Functional Analysis Of SPL13 And COL1,the Upstream Regulatory Factors Of Tomato Flowering Gene SFT

Tomato(Solanum lycopersicum)is one of the most important vegetable crops cultivated in the world.It is also an important model plant for the study of inflorescence and fruit development.It has attracted wide attention of breeders.Inflorescence structure,flowering number,fruit size,quality and plant architecture of higher plants are directly related to yield.Artificial selection for related traits regulatory genes with improved flower production and yield occurred throughout the domestication and improvement of superior varieties.Tomato SINGLE-FLOWER TRUSS(SFT)gene is a homologous gene of FT in Arabidopsis thaliana.The mutation of SFT gene directly affects inflorescence structure and flowering number.Therefore,it is very important to understand the regulatory network of SFT gene.In our laboratory,we found that SlSPL13 and SLCOL1 genes can directly bind to the promoter of SFT and regulate its expression to affect the development of inflorescence and yield of tomato plants.We have verified the transgenic function of the two genes and carried out the related genetic and biological analysis.1.The inflorescence and lateral branches of higher plants are generated by lateral meristems.The structure of the inflorescence has a direct effect on fruit yield in tomato(Solanum lycopersicum).We previously demonstrated that miR156 a plays important roles in determining the structures of the inflorescence and lateral branches in tomato by suppressing the expression of the SQUAMOSA PROMOTER BINDING PROTEIN LIKE(SPL)transcription factor gene family.In this study,we confirmed that SPL13 is the major SPL of the 7 targeted SPLs involved in miR156a-regulated tomato inflorescence structure determination and lateral branch production.Suppressing the expression of SPL13 in tomato reduces fruit yield,increases vegetative inflorescence and lateral branch number,decreases flower and fruit number,and reduces fruit size.Yeast one-hybrid,LUC/RLU ratio assay,GAL4/UAS-based assays electrophoretic mobility shift assay(EMSA)and ChIPPCR experiments evidences indicate that SPL13 controls inflorescence development by positively regulating the expression of the tomato inflorescence-associated gene SINGLE FLOWER TRUSS(SFT)by directly binding to its promoter region.Additionally,we found that SPL13 can negatively regulate the expression of the tomato lateral branch development related gene TEOSINTE BRANCHED1(TB1)and BARREN STALK1(BA1).Overexpression of either TB1 or BA1 can significantly increase lateral branch number in tomato.Additionally,we found that tomato Lateral suppressor(Ls)can interact with SPL13and represses its activity in vivo by using CoIP and GAL4/UAS-based assays methods,indicating that Ls can regulate lateral branching in tomato by affecting the SPL13-TB1/BA1 pathway.In summary,our findings provide a major conceptual advance to the miR156a-SlSPLs mechanism that regulates plant architecture and yield in tomato.2.CONSTANS(CO)and CONSTANS-LIKE(COL)transcription factors controlling flowering have been reported in many species.We found overexpression of the COL1would decrease tomato flower and fruit number.Yeast one-hybrid and GAL4/UAS-based assays biochemical evidences support that COL1 controls inflorescence development through negatively regulating the expression of tomato inflorescence associated gene SINGLE FLOWER TRUSS(SFT)by directly binding to CCAAT cis-element of its promoter region without NFYB/NFYC complex.However,there are few reports its play an important role in improving tomato fruit quality as high pigment genes.The nutritional quality of tomato fruit is mainly determined by carotenoids,soluble solids,ascorbic acid and so on.We found that CONSTANS-LIKE 1(COL1),a tomato(Solanum lycopersicum)high pigment gene,can positively regulate chlorophyll contents of fruits and leaves.Overexpression of the COL1 would lead tomato fruits and leaves to high pigment phenotypes.The green mature fruit of COL1 overexpressed transgenic lines would be dark green for more chlorophyll accumulation,and the red ripe fruit was dark red and the ascorbic acid content was significantly higher than the wild type.Otherwise,suppress the expression of COL1 would decrease the accumulation of chlorophyll in the green mature fruit,and the color of the fruit was lighter.Further analysis by GST pull-down and coimmunoprecipitation(CoIP)assays showed that COL1 can directly interact with GOLDEN2-LIKE(GLK2)and promote the stability of it.Our results demonstrate that COL1,as a new high pigment gene,interacts with GLKs and promote their stability,thereby improve tomato fruit quality.