Phosphorylation Modulates The Activity Of HLH Transcription Factor PRE1

Cell elongation in higher plants is regulated by mutiple hormonal and enviromental signals,including particularly auxin,gibberellin,brassinosteroid,ethylene,light and temperature.Paclobutrazol-Resistant(PRE)family helix-loop-helix(HLH)factors were reported to integrated diverse signals to promote cell elongation,but the mechanism by which hormonal and environmental signals regulate PREs activity in the post-transcriptional level has remained unknown.To reveal whether PRE family factors have modification at protein level,we search the online proteomic database,and found PRE proteins exhibit phosphorylation and de-phosphorylation states in two conserved Ser residues.To analyze how phosphorylation regulates the activity of PREs,Ser46 and Ser67 of PRE1 were substituted with alanien acid(PRE1S46A and PRE1S67A)or glutamic acid(PRE1S46E and PRE1S67E)to mimic the states of constitutive de-phosphorylation and phosphorylation,respectively.We found that overexpression of PRE1S46A,PRE1S46E and PRE1S67A caused long-hypocotyl phenotypes,which were similar to PRE1 overexpressing transgenic plants.Whereas overexpression of PRE1S67E showed weak cell elongation phenotype,indicating phosphorylation at Ser67 repressed the PRE1 function in the promotion of cell elongation.Further studies showed that phosphorylation had no effect on the subcellular location of PRE1,but phosphomimic PRE1S67E had low binding ability to IBH1 that antagonistically interact with PREs to regulate cell elongation.To identify the protein kinase that phosphorylate PRE1,Y2H,IP-MS and Co-expression methods were adopted.We choose nine candidate kinases and construct vectors to test whether these protein kinases interact with PRE1 by Y2H and BiFC.We also construct the gain of function and loss of function transgenic plants.In this study we prove that Ser67 is a key phosphorylation site of PRE1 and it can repress the binding ability with other proteins.Moreover,we screen several protein kinases that may phosphorylate PRE1,and we also do some researches about these kinases.This study established strong foundation for us to explore the function of PRE1.At the same time,this will help us to clarify how endogenous and exogenous signals regulate plant growth by control kinase activity and provide a strong basic theory for the improvement of crop yield and quality.