinolytic protease (Clp) of higher plants is an evolutionarily conserved protein degradation apparatus composed of a proteolytic core complex (the P and R rings) and a set of accessory proteins (ClpT, ClpC, and ClpS).<ref name="ref4" />
Rice yellow leaf mutant vyl, the performance of the entire growth period, new leaves chlorotic phenotype, then gradually turn green from the top down.<ref name="ref4" /> VYL Arabidopsis Clp protease subunit ClpP6 homologous protein in rice, is one of the subunits of the chloroplast Clp protease, with the Clp protease subunit interactions OsClpP3 and OsClpP4 respectively, play an important role in the biosynthesis of rice chloroplast.<ref name="ref4" />
What's more, the gene D53 product shares predicted features with the class I Clp ATPase proteins and can form a complex with the a/b hydrolase protein DWARF 14 (D14) and the F-box protein DWARF 3 (D3), two previously identified signalling components potentially responsible for SL(Strigolactones) perception, which means, in a D14- and D3-dependentmanner, SLs induce D53 degradation by the proteasome and abrogate its activity in promoting axillary bud outgrowth.<ref name="ref5" />
The role and molecular composition of Clps in higher plants has just begun to be unraveled, mostly from studies with the model dicotyledonous plant Arabidopsis (Arabidopsis thaliana).Some researchers isolated a virescent yellow leaf (vyl) mutant in rice (Oryza sativa), which produces chlorotic leaves throughout the entire growth period.<ref name="ref4" />
====1.The vyl Mutant Displays Reduced Chlorophyll Accumulation====
The vyl mutant was derived by transforming tissue cultures of the japonica rice variety Kita-ake. When grown under an alternating light/dark cycle (12 h of light at 30°C/12 h of darkness at 20°C) in a growth chamber, vyl mutant plants displayed a virescent yellow leaf pheno-type, and during development, leaves gradually turned green from their tips (more developed) to their bases (less developed; Fig. 1, A and B). At maturity, the vyl mutant plants also had reduced height and smaller seeds (Fig. 1, C and D). Mutant leaves also contained less chlorophyll than the wild type at various growth stages (Fig. 1E).
Additionally, vyl mutants developed chlorotic leaves under different temperature conditions and light/dark cycles, suggesting that the virescent yellow phenotype of the vyl mutant was developmentally regulated but independent of external cues (such as temperature and light)
[[File:figure 1.png]]

====2.The vyl Mutant Has Impaired Chloroplast Development====
Next, researchers investigated whether the virescent yellow phenotype of the vyl mutant was associated with ultra-structural changes in the chloroplasts. Leaf samples of L3U (upper half of the third leaf), L3L (basal half of the third leaf), and L4 (fourth leaf above the shoot base) were collected from wild-type and vyl mutant seedlings and compared (Fig. 2A). Normally, when the third leaf has fully emerged from the shoot base of a rice plant, the shoot also contains the fourth to the seventh immature leaves. The leaf cells in the L3L and L3U samples contain mature chloroplasts, whereas those in the shoot base and L4 samples contain proplastids and early developing immature chloroplasts (Sugimoto et al., 2004). Similar to the wild type, the chloroplasts from the L3U green leaf sample (already turned green) of vyl mutant seedlings displayed well-developed lamellar structures and were equipped with normally stacked grana and thylakoid membranes (Fig. 2, B and C). By contrast, the chloroplasts from the L3L and L4 pale leaves (still wrapped in leaf sheath) of the vyl mutant had much reduced thylakoid membrane networks compared with wild-type plants (Fig. 2, D–G). This developmental defect is similar to the phenotype reported in the Arabidopsis CLPP6 antisense transgenic plants (Sjögren et al., 2006).

[[File:Figure 2 .png]]

====3.The vyl Mutant Has Impaired Photosynthesis====
Chloroplasts are the organelles in plant cells that perform photosynthesis; therefore, they play an essential role in plant growth. To test whether the photosynthetic apparatus was affected in vyl mutants, we compared some key parameters of PSI and PSII between vyl and wild-type plants. Distinct differences in photochemical efficiency of PSII (FPSII), electron transport rate (ETR), nonphotochemical quenching (NPQ), and photochemical
quenching (Qp) were detected between vyl mutants and the wild type. In contrast, the maximal efficiency of PSII photochemistry (F v /F m ) values was almost comparable between vyl and wild-type plants (Table I). These observations indicate that vyl mutants absorbed much less light energy, as shown by the lower NPQ values. PSII photochemistry was reduced at both the donor and acceptor sites, as indicated by the greatly decreased Qp and ETR in vyl mutants. Notably, the PSII structure appeared intact in the mutant plants (indicated by the equivalent F v /F m values), but the actual FPSII was much lower in the mutants. These differences may underlie the defects in chloroplast biogenesis and retarded growth in the vyl mutant (Fig. 1, C and D)
[[File:Table_1.png‎]]

====4.Altered Expression of Genes Associated with Chloroplast Biogenesis and Photosynthesis in vyl Mutants====
Chloroplast biogenesis and physiological changes are tightly regulated by the coordinated expression of plastid and nuclear genes during leaf development and facilitated by protein quality control (Kusumi et al., 2010; Clarke, 2012). To examine whether the expression of genes associated with chloroplast biogenesis and photosynthesis was altered in vyl mutants, quantitative real-time reverse transcription (qRT)-PCR was performed on total RNAs extracted from the L4 and
L3U leaf sections of wild-type and vyl plants. Compared with the wild type, the transcript levels of Virescent1 (V1), V2, and V3 genes and some other genes encoding components of the plastid and nuclear transcription apparatus Sigma factor 2A (OsSig2A), Ribosomal Protein S15 (rps15) and the subunit RNA polymerase (RpoA and RpoTp) that are highly expressed in early stages of chloroplast development were significantly increased in vyl mutants. In contrast, several genes encoding components of the photosynthesis apparatus RuBisCO large subunit (RbcL), the subunit of photosystem I (PsaA), a core component of Photosystem II (PsbA), light-harvesting complex protein (Lhcp2) and Chlorophyll A/B binding protein1 (Cab1) that are highly expressed in later stages of chloroplast development were significantly reduced in vyl mutants (Fig. 3). These results suggested that VYL plays an important role in regulating chloroplast biogenesis.

[[File:Figure 3 .png]]

====5.The vyl Locus Maps to a Putative Gene Encoding OsClpP6====
Genetic analysis showed that the virescent yellow leaf phenotype in vyl mutants is controlled by a single recessive nuclear locus, VYL. Using a BC 1 F 2 mapping population of the vyl mutant and 93-11 (an indica variety), we mapped the vyl gene to a 137-kb genomic region on chromosome 3 between the insertion/deletion polymorphism (Indel) markers F17 and I53. Within this region, 17 open reading frames (ORFs) were predicted from published data (http://
www.gramene.org/; Fig. 4A). Genomic sequence analysis revealed that only the 13th ORF (LOC_Os03g29810) carries a single-nucleotide transition (G→T) at the position 1,509 bp from the ATG start codon (Fig. 4B). We obtained the full-length complementary DNA (cDNA) of LOC_Os03g29810 from both the wild type and the vyl mutant by reverse transcription (RT)-PCR. Sequence analysis showed that the full-length VYL cDNA is 780 bp long in the wild type but 842 bp in the mutant.
Comparison of the genomic and cDNA sequences revealed that the single-nucleotide substitution in the vyl mutant genome created a new splicing site and the addition of a partial fourth intron sequence in the cDNA (Fig. 4, C and D). The mutant cDNA is predicted to encode a truncated vyl mutant protein (DVYL) lacking the classic Ser protease triad (Ser-His-Asp) in the active site and the polypeptide-binding site (Fig. 4, C and E). Phylogenetic analysis and protein sequence alignment showed that VYL is most closely related to the Arabidopsis ClpP6 protein with a conserved S14_ClpP_2 domain and more remotely related to several other Arabidopsis ClpP subunits, and it apparently represents a single-copy gene in the rice genome (Fig. 4F).
To verify the identity of VYL, the plasmid pGVYL, containing a 6-kb genomic DNA fragment consisting of a 2.5-kb upstream sequence, the entire VYL coding region, including nine exons and eight introns, and a 0.6-kb downstream sequence, was constructed and introduced into the vyl mutant. All five transgenic lines containing pGVYL complemented the virescent phenotype of the vyl mutant (Fig. 5). To further confirm that disruption of the VYL gene was responsible for the vyl mutant phenotype, we generated RNA interference transgenic plants in the wild-type Kita-ake background and obtained more than eight independent transgenic
lines. qRT-PCR analysis revealed that the expression of VYL was reduced in three tested transgenic lines compared with the wild-type plants. These VYL knockdown transgenic plants showed reduced accumulation of chlorophyll a and chlorophyll b. Together, these results confirmed that LOC_Os03g29810 indeed corresponds to the VYL gene.
[[File:Figure 4 .png]]

====6.Interactions between VYL, OsClpP3, OsClpP4, OsClpP5, and OsClpT====
To gain an insight into the role of VYL in the rice Clp complex assembly, researchers sought to identify the proteins that directly interact with VYL. We first used a mass spectrometry-based tandem affinity purification proteomics approach. We generated a VYL-HBH construct in
which the C terminus of VYL was fused with a Hisbiotin tag (Tagwerker et al., 2006), and the fusion gene was driven by the Ubiquitin promoter. The VYL-HBH
fusion construct was introduced into the vyl mutant via Agrobacterium tumefaciens-mediated transformation. The VYL-HBH fusion protein transgene recovered the chlorotic phenotype of the vyl mutant, indicating that the fusion protein is biologically functiona SDS-PAGE and immunoblot analyses showed that the VYL-HBH fusion protein and several additional proteins could be effectively purified from the pUbi:: VYL-HBH transgenic plants using Ni 2+ -Sepharose and streptavidin beads (Fig. 8A). The copurified protein bands were excised from the SDS-PAGE gel and analyzed by matrix-assisted laser-desorption ionization time of flight (MALDI-TOF). Three bands were identified with confidence (P , 0.05), including two putative components of the Clp complex (LOC_Os03g29810/ VYL/OsClpP6 and LOC_Os10g43050/OsClpP4) and a vacuolar ATP synthase subunit E (Loc_Oso1g46980/ v-ATP-E; Fig. 8B; Table II). This result suggested that VYL is a bona fide component of the OsClp complex in vivo.
To further investigate the role of VYL in the OsClp core proteolytic complex assembly, we cloned the rice homologs of Arabidopsis ClpR1, ClpP3, ClpP5, and ClpT and named them OsClpR1, OsClpP3, OsClpP5, and OsClpT, respectively. It is noticeable that there is only one copy of the OsClpT gene in the rice genome.
Using yeast two-hybrid assays, we found that VYL protein directly interacted with OsClpP3 and OsClpP4 but not with vacuolar ATP synthase subunit E (v-ATP-E;
Fig. 8C). Furthermore, we found that the yeast strain (Gold Saccharomyces cerevisiae) carrying BD-VYL+AD-OsClpP4 grew and developed the blue color faster than the yeast strain (Gold Saccharomyces cerevisiae) carrying BD-DVYL+AD-OsClpP4 or BD-VYL+AD-OsClpP3 on yeast growth medium containing 5-Bromo-4-chloro-3-
indoylla-galactoside (X-a-Gal). Furthermore, the yeast strain(GoldSaccharomycescerevisiae)carryingBD-DVYL+AD-OsClpP3 did not grow even after incubation for 5 d in a growth chamber (Fig. 8D). This result suggests that the C-terminal polypeptide-binding site of VYL likely plays a role in mediating the interaction between VYL with OsClpP3 and OsClpP4. In addition, they also found that OsClpP3 interacted with OsClpT, OsClpP4 interacted with OsClpP5 and OsClpT, and that both OsClpP4 and OsClpT interacted with themselves but OsClpP5 and OsClpP3 did not (Fig. 8E). Due to the self-activation activity of VYL-AD, we were not able to test whether VYL can homodimerize.
[[File:Figure 5 .png]][[File:Figure 6 .png]][[File:Figure 7 .png]][[File:Figure 8 .png]]

===Expression===
Expression is constitutive in most tissues examined (roots, stems, leaves, leaf sheath, panicle)but most abundant in leaf sections containing
chloroplasts in early stages of development,which can be light-mediated.<ref name="ref4" />
The young chlorotic leaves turn green in later developmental stages, accompanied by alterations in chlorophyll accumulation, chloroplast ultrastructure, and the expression of chloroplast development- and photosynthesis-related genes. Positional cloning revealed that the VYL gene encodes a protein homologous to the Arabidopsis ClpP6 subunit and that it is targeted to the chloroplast. VYL expression is constitutive in most tissues examined but most abundant in leaf sections containing chloroplasts in early stages of development. The mutation in vyl causes premature termination of the predicted gene product and loss of the conserved catalytic triad (serine-histidine-aspartate) and the polypeptide-binding site of VYL. Using a tandem affinity purification approach and mass spectrometry analysis, we identified OsClpP4 as a VYL-associated protein in vivo. In addition, yeast two-hybrid assays demonstrated that VYL directly interacts with OsClpP3 and OsClpP4. Furthermore, we found that OsClpP3 directly interacts with OsClpT, that OsClpP4 directly interacts with OsClpP5 and OsClpT, and that both OsClpP4 and OsClpT can homodimerize. Together, our data provide new insights into the function, assembly, and regulation of Clps in higher plants.<ref name="ref4" />
To investigate the expression patterns of VYL during chloroplast and leaf development, researchers analyzed VYL expression in different sections of leaves at various leaf developmental stages by qRT-PCR (Fig. 2A). They found that in wild-type plants, VYL was most highly expressed in the L4 section at the early chloroplast and leaf development stage (Fig. 6A). Furthermore, qRT-PCR analysis and histochemical staining of the pVYL::GUS reporter gene transgenic plants showed that VYL was constitutively expressed in young buds, young roots, stems, leaves, leaf shoots, and panicles (Fig. 6, B and C). To test whether VYL expression is regulated by light, They analyzed VYL expression during greening of etiolated seedlings. Wild-type rice plants were grown in continuous darkness for 10 d and subsequently exposed to light for 3, 6, 9, 12, 15, 18, 21, or 24 h. The expression of VYL was highly induced after 3 h of illumination and peaked after 6 h of illumination, then its expression gradually decreased over time, and by 15 h after illumination, its expression returned to the preillumination basal level (Fig. 6D). These observations suggested that VYL likely plays a role in the light regulation of chloroplast development. We next conducted qRT-PCR analysis to examine a possible effect of the vyl mutation on the expression of other genes encoding various components of the rice Clp. We
found that OsClpPs, OsClpT, and OsClpR4 all had similar expression patterns to VYL, with a peak accumulation at the early stage of chloroplast development, but the expression levels of these genes were higher in the vyl mutants compared with the wild type (Fig. 7). This observation suggested that there may be a compensatory mechanism to increase the expression of OsClpPs, OsClpT, and OsClpR4 in vyl mutants.

==Evolution==
ATP-dependent Clp protease proteolytic subunit is an enzyme that in humans is encoded by the CLPP gene.<ref name="ref1" /><ref name="ref2" /> It is found in mitochondria and is widely distributed in bacterial species.
In several bacteria, such as E. coli, proteins tagged with the SsrA peptide (ANDENYALAA) encoded by tmRNA are digested by Clp proteases.<ref name="ref3" />

[[File:612px-Protein_CLPP_PDB_1tg6.png]][[File:1867.png]]

==Labs working on this gene==
National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China

==References==
<references>
<ref name="ref1">
Bross P, Andresen BS, Knudsen I, Kruse TA, Gregersen N (Feb 1996). "Human ClpP protease: cDNA sequence, tissue-specific expression and chromosomal assignment of the gene". FEBS Lett 377 (2): 249–52. doi:10.1016/0014-5793(95)01353-9. PMID 8543061.
</ref>
<ref name="ref2">
a b "Entrez Gene: CLPP ClpP caseinolytic peptidase, ATP-dependent, proteolytic subunit homolog (E. coli)".
</ref>
<ref name="ref3">
Gottesman S, Roche E, Zhou Y, Sauer RT (1998). "The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system". Genes Dev 12 (9): 1338–47. doi:10.1101/gad.12.9.1338. PMC 316764. PMID 9573050
</ref>
<ref name="ref4">
Hui Dong et al. A Rice Virescent-Yellow Leaf Mutant Reveals New Insights into the Role and Assembly of Plastid Caseinolytic Protease in Higher Plants. Plant Physiology, 2013, 162(4): 1867-1880
</ref>
<ref name="ref5">
Zhou Feng et al.D14–SCFD3-dependent degradation of D53 regulates strigolactone signaling. Nature,2013. doi:10.1038/nature12878
</ref>
</references>
==Structured Information==
{{JaponicaGene|
GeneName = Os03g0411500|
Description = Peptidase S14, ClpP family protein|
Version = NM_001056884.1 GI:115453496 GeneID:4333096|
Length = 3448 bp|
Definition = Oryza sativa Japonica Group Os03g0411500, complete gene.|
Source = Oryza sativa Japonica Group

ORGANISM Oryza sativa Japonica Group
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP
clade; Ehrhartoideae; Oryzeae; Oryza.
|
Chromosome = [[:category:Japonica Chromosome 3|Chromosome 3]]|
AP = Chromosome 3:17630235..17633682|
CDS = 17630290..17630357,17630478..17630512,17631200..17631269,17631410..17631614,17631776..17631841 ,17632451..17632591,17632803..17632856,17632992..17633084,17633183..17633230 |
GCID = <gbrowseImage1>
name=NC_008396:17630235..17633682
source=RiceChromosome03
preset=GeneLocation
</gbrowseImage1>|
GSID = <gbrowseImage2>
name=NC_008396:17630235..17633682
source=RiceChromosome03
preset=GeneLocation
</gbrowseImage2>|
CDNA = <cdnaseq>atggcgcctatggccatctccaccccgctcgccctccgcgcctccccgacccgcctcctctcccgcaggcggagcggagccaaatcaggcgtggctctcccaggtccacaatttgtaccacctggtatttcttcaaagttggacgagaggatacattgtcattcttctctgaggaaaaatacaattgtagcatcagagaatgaaaatccacctttaatgcctgccataatgactcctgctggtgctcttgatctggcaactgtattgttggggaaccgcattatcttcattggtcaatatattaactcgcaagtagcacagcgtgtaatatcacagcttgtcacacttgctgctgttgatgaagaggctgatattctgatctacctgaactgccccggcggaagtctctactccatcttagcaatttatgattgcatgtcctggatcaagcccaaagttggaacagtgtgctttggtgttgttgctagccaggcagcaattatacttgctggcggtgagaagggaatgcgttatgccatgccaaatgctagagtaatgattcatcaacctcaaggtgtatcagagggtaatgtggaggaggtgaggcgacaggttggggaaaccatttatgctcgtgataaagttgataagatgtttgctgcttttactgggcaaaccttggatatggtacaacagtggacagagagggatcgtttcatgtcttcatctgaagccatggactttggactagttgatgccctgctggaaacaagatactaa</cdnaseq>|
AA = <aaseq>MAPMAISTPLALRASPTRLLSRRRSGAKSGVALPGPQFVPPGIS SKLDERIHCHSSLRKNTIVASENENPPLMPAIMTPAGALDLATVLLGNRIIFIGQYIN SQVAQRVISQLVTLAAVDEEADILIYLNCPGGSLYSILAIYDCMSWIKPKVGTVCFGV VASQAAIILAGGEKGMRYAMPNARVMIHQPQGVSEGNVEEVRRQVGETIYARDKVDKM FAAFTGQTLDMVQQWTERDRFMSSSEAMDFGLVDALLETRY</aaseq>|
DNA = <dnaseqindica>56..123#244..278#966..1035#1176..1380#1542..1607#2217..2357#2569..2622#2758..2850#2949..2996#actcctcagtcctcgcctcggctcggctccctcccacgctccagctccgcctccaatggcgcctatggccatctccaccccgctcgccctccgcgcctccccgacccgcctcctctcccgcaggtgagctccacggaactacaacttccacctccttcgctcgctcgctcgccccgcgcttctctcttcatggattcccccgttcttgtcccctcaccctctgtctggtccttctttcttcaggcggagcggagccaaatcaggcgtggctctcccaggtgagatttcctaacccttggtttagcaaaccatttcccttggtcagctcgttaggccaggactgtttggtggaatttgatcgttgatttgataagctttactgagatgttgtccatggtggtgcacatattagaacatgaccatttgggcagccgtcccatcagctcctagttgtctttctctgtgccaattttttatggagtcgtcattggtaggttttgcaaagcatatagaacctctgaatgtcggcattatccaagagtatgccgacctggggttatctgaaccagtgtcaactccttcctgggccaatgtctggtatgcatcagcattagctcactaagtacacgaaaaatggatgtgcttggttgaacggatatgtataaaaacgataacctgcatataacatatcacctcagtttggtctgattctgaaattagtttagggccttttagcaaactgctgaatgagatttccagactgtatatgtgttattgtgtttgtcagtaactcagtatggtgtattagcacaactcaacatgccataatgacaggatatgcggagcacaaacttttctttggacatgttttttggattcctttactgtttagtccatctgtctttcacatgatatattgctgcaaatgtgctgagttgcattctcactcaaatttccacacctaggtccacaatttgtaccacctggtatttcttcaaagttggacgagaggatacattgtcattcttctctgaggtgatatatttgaaactgtcatgcctgatatacaatgaggatacattgcctgatttgaaactgctcatctaggttttatttgtgctgtgtatcagaatcctgttttattcattcgtaatattgtaaatattttttcacaggaaaaatacaattgtagcatcagagaatgaaaatccacctttaatgcctgccataatgactcctgctggtgctcttgatctggcaactgtattgttggggaaccgcattatcttcattggtcaatatattaactcgcaagtagcacagcgtgtaatatcacagcttgtcacacttgctgctgttgatgaagaggctgatattctggttagtgtttatttttgtgtttttcagatcataacagttaccctattgttcactgcagcagctcttgattgctcaacttcactcccttggcttgctcctttagctcacaggtgtgttgctctatatcttataactccttttgtataattctgttttgccagatctacctgaactgccccggcggaagtctctactccatcttagcaatttatgattgcatgtcctgggtatgccatctatgttgagctactttttccatgtccttcatgcattcaaatttcagagattgtattcacctatatttattcttgtggatgctttctgagttattctcatctaatttaatatttacattgttggatgagaacacattatagatgcatctcaacattttgtatcttccacattatgcatgcccctagctagtgaatttatatttataatatagcacagatatagcatttacaggaaagcctaatgtaatttaggcaaaaattatatctcatatcaatggtagtgcttgcaacatttgtattcttatatttttattgtagtacatactagacatgagcatttgccatgctgagactgtgctaattgggtttggtctggtactgcagacaacagatctcatttcctgaaatcatgcctgtctctaaaactggctttgagctggaccagccttgttagttgttagatttggctgtgtttttacttgttatgccagttttccataaccaaatactttatctacaatttcgcctactgataaataaatcccagaatattaatctttttttgttgttctgcactaacacatgaaccatttattgcagatcaagcccaaagttggaacagtgtgctttggtgttgttgctagccaggcagcaattatacttgctggcggtgagaagggaatgcgttatgccatgccaaatgctagagtaatgattcatcaacctcaaggtgtatcagaggtatgattctggggctttctgcctttctgagttactgcagcgggtgcattatgattttctaacattgtgactacagtaaataataatcatcatcattttagctggccacatgaaacttacaatatacagtcttgctaggacatacttgcttgcctttgtgtttgttgtacttgaagtttgttttttattctaaaaattggatgatttgcagggtaatgtggaggaggtgaggcgacaggttggggaaaccatttatgctcgtgatgtaagtgttttgtgatagataacaagttctatattttcttcagtgtacatttgaattagatgtttgatgagggtaaggaatttctcttgattctgctctctaagcgattaaagcttctgaaaaatctggatgcagaaagttgataagatgtttgctgcttttactgggcaaaccttggatatggtacaacagtggacagagagggatcgtttcatgtcttcatctgaagtaactttcatctcttaaatgtatcagaagaaagtaaatcatcatttgccatgtgaattataacttatttcccccttctttttttggttttaccctaggccatggactttggactagttgatgccctgctggaaacaagatactaacaaacaaacacttaggcacagtttgattagcagaggctggtacaaggattgtgaaactggagctgaagttgagattttcgccgtccttctagttcaaggactccaatgacaggaggctggatctgcgagacttgaatgcatcgccatcgctcctatgagcaaaacatctctgcgttgagtgtgattttttgctcttcttttttggatctggttttacatgccgccagcctatggtctcaatgcattggtcctgctaatgtttagtgtagaccagatgatcctttagacagcaaaacatcagttattactccgtagattttcccatgagctgattccagactgtgtgcaacttttgtgttccaattgcaaagtttgcaacccaacccaacccaacacatgggctgatgggctgttgacaaaggagagattttacacttcacatatgtcaaact</dnaseqindica>|
Link = [http://www.ncbi.nlm.nih.gov/nuccore/NM_001056884.1 RefSeq:Os03g0411500]|
}}
[[Category:Genes]]
[[Category:Japonica mRNA]]
[[Category:Oryza Sativa Japonica Group]]
[[Category:Japonica Genes]]
[[Category:Japonica Chromosome 3]]
[[Category:Chromosome 3]]

