Os08g0237000
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Contents
Annotated Information
Function
Xyloglucan endotransglucosylases/hydrolases (XTHs) that mediate cleavage and rejoining of the β (1-4)-xyloglucans of the primary cell wall are considered to play an important role in the construction and restructuring of xyloglucan cross-links.OsXTH8 was a single copy gene; its full-length cDNA was 1,298 bp encoding a predicted protein of 290 amino acids.OsXTH8 may play cooperative role with OsXTR1, OsXTR2, OsXTR3, OsXTR4 genes and others that are expressed in overlapping regions. OsXTH8 is involved in cell wall modification processes during rice growth and development.OsXTH8 acts to alter the structure of cell wall in response to gibberellic acid.
Expression
OsXTH8 was preferentially expressed in rice leaf sheath in response to gibberellic acid. OsXTH8 was highly expressed in vascular bundles of leaf sheath and young nodal roots where the cells are actively undergoing elongation and differentiation. OsXTH8 gene expression was up-regulated by gibberellic acid and there was very little effect of other hormones. Reduced accumulation of active GA was accompanied by preferential suppression of OsXTH8 expression, and OsXTH8 expression increased when exogenous GA3 was applied to the semidwarf mutant. In two genetic mutants of rice with abnormal height, the expression of OsXTH8 positively correlated with the height of the mutants. Transgenic rice expressing an RNAi construct of OsXTH8 exhibited repressed growth. These results indicate that OsXTH8 is differentially expressed in rice leaf sheath in relation to gibberellin and potentially involved in cell elongation processes.
OsXTH8 was expressed in growing regions such as shoot apical meristem, vascular bundles of leaf sheath, and young crown roots developing from nodes; thus, OsXTH8 exhibited a unique expression pattern in terms of organ and stage specificity during leaf sheath elongation and young nodal root development. The expression of OsXTH8 in the shoot apex meristem, vascular bundles in leaf sheath, and youngnodal roots supports its role in cell wall modification processes during active growth.
Alternative Organ- and Cell Type-Expression Profiles of OsXTH8
To examine the tissue specificity of OsXTH8, total RNAs from rice callus, root, leaf blade, and leaf sheath were hybridized with OsXTH8-specific DNA probe. A strong signal was detected in leaf sheaths but weak or no signal was observed in leaf blades, roots, and calli (Fig. 4A). These expressions were enhanced by GA3 treatment (Fig. 4A). When 1-month-old seedlings were used to characterize the expression in leaf sheath, the expression was mainly found in the three basal parts of leaf sheath (Fig. 4B). Enhanced expression of OsXTH8 in the third part of 1-month-old rice seedling, which corresponded to the second internode of leaf sheath, compared to two basal parts of leaf sheath showed that OsXTH8 is differentially expressed in leaf sheath.
In situ hybridization was performed using the basal part (culm) of 2-week-old rice seedlings to learn more about the expression pattern. On hybridization with gene-specific OsXTH8 antisense probe, the cross and longitudinal sections of culm tissue revealed an accumulation of OsXTH8 mRNA in shoot apex meristem, vascular tissues, and young leaves (Fig. 5A). Although the expression did not seem to be delimited to specific cell types, significant hybridization was observed in large and small vascular bundles of leaf sheath and peripheral cylinder of the vascular bundles and fibers in the nodal region (Fig. 5A). No significant signal was visible when sense probe was used.
To further characterize the spatial distribution patterns of OsXTH8 gene expression, 2,325 bp of the OsXTH8 upstream region from the proposed first translational start site was fused to the β-glucuronidase (GUS) reporter gene. This fusion gene was introduced into rice cells, and transgenic plants were regenerated. Putative OsXTH8::GUS transgenic plants were screened by PCR. Only PCR-confirmed, transgenic lines were used for GUS staining and GUS assay. To assess whether the GUS staining patterns were consistent with the result of in situ hybridization, similartissue sections of leaf sheath were used. Figure 5Bshows the GUS expression pattern driven by OsXTH8 promoter in the basal part of leaf sheath and young nodal roots of 2-week-old rice seedlings. In the case of leaf sheath, strong GUS staining was observed in shoot apex meristem and vascular bundles, very much similar to the result of in situ hybridization (Fig. 5, A and B). Microscopic observation of vascular bundles revealed GUS expression in vascular bundle sheath and mesotomic sheath surrounding xylem and phloem. Stele or vascular cylinder region in young nodal roots of the coleoptile node and roots arising from nodal roots also showed GUS staining (Fig. 5B). Weak GUS staining was found in the sclerenchyma cells lining the epidermis of young leaves (Fig. 5B).
