Functional Study Of Fruit Ripening-specific Transcription Factors SlbHLH95 And SlFSR In Tomato

Tomato（Solanum lycopersicum Mill.var）is an edible fruit vegetable cultivated and grown worldwide.The typical horticultural trait is the unripe-to-ripe transition leading to generation of the edible fleshy fruit.Tomato fruits are rich in vitamins,fiber,minerals,and antioxidants,which are key components of human nutrition.However,the unripening fruits have lower commodity value because it is inedible.Meanwhile,the commodity value of ripening fruit is greatly reduced because it has a direct effect on susceptibility to post-harvest pathogen infection and poor shelf-life.Therefore,the study on tomato fruit should not only focus on the change of pericarp color,but also focus on the improvement of post-harvest shelf-life.Tomato fruit ripening is a stepwise growth process that involves complex changes in physiological and metabolic processes,such as fruit softening,carotenoid accumulation,chlorophyll degradation,and flavor biosynthesis.However,the mechanisms underlying the transcriptional regulation remain unclear.In this study,two differentially expressed genes,SlbHLH95 and SlFSR,were identified as potential candidate by comparative transcriptome analysis of wild-type and mutant rin fruits.The biological function of SlbHLH95 and SlFSR were investigated by real-time quantitative PCR,anatomical study,dual-luciferase reporter assay and tomato transformation in wild-type and mutant rin.The main results were shown as below:（1）Through the analysis of transcriptome data from wild-type and rin（ripening inhibitor）fruits,5 648 genes have differentially occurred among the libraries of these two groups of samples,and 2 318 genes were up-regulated in rin,and 3 330 genes were down-regulated in wild-type.Among them,we noticed that the expression of SlbHLH95was up-regulated by～6.80-folds（log₂Fold Change）and SlFSR was up-regulated by～5.42-folds（log₂Fold Change）in wild-type.Meanwhile,the expression of SlbHLH95and SlFSR were significantly down-regulated in rin fruits.These results indicated that SlbHLH95 and SlFSR are closely related to fruit ripening in tomato.（2）We investigated the expression pattern of SlbHLH95 in different tissues of wild-type tomato by qRT-PCR technology.The transcripts of SlbHLH95 were low in stem,young leaves,IMG fruits,and moderate in root,sepal,MG fruits,and high in mature and senescent leaves,flowers and ripening fruits.Subsequently,we checked the transcripts of SlbHLH95 in ripening mutants Nr（never ripe）and rin.It was clearly observed that SlbHLH95 expression was significantly down-regulated in mutant fruits,especially in rin mutants.Furthermore,we observed a significantly reduced transcripts of SlbHLH95 by treated with ethylene receptor inhibitor 1-methylcyclopropene（1-MCP）in the MG fruits of wild-type tomato.Indeed,five putative CAr G-box elements were found in the promoter sequence of SlbHLH95,and the LUC/RLU ratio was increased by approximately two-fold compared with control in the presence of MADS-RIN.These results indicated that SlbHLH95 is involved in ethylene signaling and fruit ripening.（3）Suppression of SlbHLH95 resulted in reduced sensitivity to ethylene,decreased accumulation of the total carotenoid and lycopene,declined GSH content,and inhibited the expression of fruit ripening-and glutathione metabolism-related genes.Conversely,up-regulation of SlbHLH95 in wild-type tomato resulted in higher sensitivity to ethylene and increased accumulation of the total carotenoids and lycopene,and more GSH,soluble sugar and starch content.Notably,overexpressing of SlbHLH95 in rin also gave rise to up-regulated expression of fruit ripening related genes（FUL1,FUL2,SAUR69,ERF4 and CNR）and multiple glutathione metabolism-related genes（GSH1,GSH2,GSTF1 and GSTF5）.Moreover,multiple E-box elements which can be directly bound by bHLH protein were found in the promoter sequence of SAUR69 and CNR,and the activity of their promoter was directly regulated by SlbHLH95.These results clarified that SlbHLH95 participates in regulating fruit ripening,affects ethylene sensitivity and GSH metabolisms in tomato.（4）The expression profiles of the SlFSR gene in different tissues of wild-type tomato were detected by qRT–PCR.The results showed that SlFSR m RNA was predominantly expressed in the fruit during the ripening stages（B,B+4,and B+7）,but little or no expression was observed in all other tissues.Additionally,we also checked the transcripts of SlFSR in ripening mutants Nr and rin.It was shown that the expression of SlFSR was significantly down-regulated in both rin and Nr.As in wild-type fruits,almost undetectable SlFSR gene transcription were observed in IMG and MG fruits of rin and Nr mutants.Then,we found that four putative CAr G motifs were enriched in the promoter of SlFSR gene,and the activity of SlFSR promoter was directly regulated by MADS-RIN.Consequently,we speculated that higher expression of SlFSR at the ripening stages is closely related to fruit ripening in tomato.（5）Overexpression of SlFSR cannot restore the course of fruit ripening in the rin mutant,including the accumulation of total carotenoid,sugar,malic acid,and citric acid content.But it can gave rise to up-regulated expression of multiple cell wall modification-related genes,such as PG,TBG4,CEL2,XYL1,PL,PE,MAN1,EXP1,and XTH5,and significantly shortened the fruit shelf-life.In addition,the activity of the protein products of some of these genes were assessed:PG,TBG,CEL,and XYL,which showed a significant increase in activity in wild-type and SlFSR-OE-rin fruits,relative to the activity in rin fruits.The total pectin content in SlFSR-OE-rin fruits was slightly lower than that in rin.By contrast,the water-soluble pectin content was higher in SlFSR-OE-rin fruits than in rin.Moreover,the cellulose and hemicellulose contents in SlFSR-OE-rin fruits were significantly lower than in rin.These results indicated that the overexpression of SlFSR in rin indeed accelerates the degradation of the fruit cell wall.In summary,the direct target genes of the MADS-RIN protein,SlbHLH95 affects tomato fruit ripening and sensitivity to ethylene,In addition,the role of SlFSR（the direct target genes of the MADS-RIN protein）controls the shelf-life by affects cell wall metabolisms By studying the function of these two genes,a possible biotechnological approach to breeding varieties with excellent traits by altering fruit ripening and extend fruit shelf-life may be easy to obtain in practice.