File:Figure 8 .png

2014-06-08T08:36:55Z

Xiaoyanjie2012:

File:Figure 7 .png

2014-06-08T08:36:35Z

Xiaoyanjie2012:

File:Figure 6 .png

2014-06-08T08:36:17Z

Xiaoyanjie2012:

File:Figure 5 .png

2014-06-08T08:35:10Z

Xiaoyanjie2012:

Os03g0411500

2014-06-08T08:30:28Z

Xiaoyanjie2012: /* 6.Interactions between VYL, OsClpP3, OsClpP4, OsClpP5, and OsClpT */

A nuclear gene coding caseinolyse positioned in plastid or mitochondrion regulating early morphogenesis.
VYL is a gene in rice.Its RAP ID is Os03g0411500 and its MSU ID is LOC_Os03g29810. The mutant of this gene produces chlorotic leaves throughout the entire growth period.
==Annotated Information==
===Function===
The protein encoded by this gene belongs to the peptidase family S14 and hydrolyzes proteins into small peptides in the presence of ATP and magnesium. The protein is transported into mitochondrial matrix and is associated with the inner mitochondrial membrane<ref name="ref2" />, or plastid inner membrane in plants<ref name="ref4" />.
The plastidic caseinolytic protease (Clp) of higher plants is an evolutionarily conserved protein degradation apparatus composed of a proteolytic core complex (the P and R rings) and a set of accessory proteins (ClpT, ClpC, and ClpS).<ref name="ref4" />
Rice yellow leaf mutant vyl, the performance of the entire growth period, new leaves chlorotic phenotype, then gradually turn green from the top down.<ref name="ref4" /> VYL Arabidopsis Clp protease subunit ClpP6 homologous protein in rice, is one of the subunits of the chloroplast Clp protease, with the Clp protease subunit interactions OsClpP3 and OsClpP4 respectively, play an important role in the biosynthesis of rice chloroplast.<ref name="ref4" />
What's more, the gene D53 product shares predicted features with the class I Clp ATPase proteins and can form a complex with the a/b hydrolase protein DWARF 14 (D14) and the F-box protein DWARF 3 (D3), two previously identified signalling components potentially responsible for SL(Strigolactones) perception, which means, in a D14- and D3-dependentmanner, SLs induce D53 degradation by the proteasome and abrogate its activity in promoting axillary bud outgrowth.<ref name="ref5" />
The role and molecular composition of Clps in higher plants has just begun to be unraveled, mostly from studies with the model dicotyledonous plant Arabidopsis (Arabidopsis thaliana).Some researchers isolated a virescent yellow leaf (vyl) mutant in rice (Oryza sativa), which produces chlorotic leaves throughout the entire growth period.<ref name="ref4" />
====1.The vyl Mutant Displays Reduced Chlorophyll Accumulation====
The vyl mutant was derived by transforming tissue cultures of the japonica rice variety Kita-ake. When grown under an alternating light/dark cycle (12 h of light at 30°C/12 h of darkness at 20°C) in a growth chamber, vyl mutant plants displayed a virescent yellow leaf pheno-type, and during development, leaves gradually turned green from their tips (more developed) to their bases (less developed; Fig. 1, A and B). At maturity, the vyl mutant plants also had reduced height and smaller seeds (Fig. 1, C and D). Mutant leaves also contained less chlorophyll than the wild type at various growth stages (Fig. 1E).
Additionally, vyl mutants developed chlorotic leaves under different temperature conditions and light/dark cycles, suggesting that the virescent yellow phenotype of the vyl mutant was developmentally regulated but independent of external cues (such as temperature and light)
[[File:figure 1.png]]

====2.The vyl Mutant Has Impaired Chloroplast Development====
Next, researchers investigated whether the virescent yellow phenotype of the vyl mutant was associated with ultra-structural changes in the chloroplasts. Leaf samples of L3U (upper half of the third leaf), L3L (basal half of the third leaf), and L4 (fourth leaf above the shoot base) were collected from wild-type and vyl mutant seedlings and compared (Fig. 2A). Normally, when the third leaf has fully emerged from the shoot base of a rice plant, the shoot also contains the fourth to the seventh immature leaves. The leaf cells in the L3L and L3U samples contain mature chloroplasts, whereas those in the shoot base and L4 samples contain proplastids and early developing immature chloroplasts (Sugimoto et al., 2004). Similar to the wild type, the chloroplasts from the L3U green leaf sample (already turned green) of vyl mutant seedlings displayed well-developed lamellar structures and were equipped with normally stacked grana and thylakoid membranes (Fig. 2, B and C). By contrast, the chloroplasts from the L3L and L4 pale leaves (still wrapped in leaf sheath) of the vyl mutant had much reduced thylakoid membrane networks compared with wild-type plants (Fig. 2, D–G). This developmental defect is similar to the phenotype reported in the Arabidopsis CLPP6 antisense transgenic plants (Sjögren et al., 2006).

[[File:Figure 2 .png]]

====3.The vyl Mutant Has Impaired Photosynthesis====
Chloroplasts are the organelles in plant cells that perform photosynthesis; therefore, they play an essential role in plant growth. To test whether the photosynthetic apparatus was affected in vyl mutants, we compared some key parameters of PSI and PSII between vyl and wild-type plants. Distinct differences in photochemical efficiency of PSII (FPSII), electron transport rate (ETR), nonphotochemical quenching (NPQ), and photochemical
quenching (Qp) were detected between vyl mutants and the wild type. In contrast, the maximal efficiency of PSII photochemistry (F v /F m ) values was almost comparable between vyl and wild-type plants (Table I). These observations indicate that vyl mutants absorbed much less light energy, as shown by the lower NPQ values. PSII photochemistry was reduced at both the donor and acceptor sites, as indicated by the greatly decreased Qp and ETR in vyl mutants. Notably, the PSII structure appeared intact in the mutant plants (indicated by the equivalent F v /F m values), but the actual FPSII was much lower in the mutants. These differences may underlie the defects in chloroplast biogenesis and retarded growth in the vyl mutant (Fig. 1, C and D)
[[File:Table_1.png‎]]

====4.Altered Expression of Genes Associated with Chloroplast Biogenesis and Photosynthesis in vyl Mutants====
Chloroplast biogenesis and physiological changes are tightly regulated by the coordinated expression of plastid and nuclear genes during leaf development and facilitated by protein quality control (Kusumi et al., 2010; Clarke, 2012). To examine whether the expression of genes associated with chloroplast biogenesis and photosynthesis was altered in vyl mutants, quantitative real-time reverse transcription (qRT)-PCR was performed on total RNAs extracted from the L4 and
L3U leaf sections of wild-type and vyl plants. Compared with the wild type, the transcript levels of Virescent1 (V1), V2, and V3 genes and some other genes encoding components of the plastid and nuclear transcription apparatus Sigma factor 2A (OsSig2A), Ribosomal Protein S15 (rps15) and the subunit RNA polymerase (RpoA and RpoTp) that are highly expressed in early stages of chloroplast development were significantly increased in vyl mutants. In contrast, several genes encoding components of the photosynthesis apparatus RuBisCO large subunit (RbcL), the subunit of photosystem I (PsaA), a core component of Photosystem II (PsbA), light-harvesting complex protein (Lhcp2) and Chlorophyll A/B binding protein1 (Cab1) that are highly expressed in later stages of chloroplast development were significantly reduced in vyl mutants (Fig. 3). These results suggested that VYL plays an important role in regulating chloroplast biogenesis.

[[File:Figure 3 .png]]

====5.The vyl Locus Maps to a Putative Gene Encoding OsClpP6====
Genetic analysis showed that the virescent yellow leaf phenotype in vyl mutants is controlled by a single recessive nuclear locus, VYL. Using a BC 1 F 2 mapping population of the vyl mutant and 93-11 (an indica variety), we mapped the vyl gene to a 137-kb genomic region on chromosome 3 between the insertion/deletion polymorphism (Indel) markers F17 and I53. Within this region, 17 open reading frames (ORFs) were predicted from published data (http://
www.gramene.org/; Fig. 4A). Genomic sequence analysis revealed that only the 13th ORF (LOC_Os03g29810) carries a single-nucleotide transition (G→T) at the position 1,509 bp from the ATG start codon (Fig. 4B). We obtained the full-length complementary DNA (cDNA) of LOC_Os03g29810 from both the wild type and the vyl mutant by reverse transcription (RT)-PCR. Sequence analysis showed that the full-length VYL cDNA is 780 bp long in the wild type but 842 bp in the mutant.
Comparison of the genomic and cDNA sequences revealed that the single-nucleotide substitution in the vyl mutant genome created a new splicing site and the addition of a partial fourth intron sequence in the cDNA (Fig. 4, C and D). The mutant cDNA is predicted to encode a truncated vyl mutant protein (DVYL) lacking the classic Ser protease triad (Ser-His-Asp) in the active site and the polypeptide-binding site (Fig. 4, C and E). Phylogenetic analysis and protein sequence alignment showed that VYL is most closely related to the Arabidopsis ClpP6 protein with a conserved S14_ClpP_2 domain and more remotely related to several other Arabidopsis ClpP subunits, and it apparently represents a single-copy gene in the rice genome (Fig. 4F).
To verify the identity of VYL, the plasmid pGVYL, containing a 6-kb genomic DNA fragment consisting of a 2.5-kb upstream sequence, the entire VYL coding region, including nine exons and eight introns, and a 0.6-kb downstream sequence, was constructed and introduced into the vyl mutant. All five transgenic lines containing pGVYL complemented the virescent phenotype of the vyl mutant (Fig. 5). To further confirm that disruption of the VYL gene was responsible for the vyl mutant phenotype, we generated RNA interference transgenic plants in the wild-type Kita-ake background and obtained more than eight independent transgenic
lines. qRT-PCR analysis revealed that the expression of VYL was reduced in three tested transgenic lines compared with the wild-type plants. These VYL knockdown transgenic plants showed reduced accumulation of chlorophyll a and chlorophyll b. Together, these results confirmed that LOC_Os03g29810 indeed corresponds to the VYL gene.
[[File:Figure 4 .png]]

====6.Interactions between VYL, OsClpP3, OsClpP4, OsClpP5, and OsClpT====
To gain an insight into the role of VYL in the rice Clp complex assembly, researchers sought to identify the proteins that directly interact with VYL. We first used a mass spectrometry-based tandem affinity purification proteomics approach. We generated a VYL-HBH construct in
which the C terminus of VYL was fused with a Hisbiotin tag (Tagwerker et al., 2006), and the fusion gene was driven by the Ubiquitin promoter. The VYL-HBH
fusion construct was introduced into the vyl mutant via Agrobacterium tumefaciens-mediated transformation. The VYL-HBH fusion protein transgene recovered the chlorotic phenotype of the vyl mutant, indicating that the fusion protein is biologically functiona SDS-PAGE and immunoblot analyses showed that the VYL-HBH fusion protein and several additional proteins could be effectively purified from the pUbi:: VYL-HBH transgenic plants using Ni 2+ -Sepharose and streptavidin beads (Fig. 8A). The copurified protein bands were excised from the SDS-PAGE gel and analyzed by matrix-assisted laser-desorption ionization time of flight (MALDI-TOF). Three bands were identified with confidence (P , 0.05), including two putative components of the Clp complex (LOC_Os03g29810/ VYL/OsClpP6 and LOC_Os10g43050/OsClpP4) and a vacuolar ATP synthase subunit E (Loc_Oso1g46980/ v-ATP-E; Fig. 8B; Table II). This result suggested that VYL is a bona fide component of the OsClp complex in vivo.
To further investigate the role of VYL in the OsClp core proteolytic complex assembly, we cloned the rice homologs of Arabidopsis ClpR1, ClpP3, ClpP5, and ClpT and named them OsClpR1, OsClpP3, OsClpP5, and OsClpT, respectively. It is noticeable that there is only one copy of the OsClpT gene in the rice genome.
Using yeast two-hybrid assays, we found that VYL protein directly interacted with OsClpP3 and OsClpP4 but not with vacuolar ATP synthase subunit E (v-ATP-E;
Fig. 8C). Furthermore, we found that the yeast strain (Gold Saccharomyces cerevisiae) carrying BD-VYL+AD-OsClpP4 grew and developed the blue color faster than the yeast strain (Gold Saccharomyces cerevisiae) carrying BD-DVYL+AD-OsClpP4 or BD-VYL+AD-OsClpP3 on yeast growth medium containing 5-Bromo-4-chloro-3-
indoylla-galactoside (X-a-Gal). Furthermore, the yeast strain(GoldSaccharomycescerevisiae)carryingBD-DVYL+AD-OsClpP3 did not grow even after incubation for 5 d in a growth chamber (Fig. 8D). This result suggests that the C-terminal polypeptide-binding site of VYL likely plays a role in mediating the interaction between VYL with OsClpP3 and OsClpP4. In addition, they also found that OsClpP3 interacted with OsClpT, OsClpP4 interacted with OsClpP5 and OsClpT, and that both OsClpP4 and OsClpT interacted with themselves but OsClpP5 and OsClpP3 did not (Fig. 8E). Due to the self-activation activity of VYL-AD, we were not able to test whether VYL can homodimerize.

===Expression===
Expression is constitutive in most tissues examined (roots, stems, leaves, leaf sheath, panicle)but most abundant in leaf sections containing
chloroplasts in early stages of development,which can be light-mediated.<ref name="ref4" />
The young chlorotic leaves turn green in later developmental stages, accompanied by alterations in chlorophyll accumulation, chloroplast ultrastructure, and the expression of chloroplast development- and photosynthesis-related genes. Positional cloning revealed that the VYL gene encodes a protein homologous to the Arabidopsis ClpP6 subunit and that it is targeted to the chloroplast. VYL expression is constitutive in most tissues examined but most abundant in leaf sections containing chloroplasts in early stages of development. The mutation in vyl causes premature termination of the predicted gene product and loss of the conserved catalytic triad (serine-histidine-aspartate) and the polypeptide-binding site of VYL. Using a tandem affinity purification approach and mass spectrometry analysis, we identified OsClpP4 as a VYL-associated protein in vivo. In addition, yeast two-hybrid assays demonstrated that VYL directly interacts with OsClpP3 and OsClpP4. Furthermore, we found that OsClpP3 directly interacts with OsClpT, that OsClpP4 directly interacts with OsClpP5 and OsClpT, and that both OsClpP4 and OsClpT can homodimerize. Together, our data provide new insights into the function, assembly, and regulation of Clps in higher plants.<ref name="ref4" />
To investigate the expression patterns of VYL during chloroplast and leaf development, researchers analyzed VYL expression in different sections of leaves at various leaf developmental stages by qRT-PCR (Fig. 2A). They found that in wild-type plants, VYL was most highly expressed in the L4 section at the early chloroplast and leaf development stage (Fig. 6A). Furthermore, qRT-PCR analysis and histochemical staining of the pVYL::GUS reporter gene transgenic plants showed that VYL was constitutively expressed in young buds, young roots, stems, leaves, leaf shoots, and panicles (Fig. 6, B and C). To test whether VYL expression is regulated by light, They analyzed VYL expression during greening of etiolated seedlings. Wild-type rice plants were grown in continuous darkness for 10 d and subsequently exposed to light for 3, 6, 9, 12, 15, 18, 21, or 24 h. The expression of VYL was highly induced after 3 h of illumination and peaked after 6 h of illumination, then its expression gradually decreased over time, and by 15 h after illumination, its expression returned to the preillumination basal level (Fig. 6D). These observations suggested that VYL likely plays a role in the light regulation of chloroplast development. We next conducted qRT-PCR analysis to examine a possible effect of the vyl mutation on the expression of other genes encoding various components of the rice Clp. We
found that OsClpPs, OsClpT, and OsClpR4 all had similar expression patterns to VYL, with a peak accumulation at the early stage of chloroplast development, but the expression levels of these genes were higher in the vyl mutants compared with the wild type (Fig. 7). This observation suggested that there may be a compensatory mechanism to increase the expression of OsClpPs, OsClpT, and OsClpR4 in vyl mutants.

==Evolution==
ATP-dependent Clp protease proteolytic subunit is an enzyme that in humans is encoded by the CLPP gene.<ref name="ref1" /><ref name="ref2" /> It is found in mitochondria and is widely distributed in bacterial species.
In several bacteria, such as E. coli, proteins tagged with the SsrA peptide (ANDENYALAA) encoded by tmRNA are digested by Clp proteases.<ref name="ref3" />

[[File:612px-Protein_CLPP_PDB_1tg6.png]][[File:1867.png]]

==Labs working on this gene==
National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China

==References==
<references>
<ref name="ref1">
Bross P, Andresen BS, Knudsen I, Kruse TA, Gregersen N (Feb 1996). "Human ClpP protease: cDNA sequence, tissue-specific expression and chromosomal assignment of the gene". FEBS Lett 377 (2): 249–52. doi:10.1016/0014-5793(95)01353-9. PMID 8543061.
</ref>
<ref name="ref2">
a b "Entrez Gene: CLPP ClpP caseinolytic peptidase, ATP-dependent, proteolytic subunit homolog (E. coli)".
</ref>
<ref name="ref3">
Gottesman S, Roche E, Zhou Y, Sauer RT (1998). "The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system". Genes Dev 12 (9): 1338–47. doi:10.1101/gad.12.9.1338. PMC 316764. PMID 9573050
</ref>
<ref name="ref4">
Hui Dong et al. A Rice Virescent-Yellow Leaf Mutant Reveals New Insights into the Role and Assembly of Plastid Caseinolytic Protease in Higher Plants. Plant Physiology, 2013, 162(4): 1867-1880
</ref>
<ref name="ref5">
Zhou Feng et al.D14–SCFD3-dependent degradation of D53 regulates strigolactone signaling. Nature,2013. doi:10.1038/nature12878
</ref>
</references>
==Structured Information==
{{JaponicaGene|
GeneName = Os03g0411500|
Description = Peptidase S14, ClpP family protein|
Version = NM_001056884.1 GI:115453496 GeneID:4333096|
Length = 3448 bp|
Definition = Oryza sativa Japonica Group Os03g0411500, complete gene.|
Source = Oryza sativa Japonica Group

