Abstract
Brassinosteroids (BRs) are plant hormones that are perceived at the cell surface by a membrane-bound receptor kinase, BRASSINOSTEROID INSENSITIVE1 (BRI1). BRI1 interacts with BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) to initiate a signal transduction pathway in which autophosphorylation and transphosphorylation of BRI1 and BAK1, as well as phosphorylation of multiple downstream substrates, play critical roles. Detailed mechanisms of BR signaling have been examined in Arabidopsis (Arabidopsis thaliana), but the role of BRI1 and BAK1 phosphorylation in crop plants is unknown. As a foundation for understanding the mechanism of BR signaling in tomato (Solanum lycopersicum), we used liquid chromatography-tandem mass spectrometry to identify multiple in vitro phosphorylation sites of the tomato BRI1 and BAK1 cytoplasmic domains. Kinase assays showed that both tomato BRI1 and BAK1 are active in autophosphorylation as well as transphosphorylation of each other and specific peptide substrates with a defined sequence motif. Site-directed mutagenesis revealed that the highly conserved kinase domain activation loop residue threonine-1054 was essential for tomato BRI1 autophosphorylation and peptide substrate phosphorylation in vitro. Furthermore, analysis of transgenic lines expressing full-length tomato BRI1-Flag constructs in the weak tomato bri1 allele, curl3(-abs1), demonstrated that threonine-1054 is also essential for normal BRI1 signaling and tomato growth in planta. Finally, we cloned the tomato ortholog of TGF-β Receptor Interacting Protein (TRIP1), which was previously shown to be a BRI1-interacting protein and kinase domain substrate in Arabidopsis, and found that tomato TRIP1 is a substrate of both tomato BRI1 and BAK1 kinases in vitro.
Highlights
Brassinosteroids (BRs) are plant hormones that are perceived at the cell surface by a membrane-bound receptor kinase, BRASSINOSTEROID INSENSITIVE1 (BRI1)
We previously identified a BR-insensitive mutant in tomato, curl3, that is phenotypically very similar to the Arabidopsis bri1-1 mutant (Koka et al, 2000), and cloning the CU3 gene from tomato showed that it encoded tomato BRI1 (SlBRI1), which binds BR directly (Montoya et al, 2002; Holton et al, 2007)
The BRI1 constructs contain amino acids 824 to 1,207 of the previously published tomato BRI1 sequence (Montoya et al, 2002), while the cytoplasmic domain of maltosebinding protein (MBP)-SlBAK-CD shares 100% sequence identity with amino acids 254 to 617 of SlSERK3B, which has been shown by phylogenetic analysis to be the most likely tomato ortholog of Arabidopsis BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) (Mantelin et al, 2011)
Summary
Brassinosteroids (BRs) are plant hormones that are perceived at the cell surface by a membrane-bound receptor kinase, BRASSINOSTEROID INSENSITIVE1 (BRI1). Site-directed mutagenesis revealed that the highly conserved kinase domain activation loop residue threonine-1054 was essential for tomato BRI1 autophosphorylation and peptide substrate phosphorylation in vitro. To better understand the mechanism of receptor kinase action in BR signaling, a series of studies using Arabidopsis (Arabidopsis thaliana) identified specific sites of serine (Ser), threonine (Thr), and tyrosine (Tyr) phosphorylation in both BRI1 and BAK1 that are critical for kinase function and effective BR signal transduction in planta, and models of the BRI1-BAK1 interaction and transphosphorylation were presented (Oh et al, 2000, 2009b, 2010; Wang et al, 2005, 2008). A detailed analysis of BRI1 or BAK1 phosphorylation has not been reported in any plant other than Arabidopsis
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