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| | ==Annotated Information== | | ==Annotated Information== |
| − | OsAt10 is a gene in rice(Oryza sativa).Its RAP ID is Os06g0594600,and its MSU ID LOC_Os06g39390. OsAT10 belongs to a grass-diverged and expanded clade of BAHD acyl-utilizing acyltransferase proteins within the Mitchell clade. Overexpression of this gene can alters rice cell wall hydroxycinnamic acid Content and saccharification.
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| − | ===Function===
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| − | 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. OsAT10 seems likely that the native function of OsAT10 is to incorporate p-CA into a precursor of GAX.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.
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| − | 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" />
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| − | ====1.The Mitchell Clade of BAHD Acyltransferases Is Expanded and Diverged in Grasses====
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| − | 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)
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| − | 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.
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| − | 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).
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| − | [[File:Table 1xxy.png]][[File:figure 1xxy.png]]
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| − | ====2.Screen of Rice Mutants for Altered Cell Wall Hydroxycinnamic Acid Content====
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| − | [[File:Figure 2xxy.png]]
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| − | ====3.The OsAT10-D1 Line Has an Increase in Cell Wall Glc Content====
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| − | Compensatory changes are often seen among the components of the cell wall (Humphrey et al., 2007). Quantification of sugars released by acid treatment of
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| − | 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.
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| − | [[File:Figure 9xxy.png]]
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| − | ====4.The OsAT10-D1 Line Shows No Alterations in Lignin Content or Composition====
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| − | OsAT10-D1 mature straw samples show no significant differences in the content of acetylbromide-soluble lignin after saponification relative to the wild type
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| − | (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.
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| − | 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.
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| − | [[File:Table 2xxy.png]]
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| − | [[File:Figure 10xxy.png]]
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| − | ====The OsAT10-D1 Line Shows an Increase in Saccharification====
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| − | 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)
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| − | 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).
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| − | [[File:Figure 11xxy.png]]
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| | ===Expression=== | | ===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
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| − | 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.
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| − | [[File:Figure 3xxy.png]][[File:Figure 4xxy.png]]
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| | ===Evolution=== | | ===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
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| − | 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.
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| | ==Labs working on this gene== | | ==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
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| − | 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.).
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| | ==References== | | ==References== |
| − | <references>
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| − | <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.
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| | ==Structured Information== | | ==Structured Information== |