ORGANISM Oryza sativa Japonica Group
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP
clade; Ehrhartoideae; Oryzeae; Oryza.
|
Chromosome = [[:category:Japonica Chromosome 3|Chromosome 3]]|
AP = Chromosome 3:17630235..17633682|
CDS = 17630290..17630357,17630478..17630512,17631200..17631269,17631410..17631614,17631776..17631841 ,17632451..17632591,17632803..17632856,17632992..17633084,17633183..17633230 |
GCID = <gbrowseImage1>
name=NC_008396:17630235..17633682
source=RiceChromosome03
preset=GeneLocation
</gbrowseImage1>|
GSID = <gbrowseImage2>
name=NC_008396:17630235..17633682
source=RiceChromosome03
preset=GeneLocation
</gbrowseImage2>|
CDNA = <cdnaseq>atggcgcctatggccatctccaccccgctcgccctccgcgcctccccgacccgcctcctctcccgcaggcggagcggagccaaatcaggcgtggctctcccaggtccacaatttgtaccacctggtatttcttcaaagttggacgagaggatacattgtcattcttctctgaggaaaaatacaattgtagcatcagagaatgaaaatccacctttaatgcctgccataatgactcctgctggtgctcttgatctggcaactgtattgttggggaaccgcattatcttcattggtcaatatattaactcgcaagtagcacagcgtgtaatatcacagcttgtcacacttgctgctgttgatgaagaggctgatattctgatctacctgaactgccccggcggaagtctctactccatcttagcaatttatgattgcatgtcctggatcaagcccaaagttggaacagtgtgctttggtgttgttgctagccaggcagcaattatacttgctggcggtgagaagggaatgcgttatgccatgccaaatgctagagtaatgattcatcaacctcaaggtgtatcagagggtaatgtggaggaggtgaggcgacaggttggggaaaccatttatgctcgtgataaagttgataagatgtttgctgcttttactgggcaaaccttggatatggtacaacagtggacagagagggatcgtttcatgtcttcatctgaagccatggactttggactagttgatgccctgctggaaacaagatactaa</cdnaseq>|
AA = <aaseq>MAPMAISTPLALRASPTRLLSRRRSGAKSGVALPGPQFVPPGIS SKLDERIHCHSSLRKNTIVASENENPPLMPAIMTPAGALDLATVLLGNRIIFIGQYIN SQVAQRVISQLVTLAAVDEEADILIYLNCPGGSLYSILAIYDCMSWIKPKVGTVCFGV VASQAAIILAGGEKGMRYAMPNARVMIHQPQGVSEGNVEEVRRQVGETIYARDKVDKM FAAFTGQTLDMVQQWTERDRFMSSSEAMDFGLVDALLETRY</aaseq>|
DNA = <dnaseqindica>56..123#244..278#966..1035#1176..1380#1542..1607#2217..2357#2569..2622#2758..2850#2949..2996#actcctcagtcctcgcctcggctcggctccctcccacgctccagctccgcctccaatggcgcctatggccatctccaccccgctcgccctccgcgcctccccgacccgcctcctctcccgcaggtgagctccacggaactacaacttccacctccttcgctcgctcgctcgccccgcgcttctctcttcatggattcccccgttcttgtcccctcaccctctgtctggtccttctttcttcaggcggagcggagccaaatcaggcgtggctctcccaggtgagatttcctaacccttggtttagcaaaccatttcccttggtcagctcgttaggccaggactgtttggtggaatttgatcgttgatttgataagctttactgagatgttgtccatggtggtgcacatattagaacatgaccatttgggcagccgtcccatcagctcctagttgtctttctctgtgccaattttttatggagtcgtcattggtaggttttgcaaagcatatagaacctctgaatgtcggcattatccaagagtatgccgacctggggttatctgaaccagtgtcaactccttcctgggccaatgtctggtatgcatcagcattagctcactaagtacacgaaaaatggatgtgcttggttgaacggatatgtataaaaacgataacctgcatataacatatcacctcagtttggtctgattctgaaattagtttagggccttttagcaaactgctgaatgagatttccagactgtatatgtgttattgtgtttgtcagtaactcagtatggtgtattagcacaactcaacatgccataatgacaggatatgcggagcacaaacttttctttggacatgttttttggattcctttactgtttagtccatctgtctttcacatgatatattgctgcaaatgtgctgagttgcattctcactcaaatttccacacctaggtccacaatttgtaccacctggtatttcttcaaagttggacgagaggatacattgtcattcttctctgaggtgatatatttgaaactgtcatgcctgatatacaatgaggatacattgcctgatttgaaactgctcatctaggttttatttgtgctgtgtatcagaatcctgttttattcattcgtaatattgtaaatattttttcacaggaaaaatacaattgtagcatcagagaatgaaaatccacctttaatgcctgccataatgactcctgctggtgctcttgatctggcaactgtattgttggggaaccgcattatcttcattggtcaatatattaactcgcaagtagcacagcgtgtaatatcacagcttgtcacacttgctgctgttgatgaagaggctgatattctggttagtgtttatttttgtgtttttcagatcataacagttaccctattgttcactgcagcagctcttgattgctcaacttcactcccttggcttgctcctttagctcacaggtgtgttgctctatatcttataactccttttgtataattctgttttgccagatctacctgaactgccccggcggaagtctctactccatcttagcaatttatgattgcatgtcctgggtatgccatctatgttgagctactttttccatgtccttcatgcattcaaatttcagagattgtattcacctatatttattcttgtggatgctttctgagttattctcatctaatttaatatttacattgttggatgagaacacattatagatgcatctcaacattttgtatcttccacattatgcatgcccctagctagtgaatttatatttataatatagcacagatatagcatttacaggaaagcctaatgtaatttaggcaaaaattatatctcatatcaatggtagtgcttgcaacatttgtattcttatatttttattgtagtacatactagacatgagcatttgccatgctgagactgtgctaattgggtttggtctggtactgcagacaacagatctcatttcctgaaatcatgcctgtctctaaaactggctttgagctggaccagccttgttagttgttagatttggctgtgtttttacttgttatgccagttttccataaccaaatactttatctacaatttcgcctactgataaataaatcccagaatattaatctttttttgttgttctgcactaacacatgaaccatttattgcagatcaagcccaaagttggaacagtgtgctttggtgttgttgctagccaggcagcaattatacttgctggcggtgagaagggaatgcgttatgccatgccaaatgctagagtaatgattcatcaacctcaaggtgtatcagaggtatgattctggggctttctgcctttctgagttactgcagcgggtgcattatgattttctaacattgtgactacagtaaataataatcatcatcattttagctggccacatgaaacttacaatatacagtcttgctaggacatacttgcttgcctttgtgtttgttgtacttgaagtttgttttttattctaaaaattggatgatttgcagggtaatgtggaggaggtgaggcgacaggttggggaaaccatttatgctcgtgatgtaagtgttttgtgatagataacaagttctatattttcttcagtgtacatttgaattagatgtttgatgagggtaaggaatttctcttgattctgctctctaagcgattaaagcttctgaaaaatctggatgcagaaagttgataagatgtttgctgcttttactgggcaaaccttggatatggtacaacagtggacagagagggatcgtttcatgtcttcatctgaagtaactttcatctcttaaatgtatcagaagaaagtaaatcatcatttgccatgtgaattataacttatttcccccttctttttttggttttaccctaggccatggactttggactagttgatgccctgctggaaacaagatactaacaaacaaacacttaggcacagtttgattagcagaggctggtacaaggattgtgaaactggagctgaagttgagattttcgccgtccttctagttcaaggactccaatgacaggaggctggatctgcgagacttgaatgcatcgccatcgctcctatgagcaaaacatctctgcgttgagtgtgattttttgctcttcttttttggatctggttttacatgccgccagcctatggtctcaatgcattggtcctgctaatgtttagtgtagaccagatgatcctttagacagcaaaacatcagttattactccgtagattttcccatgagctgattccagactgtgtgcaacttttgtgttccaattgcaaagtttgcaacccaacccaacccaacacatgggctgatgggctgttgacaaaggagagattttacacttcacatatgtcaaact</dnaseqindica>|
Link = [http://www.ncbi.nlm.nih.gov/nuccore/NM_001056884.1 RefSeq:Os03g0411500]|
}}
[[Category:Genes]]
[[Category:Japonica mRNA]]
[[Category:Oryza Sativa Japonica Group]]
[[Category:Japonica Genes]]
[[Category:Japonica Chromosome 3]]
[[Category:Chromosome 3]]

Os03g0411500

2014-06-08T08:27:45Z

Xiaoyanjie2012: /* 5.The vyl Locus Maps to a Putative Gene Encoding OsClpP6 */

A nuclear gene coding caseinolyse positioned in plastid or mitochondrion regulating early morphogenesis.
VYL is a gene in rice.Its RAP ID is Os03g0411500 and its MSU ID is LOC_Os03g29810. The mutant of this gene produces chlorotic leaves throughout the entire growth period.
==Annotated Information==
===Function===
The protein encoded by this gene belongs to the peptidase family S14 and hydrolyzes proteins into small peptides in the presence of ATP and magnesium. The protein is transported into mitochondrial matrix and is associated with the inner mitochondrial membrane<ref name="ref2" />, or plastid inner membrane in plants<ref name="ref4" />.
The plastidic caseinolytic protease (Clp) of higher plants is an evolutionarily conserved protein degradation apparatus composed of a proteolytic core complex (the P and R rings) and a set of accessory proteins (ClpT, ClpC, and ClpS).<ref name="ref4" />
Rice yellow leaf mutant vyl, the performance of the entire growth period, new leaves chlorotic phenotype, then gradually turn green from the top down.<ref name="ref4" /> VYL Arabidopsis Clp protease subunit ClpP6 homologous protein in rice, is one of the subunits of the chloroplast Clp protease, with the Clp protease subunit interactions OsClpP3 and OsClpP4 respectively, play an important role in the biosynthesis of rice chloroplast.<ref name="ref4" />
What's more, the gene D53 product shares predicted features with the class I Clp ATPase proteins and can form a complex with the a/b hydrolase protein DWARF 14 (D14) and the F-box protein DWARF 3 (D3), two previously identified signalling components potentially responsible for SL(Strigolactones) perception, which means, in a D14- and D3-dependentmanner, SLs induce D53 degradation by the proteasome and abrogate its activity in promoting axillary bud outgrowth.<ref name="ref5" />
The role and molecular composition of Clps in higher plants has just begun to be unraveled, mostly from studies with the model dicotyledonous plant Arabidopsis (Arabidopsis thaliana).Some researchers isolated a virescent yellow leaf (vyl) mutant in rice (Oryza sativa), which produces chlorotic leaves throughout the entire growth period.<ref name="ref4" />
====1.The vyl Mutant Displays Reduced Chlorophyll Accumulation====
The vyl mutant was derived by transforming tissue cultures of the japonica rice variety Kita-ake. When grown under an alternating light/dark cycle (12 h of light at 30°C/12 h of darkness at 20°C) in a growth chamber, vyl mutant plants displayed a virescent yellow leaf pheno-type, and during development, leaves gradually turned green from their tips (more developed) to their bases (less developed; Fig. 1, A and B). At maturity, the vyl mutant plants also had reduced height and smaller seeds (Fig. 1, C and D). Mutant leaves also contained less chlorophyll than the wild type at various growth stages (Fig. 1E).
Additionally, vyl mutants developed chlorotic leaves under different temperature conditions and light/dark cycles, suggesting that the virescent yellow phenotype of the vyl mutant was developmentally regulated but independent of external cues (such as temperature and light)
[[File:figure 1.png]]

====2.The vyl Mutant Has Impaired Chloroplast Development====
Next, researchers investigated whether the virescent yellow phenotype of the vyl mutant was associated with ultra-structural changes in the chloroplasts. Leaf samples of L3U (upper half of the third leaf), L3L (basal half of the third leaf), and L4 (fourth leaf above the shoot base) were collected from wild-type and vyl mutant seedlings and compared (Fig. 2A). Normally, when the third leaf has fully emerged from the shoot base of a rice plant, the shoot also contains the fourth to the seventh immature leaves. The leaf cells in the L3L and L3U samples contain mature chloroplasts, whereas those in the shoot base and L4 samples contain proplastids and early developing immature chloroplasts (Sugimoto et al., 2004). Similar to the wild type, the chloroplasts from the L3U green leaf sample (already turned green) of vyl mutant seedlings displayed well-developed lamellar structures and were equipped with normally stacked grana and thylakoid membranes (Fig. 2, B and C). By contrast, the chloroplasts from the L3L and L4 pale leaves (still wrapped in leaf sheath) of the vyl mutant had much reduced thylakoid membrane networks compared with wild-type plants (Fig. 2, D–G). This developmental defect is similar to the phenotype reported in the Arabidopsis CLPP6 antisense transgenic plants (Sjögren et al., 2006).

[[File:Figure 2 .png]]

====3.The vyl Mutant Has Impaired Photosynthesis====
Chloroplasts are the organelles in plant cells that perform photosynthesis; therefore, they play an essential role in plant growth. To test whether the photosynthetic apparatus was affected in vyl mutants, we compared some key parameters of PSI and PSII between vyl and wild-type plants. Distinct differences in photochemical efficiency of PSII (FPSII), electron transport rate (ETR), nonphotochemical quenching (NPQ), and photochemical
quenching (Qp) were detected between vyl mutants and the wild type. In contrast, the maximal efficiency of PSII photochemistry (F v /F m ) values was almost comparable between vyl and wild-type plants (Table I). These observations indicate that vyl mutants absorbed much less light energy, as shown by the lower NPQ values. PSII photochemistry was reduced at both the donor and acceptor sites, as indicated by the greatly decreased Qp and ETR in vyl mutants. Notably, the PSII structure appeared intact in the mutant plants (indicated by the equivalent F v /F m values), but the actual FPSII was much lower in the mutants. These differences may underlie the defects in chloroplast biogenesis and retarded growth in the vyl mutant (Fig. 1, C and D)
[[File:Table_1.png‎]]

====4.Altered Expression of Genes Associated with Chloroplast Biogenesis and Photosynthesis in vyl Mutants====
Chloroplast biogenesis and physiological changes are tightly regulated by the coordinated expression of plastid and nuclear genes during leaf development and facilitated by protein quality control (Kusumi et al., 2010; Clarke, 2012). To examine whether the expression of genes associated with chloroplast biogenesis and photosynthesis was altered in vyl mutants, quantitative real-time reverse transcription (qRT)-PCR was performed on total RNAs extracted from the L4 and
L3U leaf sections of wild-type and vyl plants. Compared with the wild type, the transcript levels of Virescent1 (V1), V2, and V3 genes and some other genes encoding components of the plastid and nuclear transcription apparatus Sigma factor 2A (OsSig2A), Ribosomal Protein S15 (rps15) and the subunit RNA polymerase (RpoA and RpoTp) that are highly expressed in early stages of chloroplast development were significantly increased in vyl mutants. In contrast, several genes encoding components of the photosynthesis apparatus RuBisCO large subunit (RbcL), the subunit of photosystem I (PsaA), a core component of Photosystem II (PsbA), light-harvesting complex protein (Lhcp2) and Chlorophyll A/B binding protein1 (Cab1) that are highly expressed in later stages of chloroplast development were significantly reduced in vyl mutants (Fig. 3). These results suggested that VYL plays an important role in regulating chloroplast biogenesis.

[[File:Figure 3 .png]]

====5.The vyl Locus Maps to a Putative Gene Encoding OsClpP6====
Genetic analysis showed that the virescent yellow leaf phenotype in vyl mutants is controlled by a single recessive nuclear locus, VYL. Using a BC 1 F 2 mapping population of the vyl mutant and 93-11 (an indica variety), we mapped the vyl gene to a 137-kb genomic region on chromosome 3 between the insertion/deletion polymorphism (Indel) markers F17 and I53. Within this region, 17 open reading frames (ORFs) were predicted from published data (http://
www.gramene.org/; Fig. 4A). Genomic sequence analysis revealed that only the 13th ORF (LOC_Os03g29810) carries a single-nucleotide transition (G→T) at the position 1,509 bp from the ATG start codon (Fig. 4B). We obtained the full-length complementary DNA (cDNA) of LOC_Os03g29810 from both the wild type and the vyl mutant by reverse transcription (RT)-PCR. Sequence analysis showed that the full-length VYL cDNA is 780 bp long in the wild type but 842 bp in the mutant.
Comparison of the genomic and cDNA sequences revealed that the single-nucleotide substitution in the vyl mutant genome created a new splicing site and the addition of a partial fourth intron sequence in the cDNA (Fig. 4, C and D). The mutant cDNA is predicted to encode a truncated vyl mutant protein (DVYL) lacking the classic Ser protease triad (Ser-His-Asp) in the active site and the polypeptide-binding site (Fig. 4, C and E). Phylogenetic analysis and protein sequence alignment showed that VYL is most closely related to the Arabidopsis ClpP6 protein with a conserved S14_ClpP_2 domain and more remotely related to several other Arabidopsis ClpP subunits, and it apparently represents a single-copy gene in the rice genome (Fig. 4F).
To verify the identity of VYL, the plasmid pGVYL, containing a 6-kb genomic DNA fragment consisting of a 2.5-kb upstream sequence, the entire VYL coding region, including nine exons and eight introns, and a 0.6-kb downstream sequence, was constructed and introduced into the vyl mutant. All five transgenic lines containing pGVYL complemented the virescent phenotype of the vyl mutant (Fig. 5). To further confirm that disruption of the VYL gene was responsible for the vyl mutant phenotype, we generated RNA interference transgenic plants in the wild-type Kita-ake background and obtained more than eight independent transgenic
lines. qRT-PCR analysis revealed that the expression of VYL was reduced in three tested transgenic lines compared with the wild-type plants. These VYL knockdown transgenic plants showed reduced accumulation of chlorophyll a and chlorophyll b. Together, these results confirmed that LOC_Os03g29810 indeed corresponds to the VYL gene.
[[File:Figure 4 .png]]

====6.Interactions between VYL, OsClpP3, OsClpP4, OsClpP5, and OsClpT====
To gain an insight into the role of VYL in the rice Clp complex assembly, researchers sought to identify the proteins that directly interact with VYL. We first used a mass spectrometry-based tandem affinity purification proteomics approach. We generated a VYL-HBH construct in
which the C terminus of VYL was fused with a Hisbiotin tag (Tagwerker et al., 2006), and the fusion gene was driven by the Ubiquitin promoter. The VYL-HBH
fusion construct was introduced into the vyl mutant via Agrobacterium tumefaciens-mediated transformation. The VYL-HBH fusion protein transgene recovered the chlorotic phenotype of the vyl mutant, indicating that the fusion protein is biologically functiona SDS-PAGE and immunoblot analyses showed that the VYL-HBH fusion protein and several additional proteins could be effectively purified from the pUbi:: VYL-HBH transgenic plants using Ni 2+ -Sepharose and streptavidin beads (Fig. 8A). The copurified protein bands were excised from the SDS-PAGE gel and analyzed by matrix-assisted laser-desorption ionization time of flight (MALDI-TOF). Three bands were identified with confidence (P , 0.05), including two putative components of the Clp complex (LOC_Os03g29810/ VYL/OsClpP6 and LOC_Os10g43050/OsClpP4) and a vacuolar ATP synthase subunit E (Loc_Oso1g46980/ v-ATP-E; Fig. 8B; Table II). This result suggested that VYL is a bona fide component of the OsClp complex in vivo.

===Expression===
Expression is constitutive in most tissues examined (roots, stems, leaves, leaf sheath, panicle)but most abundant in leaf sections containing
chloroplasts in early stages of development,which can be light-mediated.<ref name="ref4" />
The young chlorotic leaves turn green in later developmental stages, accompanied by alterations in chlorophyll accumulation, chloroplast ultrastructure, and the expression of chloroplast development- and photosynthesis-related genes. Positional cloning revealed that the VYL gene encodes a protein homologous to the Arabidopsis ClpP6 subunit and that it is targeted to the chloroplast. VYL expression is constitutive in most tissues examined but most abundant in leaf sections containing chloroplasts in early stages of development. The mutation in vyl causes premature termination of the predicted gene product and loss of the conserved catalytic triad (serine-histidine-aspartate) and the polypeptide-binding site of VYL. Using a tandem affinity purification approach and mass spectrometry analysis, we identified OsClpP4 as a VYL-associated protein in vivo. In addition, yeast two-hybrid assays demonstrated that VYL directly interacts with OsClpP3 and OsClpP4. Furthermore, we found that OsClpP3 directly interacts with OsClpT, that OsClpP4 directly interacts with OsClpP5 and OsClpT, and that both OsClpP4 and OsClpT can homodimerize. Together, our data provide new insights into the function, assembly, and regulation of Clps in higher plants.<ref name="ref4" />
To investigate the expression patterns of VYL during chloroplast and leaf development, researchers analyzed VYL expression in different sections of leaves at various leaf developmental stages by qRT-PCR (Fig. 2A). They found that in wild-type plants, VYL was most highly expressed in the L4 section at the early chloroplast and leaf development stage (Fig. 6A). Furthermore, qRT-PCR analysis and histochemical staining of the pVYL::GUS reporter gene transgenic plants showed that VYL was constitutively expressed in young buds, young roots, stems, leaves, leaf shoots, and panicles (Fig. 6, B and C). To test whether VYL expression is regulated by light, They analyzed VYL expression during greening of etiolated seedlings. Wild-type rice plants were grown in continuous darkness for 10 d and subsequently exposed to light for 3, 6, 9, 12, 15, 18, 21, or 24 h. The expression of VYL was highly induced after 3 h of illumination and peaked after 6 h of illumination, then its expression gradually decreased over time, and by 15 h after illumination, its expression returned to the preillumination basal level (Fig. 6D). These observations suggested that VYL likely plays a role in the light regulation of chloroplast development. We next conducted qRT-PCR analysis to examine a possible effect of the vyl mutation on the expression of other genes encoding various components of the rice Clp. We
found that OsClpPs, OsClpT, and OsClpR4 all had similar expression patterns to VYL, with a peak accumulation at the early stage of chloroplast development, but the expression levels of these genes were higher in the vyl mutants compared with the wild type (Fig. 7). This observation suggested that there may be a compensatory mechanism to increase the expression of OsClpPs, OsClpT, and OsClpR4 in vyl mutants.

==Evolution==
ATP-dependent Clp protease proteolytic subunit is an enzyme that in humans is encoded by the CLPP gene.<ref name="ref1" /><ref name="ref2" /> It is found in mitochondria and is widely distributed in bacterial species.
In several bacteria, such as E. coli, proteins tagged with the SsrA peptide (ANDENYALAA) encoded by tmRNA are digested by Clp proteases.<ref name="ref3" />

[[File:612px-Protein_CLPP_PDB_1tg6.png]][[File:1867.png]]

==Labs working on this gene==
National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China

==References==
<references>
<ref name="ref1">
Bross P, Andresen BS, Knudsen I, Kruse TA, Gregersen N (Feb 1996). "Human ClpP protease: cDNA sequence, tissue-specific expression and chromosomal assignment of the gene". FEBS Lett 377 (2): 249–52. doi:10.1016/0014-5793(95)01353-9. PMID 8543061.
</ref>
<ref name="ref2">
a b "Entrez Gene: CLPP ClpP caseinolytic peptidase, ATP-dependent, proteolytic subunit homolog (E. coli)".
</ref>
<ref name="ref3">
Gottesman S, Roche E, Zhou Y, Sauer RT (1998). "The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system". Genes Dev 12 (9): 1338–47. doi:10.1101/gad.12.9.1338. PMC 316764. PMID 9573050
</ref>
<ref name="ref4">
Hui Dong et al. A Rice Virescent-Yellow Leaf Mutant Reveals New Insights into the Role and Assembly of Plastid Caseinolytic Protease in Higher Plants. Plant Physiology, 2013, 162(4): 1867-1880
</ref>
<ref name="ref5">
Zhou Feng et al.D14–SCFD3-dependent degradation of D53 regulates strigolactone signaling. Nature,2013. doi:10.1038/nature12878
</ref>
</references>
==Structured Information==
{{JaponicaGene|
GeneName = Os03g0411500|
Description = Peptidase S14, ClpP family protein|
Version = NM_001056884.1 GI:115453496 GeneID:4333096|
Length = 3448 bp|
Definition = Oryza sativa Japonica Group Os03g0411500, complete gene.|
Source = Oryza sativa Japonica Group

