The Mechanism Of Phosphorylation Of MDP40by BRI1Receptor Kinase Involved In Regulation Of Hypocotyl Cell Elongation In Arabidopsis

Brassinosteroids (BRs) phytohormones play crucial roles in mediating plant cell growth and morphogenesis. Microtubule cytoskeleton and microtubule regulatory proteins also participate in regulating plant cell growth and morphogenesis. The previous study showed that BRs mediated hypocotyl elongation through the microtubule cytoskeleton. We identified a Arabidopsis microtubule-destablizing protein40(MDP40) which was directly targeted by BZR1and involved in BR-mediating hypocotyl elongation on transcriptional level. Besides transcriptional level regulation, however, it is unclear if BRs can regulate microtubule regulatory proteins in other ways.In this study, MDP40was identified to be phosphorlated by BRs receptor kinase BRI1. Mass spectrometry analysis showed that BRI1phosphorylated MDP40at Ser-224and Thr-227. Overexpression of MDP40mutation mimicking constitutive phosphorylation (MDP40Ser224AspThr227Asp, abbreviated as MDP40DD), but not wild-type MDP40(wild-type MDP40, abbreviated as MDP40WT), partially rescued the shorter etiolated hypocotyl phenotype in a weak allele BRI1mutant (bril-5). The length of hypocotyl cells was increased in MDP40DD-GFP/bril-5transgenic seedlings, but not in the MDP40WT-GFP/bril-5transgenic seedlings. This result demonstrates that the phosphorylation sites in MDP40are essential for its full activity in vivo.Transiently co-expressed with the microtubule maker gene MBD-mCherry, both MDP40DD-GFP and MDP40WT-GFP co-localized with cortical microtubules in the cells of bril-5mutant, demonstrating that BRI1phosphorylation of MDP40does not alter its localization with cortical microtubules in vivo. The organization and stabilization of cortical microtubules were altered in the etiolated hypocotyl epidermal cells from MDP40DD/bril-5seedlings, comparing with that of bril-5mutant or MDP0WT/bril-5seedlings. Parallel arrays of cortical microtubules were generally transversely oriented to the longitudinal hypocotyl growth axis in the epidermal cells of the upper and middle regions from the etiolated hypocotyls of MDP40DD/bril-5mutant, which is consistent with the significantly promoting etiolated hypocotyl cell elongation and increased expression of MDP0DD. Pharmacological assay using microtubule-disrupting drug oryzalin showed that cortical microtubules were more destabilized in MDP40DD/bril-5hypocotyl epidermal cells.These results demonstrate that phosphorylation of MDP40by BRI1is essential in BRs regulation of microtubules through MDP40to mediate hypocotyl elongation. This study reveals a mechanism involving BRI1regulation of microtubules through phosphorylation of MDP40to mediate hypocotyl cell elongation.