|其他摘要||As the most abundant renewable resource on the earth, plant cell wall has numerous applications in various aspects of human living and industry servering as the main source of energy, fiber, building materials and papermaking materials. Thus, in-depth analysis of the composition, structure and biosynthesis mechanisms of plant cell wall, and optimization of the composition, structure and connections between different components by genetic engineering to improve the saccharification efficiency , is hot topics in the research of plant cell wall.
Arabidopsis thaliana seed coat cells synthesize and secrete large amounts of mucilage ploysaccarides (mainly composed of rhamnogalactoside I, RG I) to the extracellular at specific satges during the seed coat differentiation.Besides pectic RG I, mucilage also consists a small amount of cellulose and hemicellulose (e.g. heteroxylansand glactoglucomannan) components, thus mucilage represnet a specialized cell wall. Seed coat mucilage is not necessary for the germination or growth of plants, in addition, seed coat mucilage can be easily extracted and the defect phenotype can be readily identified, so it can be utilized as an ideal model system for the study of biosynthesis, modification and regulation of cell wall polysaccharides.
Previous study identified a transcription factor gene, HOMODOMAIN GLABROUS 2 (HDG2) . HDG2 belongs to homeobox subfamily HD-Zip IV. This study mainly focused on the mechanism of HDG2 in the regulation of seed coat mucilage structure.The main results are as follows: 1) Real-time quantitative PCR (qRT-PCR), in situ hybridization and promoter GUS activity analysis showed that HDG2 was specifically expressed in the seed coat, and its peak was observed at 10 days after pollination (10 DPA). 2) The transcriptional activation activity of HDG2 and its transcriptional activation domain was analyzed in yeast cell. The truncation test was performed according to the conserved domain of HDG2 protein (HD, LZ, START and SAD). The results indicated that HDG2 had transcriptional activation activity, and its transcriptional activation is located in the leucine zipper (LZ) domain. 3) Ruthenium red staining was carried out for T-DNA insertion mutants of HDG2(hdg2-2 and hdg2-3). The results showed that seed coat mucilage of hdg2 can be normally released, and no significant changes in total amount was observed. However, the mucilage layers of hdg2 was easily shaked off, leaving thinner adherent inner mucilage than that of the wild type (WT). 4) The hdg2 and WT seeds were dissected from 4 DPA-13 DPA and observed by resin embedding and slicing, the results showed that epidermal cells of hdg2-3 seed were normally developed and the secretion and the amount of mucilage displayed no significant differences compared to the WT. 5) The content, composition and structure of hdg2-3 mucilage was analyzed, and it was found that the total amount of hdg2-3 mucilage had no significant differences with that of the WT. The composition of monosaccharide showed that the contents of monosaccharides，which affect the mucilage structure (e.g. xylose, mannose) had no significant differences with the WT. 6) In situ immunohistochemical analysis showed that the crystalline cellulose content was significantly reduced in hdg2-3 compare with the WT, which was confirmed by the quantification of crystalline cellulose contents. Thus it can be conclueded that the decrease of crystalline cellulose led to the mucilage defect in hdg2-3. 7) To further confirm if HDG2 is involved in the modulation of crystalline cellulose, qRT-PCR analysis of hdg2 and WT seeds at different stages was performed. The results showed that CESA5, a key gene affecting cellulose synthesis in seed coat mucilage, was significantly decreased in hdg2-3. EMSA and yeast one hybridization analysis confirmed that HDG2 protein could bind to the L1-box motif located in CESA5 promoter. In addition, protoplast transcriptional activation assay showed that HDG2 could activate the expression of CESA5.
In summary, the molecular mechanisms of HDG2 transcription factor in the regulation of mucilage structure were characterzide in this study. A series of physiological and biochemical evidence suggested that HDG2 regulates the synthesis or assembly of cellulose by direct activation of CESA5 to maintain the normal structure of the seed coat mucilage. In contrast, HDG2 mutations inhibit the expression of CESA5, the synthesis or assembly of crystalline cellulose is blocked and finally showing a defective phenotype of the seed coat mucilage. The results obtained will help to deepen our understanding of the molecular mechanisms and regulation networks underlying cellulose synthesis and assembly, which lay a theoretical foundation for the future custom-design of the cell wall by genetic engineering means.|