ORGANISM Oryza sativa Japonica Group
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP
clade; Ehrhartoideae; Oryzeae; Oryza.
|
Chromosome = [[:category:Japonica Chromosome 3|Chromosome 3]]|
AP = Chromosome 3:17630235..17633682|
CDS = 17630290..17630357,17630478..17630512,17631200..17631269,17631410..17631614,17631776..17631841 ,17632451..17632591,17632803..17632856,17632992..17633084,17633183..17633230 |
GCID = <gbrowseImage1>
name=NC_008396:17630235..17633682
source=RiceChromosome03
preset=GeneLocation
</gbrowseImage1>|
GSID = <gbrowseImage2>
name=NC_008396:17630235..17633682
source=RiceChromosome03
preset=GeneLocation
</gbrowseImage2>|
CDNA = <cdnaseq>atggcgcctatggccatctccaccccgctcgccctccgcgcctccccgacccgcctcctctcccgcaggcggagcggagccaaatcaggcgtggctctcccaggtccacaatttgtaccacctggtatttcttcaaagttggacgagaggatacattgtcattcttctctgaggaaaaatacaattgtagcatcagagaatgaaaatccacctttaatgcctgccataatgactcctgctggtgctcttgatctggcaactgtattgttggggaaccgcattatcttcattggtcaatatattaactcgcaagtagcacagcgtgtaatatcacagcttgtcacacttgctgctgttgatgaagaggctgatattctgatctacctgaactgccccggcggaagtctctactccatcttagcaatttatgattgcatgtcctggatcaagcccaaagttggaacagtgtgctttggtgttgttgctagccaggcagcaattatacttgctggcggtgagaagggaatgcgttatgccatgccaaatgctagagtaatgattcatcaacctcaaggtgtatcagagggtaatgtggaggaggtgaggcgacaggttggggaaaccatttatgctcgtgataaagttgataagatgtttgctgcttttactgggcaaaccttggatatggtacaacagtggacagagagggatcgtttcatgtcttcatctgaagccatggactttggactagttgatgccctgctggaaacaagatactaa</cdnaseq>|
AA = <aaseq>MAPMAISTPLALRASPTRLLSRRRSGAKSGVALPGPQFVPPGIS SKLDERIHCHSSLRKNTIVASENENPPLMPAIMTPAGALDLATVLLGNRIIFIGQYIN SQVAQRVISQLVTLAAVDEEADILIYLNCPGGSLYSILAIYDCMSWIKPKVGTVCFGV VASQAAIILAGGEKGMRYAMPNARVMIHQPQGVSEGNVEEVRRQVGETIYARDKVDKM FAAFTGQTLDMVQQWTERDRFMSSSEAMDFGLVDALLETRY</aaseq>|
DNA = <dnaseqindica>56..123#244..278#966..1035#1176..1380#1542..1607#2217..2357#2569..2622#2758..2850#2949..2996#actcctcagtcctcgcctcggctcggctccctcccacgctccagctccgcctccaatggcgcctatggccatctccaccccgctcgccctccgcgcctccccgacccgcctcctctcccgcaggtgagctccacggaactacaacttccacctccttcgctcgctcgctcgccccgcgcttctctcttcatggattcccccgttcttgtcccctcaccctctgtctggtccttctttcttcaggcggagcggagccaaatcaggcgtggctctcccaggtgagatttcctaacccttggtttagcaaaccatttcccttggtcagctcgttaggccaggactgtttggtggaatttgatcgttgatttgataagctttactgagatgttgtccatggtggtgcacatattagaacatgaccatttgggcagccgtcccatcagctcctagttgtctttctctgtgccaattttttatggagtcgtcattggtaggttttgcaaagcatatagaacctctgaatgtcggcattatccaagagtatgccgacctggggttatctgaaccagtgtcaactccttcctgggccaatgtctggtatgcatcagcattagctcactaagtacacgaaaaatggatgtgcttggttgaacggatatgtataaaaacgataacctgcatataacatatcacctcagtttggtctgattctgaaattagtttagggccttttagcaaactgctgaatgagatttccagactgtatatgtgttattgtgtttgtcagtaactcagtatggtgtattagcacaactcaacatgccataatgacaggatatgcggagcacaaacttttctttggacatgttttttggattcctttactgtttagtccatctgtctttcacatgatatattgctgcaaatgtgctgagttgcattctcactcaaatttccacacctaggtccacaatttgtaccacctggtatttcttcaaagttggacgagaggatacattgtcattcttctctgaggtgatatatttgaaactgtcatgcctgatatacaatgaggatacattgcctgatttgaaactgctcatctaggttttatttgtgctgtgtatcagaatcctgttttattcattcgtaatattgtaaatattttttcacaggaaaaatacaattgtagcatcagagaatgaaaatccacctttaatgcctgccataatgactcctgctggtgctcttgatctggcaactgtattgttggggaaccgcattatcttcattggtcaatatattaactcgcaagtagcacagcgtgtaatatcacagcttgtcacacttgctgctgttgatgaagaggctgatattctggttagtgtttatttttgtgtttttcagatcataacagttaccctattgttcactgcagcagctcttgattgctcaacttcactcccttggcttgctcctttagctcacaggtgtgttgctctatatcttataactccttttgtataattctgttttgccagatctacctgaactgccccggcggaagtctctactccatcttagcaatttatgattgcatgtcctgggtatgccatctatgttgagctactttttccatgtccttcatgcattcaaatttcagagattgtattcacctatatttattcttgtggatgctttctgagttattctcatctaatttaatatttacattgttggatgagaacacattatagatgcatctcaacattttgtatcttccacattatgcatgcccctagctagtgaatttatatttataatatagcacagatatagcatttacaggaaagcctaatgtaatttaggcaaaaattatatctcatatcaatggtagtgcttgcaacatttgtattcttatatttttattgtagtacatactagacatgagcatttgccatgctgagactgtgctaattgggtttggtctggtactgcagacaacagatctcatttcctgaaatcatgcctgtctctaaaactggctttgagctggaccagccttgttagttgttagatttggctgtgtttttacttgttatgccagttttccataaccaaatactttatctacaatttcgcctactgataaataaatcccagaatattaatctttttttgttgttctgcactaacacatgaaccatttattgcagatcaagcccaaagttggaacagtgtgctttggtgttgttgctagccaggcagcaattatacttgctggcggtgagaagggaatgcgttatgccatgccaaatgctagagtaatgattcatcaacctcaaggtgtatcagaggtatgattctggggctttctgcctttctgagttactgcagcgggtgcattatgattttctaacattgtgactacagtaaataataatcatcatcattttagctggccacatgaaacttacaatatacagtcttgctaggacatacttgcttgcctttgtgtttgttgtacttgaagtttgttttttattctaaaaattggatgatttgcagggtaatgtggaggaggtgaggcgacaggttggggaaaccatttatgctcgtgatgtaagtgttttgtgatagataacaagttctatattttcttcagtgtacatttgaattagatgtttgatgagggtaaggaatttctcttgattctgctctctaagcgattaaagcttctgaaaaatctggatgcagaaagttgataagatgtttgctgcttttactgggcaaaccttggatatggtacaacagtggacagagagggatcgtttcatgtcttcatctgaagtaactttcatctcttaaatgtatcagaagaaagtaaatcatcatttgccatgtgaattataacttatttcccccttctttttttggttttaccctaggccatggactttggactagttgatgccctgctggaaacaagatactaacaaacaaacacttaggcacagtttgattagcagaggctggtacaaggattgtgaaactggagctgaagttgagattttcgccgtccttctagttcaaggactccaatgacaggaggctggatctgcgagacttgaatgcatcgccatcgctcctatgagcaaaacatctctgcgttgagtgtgattttttgctcttcttttttggatctggttttacatgccgccagcctatggtctcaatgcattggtcctgctaatgtttagtgtagaccagatgatcctttagacagcaaaacatcagttattactccgtagattttcccatgagctgattccagactgtgtgcaacttttgtgttccaattgcaaagtttgcaacccaacccaacccaacacatgggctgatgggctgttgacaaaggagagattttacacttcacatatgtcaaact</dnaseqindica>|
Link = [http://www.ncbi.nlm.nih.gov/nuccore/NM_001056884.1 RefSeq:Os03g0411500]|
}}
[[Category:Genes]]
[[Category:Japonica mRNA]]
[[Category:Oryza Sativa Japonica Group]]
[[Category:Japonica Genes]]
[[Category:Japonica Chromosome 3]]
[[Category:Chromosome 3]]

Os03g0411500

2014-06-08T08:24:09Z

Xiaoyanjie2012: /* Expression */

A nuclear gene coding caseinolyse positioned in plastid or mitochondrion regulating early morphogenesis.
VYL is a gene in rice.Its RAP ID is Os03g0411500 and its MSU ID is LOC_Os03g29810. The mutant of this gene produces chlorotic leaves throughout the entire growth period.
==Annotated Information==
===Function===
The protein encoded by this gene belongs to the peptidase family S14 and hydrolyzes proteins into small peptides in the presence of ATP and magnesium. The protein is transported into mitochondrial matrix and is associated with the inner mitochondrial membrane<ref name="ref2" />, or plastid inner membrane in plants<ref name="ref4" />.
The plastidic caseinolytic protease (Clp) of higher plants is an evolutionarily conserved protein degradation apparatus composed of a proteolytic core complex (the P and R rings) and a set of accessory proteins (ClpT, ClpC, and ClpS).<ref name="ref4" />
Rice yellow leaf mutant vyl, the performance of the entire growth period, new leaves chlorotic phenotype, then gradually turn green from the top down.<ref name="ref4" /> VYL Arabidopsis Clp protease subunit ClpP6 homologous protein in rice, is one of the subunits of the chloroplast Clp protease, with the Clp protease subunit interactions OsClpP3 and OsClpP4 respectively, play an important role in the biosynthesis of rice chloroplast.<ref name="ref4" />
What's more, the gene D53 product shares predicted features with the class I Clp ATPase proteins and can form a complex with the a/b hydrolase protein DWARF 14 (D14) and the F-box protein DWARF 3 (D3), two previously identified signalling components potentially responsible for SL(Strigolactones) perception, which means, in a D14- and D3-dependentmanner, SLs induce D53 degradation by the proteasome and abrogate its activity in promoting axillary bud outgrowth.<ref name="ref5" />
The role and molecular composition of Clps in higher plants has just begun to be unraveled, mostly from studies with the model dicotyledonous plant Arabidopsis (Arabidopsis thaliana).Some researchers isolated a virescent yellow leaf (vyl) mutant in rice (Oryza sativa), which produces chlorotic leaves throughout the entire growth period.<ref name="ref4" />
====1.The vyl Mutant Displays Reduced Chlorophyll Accumulation====
The vyl mutant was derived by transforming tissue cultures of the japonica rice variety Kita-ake. When grown under an alternating light/dark cycle (12 h of light at 30°C/12 h of darkness at 20°C) in a growth chamber, vyl mutant plants displayed a virescent yellow leaf pheno-type, and during development, leaves gradually turned green from their tips (more developed) to their bases (less developed; Fig. 1, A and B). At maturity, the vyl mutant plants also had reduced height and smaller seeds (Fig. 1, C and D). Mutant leaves also contained less chlorophyll than the wild type at various growth stages (Fig. 1E).
Additionally, vyl mutants developed chlorotic leaves under different temperature conditions and light/dark cycles, suggesting that the virescent yellow phenotype of the vyl mutant was developmentally regulated but independent of external cues (such as temperature and light)
[[File:figure 1.png]]

====2.The vyl Mutant Has Impaired Chloroplast Development====
Next, researchers investigated whether the virescent yellow phenotype of the vyl mutant was associated with ultra-structural changes in the chloroplasts. Leaf samples of L3U (upper half of the third leaf), L3L (basal half of the third leaf), and L4 (fourth leaf above the shoot base) were collected from wild-type and vyl mutant seedlings and compared (Fig. 2A). Normally, when the third leaf has fully emerged from the shoot base of a rice plant, the shoot also contains the fourth to the seventh immature leaves. The leaf cells in the L3L and L3U samples contain mature chloroplasts, whereas those in the shoot base and L4 samples contain proplastids and early developing immature chloroplasts (Sugimoto et al., 2004). Similar to the wild type, the chloroplasts from the L3U green leaf sample (already turned green) of vyl mutant seedlings displayed well-developed lamellar structures and were equipped with normally stacked grana and thylakoid membranes (Fig. 2, B and C). By contrast, the chloroplasts from the L3L and L4 pale leaves (still wrapped in leaf sheath) of the vyl mutant had much reduced thylakoid membrane networks compared with wild-type plants (Fig. 2, D–G). This developmental defect is similar to the phenotype reported in the Arabidopsis CLPP6 antisense transgenic plants (Sjögren et al., 2006).

[[File:Figure 2 .png]]

====3.The vyl Mutant Has Impaired Photosynthesis====
Chloroplasts are the organelles in plant cells that perform photosynthesis; therefore, they play an essential role in plant growth. To test whether the photosynthetic apparatus was affected in vyl mutants, we compared some key parameters of PSI and PSII between vyl and wild-type plants. Distinct differences in photochemical efficiency of PSII (FPSII), electron transport rate (ETR), nonphotochemical quenching (NPQ), and photochemical
quenching (Qp) were detected between vyl mutants and the wild type. In contrast, the maximal efficiency of PSII photochemistry (F v /F m ) values was almost comparable between vyl and wild-type plants (Table I). These observations indicate that vyl mutants absorbed much less light energy, as shown by the lower NPQ values. PSII photochemistry was reduced at both the donor and acceptor sites, as indicated by the greatly decreased Qp and ETR in vyl mutants. Notably, the PSII structure appeared intact in the mutant plants (indicated by the equivalent F v /F m values), but the actual FPSII was much lower in the mutants. These differences may underlie the defects in chloroplast biogenesis and retarded growth in the vyl mutant (Fig. 1, C and D)
[[File:Table_1.png‎]]

====4.Altered Expression of Genes Associated with Chloroplast Biogenesis and Photosynthesis in vyl Mutants====
Chloroplast biogenesis and physiological changes are tightly regulated by the coordinated expression of plastid and nuclear genes during leaf development and facilitated by protein quality control (Kusumi et al., 2010; Clarke, 2012). To examine whether the expression of genes associated with chloroplast biogenesis and photosynthesis was altered in vyl mutants, quantitative real-time reverse transcription (qRT)-PCR was performed on total RNAs extracted from the L4 and
L3U leaf sections of wild-type and vyl plants. Compared with the wild type, the transcript levels of Virescent1 (V1), V2, and V3 genes and some other genes encoding components of the plastid and nuclear transcription apparatus Sigma factor 2A (OsSig2A), Ribosomal Protein S15 (rps15) and the subunit RNA polymerase (RpoA and RpoTp) that are highly expressed in early stages of chloroplast development were significantly increased in vyl mutants. In contrast, several genes encoding components of the photosynthesis apparatus RuBisCO large subunit (RbcL), the subunit of photosystem I (PsaA), a core component of Photosystem II (PsbA), light-harvesting complex protein (Lhcp2) and Chlorophyll A/B binding protein1 (Cab1) that are highly expressed in later stages of chloroplast development were significantly reduced in vyl mutants (Fig. 3). These results suggested that VYL plays an important role in regulating chloroplast biogenesis.

[[File:Figure 3 .png]]

====5.The vyl Locus Maps to a Putative Gene Encoding OsClpP6====
Genetic analysis showed that the virescent yellow leaf phenotype in vyl mutants is controlled by a single recessive nuclear locus, VYL. Using a BC 1 F 2 mapping population of the vyl mutant and 93-11 (an indica variety), we mapped the vyl gene to a 137-kb genomic region on chromosome 3 between the insertion/deletion polymorphism (Indel) markers F17 and I53. Within this region, 17 open reading frames (ORFs) were predicted from published data (http://
www.gramene.org/; Fig. 4A). Genomic sequence analysis revealed that only the 13th ORF (LOC_Os03g29810) carries a single-nucleotide transition (G→T) at the position 1,509 bp from the ATG start codon (Fig. 4B). We obtained the full-length complementary DNA (cDNA) of LOC_Os03g29810 from both the wild type and the vyl mutant by reverse transcription (RT)-PCR. Sequence analysis showed that the full-length VYL cDNA is 780 bp long in the wild type but 842 bp in the mutant.
Comparison of the genomic and cDNA sequences revealed that the single-nucleotide substitution in the vyl mutant genome created a new splicing site and the addition of a partial fourth intron sequence in the cDNA (Fig. 4, C and D). The mutant cDNA is predicted to encode a truncated vyl mutant protein (DVYL) lacking the classic Ser protease triad (Ser-His-Asp) in the active site and the polypeptide-binding site (Fig. 4, C and E). Phylogenetic analysis and protein sequence alignment showed that VYL is most closely related to the Arabidopsis ClpP6 protein with a conserved S14_ClpP_2 domain and more remotely related to several other Arabidopsis ClpP subunits, and it apparently represents a single-copy gene in the rice genome (Fig. 4F).
To verify the identity of VYL, the plasmid pGVYL, containing a 6-kb genomic DNA fragment consisting of a 2.5-kb upstream sequence, the entire VYL coding region, including nine exons and eight introns, and a 0.6-kb downstream sequence, was constructed and introduced into the vyl mutant. All five transgenic lines containing pGVYL complemented the virescent phenotype of the vyl mutant (Fig. 5). To further confirm that disruption of the VYL gene was responsible for the vyl mutant phenotype, we generated RNA interference transgenic plants in the wild-type Kita-ake background and obtained more than eight independent transgenic
lines. qRT-PCR analysis revealed that the expression of VYL was reduced in three tested transgenic lines compared with the wild-type plants. These VYL knockdown transgenic plants showed reduced accumulation of chlorophyll a and chlorophyll b. Together, these results confirmed that LOC_Os03g29810 indeed corresponds to the VYL gene.
[[File:Figure 4 .png]]

===Expression===
Expression is constitutive in most tissues examined (roots, stems, leaves, leaf sheath, panicle)but most abundant in leaf sections containing
chloroplasts in early stages of development,which can be light-mediated.<ref name="ref4" />
The young chlorotic leaves turn green in later developmental stages, accompanied by alterations in chlorophyll accumulation, chloroplast ultrastructure, and the expression of chloroplast development- and photosynthesis-related genes. Positional cloning revealed that the VYL gene encodes a protein homologous to the Arabidopsis ClpP6 subunit and that it is targeted to the chloroplast. VYL expression is constitutive in most tissues examined but most abundant in leaf sections containing chloroplasts in early stages of development. The mutation in vyl causes premature termination of the predicted gene product and loss of the conserved catalytic triad (serine-histidine-aspartate) and the polypeptide-binding site of VYL. Using a tandem affinity purification approach and mass spectrometry analysis, we identified OsClpP4 as a VYL-associated protein in vivo. In addition, yeast two-hybrid assays demonstrated that VYL directly interacts with OsClpP3 and OsClpP4. Furthermore, we found that OsClpP3 directly interacts with OsClpT, that OsClpP4 directly interacts with OsClpP5 and OsClpT, and that both OsClpP4 and OsClpT can homodimerize. Together, our data provide new insights into the function, assembly, and regulation of Clps in higher plants.<ref name="ref4" />
To investigate the expression patterns of VYL during chloroplast and leaf development, researchers analyzed VYL expression in different sections of leaves at various leaf developmental stages by qRT-PCR (Fig. 2A). They found that in wild-type plants, VYL was most highly expressed in the L4 section at the early chloroplast and leaf development stage (Fig. 6A). Furthermore, qRT-PCR analysis and histochemical staining of the pVYL::GUS reporter gene transgenic plants showed that VYL was constitutively expressed in young buds, young roots, stems, leaves, leaf shoots, and panicles (Fig. 6, B and C). To test whether VYL expression is regulated by light, They analyzed VYL expression during greening of etiolated seedlings. Wild-type rice plants were grown in continuous darkness for 10 d and subsequently exposed to light for 3, 6, 9, 12, 15, 18, 21, or 24 h. The expression of VYL was highly induced after 3 h of illumination and peaked after 6 h of illumination, then its expression gradually decreased over time, and by 15 h after illumination, its expression returned to the preillumination basal level (Fig. 6D). These observations suggested that VYL likely plays a role in the light regulation of chloroplast development. We next conducted qRT-PCR analysis to examine a possible effect of the vyl mutation on the expression of other genes encoding various components of the rice Clp. We
found that OsClpPs, OsClpT, and OsClpR4 all had similar expression patterns to VYL, with a peak accumulation at the early stage of chloroplast development, but the expression levels of these genes were higher in the vyl mutants compared with the wild type (Fig. 7). This observation suggested that there may be a compensatory mechanism to increase the expression of OsClpPs, OsClpT, and OsClpR4 in vyl mutants.

==Evolution==
ATP-dependent Clp protease proteolytic subunit is an enzyme that in humans is encoded by the CLPP gene.<ref name="ref1" /><ref name="ref2" /> It is found in mitochondria and is widely distributed in bacterial species.
In several bacteria, such as E. coli, proteins tagged with the SsrA peptide (ANDENYALAA) encoded by tmRNA are digested by Clp proteases.<ref name="ref3" />

[[File:612px-Protein_CLPP_PDB_1tg6.png]][[File:1867.png]]

==Labs working on this gene==
National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China

==References==
<references>
<ref name="ref1">
Bross P, Andresen BS, Knudsen I, Kruse TA, Gregersen N (Feb 1996). "Human ClpP protease: cDNA sequence, tissue-specific expression and chromosomal assignment of the gene". FEBS Lett 377 (2): 249–52. doi:10.1016/0014-5793(95)01353-9. PMID 8543061.
</ref>
<ref name="ref2">
a b "Entrez Gene: CLPP ClpP caseinolytic peptidase, ATP-dependent, proteolytic subunit homolog (E. coli)".
</ref>
<ref name="ref3">
Gottesman S, Roche E, Zhou Y, Sauer RT (1998). "The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system". Genes Dev 12 (9): 1338–47. doi:10.1101/gad.12.9.1338. PMC 316764. PMID 9573050
</ref>
<ref name="ref4">
Hui Dong et al. A Rice Virescent-Yellow Leaf Mutant Reveals New Insights into the Role and Assembly of Plastid Caseinolytic Protease in Higher Plants. Plant Physiology, 2013, 162(4): 1867-1880
</ref>
<ref name="ref5">
Zhou Feng et al.D14–SCFD3-dependent degradation of D53 regulates strigolactone signaling. Nature,2013. doi:10.1038/nature12878
</ref>
</references>
==Structured Information==
{{JaponicaGene|
GeneName = Os03g0411500|
Description = Peptidase S14, ClpP family protein|
Version = NM_001056884.1 GI:115453496 GeneID:4333096|
Length = 3448 bp|
Definition = Oryza sativa Japonica Group Os03g0411500, complete gene.|
Source = Oryza sativa Japonica Group

