Functional characteristics of genes involved in brassinosteroid signaling in cotton

Brassinosteroids (BR) 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. In this study, cotton ovule culture with BR and BR biosynthesis inhibitor brassinazole has shown that BR signaling is necessary for cotton fiber initiation and elongation. The GhBRI1 cDNA was cloned from a cotton cDNA library. This 3561 by gene contains no introns and encodes a protein of 1187 amino acids. Database analyses showed that the predicted GhBRI1 protein has all the distinct domains characteristic of BRI1. Four GhBIN2 cDNAs were also cloned. They are GhBIN2-B, GhBIN2-C, GhBIN2-D, and GhBIN2-E. They are all 1146 base pairs encoding derived proteins of 381 amino acids. Sequence comparison with mammalian GSK3beta and Drosophila SHAGGY protein kinase showed that GhBIN2s share conservative regions to these two GSK3/SHAGGY-like kinases. Analysis of the expression patterns of the GhBRI1 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 GhBRI1 and GhBIN2 under control of a CaMV 35S promoter were developed. Expression of the GhBRI1 transgene in the dwarf bri1--5 mutant Arabidopsis plants restored them to normal height. The expression analysis showed that the heights of the transgenic plants correlated strongly (r = 0.9655) with the GhBRI1 expression level. These results strongly suggest that the GhBRI1 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 correlated with the GhBIN2 expression levels (the average correlation level r = -0.9025). These results indicate that the GhBIN2 genes function as negative regulators of BR signal transduction pathway. These results confirm that the GhBRI1 and GhBIN2 cDNAs encode proteins that are capable of functioning in the BR signaling pathway.