Functional characteristics of genes involved in brassinosteroid signaling in cotton
Cotton fibers are highly elongated single celled trichomes that grow from the seed integument. Elongation of fiber cells begins almost after ovule fertilization and continues for approximately 20 days. In this study, I have shown that BR signaling is necessary for cotton fiber initiation and elongation, using in vitro cultured cotton ovule with Brassinosteroids (BR) and brassinazole (Brz, BR biosynthesis inhibitor). BRs are polyhydroxylated sterol derivatives of plant origin that are required for normal plant development. Several Arabidopsis genes that encode critical components of this pathway have been identified through genetic screening. BRM encodes a membrane-bound leucine-rich receptor-like kinase that apparently acts as the BR receptor. BIN2, which acts downstream of BRM in this pathway, encodes a GSK3/SHAGGY-like kinase that down-regulates BR signaling. To understand the role of cotton orthologous genes in fiber development, cotton ESTs similar to the Arabidopsis BRM and BIN2 genes were identified. These ESTs were used to clone the corresponding full-length GhBRM and GhBIN2 cDNAs. The GhBRM was cloned from a cotton cDNA library and then amplified from cotton genomic DNA. This 3561 bp gene contains no introns and encodes a protein with 1187 amino acids. Database analyses shows that the GhBRM protein has all the distinct domains characteristic of BRM. Four GhBIN2 cDNAs were also cloned. They all include a coding sequence of 1146 base pairs in length and encode derived proteins of 381 amino acids. Sequence comparison with mammalian GSK3p and Drosophila GSK3/SHAGGY-like kinase showed that GhBIN2 proteins share many conservative regions with these two GSK3/SHAGGY-like kinases. Analysis of the expression patterns of the GhBRM and GhBIN2s genes using quantitative real-time PCR showed that they are expressed throughout cotton plants, including leaves, buds, hypocotyls, roots, sepals, ovules, bolls, and fibers. To identify the functions of these genes, gene constructs that express GhBRM and GhBIN2 under control of a CaMV 35S promoter were developed. Expression of the GhBRM transgene in the dwarf bri1-5 mutant Arabidopsis plants restored them to normal height. Expression analysis showed that the heights of the transgenic plants were significantly correlated with the GhBRH expression level (r = 0.97). These results strongly suggest that the GhBRH gene encodes a functional BR receptor protein. Conversely, expression of the GhBIN2 transgene in wildtype Arabidopsis plants resulted in severe stunting similar to strong BR deficient or insensitive mutants. Expression analysis showed that the heights of the transgenic plants were inversely correlated with these GhBlN2 expression levels (the average correlation level r= -0.90). These results indicate that the GhBlN2 genes function as negative regulators of BR signal transduction pathway. These results confirm that the GhBRM and GhBIN2 cDNAs encode proteins that are capable of functioning in the BR signaling pathway. BR signal transduction pathway could provide the basis for genetic modification of fiber development.