ORGANISM Oryza sativa Japonica Group
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP
clade; Ehrhartoideae; Oryzeae; Oryza.
|
Chromosome = [[:category:Japonica Chromosome 3|Chromosome 3]]|
AP = Chromosome 3:17630235..17633682|
CDS = 17630290..17630357,17630478..17630512,17631200..17631269,17631410..17631614,17631776..17631841 ,17632451..17632591,17632803..17632856,17632992..17633084,17633183..17633230 |
GCID = <gbrowseImage1>
name=NC_008396:17630235..17633682
source=RiceChromosome03
preset=GeneLocation
</gbrowseImage1>|
GSID = <gbrowseImage2>
name=NC_008396:17630235..17633682
source=RiceChromosome03
preset=GeneLocation
</gbrowseImage2>|
CDNA = <cdnaseq>atggcgcctatggccatctccaccccgctcgccctccgcgcctccccgacccgcctcctctcccgcaggcggagcggagccaaatcaggcgtggctctcccaggtccacaatttgtaccacctggtatttcttcaaagttggacgagaggatacattgtcattcttctctgaggaaaaatacaattgtagcatcagagaatgaaaatccacctttaatgcctgccataatgactcctgctggtgctcttgatctggcaactgtattgttggggaaccgcattatcttcattggtcaatatattaactcgcaagtagcacagcgtgtaatatcacagcttgtcacacttgctgctgttgatgaagaggctgatattctgatctacctgaactgccccggcggaagtctctactccatcttagcaatttatgattgcatgtcctggatcaagcccaaagttggaacagtgtgctttggtgttgttgctagccaggcagcaattatacttgctggcggtgagaagggaatgcgttatgccatgccaaatgctagagtaatgattcatcaacctcaaggtgtatcagagggtaatgtggaggaggtgaggcgacaggttggggaaaccatttatgctcgtgataaagttgataagatgtttgctgcttttactgggcaaaccttggatatggtacaacagtggacagagagggatcgtttcatgtcttcatctgaagccatggactttggactagttgatgccctgctggaaacaagatactaa</cdnaseq>|
AA = <aaseq>MAPMAISTPLALRASPTRLLSRRRSGAKSGVALPGPQFVPPGIS SKLDERIHCHSSLRKNTIVASENENPPLMPAIMTPAGALDLATVLLGNRIIFIGQYIN SQVAQRVISQLVTLAAVDEEADILIYLNCPGGSLYSILAIYDCMSWIKPKVGTVCFGV VASQAAIILAGGEKGMRYAMPNARVMIHQPQGVSEGNVEEVRRQVGETIYARDKVDKM FAAFTGQTLDMVQQWTERDRFMSSSEAMDFGLVDALLETRY</aaseq>|
DNA = <dnaseqindica>56..123#244..278#966..1035#1176..1380#1542..1607#2217..2357#2569..2622#2758..2850#2949..2996#actcctcagtcctcgcctcggctcggctccctcccacgctccagctccgcctccaatggcgcctatggccatctccaccccgctcgccctccgcgcctccccgacccgcctcctctcccgcaggtgagctccacggaactacaacttccacctccttcgctcgctcgctcgccccgcgcttctctcttcatggattcccccgttcttgtcccctcaccctctgtctggtccttctttcttcaggcggagcggagccaaatcaggcgtggctctcccaggtgagatttcctaacccttggtttagcaaaccatttcccttggtcagctcgttaggccaggactgtttggtggaatttgatcgttgatttgataagctttactgagatgttgtccatggtggtgcacatattagaacatgaccatttgggcagccgtcccatcagctcctagttgtctttctctgtgccaattttttatggagtcgtcattggtaggttttgcaaagcatatagaacctctgaatgtcggcattatccaagagtatgccgacctggggttatctgaaccagtgtcaactccttcctgggccaatgtctggtatgcatcagcattagctcactaagtacacgaaaaatggatgtgcttggttgaacggatatgtataaaaacgataacctgcatataacatatcacctcagtttggtctgattctgaaattagtttagggccttttagcaaactgctgaatgagatttccagactgtatatgtgttattgtgtttgtcagtaactcagtatggtgtattagcacaactcaacatgccataatgacaggatatgcggagcacaaacttttctttggacatgttttttggattcctttactgtttagtccatctgtctttcacatgatatattgctgcaaatgtgctgagttgcattctcactcaaatttccacacctaggtccacaatttgtaccacctggtatttcttcaaagttggacgagaggatacattgtcattcttctctgaggtgatatatttgaaactgtcatgcctgatatacaatgaggatacattgcctgatttgaaactgctcatctaggttttatttgtgctgtgtatcagaatcctgttttattcattcgtaatattgtaaatattttttcacaggaaaaatacaattgtagcatcagagaatgaaaatccacctttaatgcctgccataatgactcctgctggtgctcttgatctggcaactgtattgttggggaaccgcattatcttcattggtcaatatattaactcgcaagtagcacagcgtgtaatatcacagcttgtcacacttgctgctgttgatgaagaggctgatattctggttagtgtttatttttgtgtttttcagatcataacagttaccctattgttcactgcagcagctcttgattgctcaacttcactcccttggcttgctcctttagctcacaggtgtgttgctctatatcttataactccttttgtataattctgttttgccagatctacctgaactgccccggcggaagtctctactccatcttagcaatttatgattgcatgtcctgggtatgccatctatgttgagctactttttccatgtccttcatgcattcaaatttcagagattgtattcacctatatttattcttgtggatgctttctgagttattctcatctaatttaatatttacattgttggatgagaacacattatagatgcatctcaacattttgtatcttccacattatgcatgcccctagctagtgaatttatatttataatatagcacagatatagcatttacaggaaagcctaatgtaatttaggcaaaaattatatctcatatcaatggtagtgcttgcaacatttgtattcttatatttttattgtagtacatactagacatgagcatttgccatgctgagactgtgctaattgggtttggtctggtactgcagacaacagatctcatttcctgaaatcatgcctgtctctaaaactggctttgagctggaccagccttgttagttgttagatttggctgtgtttttacttgttatgccagttttccataaccaaatactttatctacaatttcgcctactgataaataaatcccagaatattaatctttttttgttgttctgcactaacacatgaaccatttattgcagatcaagcccaaagttggaacagtgtgctttggtgttgttgctagccaggcagcaattatacttgctggcggtgagaagggaatgcgttatgccatgccaaatgctagagtaatgattcatcaacctcaaggtgtatcagaggtatgattctggggctttctgcctttctgagttactgcagcgggtgcattatgattttctaacattgtgactacagtaaataataatcatcatcattttagctggccacatgaaacttacaatatacagtcttgctaggacatacttgcttgcctttgtgtttgttgtacttgaagtttgttttttattctaaaaattggatgatttgcagggtaatgtggaggaggtgaggcgacaggttggggaaaccatttatgctcgtgatgtaagtgttttgtgatagataacaagttctatattttcttcagtgtacatttgaattagatgtttgatgagggtaaggaatttctcttgattctgctctctaagcgattaaagcttctgaaaaatctggatgcagaaagttgataagatgtttgctgcttttactgggcaaaccttggatatggtacaacagtggacagagagggatcgtttcatgtcttcatctgaagtaactttcatctcttaaatgtatcagaagaaagtaaatcatcatttgccatgtgaattataacttatttcccccttctttttttggttttaccctaggccatggactttggactagttgatgccctgctggaaacaagatactaacaaacaaacacttaggcacagtttgattagcagaggctggtacaaggattgtgaaactggagctgaagttgagattttcgccgtccttctagttcaaggactccaatgacaggaggctggatctgcgagacttgaatgcatcgccatcgctcctatgagcaaaacatctctgcgttgagtgtgattttttgctcttcttttttggatctggttttacatgccgccagcctatggtctcaatgcattggtcctgctaatgtttagtgtagaccagatgatcctttagacagcaaaacatcagttattactccgtagattttcccatgagctgattccagactgtgtgcaacttttgtgttccaattgcaaagtttgcaacccaacccaacccaacacatgggctgatgggctgttgacaaaggagagattttacacttcacatatgtcaaact</dnaseqindica>|
Link = [http://www.ncbi.nlm.nih.gov/nuccore/NM_001056884.1 RefSeq:Os03g0411500]|
}}
[[Category:Genes]]
[[Category:Japonica mRNA]]
[[Category:Oryza Sativa Japonica Group]]
[[Category:Japonica Genes]]
[[Category:Japonica Chromosome 3]]
[[Category:Chromosome 3]]

Os03g0411500

2014-06-08T08:19:55Z

Xiaoyanjie2012: /* 5.The vyl Locus Maps to a Putative Gene Encoding OsClpP6 */

A nuclear gene coding caseinolyse positioned in plastid or mitochondrion regulating early morphogenesis.
VYL is a gene in rice.Its RAP ID is Os03g0411500 and its MSU ID is LOC_Os03g29810. The mutant of this gene produces chlorotic leaves throughout the entire growth period.
==Annotated Information==
===Function===
The protein encoded by this gene belongs to the peptidase family S14 and hydrolyzes proteins into small peptides in the presence of ATP and magnesium. The protein is transported into mitochondrial matrix and is associated with the inner mitochondrial membrane<ref name="ref2" />, or plastid inner membrane in plants<ref name="ref4" />.
The plastidic caseinolytic protease (Clp) of higher plants is an evolutionarily conserved protein degradation apparatus composed of a proteolytic core complex (the P and R rings) and a set of accessory proteins (ClpT, ClpC, and ClpS).<ref name="ref4" />
Rice yellow leaf mutant vyl, the performance of the entire growth period, new leaves chlorotic phenotype, then gradually turn green from the top down.<ref name="ref4" /> VYL Arabidopsis Clp protease subunit ClpP6 homologous protein in rice, is one of the subunits of the chloroplast Clp protease, with the Clp protease subunit interactions OsClpP3 and OsClpP4 respectively, play an important role in the biosynthesis of rice chloroplast.<ref name="ref4" />
What's more, the gene D53 product shares predicted features with the class I Clp ATPase proteins and can form a complex with the a/b hydrolase protein DWARF 14 (D14) and the F-box protein DWARF 3 (D3), two previously identified signalling components potentially responsible for SL(Strigolactones) perception, which means, in a D14- and D3-dependentmanner, SLs induce D53 degradation by the proteasome and abrogate its activity in promoting axillary bud outgrowth.<ref name="ref5" />
The role and molecular composition of Clps in higher plants has just begun to be unraveled, mostly from studies with the model dicotyledonous plant Arabidopsis (Arabidopsis thaliana).Some researchers isolated a virescent yellow leaf (vyl) mutant in rice (Oryza sativa), which produces chlorotic leaves throughout the entire growth period.<ref name="ref4" />
====1.The vyl Mutant Displays Reduced Chlorophyll Accumulation====
The vyl mutant was derived by transforming tissue cultures of the japonica rice variety Kita-ake. When grown under an alternating light/dark cycle (12 h of light at 30°C/12 h of darkness at 20°C) in a growth chamber, vyl mutant plants displayed a virescent yellow leaf pheno-type, and during development, leaves gradually turned green from their tips (more developed) to their bases (less developed; Fig. 1, A and B). At maturity, the vyl mutant plants also had reduced height and smaller seeds (Fig. 1, C and D). Mutant leaves also contained less chlorophyll than the wild type at various growth stages (Fig. 1E).
Additionally, vyl mutants developed chlorotic leaves under different temperature conditions and light/dark cycles, suggesting that the virescent yellow phenotype of the vyl mutant was developmentally regulated but independent of external cues (such as temperature and light)
[[File:figure 1.png]]

====2.The vyl Mutant Has Impaired Chloroplast Development====
Next, researchers investigated whether the virescent yellow phenotype of the vyl mutant was associated with ultra-structural changes in the chloroplasts. Leaf samples of L3U (upper half of the third leaf), L3L (basal half of the third leaf), and L4 (fourth leaf above the shoot base) were collected from wild-type and vyl mutant seedlings and compared (Fig. 2A). Normally, when the third leaf has fully emerged from the shoot base of a rice plant, the shoot also contains the fourth to the seventh immature leaves. The leaf cells in the L3L and L3U samples contain mature chloroplasts, whereas those in the shoot base and L4 samples contain proplastids and early developing immature chloroplasts (Sugimoto et al., 2004). Similar to the wild type, the chloroplasts from the L3U green leaf sample (already turned green) of vyl mutant seedlings displayed well-developed lamellar structures and were equipped with normally stacked grana and thylakoid membranes (Fig. 2, B and C). By contrast, the chloroplasts from the L3L and L4 pale leaves (still wrapped in leaf sheath) of the vyl mutant had much reduced thylakoid membrane networks compared with wild-type plants (Fig. 2, D–G). This developmental defect is similar to the phenotype reported in the Arabidopsis CLPP6 antisense transgenic plants (Sjögren et al., 2006).

[[File:Figure 2 .png]]

====3.The vyl Mutant Has Impaired Photosynthesis====
Chloroplasts are the organelles in plant cells that perform photosynthesis; therefore, they play an essential role in plant growth. To test whether the photosynthetic apparatus was affected in vyl mutants, we compared some key parameters of PSI and PSII between vyl and wild-type plants. Distinct differences in photochemical efficiency of PSII (FPSII), electron transport rate (ETR), nonphotochemical quenching (NPQ), and photochemical
quenching (Qp) were detected between vyl mutants and the wild type. In contrast, the maximal efficiency of PSII photochemistry (F v /F m ) values was almost comparable between vyl and wild-type plants (Table I). These observations indicate that vyl mutants absorbed much less light energy, as shown by the lower NPQ values. PSII photochemistry was reduced at both the donor and acceptor sites, as indicated by the greatly decreased Qp and ETR in vyl mutants. Notably, the PSII structure appeared intact in the mutant plants (indicated by the equivalent F v /F m values), but the actual FPSII was much lower in the mutants. These differences may underlie the defects in chloroplast biogenesis and retarded growth in the vyl mutant (Fig. 1, C and D)
[[File:Table_1.png‎]]

====4.Altered Expression of Genes Associated with Chloroplast Biogenesis and Photosynthesis in vyl Mutants====
Chloroplast biogenesis and physiological changes are tightly regulated by the coordinated expression of plastid and nuclear genes during leaf development and facilitated by protein quality control (Kusumi et al., 2010; Clarke, 2012). To examine whether the expression of genes associated with chloroplast biogenesis and photosynthesis was altered in vyl mutants, quantitative real-time reverse transcription (qRT)-PCR was performed on total RNAs extracted from the L4 and
L3U leaf sections of wild-type and vyl plants. Compared with the wild type, the transcript levels of Virescent1 (V1), V2, and V3 genes and some other genes encoding components of the plastid and nuclear transcription apparatus Sigma factor 2A (OsSig2A), Ribosomal Protein S15 (rps15) and the subunit RNA polymerase (RpoA and RpoTp) that are highly expressed in early stages of chloroplast development were significantly increased in vyl mutants. In contrast, several genes encoding components of the photosynthesis apparatus RuBisCO large subunit (RbcL), the subunit of photosystem I (PsaA), a core component of Photosystem II (PsbA), light-harvesting complex protein (Lhcp2) and Chlorophyll A/B binding protein1 (Cab1) that are highly expressed in later stages of chloroplast development were significantly reduced in vyl mutants (Fig. 3). These results suggested that VYL plays an important role in regulating chloroplast biogenesis.

[[File:Figure 3 .png]]

====5.The vyl Locus Maps to a Putative Gene Encoding OsClpP6====
Genetic analysis showed that the virescent yellow leaf phenotype in vyl mutants is controlled by a single recessive nuclear locus, VYL. Using a BC 1 F 2 mapping population of the vyl mutant and 93-11 (an indica variety), we mapped the vyl gene to a 137-kb genomic region on chromosome 3 between the insertion/deletion polymorphism (Indel) markers F17 and I53. Within this region, 17 open reading frames (ORFs) were predicted from published data (http://
www.gramene.org/; Fig. 4A). Genomic sequence analysis revealed that only the 13th ORF (LOC_Os03g29810) carries a single-nucleotide transition (G→T) at the position 1,509 bp from the ATG start codon (Fig. 4B). We obtained the full-length complementary DNA (cDNA) of LOC_Os03g29810 from both the wild type and the vyl mutant by reverse transcription (RT)-PCR. Sequence analysis showed that the full-length VYL cDNA is 780 bp long in the wild type but 842 bp in the mutant.
Comparison of the genomic and cDNA sequences revealed that the single-nucleotide substitution in the vyl mutant genome created a new splicing site and the addition of a partial fourth intron sequence in the cDNA (Fig. 4, C and D). The mutant cDNA is predicted to encode a truncated vyl mutant protein (DVYL) lacking the classic Ser protease triad (Ser-His-Asp) in the active site and the polypeptide-binding site (Fig. 4, C and E). Phylogenetic analysis and protein sequence alignment showed that VYL is most closely related to the Arabidopsis ClpP6 protein with a conserved S14_ClpP_2 domain and more remotely related to several other Arabidopsis ClpP subunits, and it apparently represents a single-copy gene in the rice genome (Fig. 4F).
To verify the identity of VYL, the plasmid pGVYL, containing a 6-kb genomic DNA fragment consisting of a 2.5-kb upstream sequence, the entire VYL coding region, including nine exons and eight introns, and a 0.6-kb downstream sequence, was constructed and introduced into the vyl mutant. All five transgenic lines containing pGVYL complemented the virescent phenotype of the vyl mutant (Fig. 5). To further confirm that disruption of the VYL gene was responsible for the vyl mutant phenotype, we generated RNA interference transgenic plants in the wild-type Kita-ake background and obtained more than eight independent transgenic
lines. qRT-PCR analysis revealed that the expression of VYL was reduced in three tested transgenic lines compared with the wild-type plants. These VYL knockdown transgenic plants showed reduced accumulation of chlorophyll a and chlorophyll b. Together, these results confirmed that LOC_Os03g29810 indeed corresponds to the VYL gene.
[[File:Figure 4 .png]]

===Expression===
Expression is constitutive in most tissues examined (roots, stems, leaves, leaf sheath, panicle)but most abundant in leaf sections containing
chloroplasts in early stages of development,which can be light-mediated.<ref name="ref4" />
The young chlorotic leaves turn green in later developmental stages, accompanied by alterations in chlorophyll accumulation, chloroplast ultrastructure, and the expression of chloroplast development- and photosynthesis-related genes. Positional cloning revealed that the VYL gene encodes a protein homologous to the Arabidopsis ClpP6 subunit and that it is targeted to the chloroplast. VYL expression is constitutive in most tissues examined but most abundant in leaf sections containing chloroplasts in early stages of development. The mutation in vyl causes premature termination of the predicted gene product and loss of the conserved catalytic triad (serine-histidine-aspartate) and the polypeptide-binding site of VYL. Using a tandem affinity purification approach and mass spectrometry analysis, we identified OsClpP4 as a VYL-associated protein in vivo. In addition, yeast two-hybrid assays demonstrated that VYL directly interacts with OsClpP3 and OsClpP4. Furthermore, we found that OsClpP3 directly interacts with OsClpT, that OsClpP4 directly interacts with OsClpP5 and OsClpT, and that both OsClpP4 and OsClpT can homodimerize. Together, our data provide new insights into the function, assembly, and regulation of Clps in higher plants.<ref name="ref4" />

==Evolution==
ATP-dependent Clp protease proteolytic subunit is an enzyme that in humans is encoded by the CLPP gene.<ref name="ref1" /><ref name="ref2" /> It is found in mitochondria and is widely distributed in bacterial species.
In several bacteria, such as E. coli, proteins tagged with the SsrA peptide (ANDENYALAA) encoded by tmRNA are digested by Clp proteases.<ref name="ref3" />

[[File:612px-Protein_CLPP_PDB_1tg6.png]][[File:1867.png]]

==Labs working on this gene==
National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China

==References==
<references>
<ref name="ref1">
Bross P, Andresen BS, Knudsen I, Kruse TA, Gregersen N (Feb 1996). "Human ClpP protease: cDNA sequence, tissue-specific expression and chromosomal assignment of the gene". FEBS Lett 377 (2): 249–52. doi:10.1016/0014-5793(95)01353-9. PMID 8543061.
</ref>
<ref name="ref2">
a b "Entrez Gene: CLPP ClpP caseinolytic peptidase, ATP-dependent, proteolytic subunit homolog (E. coli)".
</ref>
<ref name="ref3">
Gottesman S, Roche E, Zhou Y, Sauer RT (1998). "The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system". Genes Dev 12 (9): 1338–47. doi:10.1101/gad.12.9.1338. PMC 316764. PMID 9573050
</ref>
<ref name="ref4">
Hui Dong et al. A Rice Virescent-Yellow Leaf Mutant Reveals New Insights into the Role and Assembly of Plastid Caseinolytic Protease in Higher Plants. Plant Physiology, 2013, 162(4): 1867-1880
</ref>
<ref name="ref5">
Zhou Feng et al.D14–SCFD3-dependent degradation of D53 regulates strigolactone signaling. Nature,2013. doi:10.1038/nature12878
</ref>
</references>
==Structured Information==
{{JaponicaGene|
GeneName = Os03g0411500|
Description = Peptidase S14, ClpP family protein|
Version = NM_001056884.1 GI:115453496 GeneID:4333096|
Length = 3448 bp|
Definition = Oryza sativa Japonica Group Os03g0411500, complete gene.|
Source = Oryza sativa Japonica Group

ORGANISM Oryza sativa Japonica Group
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP
clade; Ehrhartoideae; Oryzeae; Oryza.
|
Chromosome = [[:category:Japonica Chromosome 3|Chromosome 3]]|
AP = Chromosome 3:17630235..17633682|
CDS = 17630290..17630357,17630478..17630512,17631200..17631269,17631410..17631614,17631776..17631841 ,17632451..17632591,17632803..17632856,17632992..17633084,17633183..17633230 |
GCID = <gbrowseImage1>
name=NC_008396:17630235..17633682
source=RiceChromosome03
preset=GeneLocation
</gbrowseImage1>|
GSID = <gbrowseImage2>
name=NC_008396:17630235..17633682
source=RiceChromosome03
preset=GeneLocation
</gbrowseImage2>|
CDNA = <cdnaseq>atggcgcctatggccatctccaccccgctcgccctccgcgcctccccgacccgcctcctctcccgcaggcggagcggagccaaatcaggcgtggctctcccaggtccacaatttgtaccacctggtatttcttcaaagttggacgagaggatacattgtcattcttctctgaggaaaaatacaattgtagcatcagagaatgaaaatccacctttaatgcctgccataatgactcctgctggtgctcttgatctggcaactgtattgttggggaaccgcattatcttcattggtcaatatattaactcgcaagtagcacagcgtgtaatatcacagcttgtcacacttgctgctgttgatgaagaggctgatattctgatctacctgaactgccccggcggaagtctctactccatcttagcaatttatgattgcatgtcctggatcaagcccaaagttggaacagtgtgctttggtgttgttgctagccaggcagcaattatacttgctggcggtgagaagggaatgcgttatgccatgccaaatgctagagtaatgattcatcaacctcaaggtgtatcagagggtaatgtggaggaggtgaggcgacaggttggggaaaccatttatgctcgtgataaagttgataagatgtttgctgcttttactgggcaaaccttggatatggtacaacagtggacagagagggatcgtttcatgtcttcatctgaagccatggactttggactagttgatgccctgctggaaacaagatactaa</cdnaseq>|
AA = <aaseq>MAPMAISTPLALRASPTRLLSRRRSGAKSGVALPGPQFVPPGIS SKLDERIHCHSSLRKNTIVASENENPPLMPAIMTPAGALDLATVLLGNRIIFIGQYIN SQVAQRVISQLVTLAAVDEEADILIYLNCPGGSLYSILAIYDCMSWIKPKVGTVCFGV VASQAAIILAGGEKGMRYAMPNARVMIHQPQGVSEGNVEEVRRQVGETIYARDKVDKM FAAFTGQTLDMVQQWTERDRFMSSSEAMDFGLVDALLETRY</aaseq>|
DNA = <dnaseqindica>56..123#244..278#966..1035#1176..1380#1542..1607#2217..2357#2569..2622#2758..2850#2949..2996#actcctcagtcctcgcctcggctcggctccctcccacgctccagctccgcctccaatggcgcctatggccatctccaccccgctcgccctccgcgcctccccgacccgcctcctctcccgcaggtgagctccacggaactacaacttccacctccttcgctcgctcgctcgccccgcgcttctctcttcatggattcccccgttcttgtcccctcaccctctgtctggtccttctttcttcaggcggagcggagccaaatcaggcgtggctctcccaggtgagatttcctaacccttggtttagcaaaccatttcccttggtcagctcgttaggccaggactgtttggtggaatttgatcgttgatttgataagctttactgagatgttgtccatggtggtgcacatattagaacatgaccatttgggcagccgtcccatcagctcctagttgtctttctctgtgccaattttttatggagtcgtcattggtaggttttgcaaagcatatagaacctctgaatgtcggcattatccaagagtatgccgacctggggttatctgaaccagtgtcaactccttcctgggccaatgtctggtatgcatcagcattagctcactaagtacacgaaaaatggatgtgcttggttgaacggatatgtataaaaacgataacctgcatataacatatcacctcagtttggtctgattctgaaattagtttagggccttttagcaaactgctgaatgagatttccagactgtatatgtgttattgtgtttgtcagtaactcagtatggtgtattagcacaactcaacatgccataatgacaggatatgcggagcacaaacttttctttggacatgttttttggattcctttactgtttagtccatctgtctttcacatgatatattgctgcaaatgtgctgagttgcattctcactcaaatttccacacctaggtccacaatttgtaccacctggtatttcttcaaagttggacgagaggatacattgtcattcttctctgaggtgatatatttgaaactgtcatgcctgatatacaatgaggatacattgcctgatttgaaactgctcatctaggttttatttgtgctgtgtatcagaatcctgttttattcattcgtaatattgtaaatattttttcacaggaaaaatacaattgtagcatcagagaatgaaaatccacctttaatgcctgccataatgactcctgctggtgctcttgatctggcaactgtattgttggggaaccgcattatcttcattggtcaatatattaactcgcaagtagcacagcgtgtaatatcacagcttgtcacacttgctgctgttgatgaagaggctgatattctggttagtgtttatttttgtgtttttcagatcataacagttaccctattgttcactgcagcagctcttgattgctcaacttcactcccttggcttgctcctttagctcacaggtgtgttgctctatatcttataactccttttgtataattctgttttgccagatctacctgaactgccccggcggaagtctctactccatcttagcaatttatgattgcatgtcctgggtatgccatctatgttgagctactttttccatgtccttcatgcattcaaatttcagagattgtattcacctatatttattcttgtggatgctttctgagttattctcatctaatttaatatttacattgttggatgagaacacattatagatgcatctcaacattttgtatcttccacattatgcatgcccctagctagtgaatttatatttataatatagcacagatatagcatttacaggaaagcctaatgtaatttaggcaaaaattatatctcatatcaatggtagtgcttgcaacatttgtattcttatatttttattgtagtacatactagacatgagcatttgccatgctgagactgtgctaattgggtttggtctggtactgcagacaacagatctcatttcctgaaatcatgcctgtctctaaaactggctttgagctggaccagccttgttagttgttagatttggctgtgtttttacttgttatgccagttttccataaccaaatactttatctacaatttcgcctactgataaataaatcccagaatattaatctttttttgttgttctgcactaacacatgaaccatttattgcagatcaagcccaaagttggaacagtgtgctttggtgttgttgctagccaggcagcaattatacttgctggcggtgagaagggaatgcgttatgccatgccaaatgctagagtaatgattcatcaacctcaaggtgtatcagaggtatgattctggggctttctgcctttctgagttactgcagcgggtgcattatgattttctaacattgtgactacagtaaataataatcatcatcattttagctggccacatgaaacttacaatatacagtcttgctaggacatacttgcttgcctttgtgtttgttgtacttgaagtttgttttttattctaaaaattggatgatttgcagggtaatgtggaggaggtgaggcgacaggttggggaaaccatttatgctcgtgatgtaagtgttttgtgatagataacaagttctatattttcttcagtgtacatttgaattagatgtttgatgagggtaaggaatttctcttgattctgctctctaagcgattaaagcttctgaaaaatctggatgcagaaagttgataagatgtttgctgcttttactgggcaaaccttggatatggtacaacagtggacagagagggatcgtttcatgtcttcatctgaagtaactttcatctcttaaatgtatcagaagaaagtaaatcatcatttgccatgtgaattataacttatttcccccttctttttttggttttaccctaggccatggactttggactagttgatgccctgctggaaacaagatactaacaaacaaacacttaggcacagtttgattagcagaggctggtacaaggattgtgaaactggagctgaagttgagattttcgccgtccttctagttcaaggactccaatgacaggaggctggatctgcgagacttgaatgcatcgccatcgctcctatgagcaaaacatctctgcgttgagtgtgattttttgctcttcttttttggatctggttttacatgccgccagcctatggtctcaatgcattggtcctgctaatgtttagtgtagaccagatgatcctttagacagcaaaacatcagttattactccgtagattttcccatgagctgattccagactgtgtgcaacttttgtgttccaattgcaaagtttgcaacccaacccaacccaacacatgggctgatgggctgttgacaaaggagagattttacacttcacatatgtcaaact</dnaseqindica>|
Link = [http://www.ncbi.nlm.nih.gov/nuccore/NM_001056884.1 RefSeq:Os03g0411500]|
}}
[[Category:Genes]]
[[Category:Japonica mRNA]]
[[Category:Oryza Sativa Japonica Group]]
[[Category:Japonica Genes]]
[[Category:Japonica Chromosome 3]]
[[Category:Chromosome 3]]

