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

Brassinosteroids (BRs) are essential plant hormones required for regulating numerous aspects of plant growth and development. BR-deficient and BR-insensitive mutants displaying a characteristic dwarf phenotype were instrumental in understanding the molecular mechanisms of BR biosynthesis and signaling in the experimental plant Arabidopsis thaliana. Isolation of BR-mutants from tomato, rice, barley and pea, expands the importance of these compounds from a model system to crops and the detailed genetic map, EST sequence database and relatively high transformation efficiency of tomato makes this plant an excellent model system for studying the molecular biology of horticultural crops. Our experiments examined the conservation in BR signaling mechanisms between Arabidopsis and tomato.;In Arabidopsis, BRs are perceived at the cell surface by a membrane bound leucine-rich repeat receptor-like kinase, BRASSINOSTEROID INSENSITIVE 1 (BRI1). A second LRR-RLK known as BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) interacts with BRI1 in vivo, suggesting an important role of receptor kinase heterodimerization and transphosphorylation in BR signaling. The cytoplasmic kinase domains (CDs) of tomato and Arabidopsis BRI1 and BAK1 show high sequence similarity with conservation of numerous Ser and Thr residues. Kinase assays of affinity purified CDs of tomato BRI1 (tBRI1-CD) and BAK1 (tBAK1-CD) revealed that both kinases autophosphorylate independently, and can also transphosphorylate each other. Tomato BRI-CD also phosphorylates peptide substrates with a similar sequence motif required for optimal phosphorylation by Arabidopsis BRI1. We further determined specific phosphorylation sites of tBRI1-CD and tBAK1-CD using liquid chromatography-tandem mass spectrometry (LC/MS/MS and/or LC/MSE) and were able to identify 12 in vitro phosphorylation sites in tBRI1 and seven in tBAK1. Interestingly, 10 of the tBRI1 sites and five of the tBAK1 sites were conserved in Arabidopsis, but two sites each of tBRI1 and tBAK1 were not, suggesting significant conservation but also possible differences in BR signaling events between these species.;Site-directed mutagenesis of identified and predicted tBRI1 phosphorylation sites revealed that phosphorylation of highly conserved residues in the kinase domain activation loop of tBRI1, was essential for kinase function in vitro. Furthermore, mutations in specific juxtamembrane residues to their unphosphorylared counterparts, increased tBRI1 kinase activity. Analysis of tomato transgenic lines expressing full-length 35S::tBRI1-Flag wild-type and mutated constructs in the weak tomato bri1 allele, curl3-abs1, showed that the highly conserved residue T-1054 is essential for normal BRI1 signaling in planta. We attempted to identify in vivo tBRI1 phosphorylation sites using transgenic tomato lines expressing full-length tBRI1-Flag, and while we did find peptides for tBR1-FLAG and its in vivo interacting proteins by LC/MSE analysis, no in vivo phosphorylation sites of tBRI1 have been found.;Finally, we cloned the tomato ortholog of Transforming Growth Factor-beta Interacting Protein TRIP1 (tTRIP1), which was previously shown to be a BRI1 interacting protein and kinase domain substrate in Arabidopsis. In vitro kinase assays demonstrated that tTRIP-1 is a substrate of tBRI1 and tBAK1 kinases and that tBRI1 can phosphorylate the same TRIP1 peptide substrates recognized by Arabidopsis BRI1. Tomato plants constitutively overexpressing tTRIP1 (containing N and C terminal epitope tags) were also generated for future studies of in vivo function of TRIP1 in tomato.;In summary, we examined early events in tomato BR signal transduction mechanisms using molecular genetics, proteomics, kinase biochemistry and mass spectrometry. The comparative functional analysis of tomato BRI1 and BAK1 kinase activity and phosphorylation sites with their Arabidopsis counterparts revealed significant inter-species conservation but also possible divergence in receptor kinase mechanisms involved in BR signaling regulating plant growth and development.