Effects Of SlBRI1 Phosphorylation Site Thr-1050 And Downstream Components On Growth And Development In Tomato

Brassinolides?BRs?are important endogenous plant hormones,which play a vital role in plant growth and development.Tomato is one of the most widely planted vegetable crops in the world.Appropriate brassinosteroid?BR?signal strength caused by the exogenous application or endogenous regulation of BR-related genes can promote the formation of tomato agronomic traits.Previous study demonstrated that modifying BRI1phosphorylation sites could lead to various BR signaling strengths and botanical characteristics in Arabidopsis.As an important horticultural crop,only a few tomato BR signaling components were studied,such as Sl DWARF,Sl BRI1,Sl SERK3A,Sl SERK3B and Sl BZR1.To further to elucidate the effect of BR signaling to agronomic characters of tomato,the phosphorylation site threonine-1050 of Sl BRI1 was first used as the research object and tested from several aspects such as BRI1 autophosphorylation level,the strength of BR signaling,the response of plant hormone,agronomic traits in the vegetative and reproductive period.Next,Sl BIN2.1,Sl BIN2.2,and Sl BIN2.3,which are homologous genes of At BIN2 in tomato,were cloned,and their functions as negative regulators of BR signaling were clarified in Arabidopsis and tomato.Finally,the Sl BIN2.1 downstream substrate Sl BIML1 was screened,and its biological function was preliminary studied.The main results as follows.1. The Sl BRI1 phosphorylation site Thr-1050 was taken as the research object and was simulated in phosphorylated and dephosphorylated states by site-directed mutation.The mimic dephosphorylation of Thr-1050 can increase the autophosphorylation level of Sl BRI1 in vitro and in vivo,which indicated that the Thr-1050 negatively regulates the autophosphorylation level of Sl BRI1.As a Sl BRI1 weak mutant,BR signaling deficiency phenotype of cu3^-abs1 can restore by Sl BRI1 or T1050A overexpression.Based on the evaluation of the hypocotyl length in dark,BR content,the expression level of BR signaling marker genes Sl CPD and Sl DWARF in T1050A,Sl BRI1-1 and cu3^-abs1,we conclude that both T1050A and Sl BRI1-1 can recover the BR signaling of cu3^-abs1,and the BR signaling strength of T1050A is stronger than Sl BRI1-1.It was revealed that the Thr-1050 is a negative regulatory site of Sl BRI1 mediated BR signal transmission.The agronomic character statistics show that plant height,stem diameter,and internodal distance were similar between the transgenic plants,but cu3^-abs1 harboring T1050A promoting yield via increased plant expansion,leaf area,fruit weight and fruit number per cluster but reducing the nutrients,including ascorbic acid and soluble sugars.Our results reveal the phosphorylation site Thr-1050 has a negative regulatory function in formation of yield traits in tomato,and its regulatory of tomato quality is different from Sl BRI1.2. The homolog gene of At BIN2 in tomato,named Sl BIN2.1?Solyc02g072300?,Sl BIN2.2?Sl SK?and Sl BIN2.3?Solyc03g006070?were cloned through homology cloning technique,all of which are evolutionally on the GSK3 class protein Clad II subgroup.The subcellular localization and kinase activity assays showed that Sl BIN2s are cytoplasm and nucleus-localized kinase.Heterologous expression of Sl BIN2s can enhance the defective phenotype of bri1-5 by inhibiting BR signaling.According to the phenotype and the expression level of BR signaling marker genes Sl CPD and Sl DWF in Sl BIN2s overexpressing lines and knockout lines,the result revealed that Sl BIN2s play negative regulatory functions in tomato BR signaling,and have functional redundancy.In addition,Sl BIN2s has balanced regulation in tomato,which leads to the inhibition of the growth and development of Sl BIN2s-overexpressing lines and triple mutant slbin2-t.3. Sl BIML1,a G2-like transcription factor homologous to At EFM,was screened by yeast library.Sl BIML1 has a conserved Myb domain and is shown to be distributed in both nucleus and cytoplasm.Promoter analysis showed that Sl BIML1 was mainly expressed in the vascular system of tomato,and can be induced by BL and nitrate nitrogen.Yeast two-hybrid,GST pull-down and Bi FC demonstrated that Sl BIML1 interacts with Sl BIN2.1in vivo and in vivo,and its'phosphorylated by Sl BIN2.1.According to the phenotype and the expression level of BR signaling marker genes Sl CPD and Sl DWF in Sl BIML1-overexpressing and knockout lines,the conclude that Sl BIML1 has no effect the BR signaling pathway and have a balance regulation in tomato cv.Micro-Tom.Moreover,hypocotyl length of Sl BIML1-overexpressing lines was promoted by cell elongation.Sl BIML1 knocout line showed low chlorophyll content and apical necrosis.Genetic experiment confirmed that Sl BIN2.3-overexpressing could partially restore the phenotype of slbiml1 chlorophyll deficiency and completely suppress apical necrosis.Besides,20°C culture could partially suppress apical necrosis of slbiml1.In conclusion,our study revealed that all of the phosphorylation site Thr-1050 of Sl BRI1,the negative regulator of BR signaling Sl BIN2s and its downstream transcription factor Sl BIML1 play important roles in BR signaling-mediated growth,development and agronomic trait formationcan in tomato.