File:Figure 4 .png

2014-06-08T08:19:24Z

Xiaoyanjie2012:

Os03g0411500

2014-06-08T08:18:20Z

Xiaoyanjie2012: /* 4.Altered Expression of Genes Associated with Chloroplast Biogenesis and Photosynthesis in vyl Mutants */

A nuclear gene coding caseinolyse positioned in plastid or mitochondrion regulating early morphogenesis.
VYL is a gene in rice.Its RAP ID is Os03g0411500 and its MSU ID is LOC_Os03g29810. The mutant of this gene produces chlorotic leaves throughout the entire growth period.
==Annotated Information==
===Function===
The protein encoded by this gene belongs to the peptidase family S14 and hydrolyzes proteins into small peptides in the presence of ATP and magnesium. The protein is transported into mitochondrial matrix and is associated with the inner mitochondrial membrane<ref name="ref2" />, or plastid inner membrane in plants<ref name="ref4" />.
The plastidic caseinolytic protease (Clp) of higher plants is an evolutionarily conserved protein degradation apparatus composed of a proteolytic core complex (the P and R rings) and a set of accessory proteins (ClpT, ClpC, and ClpS).<ref name="ref4" />
Rice yellow leaf mutant vyl, the performance of the entire growth period, new leaves chlorotic phenotype, then gradually turn green from the top down.<ref name="ref4" /> VYL Arabidopsis Clp protease subunit ClpP6 homologous protein in rice, is one of the subunits of the chloroplast Clp protease, with the Clp protease subunit interactions OsClpP3 and OsClpP4 respectively, play an important role in the biosynthesis of rice chloroplast.<ref name="ref4" />
What's more, the gene D53 product shares predicted features with the class I Clp ATPase proteins and can form a complex with the a/b hydrolase protein DWARF 14 (D14) and the F-box protein DWARF 3 (D3), two previously identified signalling components potentially responsible for SL(Strigolactones) perception, which means, in a D14- and D3-dependentmanner, SLs induce D53 degradation by the proteasome and abrogate its activity in promoting axillary bud outgrowth.<ref name="ref5" />
The role and molecular composition of Clps in higher plants has just begun to be unraveled, mostly from studies with the model dicotyledonous plant Arabidopsis (Arabidopsis thaliana).Some researchers isolated a virescent yellow leaf (vyl) mutant in rice (Oryza sativa), which produces chlorotic leaves throughout the entire growth period.<ref name="ref4" />
====1.The vyl Mutant Displays Reduced Chlorophyll Accumulation====
The vyl mutant was derived by transforming tissue cultures of the japonica rice variety Kita-ake. When grown under an alternating light/dark cycle (12 h of light at 30°C/12 h of darkness at 20°C) in a growth chamber, vyl mutant plants displayed a virescent yellow leaf pheno-type, and during development, leaves gradually turned green from their tips (more developed) to their bases (less developed; Fig. 1, A and B). At maturity, the vyl mutant plants also had reduced height and smaller seeds (Fig. 1, C and D). Mutant leaves also contained less chlorophyll than the wild type at various growth stages (Fig. 1E).
Additionally, vyl mutants developed chlorotic leaves under different temperature conditions and light/dark cycles, suggesting that the virescent yellow phenotype of the vyl mutant was developmentally regulated but independent of external cues (such as temperature and light)
[[File:figure 1.png]]

====2.The vyl Mutant Has Impaired Chloroplast Development====
Next, researchers investigated whether the virescent yellow phenotype of the vyl mutant was associated with ultra-structural changes in the chloroplasts. Leaf samples of L3U (upper half of the third leaf), L3L (basal half of the third leaf), and L4 (fourth leaf above the shoot base) were collected from wild-type and vyl mutant seedlings and compared (Fig. 2A). Normally, when the third leaf has fully emerged from the shoot base of a rice plant, the shoot also contains the fourth to the seventh immature leaves. The leaf cells in the L3L and L3U samples contain mature chloroplasts, whereas those in the shoot base and L4 samples contain proplastids and early developing immature chloroplasts (Sugimoto et al., 2004). Similar to the wild type, the chloroplasts from the L3U green leaf sample (already turned green) of vyl mutant seedlings displayed well-developed lamellar structures and were equipped with normally stacked grana and thylakoid membranes (Fig. 2, B and C). By contrast, the chloroplasts from the L3L and L4 pale leaves (still wrapped in leaf sheath) of the vyl mutant had much reduced thylakoid membrane networks compared with wild-type plants (Fig. 2, D–G). This developmental defect is similar to the phenotype reported in the Arabidopsis CLPP6 antisense transgenic plants (Sjögren et al., 2006).

[[File:Figure 2 .png]]

====3.The vyl Mutant Has Impaired Photosynthesis====
Chloroplasts are the organelles in plant cells that perform photosynthesis; therefore, they play an essential role in plant growth. To test whether the photosynthetic apparatus was affected in vyl mutants, we compared some key parameters of PSI and PSII between vyl and wild-type plants. Distinct differences in photochemical efficiency of PSII (FPSII), electron transport rate (ETR), nonphotochemical quenching (NPQ), and photochemical
quenching (Qp) were detected between vyl mutants and the wild type. In contrast, the maximal efficiency of PSII photochemistry (F v /F m ) values was almost comparable between vyl and wild-type plants (Table I). These observations indicate that vyl mutants absorbed much less light energy, as shown by the lower NPQ values. PSII photochemistry was reduced at both the donor and acceptor sites, as indicated by the greatly decreased Qp and ETR in vyl mutants. Notably, the PSII structure appeared intact in the mutant plants (indicated by the equivalent F v /F m values), but the actual FPSII was much lower in the mutants. These differences may underlie the defects in chloroplast biogenesis and retarded growth in the vyl mutant (Fig. 1, C and D)
[[File:Table_1.png‎]]

====4.Altered Expression of Genes Associated with Chloroplast Biogenesis and Photosynthesis in vyl Mutants====
Chloroplast biogenesis and physiological changes are tightly regulated by the coordinated expression of plastid and nuclear genes during leaf development and facilitated by protein quality control (Kusumi et al., 2010; Clarke, 2012). To examine whether the expression of genes associated with chloroplast biogenesis and photosynthesis was altered in vyl mutants, quantitative real-time reverse transcription (qRT)-PCR was performed on total RNAs extracted from the L4 and
L3U leaf sections of wild-type and vyl plants. Compared with the wild type, the transcript levels of Virescent1 (V1), V2, and V3 genes and some other genes encoding components of the plastid and nuclear transcription apparatus Sigma factor 2A (OsSig2A), Ribosomal Protein S15 (rps15) and the subunit RNA polymerase (RpoA and RpoTp) that are highly expressed in early stages of chloroplast development were significantly increased in vyl mutants. In contrast, several genes encoding components of the photosynthesis apparatus RuBisCO large subunit (RbcL), the subunit of photosystem I (PsaA), a core component of Photosystem II (PsbA), light-harvesting complex protein (Lhcp2) and Chlorophyll A/B binding protein1 (Cab1) that are highly expressed in later stages of chloroplast development were significantly reduced in vyl mutants (Fig. 3). These results suggested that VYL plays an important role in regulating chloroplast biogenesis.

[[File:Figure 3 .png]]

====5.The vyl Locus Maps to a Putative Gene Encoding OsClpP6====
Genetic analysis showed that the virescent yellow leaf phenotype in vyl mutants is controlled by a single recessive nuclear locus, VYL. Using a BC 1 F 2 mapping population of the vyl mutant and 93-11 (an indica variety), we mapped the vyl gene to a 137-kb genomic region on chromosome 3 between the insertion/deletion polymorphism (Indel) markers F17 and I53. Within this region, 17 open reading frames (ORFs) were predicted from published data (http://
www.gramene.org/; Fig. 4A). Genomic sequence analysis revealed that only the 13th ORF (LOC_Os03g29810) carries a single-nucleotide transition (G→T) at the position 1,509 bp from the ATG start codon (Fig. 4B). We obtained the full-length complementary DNA (cDNA) of LOC_Os03g29810 from both the wild type and the vyl mutant by reverse transcription (RT)-PCR. Sequence analysis showed that the full-length VYL cDNA is 780 bp long in the wild type but 842 bp in the mutant.
Comparison of the genomic and cDNA sequences revealed that the single-nucleotide substitution in the vyl mutant genome created a new splicing site and the addition of a partial fourth intron sequence in the cDNA (Fig. 4, C and D). The mutant cDNA is predicted to encode a truncated vyl mutant protein (DVYL) lacking the classic Ser protease triad (Ser-His-Asp) in the active site and the polypeptide-binding site (Fig. 4, C and E). Phylogenetic analysis and protein sequence alignment showed that VYL is most closely related to the Arabidopsis ClpP6 protein with a conserved S14_ClpP_2 domain and more remotely related to several other Arabidopsis ClpP subunits, and it apparently represents a single-copy gene in the rice genome (Fig. 4F).
To verify the identity of VYL, the plasmid pGVYL, containing a 6-kb genomic DNA fragment consisting of a 2.5-kb upstream sequence, the entire VYL coding region, including nine exons and eight introns, and a 0.6-kb downstream sequence, was constructed and introduced into the vyl mutant. All five transgenic lines containing pGVYL complemented the virescent phenotype of the vyl mutant (Fig. 5). To further confirm that disruption of the VYL gene was responsible for the vyl mutant phenotype, we generated RNA interference transgenic plants in the wild-type Kita-ake background and obtained more than eight independent transgenic
lines. qRT-PCR analysis revealed that the expression of VYL was reduced in three tested transgenic lines compared with the wild-type plants. These VYL knockdown transgenic plants showed reduced accumulation of chlorophyll a and chlorophyll b. Together, these results confirmed that LOC_Os03g29810 indeed corresponds to the VYL gene.

===Expression===
Expression is constitutive in most tissues examined (roots, stems, leaves, leaf sheath, panicle)but most abundant in leaf sections containing
chloroplasts in early stages of development,which can be light-mediated.<ref name="ref4" />
The young chlorotic leaves turn green in later developmental stages, accompanied by alterations in chlorophyll accumulation, chloroplast ultrastructure, and the expression of chloroplast development- and photosynthesis-related genes. Positional cloning revealed that the VYL gene encodes a protein homologous to the Arabidopsis ClpP6 subunit and that it is targeted to the chloroplast. VYL expression is constitutive in most tissues examined but most abundant in leaf sections containing chloroplasts in early stages of development. The mutation in vyl causes premature termination of the predicted gene product and loss of the conserved catalytic triad (serine-histidine-aspartate) and the polypeptide-binding site of VYL. Using a tandem affinity purification approach and mass spectrometry analysis, we identified OsClpP4 as a VYL-associated protein in vivo. In addition, yeast two-hybrid assays demonstrated that VYL directly interacts with OsClpP3 and OsClpP4. Furthermore, we found that OsClpP3 directly interacts with OsClpT, that OsClpP4 directly interacts with OsClpP5 and OsClpT, and that both OsClpP4 and OsClpT can homodimerize. Together, our data provide new insights into the function, assembly, and regulation of Clps in higher plants.<ref name="ref4" />

==Evolution==
ATP-dependent Clp protease proteolytic subunit is an enzyme that in humans is encoded by the CLPP gene.<ref name="ref1" /><ref name="ref2" /> It is found in mitochondria and is widely distributed in bacterial species.
In several bacteria, such as E. coli, proteins tagged with the SsrA peptide (ANDENYALAA) encoded by tmRNA are digested by Clp proteases.<ref name="ref3" />

[[File:612px-Protein_CLPP_PDB_1tg6.png]][[File:1867.png]]

==Labs working on this gene==
National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China

==References==
<references>
<ref name="ref1">
Bross P, Andresen BS, Knudsen I, Kruse TA, Gregersen N (Feb 1996). "Human ClpP protease: cDNA sequence, tissue-specific expression and chromosomal assignment of the gene". FEBS Lett 377 (2): 249–52. doi:10.1016/0014-5793(95)01353-9. PMID 8543061.
</ref>
<ref name="ref2">
a b "Entrez Gene: CLPP ClpP caseinolytic peptidase, ATP-dependent, proteolytic subunit homolog (E. coli)".
</ref>
<ref name="ref3">
Gottesman S, Roche E, Zhou Y, Sauer RT (1998). "The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system". Genes Dev 12 (9): 1338–47. doi:10.1101/gad.12.9.1338. PMC 316764. PMID 9573050
</ref>
<ref name="ref4">
Hui Dong et al. A Rice Virescent-Yellow Leaf Mutant Reveals New Insights into the Role and Assembly of Plastid Caseinolytic Protease in Higher Plants. Plant Physiology, 2013, 162(4): 1867-1880
</ref>
<ref name="ref5">
Zhou Feng et al.D14–SCFD3-dependent degradation of D53 regulates strigolactone signaling. Nature,2013. doi:10.1038/nature12878
</ref>
</references>
==Structured Information==
{{JaponicaGene|
GeneName = Os03g0411500|
Description = Peptidase S14, ClpP family protein|
Version = NM_001056884.1 GI:115453496 GeneID:4333096|
Length = 3448 bp|
Definition = Oryza sativa Japonica Group Os03g0411500, complete gene.|
Source = Oryza sativa Japonica Group

ORGANISM Oryza sativa Japonica Group
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP
clade; Ehrhartoideae; Oryzeae; Oryza.
|
Chromosome = [[:category:Japonica Chromosome 3|Chromosome 3]]|
AP = Chromosome 3:17630235..17633682|
CDS = 17630290..17630357,17630478..17630512,17631200..17631269,17631410..17631614,17631776..17631841 ,17632451..17632591,17632803..17632856,17632992..17633084,17633183..17633230 |
GCID = <gbrowseImage1>
name=NC_008396:17630235..17633682
source=RiceChromosome03
preset=GeneLocation
</gbrowseImage1>|
GSID = <gbrowseImage2>
name=NC_008396:17630235..17633682
source=RiceChromosome03
preset=GeneLocation
</gbrowseImage2>|
CDNA = <cdnaseq>atggcgcctatggccatctccaccccgctcgccctccgcgcctccccgacccgcctcctctcccgcaggcggagcggagccaaatcaggcgtggctctcccaggtccacaatttgtaccacctggtatttcttcaaagttggacgagaggatacattgtcattcttctctgaggaaaaatacaattgtagcatcagagaatgaaaatccacctttaatgcctgccataatgactcctgctggtgctcttgatctggcaactgtattgttggggaaccgcattatcttcattggtcaatatattaactcgcaagtagcacagcgtgtaatatcacagcttgtcacacttgctgctgttgatgaagaggctgatattctgatctacctgaactgccccggcggaagtctctactccatcttagcaatttatgattgcatgtcctggatcaagcccaaagttggaacagtgtgctttggtgttgttgctagccaggcagcaattatacttgctggcggtgagaagggaatgcgttatgccatgccaaatgctagagtaatgattcatcaacctcaaggtgtatcagagggtaatgtggaggaggtgaggcgacaggttggggaaaccatttatgctcgtgataaagttgataagatgtttgctgcttttactgggcaaaccttggatatggtacaacagtggacagagagggatcgtttcatgtcttcatctgaagccatggactttggactagttgatgccctgctggaaacaagatactaa</cdnaseq>|
AA = <aaseq>MAPMAISTPLALRASPTRLLSRRRSGAKSGVALPGPQFVPPGIS SKLDERIHCHSSLRKNTIVASENENPPLMPAIMTPAGALDLATVLLGNRIIFIGQYIN SQVAQRVISQLVTLAAVDEEADILIYLNCPGGSLYSILAIYDCMSWIKPKVGTVCFGV VASQAAIILAGGEKGMRYAMPNARVMIHQPQGVSEGNVEEVRRQVGETIYARDKVDKM FAAFTGQTLDMVQQWTERDRFMSSSEAMDFGLVDALLETRY</aaseq>|
DNA = <dnaseqindica>56..123#244..278#966..1035#1176..1380#1542..1607#2217..2357#2569..2622#2758..2850#2949..2996#actcctcagtcctcgcctcggctcggctccctcccacgctccagctccgcctccaatggcgcctatggccatctccaccccgctcgccctccgcgcctccccgacccgcctcctctcccgcaggtgagctccacggaactacaacttccacctccttcgctcgctcgctcgccccgcgcttctctcttcatggattcccccgttcttgtcccctcaccctctgtctggtccttctttcttcaggcggagcggagccaaatcaggcgtggctctcccaggtgagatttcctaacccttggtttagcaaaccatttcccttggtcagctcgttaggccaggactgtttggtggaatttgatcgttgatttgataagctttactgagatgttgtccatggtggtgcacatattagaacatgaccatttgggcagccgtcccatcagctcctagttgtctttctctgtgccaattttttatggagtcgtcattggtaggttttgcaaagcatatagaacctctgaatgtcggcattatccaagagtatgccgacctggggttatctgaaccagtgtcaactccttcctgggccaatgtctggtatgcatcagcattagctcactaagtacacgaaaaatggatgtgcttggttgaacggatatgtataaaaacgataacctgcatataacatatcacctcagtttggtctgattctgaaattagtttagggccttttagcaaactgctgaatgagatttccagactgtatatgtgttattgtgtttgtcagtaactcagtatggtgtattagcacaactcaacatgccataatgacaggatatgcggagcacaaacttttctttggacatgttttttggattcctttactgtttagtccatctgtctttcacatgatatattgctgcaaatgtgctgagttgcattctcactcaaatttccacacctaggtccacaatttgtaccacctggtatttcttcaaagttggacgagaggatacattgtcattcttctctgaggtgatatatttgaaactgtcatgcctgatatacaatgaggatacattgcctgatttgaaactgctcatctaggttttatttgtgctgtgtatcagaatcctgttttattcattcgtaatattgtaaatattttttcacaggaaaaatacaattgtagcatcagagaatgaaaatccacctttaatgcctgccataatgactcctgctggtgctcttgatctggcaactgtattgttggggaaccgcattatcttcattggtcaatatattaactcgcaagtagcacagcgtgtaatatcacagcttgtcacacttgctgctgttgatgaagaggctgatattctggttagtgtttatttttgtgtttttcagatcataacagttaccctattgttcactgcagcagctcttgattgctcaacttcactcccttggcttgctcctttagctcacaggtgtgttgctctatatcttataactccttttgtataattctgttttgccagatctacctgaactgccccggcggaagtctctactccatcttagcaatttatgattgcatgtcctgggtatgccatctatgttgagctactttttccatgtccttcatgcattcaaatttcagagattgtattcacctatatttattcttgtggatgctttctgagttattctcatctaatttaatatttacattgttggatgagaacacattatagatgcatctcaacattttgtatcttccacattatgcatgcccctagctagtgaatttatatttataatatagcacagatatagcatttacaggaaagcctaatgtaatttaggcaaaaattatatctcatatcaatggtagtgcttgcaacatttgtattcttatatttttattgtagtacatactagacatgagcatttgccatgctgagactgtgctaattgggtttggtctggtactgcagacaacagatctcatttcctgaaatcatgcctgtctctaaaactggctttgagctggaccagccttgttagttgttagatttggctgtgtttttacttgttatgccagttttccataaccaaatactttatctacaatttcgcctactgataaataaatcccagaatattaatctttttttgttgttctgcactaacacatgaaccatttattgcagatcaagcccaaagttggaacagtgtgctttggtgttgttgctagccaggcagcaattatacttgctggcggtgagaagggaatgcgttatgccatgccaaatgctagagtaatgattcatcaacctcaaggtgtatcagaggtatgattctggggctttctgcctttctgagttactgcagcgggtgcattatgattttctaacattgtgactacagtaaataataatcatcatcattttagctggccacatgaaacttacaatatacagtcttgctaggacatacttgcttgcctttgtgtttgttgtacttgaagtttgttttttattctaaaaattggatgatttgcagggtaatgtggaggaggtgaggcgacaggttggggaaaccatttatgctcgtgatgtaagtgttttgtgatagataacaagttctatattttcttcagtgtacatttgaattagatgtttgatgagggtaaggaatttctcttgattctgctctctaagcgattaaagcttctgaaaaatctggatgcagaaagttgataagatgtttgctgcttttactgggcaaaccttggatatggtacaacagtggacagagagggatcgtttcatgtcttcatctgaagtaactttcatctcttaaatgtatcagaagaaagtaaatcatcatttgccatgtgaattataacttatttcccccttctttttttggttttaccctaggccatggactttggactagttgatgccctgctggaaacaagatactaacaaacaaacacttaggcacagtttgattagcagaggctggtacaaggattgtgaaactggagctgaagttgagattttcgccgtccttctagttcaaggactccaatgacaggaggctggatctgcgagacttgaatgcatcgccatcgctcctatgagcaaaacatctctgcgttgagtgtgattttttgctcttcttttttggatctggttttacatgccgccagcctatggtctcaatgcattggtcctgctaatgtttagtgtagaccagatgatcctttagacagcaaaacatcagttattactccgtagattttcccatgagctgattccagactgtgtgcaacttttgtgttccaattgcaaagtttgcaacccaacccaacccaacacatgggctgatgggctgttgacaaaggagagattttacacttcacatatgtcaaact</dnaseqindica>|
Link = [http://www.ncbi.nlm.nih.gov/nuccore/NM_001056884.1 RefSeq:Os03g0411500]|
}}
[[Category:Genes]]
[[Category:Japonica mRNA]]
[[Category:Oryza Sativa Japonica Group]]
[[Category:Japonica Genes]]
[[Category:Japonica Chromosome 3]]
[[Category:Chromosome 3]]

