Positional Cloning And Functional Study Of Brittle Sheath Gene BSH1 In Rice (Oryza Sativa L.)

Plant mechanical strength is an important agronomic trait to be considered in plant breeding program and crop production practice. Plant cell wall constitutes the skeletal structure and determines the mechanical strength of the plant body. Higher plants develop two different types of cell wall, i.e. primary and secondary cell wall. Secondary cell wall is formed inside of primary cell wall after the cessation of cell, and develops particularly in sclerenchyma tissues and xylem elements. In-depth studies of the genes functioning in cell wall biosynthesis and assembly would be beneficial to uncover the underlying mechanisms of mechanical strength formation. In our study, we identified a dwarf and brittle sheath, bsh1, in rice and isolated BSH1 via map-based strategy. Furthermore, the biological function of BSH1 was analyzed by molecular biology experiments. The main results were as follows: 1. Phenotypic characterization of the brittle sheath1 mutantCompared with the wild type, the phenotype of matured mutant plant showed a significant reduction in plant height, 1000-grain weight and yield per plant. The most obvious phenotype different from wild type plants was the brittle sheath observed at 6-week-old seedlings. To understand the alterations in cell wall structure and thickness responsible for the brittle phenotype, we next observed the sclerenchyma cell wall of sheath by TEM. The cell wall of the sclerenchyma tissues in bsh1 is apparently thinner than that in wild type. We further analyzed the cell wall composition of sheaths. The main components of cell wall were reduced in different degrees. 2. BSH1 encoding a cytochrome P450 CYP96B4The locus was delimited in the 28 kb DNA region by map-based cloning. Sequence analysis revealed an insertion of unidentified length in the promoter region of CYP96B4 in bsh1. As such, a dramatic reduction of CYP96B4 expression level was found in bsh1. Thus, we considered CYP96B4 as the candidate of BSH1. The transgenic plants with elevated expression level of CYP96B4 showed similar phenotypes to wild type, whereas the transgenic plants carrying an RNA interference vector displayed similar phenotypes in correlation with reduced expression level of CYP96B4. Therefore, these results confirm that CYP96B4 is BSH1 resulting in dwarf and brittle sheath phenotype in bsh1. 3. The spatio-temporal expression pattern of BSH1 geneThe BSH1-GFP fusion protein was co-localized with an indicator of endoplasmic reticulum membrane suggesting that BSH1 is targeted to endoplasmic reticulum membrane. Quantitative PCR analysis revealed that BSH1 was expressed in all tested tissues, with the highest level in mature sheath and the lowest in root. The GUS activity was ubiquitously detected in the coleoptile, sheath, culm, leave, root and panicle of transgenic lines. In the seedlings, GUS active straining was found in the root hair zone, coleoptile, unexpanded leaf and young sheath. Cross-sections of coleoptile, leaf and sheath showed that expression of BSH1 was preferable in vascular tissues of coleoptile and leaf, and specifically in the cells surrounding vascular tissues of sheath. The blue band in root exactly overlapped with the sclerenchyma cells. Moreover, at the end of the elongation process, GUS activity was detected in the tender part of the first internode. GUS activity was also observed in mature organs, such as flower branches and glumes, and as the glume was getting older, staining was extended to the entire glume. 4. Function analysis of BSH1The CYP96 members were categorized into two distinct groups, and in particular with monocot Os CYP96B4 in one group and dicot At CYP96A15 in another. The reported CYP96 s are functional diversity, including hydroxylation of alkane and resistance to disease. However, most members of CYP96 are function-unknown.We further investigated whether there was any change in fatty acid content of the bsh1 plants. The measurement indicated that the cuticular wax content was reduced from 950.70 μg/g in wild type to 393.16 μg/g in bsh1 sheaths. In our more detailed measurements, all individual wax constituents of the bsh1 sheath, including C30-C34 aldehydes, C29-C41 alkanes, C22-C32 fatty acids and C26-C30 primary alcohols, were reduced remarkably relative to those of wild type. Analysis of cutin, internal lipids and seed lipids showed similar level changes between bsh1 and wild type. The results suggest that BSH1 controls the biosynthesis of VLCFAs and their derivatives in wax.To investigate mechanisms of BSH1 in regulating cell wall formation, we subsequently analyzed expression level of genes related to the biosynthesis of cell wall. Almost every gene we detected was reduced remarkably,supporting that the formation of cell wall is abnormal in bsh1, caused by lowered expression level of BSH1. However, the function of very long chain fatty acids in regulating cell wall formation is unclear.