Plant glycoside hydrolase family 9 (GH9) comprises typical endo-β-1,4-glucanase (EGases, EC3.2.1.4). Although GH9A (KORRIGAN) family genes have been reported to be involved in cellulose biosynthesis in plants, much remains unknown about other GH9 subclasses. In this study, we observed a global gene co-expression profiling and conducted a correlation analysis between OsGH9 and OsCESA among 66 tissues covering most periods of life cycles in 2 rice varieties. Our results showed that OsGH9A3 and B5 possessed an extremely high co-expression with OsCESA1, 3, and 8 typical for cellulose biosynthesis in rice. Using two distinct rice non-GH9 mutants and wild type, we performed integrative analysis of gene expression level by qRT-PCR, cellulase activities in situ and in vitro, and lignocellulose crystallinity index (CrI) in four internodes of stem tissues. For the first time, OsGH9B1, 3, and 16 were characterized with the potential role in lignocellulose crystallinity alteration in rice, whereas OsGH9A3 and B5 were suggested for cellulose biosynthesis. In addition, phylogenetic analysis and gene co-expression comparison revealed GH9 function similarity in Arabidopsis and rice. Hence, the data can provide insights into GH9 function in plants and offer the potential strategy for genetic manipulation of plant cell wall using the five aforementioned novel OsGH9 genes.

Background

The cellulose synthase and cellulose synthase-like gene superfamily (CESA/CSL) is proposed to encode enzymes for cellulose and non-cellulosic matrix polysaccharide synthesis in plants. Although the rice (Oryza sativa L.) genome has been sequenced for a few years, the global expression profiling patterns and functions of the OsCESA/CSL superfamily remain largely unknown.

Results

A total of 45 identified members of OsCESA/CSL were classified into two clusters based on phylogeny and motif constitution. Duplication events contributed largely to the expansion of this superfamily, with Cluster I and II mainly attributed to tandem and segmental duplication, respectively. With microarray data of 33 tissue samples covering the entire life cycle of rice, fairly high OsCESA gene expression and rather variable OsCSL expression were observed. While some members from each CSL family (A1, C9, D2, E1, F6 and H1) were expressed in all tissues examined, many of OsCSL genes were expressed in specific tissues (stamen and radicles). The expression pattern of OsCESA/CSL and OsBC1L which extensively co-expressed with OsCESA/CSL can be divided into three major groups with ten subgroups, each showing a distinct co-expression in tissues representing typically distinct cell wall constitutions. In particular, OsCESA1, -3 & -8 and OsCESA4, -7 & -9 were strongly co-expressed in tissues typical of primary and secondary cell walls, suggesting that they form as a cellulose synthase complex; these results are similar to the findings in Arabidopsis. OsCESA5/OsCESA6 is likely partially redundant with OsCESA3 for OsCESA complex organization in the specific tissues (plumule and radicle). Moreover, the phylogenetic comparison in rice, Arabidopsis and other species can provide clues for the prediction of orthologous gene expression patterns.

Conclusions

The study characterized the CESA/CSL of rice using an integrated approach comprised of phylogeny, transcriptional profiling and co-expression analyses. These investigations revealed very useful clues on the major roles of CESA/CSL, their potentially functional complement and their associations for appropriate cell wall synthesis in higher plants.