Comparative Analysis of Early Events In Tomato and Arabidopsis Brassinosteroid Signal Transduction

Brassinosteroids (BRs) are essential plant hormones that regulate multiple aspects of plant growth and development. The identification of BR biosynthetic and insensitive mutants in tomato, rice, barley and pea, clearly extend the importance of these compounds from the experimental plant Arabidopsis thaliana to crop plants. While Arabidopsis is still the best model system for proteomic studies (which require a completely sequenced genome for optimum experimental efficiency), tomato is rapidly becoming an excellent model system for molecular studies of horticultural crops with an extensive genetic map, EST sequence database and a relatively efficient procedure for tomato transformation. Our experiments examined the conservation of molecular mechanisms between BR signaling in Arabidopsis and tomato. BRs are perceived at the cell surface by BRASSINOSTEROID INSENSITIVE 1 (BRI1) a member of the large family of leucine-rich repeat receptor-like kinases found in plants. The cytoplasmic kinase domains of tomato and Arabidopsis BRI1 are highly conserved (82% identical) and many of the Ser and Thr residues in Arabidopsis BRI1 occur in the same relative position in tomato BRI1, based on sequence alignment. We expressed the cytoplasmic kinase domain of tomato BRI1 in E. coli, autophosphorylated the recombinant protein and determined the phosphorylation sites in tryptic peptides using a Nano Acuity Premier Q-ToF liquid chromatography-tandem mass spectrometry (LC/MS/MS) system. We identified 8 in vitro phosphorylation sites in tomato BRI1  compared to the 11 sites we previously identified in Arabidopsis. Interestingly, five of the tomato sites were conserved in Arabidopsis, but three were not, suggesting significant conservation but also possible differences in BRI1 downstream signaling between the two species. We also have bulked seeds of an advanced transgenic tomato line expressing full-length tomato BRI1-Flag and have begun in vivo phosphorylation site analysis by immunoprecipitation of BR-treated tissue followed by LC/MS/MS analysis. The successful identification of tomato BRI1 phosphorylation sites has allowed us to initiate a comparative functional analysis of BRI1 signaling in tomato and Arabidopsis in order to determine the inter-species conservation and divergence of receptor kinase mechanisms involved in BR signaling regulating plant growth.