To examine the tissue specificity of OsXTH8, total RNAs from rice callus, root, leaf blade, and leaf sheath were hybridized with OsXTH8-specific DNA probe. A strong signal was detected in leaf sheaths but weak or no signal was observed in leaf blades, roots, and calli (Fig. 4A). These expressions were enhanced by GA3 treatment (Fig. 4A). When 1-month-old seedlings were used to characterize the expression in leaf sheath, the expression was mainly found in the three basal parts of leaf sheath (Fig. 4B). Enhanced expression of OsXTH8 in the third part of 1-month-old rice seedling, which corresponded to the second internode of leaf sheath, compared to two basal parts of leaf sheath showed that OsXTH8 is differentially expressed in leaf sheath.
In situ hybridization was performed using the basal part (culm) of 2-week-old rice seedlings to learn more about the expression pattern. On hybridization with gene-specific OsXTH8 antisense probe, the cross and longitudinal sections of culm tissue revealed an accumulation of OsXTH8 mRNA in shoot apex meristem, vascular tissues, and young leaves (Fig. 5A). Although the expression did not seem to be delimited to specific cell types, significant hybridization was observed in large and small vascular bundles of leaf sheath and peripheral cylinder of the vascular bundles and fibers in the nodal region (Fig. 5A). No significant signal was visible when sense probe was used.
To further characterize the spatial distribution patterns of OsXTH8 gene expression, 2,325 bp of the OsXTH8 upstream region from the proposed first translational start site was fused to the β-glucuronidase (GUS) reporter gene. This fusion gene was introduced into rice cells, and transgenic plants were regenerated. Putative OsXTH8::GUS transgenic plants were screened by PCR. Only PCR-confirmed, transgenic lines were used for GUS staining and GUS assay. To assess whether the GUS staining patterns were consistent with the result of in situ hybridization, similartissue sections of leaf sheath were used. Figure 5Bshows the GUS expression pattern driven by OsXTH8 promoter in the basal part of leaf sheath and young nodal roots of 2-week-old rice seedlings. In the case of leaf sheath, strong GUS staining was observed in shoot apex meristem and vascular bundles, very much similar to the result of in situ hybridization (Fig. 5, A and B). Microscopic observation of vascular bundles revealed GUS expression in vascular bundle sheath and mesotomic sheath surrounding xylem and phloem. Stele or vascular cylinder region in young nodal roots of the coleoptile node and roots arising from nodal roots also showed GUS staining (Fig. 5B). Weak GUS staining was found in the sclerenchyma cells lining the epidermis of young leaves (Fig. 5B).
Hormonal Regulation of OsXTH8 Expression
GA dose-dependent expression patterns of OsXTH8 mRNA were determined in rice leaf sheaths using different concentration of GA3 (1, 5, 10, and 50 μM). OsXTH8 expression was found to be up-regulated with the increase in GA3 concentration as there was no inhibitory effect of increase in GA3 concentration up to 50 μM; however, 5 μM GA3 induced maximum expression of OsXTH8 (Fig. 6A). To determine temporal expression patterns of OsXTH8 mRNA, leaf sheaths were treated for 1, 3, 6, 12, and 24 h. OsXTH8 mRNA accumulation in leaf sheath showed that during treatment of leaf sheath fragments with GA3, OsXTH8 expression was up-regulated and continued to increase throughout the 24-h incubation period (Fig. 6B). As 2-week-old rice seedlings were used for timecourse experiment, because of the growth of leaf sheaths, increase in OsXTH8 expression in control could also be detected.
It has been shown that XTH genes are regulated by various hormones. For example, BRU1, a soybean XTH, is regulated by BR (Zurek and Clouse, 1994), and TCH4, an Arabidopsis (Arabidopsis thaliana) XTH gene, is up-regulated by auxin and BR (Xu et al., 1995). To characterize hormonal regulation of OsXTH8 expression, the effect of several plant hormones was examined on OsXTH8 mRNA abundance (Fig. 6C). When leaf sheaths were treated with GA3, brassinolide (BL), 6-bezyladenine (BA), indole-3-acetic acid (IAA), and abscisic acid (ABA), GA3 up-regulated the expression of OsXTH8 and there was very little effect of other hormones. Uniconazole, which is a potent GA biosynthesis inhibitor, had an inhibitory effect on the OsXTH8 mRNA accumulation (Fig. 6C).
