|其他摘要||Xylans are the second most abundant polysaccharides in nature, and represent a major hemicellulosic component in secondary cell walls of dicots and all walls of grasses. Xylans consist of a linear polymer of β-(1-4)-linked xylose backbone and substituted with acetyl, glucuronic acid (GlcA), 4-O-methylglucuronic acid (Me-GlcA), and arabinose residues. Dicot xylans contain the tetrasaccharide 4-β-D-Xyl-(1–4)-β-D-Xyl-(1–3)-α-L-Rha-(1–2)-α-D-GalA-(1–4)-D-Xyl at their reducing ends. In the model plant Arabidopsis, much progress has been gained in xylan biosynthesis especially in the studies utilizing the irregular xylem (irx) mutants. Irregular Xylem 9 (IRX9)/9L and IRX14/14L from glycosyltransferase (GT) family 43, which form two functionally nonredundant groups, have been proved to play crucial roles in xylan backbone elongation. However, xylan biosynthesis in grasses especially in Miscanthus is poorly understood. Miscanthus is a perennial lignocellulosic herbaceous energy plant with superior characteristics such as high photosynthetic efficiency, low fertilizer and water demand, wide adaptability and high biomass yield. It has attracted much concern worldwide as a superior lignocellulosic feedstock for next-generation bioethanol production. Xylans are the main source of feedstock for next-gerneration biofuel production, thus it is of important theoretical value to gain a complete understanding of the biochemical mechanism underlying xylan biosynthesis and modification, which may lay the foundation for future genetic modification of lignocellulosic biomass to be better tailored for various economically important applications, including the more efficient utilization of xylan for biofuel production.
In this study, we identified seven putative GT43 genes from M. lutarioriparius, a promising bioenergy crop, in-depth studies indicated that they are functional orthologues of Arabidopsis IRX9 or IRX14. Firstly, Phylogenetic analysis of GT43 proteins from nine representative plant species and Miscanthus revealed that these proteins were classified into three major clades, namely IRX9, IRX9L and IRX14/IRX14L. Quantitative real-time RT-PCR analysis revealed that all MlGT43 genes exhibited broad expression patterns across the tissues examined. mRNA in situ hybridization experiment unambiguously demonstrated that MlGT43A-B and MlGT43F-G were preferentially expressed in cells undergoing secondary wall thickening, while MlGT43C-E were expressed in both sclerenchyma and parenchyma cells. Subcellular localization analysis indicated all seven MlGT43 proteins were localized to Golgi apparatus. Overexpression of MlGT43A-E genes but not MlGT43F and MlGT43G in Arabidopsis irx9 fully or partially rescued the mutant defects, including plant height, irregular xylem cells in stem cross sections and xylose contents, whereas overexpression of MlGT43F and MlGT43G but not MlGT43A-E complemented the defects of irx14. These results suggest that MlGT43 genes have evolved into two distinct functional groups, with MlGT43A-E are orthologous to IRX9, while MlGT43F and MlGT43G are orthologous to IRX14. Nevertheless, the functional divergence of IRX14 orthologues in M. lutarioriparius has occurred as none of MlGT43 genes could rescue the mucilage defects of irx14 seeds, suggesting that MlGT43 could not synthesize the xylan which is involved in maintaining the structure of seed coat mucilage. Furthermore, transient transactivation analyses of MlGT43A-E reporters demonstrated that MlGT43A and MlGT43B were differentially activated by MlSND1, MlMYB46 or MlVND7 orthologues, three key transcriptional switches governing secondary cell wall biosynthesis. By contrast, the Miscanthus IRX9L orthologues (MlGT43C-E) were not significantly transactivated by these transcription factors.
Our results provide the first line of genetic evidence demonstrating that Miscanthus has evolved to retain two functionally non-redundant groups of MlGT43 genes involved in xylan biosynthesis but diverged in seed coat mucilage biosynthesis. This study further deepen our understanding of xylan biosynthesis, and lay a foundation for future genetic modification the composition and structure of grass xylan.|