Comprehensive Analysis Of BES1 Gene Family And Functional Identification Of SlBES1.8 In Tomato

Brassinosteroid(BR)is a kind of steroidal hormone that is necessary for plant growth,and it is proved to involve in plant developmental and physiological processes,resistance to pathogens and acclimation to environmental stresses.The BR signal transduction pathway from the cell surface receptor kinase BRI1 to the BES1 family transcription factors has been illustrated.BES1 transcription factors regulate thousands of downstream target genes,which links BR signaling to various cellular,metabolic,and developmental processes.The related studies were mostly conducted in the model plant Arabidopsis,while the functions of BES1 transcription factors in tomato(Solanum lycopersicum),the most important model plant with fleshy fruit,are still waited to be discovered.In this paper,the comprehensive identification,characterization and expression analysis were performed for tomato BES1 family,including its bioinformatics information,protein properties,spatio-temporal expression profiles and responsiveness to plant hormones and stresses.According to the analysis,one of the tomato BES1 family member with potential important roles in tomato growth and development,SlBES1.8,was selected to be further characterized both in functions and mechanism.The main results are as follow:1.By searching the existence of BES1-type domain in tomato genome,9 BES1 family genes were identified in tomato,which were subsequently named as SlBES1.1 to SlBES1.9 according to their genomic locus.By analyzing the conserved amino acid residues in BES1-type domain,it was found that its N-terminal remained conserved at most of loci,while the C-terminal was less conserved.Phylogenetic and syntenic analysis revealed that BES1 family may play important roles to plant evolution.Gene structure and amino acids conserved motif of SlBES1 genes clustered in the same clade tended to share more similar pattern.Besides,lots of potential cis-elements related to plant development and phytohormone and stress responsiveness were found in SlBES1 gene promoters.By transiently expressing the SlBES1-GFP fused proteins,it was found that SlBES1.2,SlBES1.3,SlBES1.4,SlBES1.5,SlBES1.6,SlBES1.8 and SlBES1.9 localized both in the nucleus and cytoplasm,while SlBES1.1 and SlBES1.7 may localize in the endoplasmic reticulum.By detecting with the GAL4-responsive reporter system,SlBES1.3,SlBES1.4,SlBES1.5,SlBES1.6 and SlBES1.9 were proved to be transcriptional activators,and SlBES1.2 and SlBES1.8 were transcriptional repressors,while SlBES1.1 and SlBES1.7 had no transcriptional regulation ability.Spatio-temporal expression profiles revealed that most of SlBES1 genes expressed ubiquitously in all organs except SlBES1.8 that principally expressed in floral organs.Meanwhile,all SlBES1 genes could response to at least one kind of plant hormone,while the responsiveness to different plant hormone was distinguishing.What’s more,SlBES1 genes could be affected by multiple stresses,which principally exhibited the downregulated trend.2.Tissue expression profile detected by qRT-PCR and GUS staining showed that SlBES1.8 specifically expressed in shoot apical meristem(SAM)and floral organs.By performing immunoblot,SlBES1.8 was proved to be regulated by phosphorylation,and epi-brassinolide(EBL)treatment could transform phosphorylated SlBES1.8 into dephosphorylated form,suggesting that BR could also function through the way of SlBES1.8.By generating SlBES1.8 overexpression and knockout lines,it was found that SlBES1.8 could affect SAM size,floral organ number and fruit shape,and the functions of SlBES1.8 was unaffected by BR deficiency.The underlying regulatory mechanism of SlBES1.8 in regulating tomato SAM development was subsequently explored.Surprisingly,SlBES1.8 could not directly regulate the expression of CLV-WUS signaling related genes,while could physically interact with SlWUS protein,and the sequence between BES1-type domain and PEST domain of SlBES1.8 and the dimerization domain and EAR-like domain of SlWUS were critical for their interaction.Further experiments identified multiple SlWUS binding sites in CLV3 promoters,while the bindings could be abolished by SlBES1.8.Meanwhile,the transcriptional activation activity of SlWUS could also be repressed by SlBES1.8.Besides,RNA-sequencing determined lots of DEGs affected by SlBES1.8 that were also involved in meristem maturation and termination.Furthermore,SlWUS could bind to the promoters of some selected DEGs,and the bindings were inhibited in the presence of SlBES1.8.As a result,SlBES1.8 serves as a co-regulator of SlWUS in regulating SAM development.3.Overexpression of SlBES1.8 resulted in simpler leaf shape with smooth margin compared with wild type plants,which mimicked the effect of exogenous gibberellin(GA)application in simplifying tomato leaf complexity.Besides,OE-SlBES1.8 plants exhibited reduced sensibility to exogenous GA3 treatment whereas showed increased sensibility to the application of GA biosynthesis inhibitor,PAC.In line with the phenotypic observation,the endogenous bioactive GA contents were increased in OE-SlBES1.8 lines,which certainly promoted the degradation of the GA signaling negative regulator,SlDELLA.Moreover,transcriptomic analysis uncovered a set of overlapping genomic targets of SlBES1.8 and GA,and most of them were regulated in the same way.Further experiments determined the direct binding and repression of SlBES1.8 to the promoters of Sl GA2ox2,Sl GA2ox6,and Sl GID1b-1.Meanwhile,SlBES1.8 could physically interact with SlDELLA protein,and the PEST domain of SlBES1.8 and the DELLA motif of SlDELLA were critical for their interaction.As a result,SlDELLA interaction repressed the transcriptional regulation activity of SlBES1.8 by inhibiting its DNA binding ability.Conclusively,by mediating GA deactivation and signaling,SlBES1.8 greatly affected tomato leaf morphogenesis.In summary,this paper detailedly analyzed tomato BES1 gene family,providing valuable information for the functional characterization of this family.Furthermore,the important roles of SlBES1.8 in affecting tomato SAM development and leaf morphogenesis were determined,and the underlying regulatory mechanism was also elucidated.The outcome of this paper can not only lay the foundation for deciphering the regulation network and molecular mechanism of plant growth and development mediated by BES1 family,but also provide valuable genetic resources for molecular breeding.