File:Figure 3 .png

2014-06-08T08:17:58Z

Xiaoyanjie2012:

Os03g0411500

2014-06-08T08:14:42Z

Xiaoyanjie2012: /* 5.The vyl Locus Maps to a Putative Gene Encoding OsClpP6 */

A nuclear gene coding caseinolyse positioned in plastid or mitochondrion regulating early morphogenesis.
VYL is a gene in rice.Its RAP ID is Os03g0411500 and its MSU ID is LOC_Os03g29810. The mutant of this gene produces chlorotic leaves throughout the entire growth period.
==Annotated Information==
===Function===
The protein encoded by this gene belongs to the peptidase family S14 and hydrolyzes proteins into small peptides in the presence of ATP and magnesium. The protein is transported into mitochondrial matrix and is associated with the inner mitochondrial membrane<ref name="ref2" />, or plastid inner membrane in plants<ref name="ref4" />.
The plastidic caseinolytic protease (Clp) of higher plants is an evolutionarily conserved protein degradation apparatus composed of a proteolytic core complex (the P and R rings) and a set of accessory proteins (ClpT, ClpC, and ClpS).<ref name="ref4" />
Rice yellow leaf mutant vyl, the performance of the entire growth period, new leaves chlorotic phenotype, then gradually turn green from the top down.<ref name="ref4" /> VYL Arabidopsis Clp protease subunit ClpP6 homologous protein in rice, is one of the subunits of the chloroplast Clp protease, with the Clp protease subunit interactions OsClpP3 and OsClpP4 respectively, play an important role in the biosynthesis of rice chloroplast.<ref name="ref4" />
What's more, the gene D53 product shares predicted features with the class I Clp ATPase proteins and can form a complex with the a/b hydrolase protein DWARF 14 (D14) and the F-box protein DWARF 3 (D3), two previously identified signalling components potentially responsible for SL(Strigolactones) perception, which means, in a D14- and D3-dependentmanner, SLs induce D53 degradation by the proteasome and abrogate its activity in promoting axillary bud outgrowth.<ref name="ref5" />
The role and molecular composition of Clps in higher plants has just begun to be unraveled, mostly from studies with the model dicotyledonous plant Arabidopsis (Arabidopsis thaliana).Some researchers isolated a virescent yellow leaf (vyl) mutant in rice (Oryza sativa), which produces chlorotic leaves throughout the entire growth period.<ref name="ref4" />
====1.The vyl Mutant Displays Reduced Chlorophyll Accumulation====
The vyl mutant was derived by transforming tissue cultures of the japonica rice variety Kita-ake. When grown under an alternating light/dark cycle (12 h of light at 30°C/12 h of darkness at 20°C) in a growth chamber, vyl mutant plants displayed a virescent yellow leaf pheno-type, and during development, leaves gradually turned green from their tips (more developed) to their bases (less developed; Fig. 1, A and B). At maturity, the vyl mutant plants also had reduced height and smaller seeds (Fig. 1, C and D). Mutant leaves also contained less chlorophyll than the wild type at various growth stages (Fig. 1E).
Additionally, vyl mutants developed chlorotic leaves under different temperature conditions and light/dark cycles, suggesting that the virescent yellow phenotype of the vyl mutant was developmentally regulated but independent of external cues (such as temperature and light)
[[File:figure 1.png]]

====2.The vyl Mutant Has Impaired Chloroplast Development====
Next, researchers investigated whether the virescent yellow phenotype of the vyl mutant was associated with ultra-structural changes in the chloroplasts. Leaf samples of L3U (upper half of the third leaf), L3L (basal half of the third leaf), and L4 (fourth leaf above the shoot base) were collected from wild-type and vyl mutant seedlings and compared (Fig. 2A). Normally, when the third leaf has fully emerged from the shoot base of a rice plant, the shoot also contains the fourth to the seventh immature leaves. The leaf cells in the L3L and L3U samples contain mature chloroplasts, whereas those in the shoot base and L4 samples contain proplastids and early developing immature chloroplasts (Sugimoto et al., 2004). Similar to the wild type, the chloroplasts from the L3U green leaf sample (already turned green) of vyl mutant seedlings displayed well-developed lamellar structures and were equipped with normally stacked grana and thylakoid membranes (Fig. 2, B and C). By contrast, the chloroplasts from the L3L and L4 pale leaves (still wrapped in leaf sheath) of the vyl mutant had much reduced thylakoid membrane networks compared with wild-type plants (Fig. 2, D–G). This developmental defect is similar to the phenotype reported in the Arabidopsis CLPP6 antisense transgenic plants (Sjögren et al., 2006).

[[File:Figure 2 .png]]

====3.The vyl Mutant Has Impaired Photosynthesis====
Chloroplasts are the organelles in plant cells that perform photosynthesis; therefore, they play an essential role in plant growth. To test whether the photosynthetic apparatus was affected in vyl mutants, we compared some key parameters of PSI and PSII between vyl and wild-type plants. Distinct differences in photochemical efficiency of PSII (FPSII), electron transport rate (ETR), nonphotochemical quenching (NPQ), and photochemical
quenching (Qp) were detected between vyl mutants and the wild type. In contrast, the maximal efficiency of PSII photochemistry (F v /F m ) values was almost comparable between vyl and wild-type plants (Table I). These observations indicate that vyl mutants absorbed much less light energy, as shown by the lower NPQ values. PSII photochemistry was reduced at both the donor and acceptor sites, as indicated by the greatly decreased Qp and ETR in vyl mutants. Notably, the PSII structure appeared intact in the mutant plants (indicated by the equivalent F v /F m values), but the actual FPSII was much lower in the mutants. These differences may underlie the defects in chloroplast biogenesis and retarded growth in the vyl mutant (Fig. 1, C and D)
[[File:Table_1.png‎]]

====4.Altered Expression of Genes Associated with Chloroplast Biogenesis and Photosynthesis in vyl Mutants====
Chloroplast biogenesis and physiological changes are tightly regulated by the coordinated expression of plastid and nuclear genes during leaf development and facilitated by protein quality control (Kusumi et al., 2010; Clarke, 2012). To examine whether the expression of genes associated with chloroplast biogenesis and photosynthesis was altered in vyl mutants, quantitative real-time reverse transcription (qRT)-PCR was performed on total RNAs extracted from the L4 and
L3U leaf sections of wild-type and vyl plants. Compared with the wild type, the transcript levels of Virescent1 (V1), V2, and V3 genes and some other genes encoding components of the plastid and nuclear transcription apparatus Sigma factor 2A (OsSig2A), Ribosomal Protein S15 (rps15) and the subunit RNA polymerase (RpoA and RpoTp) that are highly expressed in early stages of chloroplast development were significantly increased in vyl mutants. In contrast, several genes encoding components of the photosynthesis apparatus RuBisCO large subunit (RbcL), the subunit of photosystem I (PsaA), a core component of Photosystem II (PsbA), light-harvesting complex protein (Lhcp2) and Chlorophyll A/B binding protein1 (Cab1) that are highly expressed in later stages of chloroplast development were significantly reduced in vyl mutants (Fig. 3). These results suggested that VYL plays an important role in regulating chloroplast biogenesis.
====5.The vyl Locus Maps to a Putative Gene Encoding OsClpP6====
Genetic analysis showed that the virescent yellow leaf phenotype in vyl mutants is controlled by a single recessive nuclear locus, VYL. Using a BC 1 F 2 mapping population of the vyl mutant and 93-11 (an indica variety), we mapped the vyl gene to a 137-kb genomic region on chromosome 3 between the insertion/deletion polymorphism (Indel) markers F17 and I53. Within this region, 17 open reading frames (ORFs) were predicted from published data (http://
www.gramene.org/; Fig. 4A). Genomic sequence analysis revealed that only the 13th ORF (LOC_Os03g29810) carries a single-nucleotide transition (G→T) at the position 1,509 bp from the ATG start codon (Fig. 4B). We obtained the full-length complementary DNA (cDNA) of LOC_Os03g29810 from both the wild type and the vyl mutant by reverse transcription (RT)-PCR. Sequence analysis showed that the full-length VYL cDNA is 780 bp long in the wild type but 842 bp in the mutant.
Comparison of the genomic and cDNA sequences revealed that the single-nucleotide substitution in the vyl mutant genome created a new splicing site and the addition of a partial fourth intron sequence in the cDNA (Fig. 4, C and D). The mutant cDNA is predicted to encode a truncated vyl mutant protein (DVYL) lacking the classic Ser protease triad (Ser-His-Asp) in the active site and the polypeptide-binding site (Fig. 4, C and E). Phylogenetic analysis and protein sequence alignment showed that VYL is most closely related to the Arabidopsis ClpP6 protein with a conserved S14_ClpP_2 domain and more remotely related to several other Arabidopsis ClpP subunits, and it apparently represents a single-copy gene in the rice genome (Fig. 4F).
To verify the identity of VYL, the plasmid pGVYL, containing a 6-kb genomic DNA fragment consisting of a 2.5-kb upstream sequence, the entire VYL coding region, including nine exons and eight introns, and a 0.6-kb downstream sequence, was constructed and introduced into the vyl mutant. All five transgenic lines containing pGVYL complemented the virescent phenotype of the vyl mutant (Fig. 5). To further confirm that disruption of the VYL gene was responsible for the vyl mutant phenotype, we generated RNA interference transgenic plants in the wild-type Kita-ake background and obtained more than eight independent transgenic
lines. qRT-PCR analysis revealed that the expression of VYL was reduced in three tested transgenic lines compared with the wild-type plants. These VYL knockdown transgenic plants showed reduced accumulation of chlorophyll a and chlorophyll b. Together, these results confirmed that LOC_Os03g29810 indeed corresponds to the VYL gene.

===Expression===
Expression is constitutive in most tissues examined (roots, stems, leaves, leaf sheath, panicle)but most abundant in leaf sections containing
chloroplasts in early stages of development,which can be light-mediated.<ref name="ref4" />
The young chlorotic leaves turn green in later developmental stages, accompanied by alterations in chlorophyll accumulation, chloroplast ultrastructure, and the expression of chloroplast development- and photosynthesis-related genes. Positional cloning revealed that the VYL gene encodes a protein homologous to the Arabidopsis ClpP6 subunit and that it is targeted to the chloroplast. VYL expression is constitutive in most tissues examined but most abundant in leaf sections containing chloroplasts in early stages of development. The mutation in vyl causes premature termination of the predicted gene product and loss of the conserved catalytic triad (serine-histidine-aspartate) and the polypeptide-binding site of VYL. Using a tandem affinity purification approach and mass spectrometry analysis, we identified OsClpP4 as a VYL-associated protein in vivo. In addition, yeast two-hybrid assays demonstrated that VYL directly interacts with OsClpP3 and OsClpP4. Furthermore, we found that OsClpP3 directly interacts with OsClpT, that OsClpP4 directly interacts with OsClpP5 and OsClpT, and that both OsClpP4 and OsClpT can homodimerize. Together, our data provide new insights into the function, assembly, and regulation of Clps in higher plants.<ref name="ref4" />

==Evolution==
ATP-dependent Clp protease proteolytic subunit is an enzyme that in humans is encoded by the CLPP gene.<ref name="ref1" /><ref name="ref2" /> It is found in mitochondria and is widely distributed in bacterial species.
In several bacteria, such as E. coli, proteins tagged with the SsrA peptide (ANDENYALAA) encoded by tmRNA are digested by Clp proteases.<ref name="ref3" />

[[File:612px-Protein_CLPP_PDB_1tg6.png]][[File:1867.png]]

==Labs working on this gene==
National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China

==References==
<references>
<ref name="ref1">
Bross P, Andresen BS, Knudsen I, Kruse TA, Gregersen N (Feb 1996). "Human ClpP protease: cDNA sequence, tissue-specific expression and chromosomal assignment of the gene". FEBS Lett 377 (2): 249–52. doi:10.1016/0014-5793(95)01353-9. PMID 8543061.
</ref>
<ref name="ref2">
a b "Entrez Gene: CLPP ClpP caseinolytic peptidase, ATP-dependent, proteolytic subunit homolog (E. coli)".
</ref>
<ref name="ref3">
Gottesman S, Roche E, Zhou Y, Sauer RT (1998). "The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system". Genes Dev 12 (9): 1338–47. doi:10.1101/gad.12.9.1338. PMC 316764. PMID 9573050
</ref>
<ref name="ref4">
Hui Dong et al. A Rice Virescent-Yellow Leaf Mutant Reveals New Insights into the Role and Assembly of Plastid Caseinolytic Protease in Higher Plants. Plant Physiology, 2013, 162(4): 1867-1880
</ref>
<ref name="ref5">
Zhou Feng et al.D14–SCFD3-dependent degradation of D53 regulates strigolactone signaling. Nature,2013. doi:10.1038/nature12878
</ref>
</references>
==Structured Information==
{{JaponicaGene|
GeneName = Os03g0411500|
Description = Peptidase S14, ClpP family protein|
Version = NM_001056884.1 GI:115453496 GeneID:4333096|
Length = 3448 bp|
Definition = Oryza sativa Japonica Group Os03g0411500, complete gene.|
Source = Oryza sativa Japonica Group

ORGANISM Oryza sativa Japonica Group
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP
clade; Ehrhartoideae; Oryzeae; Oryza.
|
Chromosome = [[:category:Japonica Chromosome 3|Chromosome 3]]|
AP = Chromosome 3:17630235..17633682|
CDS = 17630290..17630357,17630478..17630512,17631200..17631269,17631410..17631614,17631776..17631841 ,17632451..17632591,17632803..17632856,17632992..17633084,17633183..17633230 |
GCID = <gbrowseImage1>
name=NC_008396:17630235..17633682
source=RiceChromosome03
preset=GeneLocation
</gbrowseImage1>|
GSID = <gbrowseImage2>
name=NC_008396:17630235..17633682
source=RiceChromosome03
preset=GeneLocation
</gbrowseImage2>|
CDNA = <cdnaseq>atggcgcctatggccatctccaccccgctcgccctccgcgcctccccgacccgcctcctctcccgcaggcggagcggagccaaatcaggcgtggctctcccaggtccacaatttgtaccacctggtatttcttcaaagttggacgagaggatacattgtcattcttctctgaggaaaaatacaattgtagcatcagagaatgaaaatccacctttaatgcctgccataatgactcctgctggtgctcttgatctggcaactgtattgttggggaaccgcattatcttcattggtcaatatattaactcgcaagtagcacagcgtgtaatatcacagcttgtcacacttgctgctgttgatgaagaggctgatattctgatctacctgaactgccccggcggaagtctctactccatcttagcaatttatgattgcatgtcctggatcaagcccaaagttggaacagtgtgctttggtgttgttgctagccaggcagcaattatacttgctggcggtgagaagggaatgcgttatgccatgccaaatgctagagtaatgattcatcaacctcaaggtgtatcagagggtaatgtggaggaggtgaggcgacaggttggggaaaccatttatgctcgtgataaagttgataagatgtttgctgcttttactgggcaaaccttggatatggtacaacagtggacagagagggatcgtttcatgtcttcatctgaagccatggactttggactagttgatgccctgctggaaacaagatactaa</cdnaseq>|
AA = <aaseq>MAPMAISTPLALRASPTRLLSRRRSGAKSGVALPGPQFVPPGIS SKLDERIHCHSSLRKNTIVASENENPPLMPAIMTPAGALDLATVLLGNRIIFIGQYIN SQVAQRVISQLVTLAAVDEEADILIYLNCPGGSLYSILAIYDCMSWIKPKVGTVCFGV VASQAAIILAGGEKGMRYAMPNARVMIHQPQGVSEGNVEEVRRQVGETIYARDKVDKM FAAFTGQTLDMVQQWTERDRFMSSSEAMDFGLVDALLETRY</aaseq>|
DNA = <dnaseqindica>56..123#244..278#966..1035#1176..1380#1542..1607#2217..2357#2569..2622#2758..2850#2949..2996#actcctcagtcctcgcctcggctcggctccctcccacgctccagctccgcctccaatggcgcctatggccatctccaccccgctcgccctccgcgcctccccgacccgcctcctctcccgcaggtgagctccacggaactacaacttccacctccttcgctcgctcgctcgccccgcgcttctctcttcatggattcccccgttcttgtcccctcaccctctgtctggtccttctttcttcaggcggagcggagccaaatcaggcgtggctctcccaggtgagatttcctaacccttggtttagcaaaccatttcccttggtcagctcgttaggccaggactgtttggtggaatttgatcgttgatttgataagctttactgagatgttgtccatggtggtgcacatattagaacatgaccatttgggcagccgtcccatcagctcctagttgtctttctctgtgccaattttttatggagtcgtcattggtaggttttgcaaagcatatagaacctctgaatgtcggcattatccaagagtatgccgacctggggttatctgaaccagtgtcaactccttcctgggccaatgtctggtatgcatcagcattagctcactaagtacacgaaaaatggatgtgcttggttgaacggatatgtataaaaacgataacctgcatataacatatcacctcagtttggtctgattctgaaattagtttagggccttttagcaaactgctgaatgagatttccagactgtatatgtgttattgtgtttgtcagtaactcagtatggtgtattagcacaactcaacatgccataatgacaggatatgcggagcacaaacttttctttggacatgttttttggattcctttactgtttagtccatctgtctttcacatgatatattgctgcaaatgtgctgagttgcattctcactcaaatttccacacctaggtccacaatttgtaccacctggtatttcttcaaagttggacgagaggatacattgtcattcttctctgaggtgatatatttgaaactgtcatgcctgatatacaatgaggatacattgcctgatttgaaactgctcatctaggttttatttgtgctgtgtatcagaatcctgttttattcattcgtaatattgtaaatattttttcacaggaaaaatacaattgtagcatcagagaatgaaaatccacctttaatgcctgccataatgactcctgctggtgctcttgatctggcaactgtattgttggggaaccgcattatcttcattggtcaatatattaactcgcaagtagcacagcgtgtaatatcacagcttgtcacacttgctgctgttgatgaagaggctgatattctggttagtgtttatttttgtgtttttcagatcataacagttaccctattgttcactgcagcagctcttgattgctcaacttcactcccttggcttgctcctttagctcacaggtgtgttgctctatatcttataactccttttgtataattctgttttgccagatctacctgaactgccccggcggaagtctctactccatcttagcaatttatgattgcatgtcctgggtatgccatctatgttgagctactttttccatgtccttcatgcattcaaatttcagagattgtattcacctatatttattcttgtggatgctttctgagttattctcatctaatttaatatttacattgttggatgagaacacattatagatgcatctcaacattttgtatcttccacattatgcatgcccctagctagtgaatttatatttataatatagcacagatatagcatttacaggaaagcctaatgtaatttaggcaaaaattatatctcatatcaatggtagtgcttgcaacatttgtattcttatatttttattgtagtacatactagacatgagcatttgccatgctgagactgtgctaattgggtttggtctggtactgcagacaacagatctcatttcctgaaatcatgcctgtctctaaaactggctttgagctggaccagccttgttagttgttagatttggctgtgtttttacttgttatgccagttttccataaccaaatactttatctacaatttcgcctactgataaataaatcccagaatattaatctttttttgttgttctgcactaacacatgaaccatttattgcagatcaagcccaaagttggaacagtgtgctttggtgttgttgctagccaggcagcaattatacttgctggcggtgagaagggaatgcgttatgccatgccaaatgctagagtaatgattcatcaacctcaaggtgtatcagaggtatgattctggggctttctgcctttctgagttactgcagcgggtgcattatgattttctaacattgtgactacagtaaataataatcatcatcattttagctggccacatgaaacttacaatatacagtcttgctaggacatacttgcttgcctttgtgtttgttgtacttgaagtttgttttttattctaaaaattggatgatttgcagggtaatgtggaggaggtgaggcgacaggttggggaaaccatttatgctcgtgatgtaagtgttttgtgatagataacaagttctatattttcttcagtgtacatttgaattagatgtttgatgagggtaaggaatttctcttgattctgctctctaagcgattaaagcttctgaaaaatctggatgcagaaagttgataagatgtttgctgcttttactgggcaaaccttggatatggtacaacagtggacagagagggatcgtttcatgtcttcatctgaagtaactttcatctcttaaatgtatcagaagaaagtaaatcatcatttgccatgtgaattataacttatttcccccttctttttttggttttaccctaggccatggactttggactagttgatgccctgctggaaacaagatactaacaaacaaacacttaggcacagtttgattagcagaggctggtacaaggattgtgaaactggagctgaagttgagattttcgccgtccttctagttcaaggactccaatgacaggaggctggatctgcgagacttgaatgcatcgccatcgctcctatgagcaaaacatctctgcgttgagtgtgattttttgctcttcttttttggatctggttttacatgccgccagcctatggtctcaatgcattggtcctgctaatgtttagtgtagaccagatgatcctttagacagcaaaacatcagttattactccgtagattttcccatgagctgattccagactgtgtgcaacttttgtgttccaattgcaaagtttgcaacccaacccaacccaacacatgggctgatgggctgttgacaaaggagagattttacacttcacatatgtcaaact</dnaseqindica>|
Link = [http://www.ncbi.nlm.nih.gov/nuccore/NM_001056884.1 RefSeq:Os03g0411500]|
}}
[[Category:Genes]]
[[Category:Japonica mRNA]]
[[Category:Oryza Sativa Japonica Group]]
[[Category:Japonica Genes]]
[[Category:Japonica Chromosome 3]]
[[Category:Chromosome 3]]

