The Mechanisms Of Brassinosteroids And Cytokinins-regulated Shoot Branching In Tomato

Shoot branching is an important feature of plant,which plays an important role in the morphogenesis of plants.As a model for branching research,the branching habits of tomato affect the yield,quality and production efficiency of tomato.Therefore,it is of great theoretical and productive significance to study the mechanism of tomato branching and explore the efficient regulation mode.The branching of tomato is influenced by many aspects such as genetic factor,environmental factor,plant hormone and so on.Plant hormones mediate genetic and environmental signals and play an important role in the regulation of branch.Auxin,cytokinins and strigolactones are the main hormones that influence branching and three kinds of hormone regulatory networks have been improvedd in recent years.Brassinosteroid which is an important steroid hormone,plays an important role in regulating growth and development.However,the mechanism of regulating branching between BR and other hormones is not clear.This paper uses tomato(Solanum lycopersicum)as material,the effects of CK and BR on tomato branching were determined from hormone content and gene expression level by using decapitation method and different BR level plants.The regulatory function of CK and BR signal components to tomato branching was identified by using virus-induced gene silencing(VIGS)and CRISPR/Cas9 technology.The possible mechanisms of CK and BR in regulating tomato branching were explored by yeast two hybrid,biomolecular fluorescence complementation and protein immunoblotting.The main results are as follows:1.We have clarified the promoting effect of cytokinin on tomato branching.After decapitation,cytokinin content and lateral branching length increased significantly,which preliminarily proved the promotion of cytokinin content on tomato branching.In the process of decapitation,we found that the cytokinin degradation and response genes have a large difference in a short time.The VIGS technique was used to silence the cytokinin degradation gene CKX5 and CKX7.The results showed that the lateral bud length of CKX5 and CKX7 gene silenced plants increased significantly,which proved that the elevation of endogenous cytokinin increased the promotion of tomato branching.Similarly,we use VIGS technology to silence the cytokinin signal transduction positive regulator RR10,RR23,which have a weaker ability of branching.These results demonstrate that the synthesis,degradation and signal transduction of cytokinin played an important role in tomato branching.2.We have demonstrated the regulation effect of BR and the relationship with CK on tomato branching.By tissue culture technique,we obtained the DWARF-overexpressing plant which has a increase ability of BR synthesis,compared with the wild type,the lateral bud length of DWARF-overexpressing plant increased significantly.However,the branching ability of DWARF deletion mutant became weaker,thus we preliminarily proved the effect of endogenous BR content on tomato branching.At the same time,we determined the cytokinins content and relative expression of different BR levels plants,and further explored the relationship between BR and CK in regulating shoot branching.These results indicated that there was no significant correlation between the content of cytokinin,the expression of synthesis,degradation and signal response genes and the content of BR.In addition,we used cytokinin synthetic agent 6-BA to treat different BR levels plants,and found that the lateral bud length of DWARF deletion mutant was significantly shorter than the treatment of wild type plants.Taken together,our results suggest that the incresement of endogenous BR content can significantly promote tomato branching and the regulation of BR on branching is independent of the increase of cytokinin content.3.We have clarified the role of BIN2,a negative regulatory factor of BR signaling pathway,in tomato branching.Three BIN2 genes BIN2-1,BIN2-2 and BIN2-3 were screened by phylogenetic tree analysis,then those three genes were silenced by VIGS technique,and the lateral bud length of BIN2-2-silenced plants was obviously increased.By CRISPR/Cas9 technology,we obtained the homozygous mutant plant bin2-2-5# and lateral branching phenotype of mutant was consistent with the BIN2-2-silenced plant.In addition,we carried out the exogenous EBR treatment on BIN2-2-overexpressing plant and decreased the content of BIN2-2 protein.By yeast two hybrid and biomolecular fluorescence complementation,we have verified the interaction between BIN2-2 and BZR1.The results incidated that BIN2-2,the negative regulatory factor of BR signaling pathway,inhibited tomato branching.4.We have studied the role of BIN2-2 mediated interaction between CK and BR in the regulation of tomato branching.The expression level of cytokinin biosynthesis gene and the content of cytokinin in BIN2-2-silenced plants was partly down regulated or not significantly different,respectively,which indicated that BIN2-2 regulating branching was irrelevant with cytokinin content.However,the expression of cytokinin responsive genes RR2,RR3 and RR7 in BIN2-2-silenced plants was up-regulated,indicating that BIN2-2 may affect cytokinin signal transduction process.By yeast two hybrid and biomolecular fluorescence complementation,we found that BIN2-2 could interact with HP3,a histidine phsphotransfer protein of cytokinin.The gene silencing of HP3 was carried out by VIGS technology,and the results showed that the lateral branching length of HP3-silenced plants was significantly increased.These results suggest that BIN2-2 may affect the signal transduction of cytokinin by regulating HP3,and ultimately inhibit the branch of tomato.