Effects Of SlBRI1 Phosphorylation Sites Thr-848,Thr-825 And Thr-1044 On Tomato Growth And Development And Regulation Mechanism Research

Tomato is an important vegetable crop.As a vital plant hormone,Brassinosteroids（BRs）promote the growth and development of tomato and improve stress resistance.BRI1（BRASSINOSTEROID INSENSITIVE 1）,as a receptor kinase of BR signaling,received and transmitted BR signal through a series of phosphorylated cascade,thus regulating plant growth and development.Although individual phosphorylation sites of TOMATO SlBRI1have been studied,however,the research is not sufficient,and the biological functions and agronomic values of a large number of important phosphorylation sites remain to be studied.In this study,SlBRI1 phosphorylation sites Thr-848,Thr-825 and Thr-1044 were focused as research objects.Through the combination of autophosphorylation level analysis in vitro,hypocotyl sensitivity test to exogenous hormone application and BR signal intensity detection,botany and agronomic traits investigation for transgenic plants expressing SlBRI1 with simulated non-phosphorylation site,etc.To investigate the effects of SlBRI1 phosphorylation sites Thr-848,Thr-825 and Thr-1044 on SlBRI1 kinase activity,BR signal transduction,tomato growth and development,and regulation mechanism.The main research results are as follows:（1）The phosphorylation of SlBRI1 T848 site has a negative regulatory role on kinase phosphorylation and activity,and BR signaling of transgenic plants expressing SlBRI1 with a non-phosphorylation T848（T848A）was enhanced.SlBRI1 phosphorylation site Thr-848was site-specific mutated into Ala to simulate non-phosphorylation state.The autophosphorylation levels of SlBRI1 kinase region,which were expressed in Escherichia coli,were detected in vitro.The result showed that BR signaling was enhanced.Transgenic plants expressing SlBRI1 with T848A site mutation gene were obtained by genetic transformation into tomato BRI1 weak mutant cu3^-abs1.Wild-type SlBRI1 transgenic plants and the background material SlBRI1 weak mutant cu3^-abs1 as positive and negative control,respectively.The m RNA transcription levels of endogenous SlBRI1 and Sl CPD were detected.The results showed that BR signaling in T848A and wild-type SlBRI1 transgenic lines were both stronger than that in cu3^-abs1 lines,and restoring the BR signaling pathway of cu3^-abs1 lines.This is consistent with the results of BR signaling detection by hypocotyl sensitivity test to exogenous hormones.（2）Transgenic plants expressing SlBRI1 with a non-phosphorylation T848 had no significantly effect on the plant morphological traits,fruit traits and fruit quality of tomato plants,but the non-phosphorylation site T848 will significantly affect flower organ development at late growth stage of tomato,and produced a large number of long style flowers.The agronomic traits of tomato T848A transgenic lines were investigated.The results showed that T848A transgenic lines could partly rescue the BR-deficient phenotype of weak mutant cu3^-abs1,but there were no significant differences in plant height,stem diameter,internodal length,plant expansion and stem-leaf angle between T848A transgenic lines and wild-type SlBRI1 transgenic lines.Compared with wild-type SlBRI1 and cu3^-abs1lines,T848A transgenic lines increased the flower numbers of the fourth to the fifth spike and elongated pistil,but no significant on stamen growth.The flowers showed elongated styles,which disturbed pollination and resulted in lower yield.At the same time,compared with wild-type SlBRI1 transgenic lines,the fruit of T848A transgenic lines became harder,but the pericarp was thinner,and the difference in fruit shape index,fruit weight and fruit quality was not significant,indicating that the difference in fruit weight had little effect on yield reduction.（3）The unique mechanism by which the T825 phosphorylation site interacts with GA signaling may contribute to improving tomato yield by regulating phosphorylation at this site.The phenotype of fruit enlargement and yield increase for T825A transgenic lines were caused by the increased BR signaling on early fruit development,which regulated the expression of genes related to fruit development.Meanwhile,it inhibited the expression of Sl DELLA,led to enhance GA signaling,and encouraged cell division and enlargement,and enlarged ovary wall cells sizes,resulting in larger fruit.In addition,T825A can promote the seed germination and improve seed vigor.（4）Both phosphorylation and non-phosphorylation of SlBRI1 phosphorylation site T1044 significantly reduced phosphorylation of SlBRI1.Transgenic plants expressing SlBRI1 with a non-phosphorylation T1044 rescueed the phenotype of cu3^-abs1 lines,but significantly decreased BR signaling than transgenic plants expressing wild-type SlBRI1.In conclusion,different phosphorylation sites have unique biological functions and agronomic values for tomato growth and development.The study on the function of phosphorylation sites on the growth and development and regulation mechanism laid the foundation for the establishment of excellent SlBRI1 allele in tomato,provided a basis for tomato molecular breeding,and provided reference for the gene modification to improve agronomic traits through molecular technology in other crops and genes.