To understand the physiological functions of OsXTH8, its expression in rice mutants with abnormal heights was investigated. Tanginbozu is a GA-deficient semidwarf mutant and a single recessive gene controls the semidwarfism of Tanginbozu. Mutation in Tanginbozu blocks the three oxidative steps whereby ent-kaurene is converted to ent-kaurenoic acid resulting in less accumulation of active GA (Ogawa et al., 1996). Northern-blot analysis showed that the level of OsXTH8 mRNA in the mutant was lower than that in its wild-type cv Ginbozu (Fig. 6D). The expression of OsXTH8 in the mutant was induced to exceed wildtype level following treatment with GA3 for 24 h. Slender rice1 (slr1), a GA-insensitive mutant, shows a constitutive GA-response phenotype (Itoh et al., 2002). Stem of the slr1 mutant grows 2 to 3 times more than the stem of wild-type cv Nipponbare. Northernblot analysis confirmed that the level of OsXTH8 expression was higher in slr1 mutant than that of its wild type (Fig. 6D).
To analyze whether the 2,325-bp 5’-promoter regionof the OsXTH8 locus is sufficient for the induction ofits expression, an independent transgenic line (Fig.7B), which was transformed with 2,325 bp of OsXTH8 promoter fused to the GUS reporter gene, was treated with 5 μM GA3. It was observed that GA3 treatment enhanced the expression of the GUS reporter gene compared to untreated (mock) OsXTH8::GUS transgenic rice (Fig. 7B). This indicated that the 2,325-bp promoter region of OsXTH8 was sufficient for hormone- induced OsXTH8 expression. This observation was also confirmed by GUS assay using methylumbelliferylglucuronide as a substrate (Fig. 7C).
Evolution
The analysis revealed that XTHs could be loosely grouped into four distinct groups (Fig. 3). Group 1 contains genes that share a high level of sequence identity among different species and that are expressed in young developing tissues. Group 2 comprises XTH genes from several species showing diverse patterns of expression and response to hormonal or mechanical stimuli, including touchinducible, flooding-response, BR-inducible, and fruit ripening-related XTHs. Group 3 represents a divergent group of XTHs, including NXG1 from nasturtium (Tropaeolum majus) that can act as xyloglucan hydrolase and transglycosylase. OsXTH8 has the highest homology to sequences in Group 4, consisting of well-characterized barley (Hordeum vulgare) genes, HvPM2 and HvPM5,which are up-regulated by GA in barley leaf sheaths and leaves. Monocot members of group 4 revealed to have two substitutions (DEIDIEFMG) compared to the consensus sequence (DEIDFEFLG). The presence of different amino acid residues in the putative catalytic region may attribute to unique enzymatic activity of OsXTH8.
Labs working on this gene
National Institute of Agrobiological Sciences, Tsukuba 305–8602, Japan (A.J., G.Y., H.N., H.I., S.K.);
University of Tsukuba, Tsukuba 305–8572, Japan (A.J., H.M.);
Nagoya University, Nagoya 464–8601, Japan (H.K., M.M.);
National Institute of Crop Science, Tsukuba 305-8518, Japan.
References
[1] Jan A, Yang G, Nakamura H, et al. Characterization of a xyloglucan endotransglucosylase gene that is up-regulated by gibberellin in rice [J]. Plant physiology, 2004, 136(3): 3670-3681.
[2]Tanaka T, Antonio B A, Kikuchi S, et al. The rice annotation project database (RAP-DB): 2008 update[J]. Nucleic acids research, 2008, 36(Sup 1): D1028-D1033.
[3]Rice Annotation Project, et al. Curated genome annotation of Oryza sativa ssp. japonica and comparative genome analysis with Arabidopsis thaliana. Genome Res, 2007, 17: 175-183.
[4] Ohyanagi H, et al. The Rice Annotation Project Database (RAP-DB): hub for Oryza sativa ssp. japonica genome information. Nucleic Acids Research, 2006, 34: D741-D744.
[5] International Rice Genome Sequencing Project. The map-based sequence of the rice genome. Nature, 2005,436(11): 793-800.
[6] Asad Jan and Setsuko Komatsu. Functional Characterization of Gibberellin-Regulated Genes in Rice Using Microarray System. Geno. Prot. Bioinfo. 2006,4(3): 137-144.
