OsMYB103L

OsMYB103L encodes an R2R3-MYB transcription factor, it influences leaf rolling and mechanical strength in rice (Oryza sativa L.).

Annotated information

Function

OsMYB103L, which id also called Os08g05520, encodes an R2R3-MYB transcription factor. OsMYB103L may target CESA genes for regulation of cellulose synthesis thus influences leaf shape and mechanical strength in rice, which makes it a potential engineering target for desirable leaf mechanical properties. OsMYB103L is localized in the nucleus and has transactivation activity. Overexpression of OsMYB103L in rice could result in a rolled leaf phenotype, and significantly higher levels of several cellulose synthase genes (CESAs), and a significant cellulose content increase. Knockdown of OsMYB103L by RNA interference could lead to a decreased level of cellulose content thus reduced mechanical strength in leaves. Meanwhile, the expression levels of the relevant CESA genes could also be decreased.

Structural and Homology Features

According to the rice genome annotation database ([1]), LOC_Os08g05520 encodes a putative R2R3- MYB family transcription factor with a length of 359 amino acids and a molecular mass of approximately 40 kD. The Pfam database ([2]) shows that the deduced protein has two MYB DNA-binding domains (PF00249) at the N-terminus. As revealed by phylogenetic analysis of the related MYB transcription factors in Arabidopsis thaliana and rice, Os08g05520 is closely related to At1g63910 (AtMYB103)[1]. Protein sequence alignment showed that they are highly conserved in the predicted R2- and R3-MYB DNA-binding domains. That's why Os08g05520 was designated as OsMYB103L (Oryza sativa MYB103 Like).

Expression

To determine the subcellular localization of its expression, researchers fused green fluorescent protein (GFP) to the C-terminus of OsMYB103L to produce an OsMYB103L-GFP fusion protein and monitored the fluorescence of the transiently expressed fusion protein in both rice protoplasts and onion (Allium cepa L.) epidermis cells. The results indicated that OsMYB103L is a nuclearlocalized protein, just like most transcription factors.

After determining the expression level of OsMYB103L in various rice organs using quantitative real-time PCR (qRT-PCR), it was found out that The expression level of OsMYB103L was highest in culm, lower in root, leaf and mature panicle, and lowest in young panicle.(see this fugure)

Also the examination of the expression pattern of OsMYB103L by the β-glucuronidase(GUS) reporter gene driven by OsMYB103L putative promoter in three independent transgenic lines showed that the GUS signals were stronger in culm and weaker in vascular bundles of coleoptile, root and leaf. Meanwhile, GUS staining was stronger in mature panicle than young panicle. Further examination showed that GUS activity was detectable in pedicel, lemma and palea while no GUS activity was detected in sterile lemma, lodicule, pistil, and stamen in mature panicle. These results indicate OsMYB103L's diverse expressions in various tissues and organs in rice.

The most significant phenotype of OsMYB103L overexpression(OE) lines was upward curling of the leaf blades. This phenotype appeared first at the seedling stage and then maintained through the rest of the plant growth (Figure B, see this fugure)

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The leaf blade cross-sections did not show significant differences between wild type (WT) and OE in terms of organization of the sclerenchyma and vascular tissues. It suggests that there is no obvious alteration of leaf polarity in transgenic plants. However, in contrast to WT, the leaves of transgenic plants displayed smaller bulliform cells in the rolled regions (Figure D, see this figure)

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Molecular Mechanism of OsMYB103L’s Effects

The expression profile in an OE line(OE-1) and WT leaf blades was examined using Digital Gene Expression profiling analysis (DGE). Subsequently, ontological terms were assigned and the enrichment significance was analyzed by AgriGO[2] and it turned out that 21 Gene Ontology (GO) terms were significantly enriched in the up-regulated gene set, showing that up-regulated genes were involved in multiple biological processes including metabolism, biogenesis, localization, and cellular processes (see this Figure A below).

Notably, cellulose metabolic process (GO:0030243) and cellulose biosynthetic process (GO:0030244) were possibly related to the leaf rolling trait as reported in rl14 mutant [3]. The comparison data between the expression levels of the rice CESA genes[4] showed that OsCESA1, OsCESA4, OsCESA7, OsCESA8, OsCESA9, and OsCESA11 were upregulated while OsCESA5 and OsCESA6 were down-regulated. Verification of the expression levels of these genes in three independent lines(OE-1, OE-4 and OE-8) through qRT-PCR showed that the transcription of four CESA genes, OsCESA4, OsCESA7, OsCESA8, and OsCESA9, was significantly increased while only OsCESA6 was downregulated(see this Figure B below).

CESAs are proteins responsible for cellulose synthesis in plants[4-6]. And the measuring results of cellulose content in leaf blades of WT and OE plants showed indeed that OE leaf blades had 13% higher cellulose content compared to WT. In summary, OsMYB103L regulates CESA gene expression and may consequently influence cellulose synthesis in rice.

RNAi-knockdown plants for OsMYB103L were created and the leaves appeared brittle in the heading stage although there were no obvious morphological alterations compared to wild type (see this Figure B below).

Results of cellulose content measurement showed that compared with WT, the levels of cellulose content in RNAi lines significantly decreased (see this Figure D below).

Therefore, the decreased mechanical strength of RNAi plants might result from the decreased cellulose content. qRT-PCR results also showed that three CESA genes, OsCESA4, OsCESA7, and OsCESA9 were significantly down-regulated in the RNAi lines (see this Figure E below).

In summary, the down-regulated CESA genes in OsMYB103L RNAi plants led to decrease in cellulose content and weakened mechanical strength of transgenic leaf blades.

References

1 Zhong R, Lee C, Zhou J, McCarthy RL, Ye ZH: A battery of transcription factors involved in the regulation of secondary cell wall biosynthesis in Arabidopsis. Plant Cell 2008, 20(10):2763–2782.

2 Du Z, Zhou X, Ling Y, Zhang Z, Su Z: agriGO: a GO analysis toolkit for the agricultural community. Nucleic Acids Res 2010, 38(Web Server issue):W64–W70.

3 Fang L, Zhao F, Cong Y, Sang X, Du Q, Wang D, Li Y, Ling Y, Yang Z, He G: Rollingleaf14 is a 2OG-Fe (II) oxygenase family protein that modulates rice leaf rolling by affecting secondary cell wall formation in leaves. Plant Biotechnol J 2012, 10(5):524–532.

4 Wang L, Guo K, Li Y, Tu Y, Hu H, Wang B, Cui X, Peng L: Expression profiling and integrative analysis of the CESA/CSL superfamily in rice. BMC Plant Biol 2010, 10:282.

5 Somerville C: Cellulose synthesis in higher plants. Annu Rev Cell Dev Biol 2006, 22:53–78.

6 Richmond T: Higher plant cellulose synthases. Genome Biol 2000, 1(4):REVIEWS3001.