Os03g0411500

2014-06-08T08:12:36Z

Xiaoyanjie2012: /* 5.The vyl Locus Maps to a Putative Gene Encoding OsClpP6 */

A nuclear gene coding caseinolyse positioned in plastid or mitochondrion regulating early morphogenesis.
VYL is a gene in rice.Its RAP ID is Os03g0411500 and its MSU ID is LOC_Os03g29810. The mutant of this gene produces chlorotic leaves throughout the entire growth period.
==Annotated Information==
===Function===
The protein encoded by this gene belongs to the peptidase family S14 and hydrolyzes proteins into small peptides in the presence of ATP and magnesium. The protein is transported into mitochondrial matrix and is associated with the inner mitochondrial membrane<ref name="ref2" />, or plastid inner membrane in plants<ref name="ref4" />.
The plastidic caseinolytic protease (Clp) of higher plants is an evolutionarily conserved protein degradation apparatus composed of a proteolytic core complex (the P and R rings) and a set of accessory proteins (ClpT, ClpC, and ClpS).<ref name="ref4" />
Rice yellow leaf mutant vyl, the performance of the entire growth period, new leaves chlorotic phenotype, then gradually turn green from the top down.<ref name="ref4" /> VYL Arabidopsis Clp protease subunit ClpP6 homologous protein in rice, is one of the subunits of the chloroplast Clp protease, with the Clp protease subunit interactions OsClpP3 and OsClpP4 respectively, play an important role in the biosynthesis of rice chloroplast.<ref name="ref4" />
What's more, the gene D53 product shares predicted features with the class I Clp ATPase proteins and can form a complex with the a/b hydrolase protein DWARF 14 (D14) and the F-box protein DWARF 3 (D3), two previously identified signalling components potentially responsible for SL(Strigolactones) perception, which means, in a D14- and D3-dependentmanner, SLs induce D53 degradation by the proteasome and abrogate its activity in promoting axillary bud outgrowth.<ref name="ref5" />
The role and molecular composition of Clps in higher plants has just begun to be unraveled, mostly from studies with the model dicotyledonous plant Arabidopsis (Arabidopsis thaliana).Some researchers isolated a virescent yellow leaf (vyl) mutant in rice (Oryza sativa), which produces chlorotic leaves throughout the entire growth period.<ref name="ref4" />
====1.The vyl Mutant Displays Reduced Chlorophyll Accumulation====
The vyl mutant was derived by transforming tissue cultures of the japonica rice variety Kita-ake. When grown under an alternating light/dark cycle (12 h of light at 30°C/12 h of darkness at 20°C) in a growth chamber, vyl mutant plants displayed a virescent yellow leaf pheno-type, and during development, leaves gradually turned green from their tips (more developed) to their bases (less developed; Fig. 1, A and B). At maturity, the vyl mutant plants also had reduced height and smaller seeds (Fig. 1, C and D). Mutant leaves also contained less chlorophyll than the wild type at various growth stages (Fig. 1E).
Additionally, vyl mutants developed chlorotic leaves under different temperature conditions and light/dark cycles, suggesting that the virescent yellow phenotype of the vyl mutant was developmentally regulated but independent of external cues (such as temperature and light)
[[File:figure 1.png]]

====2.The vyl Mutant Has Impaired Chloroplast Development====
Next, researchers investigated whether the virescent yellow phenotype of the vyl mutant was associated with ultra-structural changes in the chloroplasts. Leaf samples of L3U (upper half of the third leaf), L3L (basal half of the third leaf), and L4 (fourth leaf above the shoot base) were collected from wild-type and vyl mutant seedlings and compared (Fig. 2A). Normally, when the third leaf has fully emerged from the shoot base of a rice plant, the shoot also contains the fourth to the seventh immature leaves. The leaf cells in the L3L and L3U samples contain mature chloroplasts, whereas those in the shoot base and L4 samples contain proplastids and early developing immature chloroplasts (Sugimoto et al., 2004). Similar to the wild type, the chloroplasts from the L3U green leaf sample (already turned green) of vyl mutant seedlings displayed well-developed lamellar structures and were equipped with normally stacked grana and thylakoid membranes (Fig. 2, B and C). By contrast, the chloroplasts from the L3L and L4 pale leaves (still wrapped in leaf sheath) of the vyl mutant had much reduced thylakoid membrane networks compared with wild-type plants (Fig. 2, D–G). This developmental defect is similar to the phenotype reported in the Arabidopsis CLPP6 antisense transgenic plants (Sjögren et al., 2006).

[[File:Figure 2 .png]]

====3.The vyl Mutant Has Impaired Photosynthesis====
Chloroplasts are the organelles in plant cells that perform photosynthesis; therefore, they play an essential role in plant growth. To test whether the photosynthetic apparatus was affected in vyl mutants, we compared some key parameters of PSI and PSII between vyl and wild-type plants. Distinct differences in photochemical efficiency of PSII (FPSII), electron transport rate (ETR), nonphotochemical quenching (NPQ), and photochemical
quenching (Qp) were detected between vyl mutants and the wild type. In contrast, the maximal efficiency of PSII photochemistry (F v /F m ) values was almost comparable between vyl and wild-type plants (Table I). These observations indicate that vyl mutants absorbed much less light energy, as shown by the lower NPQ values. PSII photochemistry was reduced at both the donor and acceptor sites, as indicated by the greatly decreased Qp and ETR in vyl mutants. Notably, the PSII structure appeared intact in the mutant plants (indicated by the equivalent F v /F m values), but the actual FPSII was much lower in the mutants. These differences may underlie the defects in chloroplast biogenesis and retarded growth in the vyl mutant (Fig. 1, C and D)
[[File:Table_1.png‎]]

====4.Altered Expression of Genes Associated with Chloroplast Biogenesis and Photosynthesis in vyl Mutants====
Chloroplast biogenesis and physiological changes are tightly regulated by the coordinated expression of plastid and nuclear genes during leaf development and facilitated by protein quality control (Kusumi et al., 2010; Clarke, 2012). To examine whether the expression of genes associated with chloroplast biogenesis and photosynthesis was altered in vyl mutants, quantitative real-time reverse transcription (qRT)-PCR was performed on total RNAs extracted from the L4 and
L3U leaf sections of wild-type and vyl plants. Compared with the wild type, the transcript levels of Virescent1 (V1), V2, and V3 genes and some other genes encoding components of the plastid and nuclear transcription apparatus Sigma factor 2A (OsSig2A), Ribosomal Protein S15 (rps15) and the subunit RNA polymerase (RpoA and RpoTp) that are highly expressed in early stages of chloroplast development were significantly increased in vyl mutants. In contrast, several genes encoding components of the photosynthesis apparatus RuBisCO large subunit (RbcL), the subunit of photosystem I (PsaA), a core component of Photosystem II (PsbA), light-harvesting complex protein (Lhcp2) and Chlorophyll A/B binding protein1 (Cab1) that are highly expressed in later stages of chloroplast development were significantly reduced in vyl mutants (Fig. 3). These results suggested that VYL plays an important role in regulating chloroplast biogenesis.
====5.The vyl Locus Maps to a Putative Gene Encoding OsClpP6====
Genetic analysis showed that the virescent yellow leaf phenotype in vyl mutants is controlled by a single recessive nuclear locus, VYL. Using a BC 1 F 2 mapping population of the vyl mutant and 93-11 (an indica variety), we mapped the vyl gene to a 137-kb genomic region on chromosome 3 between the insertion/deletion polymorphism (Indel) markers F17 and I53. Within this region, 17 open reading frames (ORFs) were predicted from published data (http://
www.gramene.org/; Fig. 4A). Genomic sequence analysis revealed that only the 13th ORF (LOC_Os03g29810) carries a single-nucleotide transition (G→T) at the position 1,509 bp from the ATG start codon (Fig. 4B). We obtained the full-length complementary DNA (cDNA) of LOC_Os03g29810 from both the wild type and the vyl mutant by reverse transcription (RT)-PCR. Sequence analysis showed that the full-length VYL cDNA is 780 bp long in the wild type but 842 bp in the mutant.
Comparison of the genomic and cDNA sequences revealed that the single-nucleotide substitution in the vyl mutant genome created a new splicing site and the addition of a partial fourth intron sequence in the cDNA (Fig. 4, C and D). The mutant cDNA is predicted to encode a truncated vyl mutant protein (DVYL) lacking the classic Ser protease triad (Ser-His-Asp) in the active site and the polypeptide-binding site (Fig. 4, C and E). Phylogenetic analysis and protein sequence alignment showed that VYL is most closely related to the Arabidopsis ClpP6 protein with a conserved S14_ClpP_2 domain and more remotely related to several other Arabidopsis ClpP subunits, and it apparently represents a single-copy gene in the rice genome (Fig. 4F).

===Expression===
Expression is constitutive in most tissues examined (roots, stems, leaves, leaf sheath, panicle)but most abundant in leaf sections containing
chloroplasts in early stages of development,which can be light-mediated.<ref name="ref4" />
The young chlorotic leaves turn green in later developmental stages, accompanied by alterations in chlorophyll accumulation, chloroplast ultrastructure, and the expression of chloroplast development- and photosynthesis-related genes. Positional cloning revealed that the VYL gene encodes a protein homologous to the Arabidopsis ClpP6 subunit and that it is targeted to the chloroplast. VYL expression is constitutive in most tissues examined but most abundant in leaf sections containing chloroplasts in early stages of development. The mutation in vyl causes premature termination of the predicted gene product and loss of the conserved catalytic triad (serine-histidine-aspartate) and the polypeptide-binding site of VYL. Using a tandem affinity purification approach and mass spectrometry analysis, we identified OsClpP4 as a VYL-associated protein in vivo. In addition, yeast two-hybrid assays demonstrated that VYL directly interacts with OsClpP3 and OsClpP4. Furthermore, we found that OsClpP3 directly interacts with OsClpT, that OsClpP4 directly interacts with OsClpP5 and OsClpT, and that both OsClpP4 and OsClpT can homodimerize. Together, our data provide new insights into the function, assembly, and regulation of Clps in higher plants.<ref name="ref4" />

==Evolution==
ATP-dependent Clp protease proteolytic subunit is an enzyme that in humans is encoded by the CLPP gene.<ref name="ref1" /><ref name="ref2" /> It is found in mitochondria and is widely distributed in bacterial species.
In several bacteria, such as E. coli, proteins tagged with the SsrA peptide (ANDENYALAA) encoded by tmRNA are digested by Clp proteases.<ref name="ref3" />

[[File:612px-Protein_CLPP_PDB_1tg6.png]][[File:1867.png]]

==Labs working on this gene==
National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China

==References==
<references>
<ref name="ref1">
Bross P, Andresen BS, Knudsen I, Kruse TA, Gregersen N (Feb 1996). "Human ClpP protease: cDNA sequence, tissue-specific expression and chromosomal assignment of the gene". FEBS Lett 377 (2): 249–52. doi:10.1016/0014-5793(95)01353-9. PMID 8543061.
</ref>
<ref name="ref2">
a b "Entrez Gene: CLPP ClpP caseinolytic peptidase, ATP-dependent, proteolytic subunit homolog (E. coli)".
</ref>
<ref name="ref3">
Gottesman S, Roche E, Zhou Y, Sauer RT (1998). "The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system". Genes Dev 12 (9): 1338–47. doi:10.1101/gad.12.9.1338. PMC 316764. PMID 9573050
</ref>
<ref name="ref4">
Hui Dong et al. A Rice Virescent-Yellow Leaf Mutant Reveals New Insights into the Role and Assembly of Plastid Caseinolytic Protease in Higher Plants. Plant Physiology, 2013, 162(4): 1867-1880
</ref>
<ref name="ref5">
Zhou Feng et al.D14–SCFD3-dependent degradation of D53 regulates strigolactone signaling. Nature,2013. doi:10.1038/nature12878
</ref>
</references>
==Structured Information==
{{JaponicaGene|
GeneName = Os03g0411500|
Description = Peptidase S14, ClpP family protein|
Version = NM_001056884.1 GI:115453496 GeneID:4333096|
Length = 3448 bp|
Definition = Oryza sativa Japonica Group Os03g0411500, complete gene.|
Source = Oryza sativa Japonica Group

ORGANISM Oryza sativa Japonica Group
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP
clade; Ehrhartoideae; Oryzeae; Oryza.
|
Chromosome = [[:category:Japonica Chromosome 3|Chromosome 3]]|
AP = Chromosome 3:17630235..17633682|
CDS = 17630290..17630357,17630478..17630512,17631200..17631269,17631410..17631614,17631776..17631841 ,17632451..17632591,17632803..17632856,17632992..17633084,17633183..17633230 |
GCID = <gbrowseImage1>
name=NC_008396:17630235..17633682
source=RiceChromosome03
preset=GeneLocation
</gbrowseImage1>|
GSID = <gbrowseImage2>
name=NC_008396:17630235..17633682
source=RiceChromosome03
preset=GeneLocation
</gbrowseImage2>|
CDNA = <cdnaseq>atggcgcctatggccatctccaccccgctcgccctccgcgcctccccgacccgcctcctctcccgcaggcggagcggagccaaatcaggcgtggctctcccaggtccacaatttgtaccacctggtatttcttcaaagttggacgagaggatacattgtcattcttctctgaggaaaaatacaattgtagcatcagagaatgaaaatccacctttaatgcctgccataatgactcctgctggtgctcttgatctggcaactgtattgttggggaaccgcattatcttcattggtcaatatattaactcgcaagtagcacagcgtgtaatatcacagcttgtcacacttgctgctgttgatgaagaggctgatattctgatctacctgaactgccccggcggaagtctctactccatcttagcaatttatgattgcatgtcctggatcaagcccaaagttggaacagtgtgctttggtgttgttgctagccaggcagcaattatacttgctggcggtgagaagggaatgcgttatgccatgccaaatgctagagtaatgattcatcaacctcaaggtgtatcagagggtaatgtggaggaggtgaggcgacaggttggggaaaccatttatgctcgtgataaagttgataagatgtttgctgcttttactgggcaaaccttggatatggtacaacagtggacagagagggatcgtttcatgtcttcatctgaagccatggactttggactagttgatgccctgctggaaacaagatactaa</cdnaseq>|
AA = <aaseq>MAPMAISTPLALRASPTRLLSRRRSGAKSGVALPGPQFVPPGIS SKLDERIHCHSSLRKNTIVASENENPPLMPAIMTPAGALDLATVLLGNRIIFIGQYIN SQVAQRVISQLVTLAAVDEEADILIYLNCPGGSLYSILAIYDCMSWIKPKVGTVCFGV VASQAAIILAGGEKGMRYAMPNARVMIHQPQGVSEGNVEEVRRQVGETIYARDKVDKM FAAFTGQTLDMVQQWTERDRFMSSSEAMDFGLVDALLETRY</aaseq>|
DNA = <dnaseqindica>56..123#244..278#966..1035#1176..1380#1542..1607#2217..2357#2569..2622#2758..2850#2949..2996#actcctcagtcctcgcctcggctcggctccctcccacgctccagctccgcctccaatggcgcctatggccatctccaccccgctcgccctccgcgcctccccgacccgcctcctctcccgcaggtgagctccacggaactacaacttccacctccttcgctcgctcgctcgccccgcgcttctctcttcatggattcccccgttcttgtcccctcaccctctgtctggtccttctttcttcaggcggagcggagccaaatcaggcgtggctctcccaggtgagatttcctaacccttggtttagcaaaccatttcccttggtcagctcgttaggccaggactgtttggtggaatttgatcgttgatttgataagctttactgagatgttgtccatggtggtgcacatattagaacatgaccatttgggcagccgtcccatcagctcctagttgtctttctctgtgccaattttttatggagtcgtcattggtaggttttgcaaagcatatagaacctctgaatgtcggcattatccaagagtatgccgacctggggttatctgaaccagtgtcaactccttcctgggccaatgtctggtatgcatcagcattagctcactaagtacacgaaaaatggatgtgcttggttgaacggatatgtataaaaacgataacctgcatataacatatcacctcagtttggtctgattctgaaattagtttagggccttttagcaaactgctgaatgagatttccagactgtatatgtgttattgtgtttgtcagtaactcagtatggtgtattagcacaactcaacatgccataatgacaggatatgcggagcacaaacttttctttggacatgttttttggattcctttactgtttagtccatctgtctttcacatgatatattgctgcaaatgtgctgagttgcattctcactcaaatttccacacctaggtccacaatttgtaccacctggtatttcttcaaagttggacgagaggatacattgtcattcttctctgaggtgatatatttgaaactgtcatgcctgatatacaatgaggatacattgcctgatttgaaactgctcatctaggttttatttgtgctgtgtatcagaatcctgttttattcattcgtaatattgtaaatattttttcacaggaaaaatacaattgtagcatcagagaatgaaaatccacctttaatgcctgccataatgactcctgctggtgctcttgatctggcaactgtattgttggggaaccgcattatcttcattggtcaatatattaactcgcaagtagcacagcgtgtaatatcacagcttgtcacacttgctgctgttgatgaagaggctgatattctggttagtgtttatttttgtgtttttcagatcataacagttaccctattgttcactgcagcagctcttgattgctcaacttcactcccttggcttgctcctttagctcacaggtgtgttgctctatatcttataactccttttgtataattctgttttgccagatctacctgaactgccccggcggaagtctctactccatcttagcaatttatgattgcatgtcctgggtatgccatctatgttgagctactttttccatgtccttcatgcattcaaatttcagagattgtattcacctatatttattcttgtggatgctttctgagttattctcatctaatttaatatttacattgttggatgagaacacattatagatgcatctcaacattttgtatcttccacattatgcatgcccctagctagtgaatttatatttataatatagcacagatatagcatttacaggaaagcctaatgtaatttaggcaaaaattatatctcatatcaatggtagtgcttgcaacatttgtattcttatatttttattgtagtacatactagacatgagcatttgccatgctgagactgtgctaattgggtttggtctggtactgcagacaacagatctcatttcctgaaatcatgcctgtctctaaaactggctttgagctggaccagccttgttagttgttagatttggctgtgtttttacttgttatgccagttttccataaccaaatactttatctacaatttcgcctactgataaataaatcccagaatattaatctttttttgttgttctgcactaacacatgaaccatttattgcagatcaagcccaaagttggaacagtgtgctttggtgttgttgctagccaggcagcaattatacttgctggcggtgagaagggaatgcgttatgccatgccaaatgctagagtaatgattcatcaacctcaaggtgtatcagaggtatgattctggggctttctgcctttctgagttactgcagcgggtgcattatgattttctaacattgtgactacagtaaataataatcatcatcattttagctggccacatgaaacttacaatatacagtcttgctaggacatacttgcttgcctttgtgtttgttgtacttgaagtttgttttttattctaaaaattggatgatttgcagggtaatgtggaggaggtgaggcgacaggttggggaaaccatttatgctcgtgatgtaagtgttttgtgatagataacaagttctatattttcttcagtgtacatttgaattagatgtttgatgagggtaaggaatttctcttgattctgctctctaagcgattaaagcttctgaaaaatctggatgcagaaagttgataagatgtttgctgcttttactgggcaaaccttggatatggtacaacagtggacagagagggatcgtttcatgtcttcatctgaagtaactttcatctcttaaatgtatcagaagaaagtaaatcatcatttgccatgtgaattataacttatttcccccttctttttttggttttaccctaggccatggactttggactagttgatgccctgctggaaacaagatactaacaaacaaacacttaggcacagtttgattagcagaggctggtacaaggattgtgaaactggagctgaagttgagattttcgccgtccttctagttcaaggactccaatgacaggaggctggatctgcgagacttgaatgcatcgccatcgctcctatgagcaaaacatctctgcgttgagtgtgattttttgctcttcttttttggatctggttttacatgccgccagcctatggtctcaatgcattggtcctgctaatgtttagtgtagaccagatgatcctttagacagcaaaacatcagttattactccgtagattttcccatgagctgattccagactgtgtgcaacttttgtgttccaattgcaaagtttgcaacccaacccaacccaacacatgggctgatgggctgttgacaaaggagagattttacacttcacatatgtcaaact</dnaseqindica>|
Link = [http://www.ncbi.nlm.nih.gov/nuccore/NM_001056884.1 RefSeq:Os03g0411500]|
}}
[[Category:Genes]]
[[Category:Japonica mRNA]]
[[Category:Oryza Sativa Japonica Group]]
[[Category:Japonica Genes]]
[[Category:Japonica Chromosome 3]]
[[Category:Chromosome 3]]

Os03g0411500

2014-06-08T08:08:51Z

Xiaoyanjie2012: /* 3.The vyl Mutant Has Impaired Photosynthesis */

Os03g0411500

2014-06-08T08:03:40Z

Xiaoyanjie2012: /* 3.The vyl Mutant Has Impaired Photosynthesis */

File:Table 1.png

2014-06-08T08:02:56Z

Xiaoyanjie2012: uploaded a new version of "File:Table 1.png"

Shibo Zhang, Calvin Chen, Lei Li, Ling Meng, Jaswinder Singh, Ning Jiang, Xingwang Deng, Zhenghui He and Peggy G. Lemaux (2005). Evolutionary Expansion, Gene Structure, and Expression of the Rice Wall-Associated Kinase Gene Family. American Society of Plant Biologists 139,07-24.

Os03g0411500

2014-06-08T08:01:04Z

Xiaoyanjie2012: /* 3.The vyl Mutant Has Impaired Photosynthesis */

Os03g0411500

2014-06-08T08:00:47Z

Xiaoyanjie2012: /* 2.The vyl Mutant Has Impaired Chloroplast Development */

Os03g0411500

2014-06-08T08:00:27Z

Xiaoyanjie2012: /* 1.The vyl Mutant Displays Reduced Chlorophyll Accumulation */

Os03g0411500

2014-06-08T08:00:00Z

Xiaoyanjie2012: /* 2.The vyl Mutant Has Impaired Chloroplast Development */

Os03g0411500

2014-06-08T07:56:12Z

Xiaoyanjie2012: /* 2.The vyl Mutant Has Impaired Chloroplast Development */

Os03g0411500

2014-06-08T07:55:48Z

Xiaoyanjie2012: /* 2.The vyl Mutant Has Impaired Chloroplast Development */

File:Figure 2 .png

2014-06-08T07:55:07Z

Xiaoyanjie2012:

Os03g0411500

2014-06-08T07:52:07Z

Xiaoyanjie2012: /* 1.The vyl Mutant Displays Reduced Chlorophyll Accumulation */

Os03g0411500

2014-06-08T07:47:49Z

Xiaoyanjie2012: /* 1.The vyl Mutant Displays Reduced Chlorophyll Accumulation */

Os03g0411500

2014-06-08T07:46:25Z

Xiaoyanjie2012: /* 1.The vyl Mutant Displays Reduced Chlorophyll Accumulation */