Structured Information
| Gene Name |
Os08g0237000 |
|---|---|
| Description |
Xyloglucan endotransglycosylase/hydrolase protein 8 precursor (EC 2.4.1.207) (End-xyloglucan transferase) (OsXTH8) (OsXRT5) |
| Version |
NM_001067854.1 GI:115475444 GeneID:4345019 |
| Length |
1378 bp |
| Definition |
Oryza sativa Japonica Group Os08g0237000, 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 | |
| Location |
Chromosome 8:8327025..8328402 |
| Sequence Coding Region |
8327064..8327545,8327644..8328034 |
| Expression | |
| Genome Context |
<gbrowseImage1> name=NC_008401:8327025..8328402 source=RiceChromosome08 preset=GeneLocation </gbrowseImage1> |
| Gene Structure |
<gbrowseImage2> name=NC_008401:8327025..8328402 source=RiceChromosome08 preset=GeneLocation </gbrowseImage2> |
| Coding Sequence |
<cdnaseq>atggcgaagcatctcgcgctgtccgtggccgccgcggtggccgtgtcgtggctggcggcgtcgtcggcggcggcggcggggttctacgagaagttcgacgtggtgggcgccggcgaccacgtgagggtggtgagcgacgacgggaagacgcagcaggtggcgctgacgctggaccggagctccgggtccgggttcacctccaaggacacctacctgttcggcgagttcagcgtccagatgaagctcgtcggcggcaactccgccggcaccgtcacctccttctacctctcctccggcgagggcgacggccacgacgagatcgacatcgagttcatgggcaacctcagcggcaacccctacgtcatgaacaccaacgtctgggctaatggcgacggcaagaaggagcaccagttctacctctggttcgaccccaccgccgacttccacacctacaagatcatctggaatccccaaaacatcatattccaggtggacgacgtgccggtgaggacgttcaagaagtacgacgacctggcgtacccgcagagcaagccgatgaggctgcacgcgacgctgtgggacggcagctactgggcgacgaggcacggcgacgtcaagatcgactggagcggcgcgccgttcgtggtgtcgtaccgcgggtacagcaccaacgcgtgcgtcaacaacaatcccgccggcgggtggtcgtcgtcgtggtgccccgagggcacgtcggcgtggatccaccgcgagctcgacggcgccgagctcggcaccgtcgcgtgggccgagcgcaactacatgtcctacaactactgcgccgacggctggcgcttcccccagggcttccccgccgagtgctaccgcaagtga</cdnaseq> |
| Protein Sequence |
<aaseq>MAKHLALSVAAAVAVSWLAASSAAAAGFYEKFDVVGAGDHVRVV SDDGKTQQVALTLDRSSGSGFTSKDTYLFGEFSVQMKLVGGNSAGTVTSFYLSSGEGD GHDEIDIEFMGNLSGNPYVMNTNVWANGDGKKEHQFYLWFDPTADFHTYKIIWNPQNI IFQVDDVPVRTFKKYDDLAYPQSKPMRLHATLWDGSYWATRHGDVKIDWSGAPFVVSY RGYSTNACVNNNPAGGWSSSWCPEGTSAWIHRELDGAELGTVAWAERNYMSYNYCADG WRFPQGFPAECYRK</aaseq> |
| Gene Sequence |
<dnaseqindica>40..521#620..1010#atccaccacccaaagacaaagcaagcaagtacagtagccatggcgaagcatctcgcgctgtccgtggccgccgcggtggccgtgtcgtggctggcggcgtcgtcggcggcggcggcggggttctacgagaagttcgacgtggtgggcgccggcgaccacgtgagggtggtgagcgacgacgggaagacgcagcaggtggcgctgacgctggaccggagctccgggtccgggttcacctccaaggacacctacctgttcggcgagttcagcgtccagatgaagctcgtcggcggcaactccgccggcaccgtcacctccttctacctctcctccggcgagggcgacggccacgacgagatcgacatcgagttcatgggcaacctcagcggcaacccctacgtcatgaacaccaacgtctgggctaatggcgacggcaagaaggagcaccagttctacctctggttcgaccccaccgccgacttccacacctacaagatcatctggaatccccaaaacatcatgtacgcatcacaaaaacgaactcaataatcctctcttcgtcaatcaaacaatcatcgtgttaatttgctgaattacgattacttccgctatgatacagattccaggtggacgacgtgccggtgaggacgttcaagaagtacgacgacctggcgtacccgcagagcaagccgatgaggctgcacgcgacgctgtgggacggcagctactgggcgacgaggcacggcgacgtcaagatcgactggagcggcgcgccgttcgtggtgtcgtaccgcgggtacagcaccaacgcgtgcgtcaacaacaatcccgccggcgggtggtcgtcgtcgtggtgccccgagggcacgtcggcgtggatccaccgcgagctcgacggcgccgagctcggcaccgtcgcgtgggccgagcgcaactacatgtcctacaactactgcgccgacggctggcgcttcccccagggcttccccgccgagtgctaccgcaagtgattttgaactcgatcgattcaaatcctcctccattgatgagttcttggcaatgatttgtaattgcttcttgttcttgttttcgtcttcgtcttcgtcttcttcttcttgatccatgtacattttgccatccattcgttctccatttgttacagttacagagacaggttgatggtgaattactattgccgccaatttttttcttcttcttgttactcgatccaattattactactagagttcatcttgcacagttgggcggtgtgaaaaactgaaaaagaaaagggtgtcaaatttgttgctgatgctgagcaagtgagcacatgcttatgctctcaggtatgaacaaaataaacagtaaaattatctgc</dnaseqindica> |
| External Link(s) |
