| Tomato(Solanum Lycopersicum L.)is a vine plant belong to Solanum of Solanaceae and is an important food in the human diet as it provides sources of vitamins A,C and lycopene et.al,and has become one of the most important fruits and vegetables in the world.Under the pressure of artificial selection,cultivated tomato production is getting higher,but meanwhile,the fruit sweetness is getting lower and resulted in fruit out of flavor.As the principle site of photosynthesis,plant leaves have been shown to have important effects on fruit yield.However,how leaf morphology effecte on fruit quality(such as sweetness)is yet little known.Therefore,in this study,two tomato bipinnata(bip)mutants(bip2 and bip0663)and their corresponding wild types(M82 and Lukullus)were used and identified,and based on study of fruit Brix and leaf shape such as leaf complexity,leaf vein density et.al,we analyzed the relationship between leaf shape and fruit sweetness in tomato and explored the genetic mechanism of leaf shape influence on fruit sweetness by using comparative analysis of transcriptome.This study deepened understanding of regulation of compound leaf development and revealed the importance of leaf shape to fruit quality in tomato and the mechanism of the leaf shape effect on fruit sweetness,and provided an important theoretical basis for the improvement of tomato fruit quality in the future.The main findings are as follows:1.Morphological studies showed that the increase in leaf complexity of two bip mutants bip2 and bip0663 was caused by the increase in the number of secondary and intercalary leaflets.The point that differences initiated during leaf developmental between the two bip mutants and its corresponding wild type was at P5～P6 stage.During P5～P6 stage,the activity of the MB region at the edge of the leaf primodia of bip mutants was prolonged,which formed more secondary and intercalary on the edge of the leaf and resulted in increasing of leaf complexity.Meanwhile,the expression of TKN1 in SAM(Shoot apical meristem)of bip2 and bip0663 was significantly upregulated.Variants and PROVEAN analysis at whole genome level showed that the increase in leaf complexity of bip2 and bip0663 were predicted to be caused by mutation of Solyc02g089940(BIP).The BIP genes in that two bip mutants were both mutated with respect to their corresponding wild-type and were both resulted in functional changes in the encoded proteins.2.Study on CRISPR/Cas9 mediated BIP gene mutagenesis indicated that the phenotype of highly leaf complex of bip mutants were caused by mutations of BIP gene,In CRISPR/Cas9 transgenic mutant CRISPR-BIP-2,BIP gene was mutated at 1841 bp with a single base deletion and resulted in translation prematurely terminated.Morphological studies showed that CRISPR-BIP-2 exhibited a leaf phenotype resembles bip mutant: leaf complexity increased significantly and caused by an increase in the number of secondary and itercalary leaflets,and intercalary leaflet primordia was initiated at site of the gap between lateral primary leaflets at P6 stage.In addition,expression of TKN1 gene was also significantly increased in CRISPR-BIP-2.3.Analysis of fruit Brix showed that the fruit Brix of bip2 and M82 was significantly lower than that of bip0663 and Lukullus,and fruit Brix of bip0663 was significantly increased compared with its corresponding wild type Lukullus,while the fruit Brix of bip2 was basically the same as its corresponding wild type M82.This indicated that changes in fruits Brix is caused by changes of leaf complexity.Grafting assay on Lukullus and bip0663 showed that the variation of fruit Brix between Lukullus and bip0663 was caused by change of leaf and revealed the importance of leaf shape to fruit Brix in tomato.Correlation analysis between leaf vein density and fruit Brix showed that there was a significant and high negative correlation between leaf vein density and fruit Brix in bip2,M82,bip0663 and Lukullus.4.Transcriptome analysis showed that the reduced leaf veins density in bip0663 and Lukullus were caused by down-regulation of G6PT2.In SAM and YL(Young leaf)of bip0663 and Lukullus,G6PT2 was significantly down regulated compared with bip2 and M82.In addition,the expression of G6PT2 in bip0663 was significantly lower than that in Lukullus,indicating that the difference of leaf vein density between bip0663 and Lukullus might be also caused by down-regulation of G6PT2.The differences in fruit Brix between bip0663,Lukullus(HB,high fruit Brix phenotype)and bip2,M82(LB,low fruit Brix phenotype)may be resulted from up regulation of carbohydrate synthesis and transport related genes in mature leaves(ML)of HB.Compared with bip2 and M82(low fruit Brix phenotype,LB),a plenty of genes that significantly up regulated in ML(Mature leaf)of bip0663 and Lukullus(high fruit Brix phenotype,HB)were involved in regulating carbohydrate synthesis and substance transport.Moreover,the expression of the glucose transporter gene Solyc09g074230 in ML of HB was up-regulated by 3-fold compared with LB phenotype.5.Co-expression network analysis of differentially expressed genes showed there was a negative correlation between genes involved in regulating leaf veins development and genes involved in sugar metabolism and transport,which explained the significantly negative correlation between leaf veins density and fruit Brix.The differentially expressed genes related to leaf veins development were down-regulated in the HB leaf,while the differentially expressed genes involved in sugar metabolism and transport were up-regulated in HB leaf.Moreover,the expression of G6PT2 gene,which has a positive regulation of leaf vein development,shows a linear negative correlation with the expression of glucose transporter gene Solyc09g074230. |
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