Functional Study Of SlBES1.8 In Regulating Tomato Shoot Apical Meristem Development And Fruit Formation

Plant growth and development is a complex and delicate process.To study the regulatory mechanism in plant growth and development especially in fruit formation can provide important clues for species improvement by using the model plant tomato(Solanum lycopersicum)as the material.As the key regulator in BR signal transduction pathway,BES1/BZR1 family transcription factors are involved in many plant process during growth and development and stress responses.While most of previous studies were performed based on Arabidopsis,and there was no report in regard to the regulation of shoot apical meristem(SAM)and fruit development mediated by this gene family.In this study,a tomato BES1/BZR1 transcription factor,SlBES1.8,is characterized.The function in regulating the development of SAM,floral organ and fruit formation is firstly studied,as well as the analysis of functional mechanism.The obtained results are showed as follow:(1)By amino acid alignment,several amino acid fragments are lacked in SlBES1.8compared with BES1/BZR1,but the characteristic regions of BES1/BZR1 gene family are retained in SlBES1.8,like BES1-type region,PEST motif,and EAR motif.Besides,the cytoplasmic and nuclear subcellular localization are observed for SlBES1.8 just like the same results for BES1/BZR1,thus it can be speculated that SlBES1.8 also presents the dephosphorylated and phosphorylated states and may has the same phosphorylation mechanism.From the analysis of the transactivation activity in yeast and dual-luciferase assay,SlBES1.8 is regarded as a transcriptional repressor,and its transcriptional repression activity is contributed by the EAR motif and BES1-type domain.(2)The analysis of tissue-specific expression profile of SlBES1.8 showed that it expressed exclusively in the SAM and floral organ.Overexpressed SlBES1.8 in tomato increased the size of SAM,and also increased the numbers of the subsequent developed floral organs.Meanwhile,the shape of SlBES1.8 overexpressed fruit is apparently changed.On the other hand,SlBES1.8 is knocked out by CRISPR/Cas9 technique,due to the screening and propagation of materials,a small number of mutant plants have been observed and no corresponding significant phenotypic changes have been found yet.Therefore,it is necessary to further obtain sufficient plant materials for biological statistical verification of relevant indicators.To confirm whether the deletion of SlBES1.8 affected the growth and development of tomato plants.(3)The development of SAM is mainly regulated by CLV-WUS signal transduction pathway.By analyzing the expression profile in different tissues,the highly consistent spatio-temporal expression profiles between SlBES1.8 and CLV-WUS signal components are observed,implying that SlBES1.8 may regulate or interact with those genes involved in CLV-WUS signaling.Dual-luciferase assay showed that SlBES1.8can repress the promoter activity of Sl CLV3,Sl HPAT3,Sl XEG113,Sl RRA3 a and Sl CLE9.Besides,the protein-protein interaction of SlBES1.8 with Sl WUS is confirmed by Bimolecular fluorescence complementation(Bi FC)and Firefly luciferase complementation imaging(LCI)assays.The results presented above indicate that SlBES1.8 regulates the development of tomato SAM by mediating the CLV-WUS signal transduction pathway.(4)The regulation to the development of tomato SAM,floral organ and fruit formation by SlBES1.8 is mainly through the CLV-WUS pathway.While beyond this,overexpression of SlBES1.8 also influences the development of tomato style and seed,and the shape of SlBES1.8 overexpressed fruit is apparently different with wild type even in those fruit with equal locule numbers.According to the analysis of RNA-sequencing,overexpression of SlBES1.8 affects many Gene Ontology(GO)terms related to fruit development.Several differentially expressed genes(DEGs)are involved in cell development,fruit shape,auxin and gibberellin signaling and metabolism.Dual-luciferase assay is used to explore the regulation relationship between SlBES1.8and those down-regulated DEGs and found that SlBES1.8 can repress the promoter activity of Sl SUN,Sl ARF18,Sl KO and Sl GA2ox3.These results suggest that SlBES1.8may also regulate the formation and development of tomato fruit in a CLV-WUS independent manner.As a conclusion,this study identified the function of SlBES1.8 in the development of tomato SAM,floral organ and fruit.The molecular mechanism of SlBES1.8functioning is also explored.The results obtained in this study provided new insight in the research of plant growth and development and particularly in fruit formation,and offered important materials for cultivar development and improvement.