Functional Study Of Two Tomato Transcription Factors SlOFP20 And SlGT16 Genes In Growth And Development

Ovate family proteins（OFPs）and Trihelix transcription factors play an important role in plant growth and development.At present,great progress has been made in the functional research of OFPs and Trihelix transcription factors,but they are mainly concentrated in Arabidopsis and rice.However,the molecular mechanism of the functions of OFPs and Trihelix transcription factors in tomato has not been clarified clearly.Tomato is not only a vegetable crop with high nutrition and high economic value,but also the best model plant to study the development and maturity of fleshy fruit.Through bioinformatics analysis,we selected SlOFP20 and SlGT16 as the target genes in this study.We used transgenic technology,quantitative RT-PCR technology,RNA-seq technology and yeast two-hybrid technology as the main research methods,and systematically discussed the function of SlOFP20 and SlGT16 genes in tomato growth and development from the aspects of anatomy,physiology,biochemistry and molecular biology.Tissue expression pattern analysis showed that SlOFP20 gene had the highest expression in root,followed by stem and flower,and relatively low expression in leaf and IMG fruit.The results of hormone-responsive expression patterns showed that SlOFP20 gene responded to different hormone treatments in different degrees,especially brassinolide（BR）and gibberellin（GA）treatments significantly reduced the expression level of SlOFP20 gene.SlGT16 gene was mainly expressed in stem,leaf,flower and fruit.And the expression patterns in different stages of flower development further indicated that the expression of SlGT16 gene was the highest in the anthesis stage.The expression of SlGT16 gene decreased with fruit ripening.Compared with wild type AC⁺⁺,the expression of SlGT16 gene in Nr and Rin mutant fruits decreased significantly.The results suggest that SlOFP20 and SlGT16 genes may be involved in the regulation of vegetative and reproductive development in tomato.SlOFP20 overexpression lines exhibited the following phenotypes:the length of main root,hypocotyls and cotyledons decreased,the number of lateral roots decreased,the plant dwarfed,the stem became thicker,the number of lateral buds decreased,and the leaf angle became smaller.These results indicate that SlOFP20 gene is involved in the regulation of tomato plant morphology.Moreover,overexpression of SlOFP20resulted in thickened and dark green leaves.the chloroplast development genes,chlorophyll positive regulatory genes and chlorophyll metabolism genes were all up-regulated in varying degrees.Secondly,by measuring the content of photosynthate,analyzing the expression level of photosynthesis and starch synthesis related genes,it showed that overexpression of SlOFP20 gene led to the increase of total chlorophyll content,and then enhanced the photosynthetic capacity of the transgenic lines.The expression of SlOFP20 gene was down-regulated in the process of leaf senescence.Combined with physiological indexes and gene expression level detection of leaf senescence,overexpression of SlOFP20 could delay leaf senescence.During the process of reproductive development,it was found that the plants with strong overexpression of SlOFP20 gene resulted in the vertical shortening and horizontal broadening of flower organs and exserted stigmas,which resulted in the failure of fruit setting,while the transgenic lines with low overexpression efficiency could seat fruit but the fruit was flat.Morphological statistics,paraffin section and SEM analysis of flower organs revealed that SlOFP20 could inhibit the vertical growth of organs and promote the horizontal growth.Pollen germination,TCC staining,cross assay,pollen development-related genes detection and promoter analysis showed that overexpression of SlOFP20 could also reduce the pollen activity of tomato,resulting in the reduction of male fertility,while female fertility might not be affected.According to the phenotypes of SlOFP20 overexpression transgenic lines,we detected the transcription level of BR and GA related genes.The results showed that the expression level of positive regulator GRAS41 of BR signaling pathway in SlOFP20-OE transgenic lines decreased,while the expression level of negative regulator KNOX1 increased significantly,and the biosynthesis and degradation genes of BR and GA were all up-regulated.The genes related to cell division and cell elongation in the downstream of BR and GA pathways were significantly inhibited.Exogenous GA₃ could partially restore the dwarfing phenotype of SlOFP20-OE lines,while the sensitivity of SlOFP20-OE lines to exogenous e BR was significantly reduced.The yeast two-hybrid experiment showed that SlOFP20 could interact with KNOX1 and GRAS41 respectively,and KNOX1 and GRAS41 could also interact directly.Our data show that SlOFP20 can influence the growth and development of tomato through the pathway of BR and GA.Overexpression of SlGT16 strongly inhibited the growth of tomato organs,including leaf size,stem diameter,four-whorl floral organ size and fruit size.Quantitative Real-time PCR analysis showed that the expression level of GA and cell elongation related genes in SlGT16-OE transgenic lines was affected to varying degrees,which indicated that SlGT16 was an important regulator of organ size.Moreover,overexpression of SlGT16 accelerated the senescence of tomato leaves.The results showed that the total chlorophyll content in the leaves of SlGT16-OE transgenic lines decreased significantly,the chloroplast development and positive chlorophyll regulatory genes（DCL,Sl GLK1 and Sl GLK2）decreased significantly,while the negative regulatory gene SGR1 increased.At the same time,overexpression of SlGT16 also led to a significant down-regulation of photosynthesis-related genes in tomato.RNA-seq analysis of mature leaves showed that overexpression of SlGT16 caused a series of phenotype related gene expression changes,including chloroplast development,chlorophyll metabolism,hormones and transcription factors.In addition,the ripening time of SlGT16 silenced transgenic tomato was significantly later than that of wild type.The accumulation of carotenoid and lycopene in SlGT16 silenced transgenic tomato fruits was significantly reduced,while the ethylene content in wild type and SlGT16silenced tomato fruits did not change significantly in B+4 stage.The expression level of carotenoid biosynthesis-related genes,ethylene synthesis and response genes and fruit ripening related genes in SlGT16 silenced transgenic fruits were significantly changed.RNA-seq data further confirmed that the silencing of SlGT16 gene affected the expression of a series of fruit ripening related genes.These results showed that SlGT16is the first key factor in trihelix transcription factor family to be found to participate in the regulation of fruit ripening,which mainly affects the synthesis of carotenoids,ethylene biosynthesis and response,and then affects the fruit ripening of tomato.In conclusion,this study explored and analyzed the function and mechanism of SlOFP20 and SlGT16 in tomato growth and development,and laid a foundation for the comprehensive elucidation of the function and mechanism of SlOFP20 and SlGT16 in plants and the future application in genetic engineering practice.