Regulation Mechanism Of Fruit Load On The Carbon Metabolism Of Grape(Vitis Vinifera L.)

Carbon metabolism is an important factor affecting plant growth and is directly relevant to grape yield and quality.Increasing the source or the sink strength can effectively improve plant productivity.Nevertheless,the source-sink relationship is a complex and comprehensive regulation mechanism,and the changes of source-sink relationship response to sink strength in grapevine have not been systematically and comprehensively studied.The present study set three fruit loads treatments through cluster thinning at the fruit set,including 1/4 remaining fruit load（C1）:7-8clusters/vine（3/4 clusters removed）;1/2 remaining fruit load（C2）:15-16 clusters/vine（1/2 clusters removed）;full fruit load（C3）:30-32 clusters per plant（no clusters removed）,with C3 as the control,to explore the mechanism of regulating grape carbon metabolism by fruit load.To measure the source（leaf）and sink（fruit）strength,The berries and leaves were sampled at pre-veraison（60 days after anthesis,DAA）,veraison（75 DAA）,post-veraison（90 DAA）,and harvest（105 DAA）,respectively.By using ¹³C labeling and biochemical indexes to measure carbon absorption,distribution,and metabolism.The full-length transcriptome sequencing analysis was performed on berries at veraison.The main results are as follows:1.C2 improved the photosynthetic rate（Pn）,intercellular CO₂concentration（Ci）,the maximum quantum yield in PS II（F_v/F_m）,and berry soluble sugar accumulation in comparison with C1 and C3.The ¹³C labeling revealed that the ¹³C content in the entire plant and clusters of C2 was higher than that in C1 and C3.C1 and C2stimulated the activity of sucrose synthase（SS）,sucrose phosphate synthase（SPS）,neutral invertase（NI）,α-amylase（α-Amy）andβ-amylase（β-Amy）in berries compared with C3.The relative expression levels of VvNI3,VvSWEET14,VvBMY1,and VvSUC27 were up-regulated in berries of C2.C2 increased soluble solid,soluble sugar,anthocyanin,total phenol,and tannin content,decreased titrable acid content at harvest.Thus,the 1/2 remaining fruit load grapevines increased the source strength by increasing photosynthesis,increased the sink strength by improving soluble sugar accumulation and the carbon allocation in berries,which helped to improve berry quality.The optimal leaf area/yield（LA:Y）ratio（1.00～1.20）was obtained by the 1/2remaining fruit load.2.Taking into account the variation trend of source-sink strength under different fruit loads（significant differences occurred at veraison stage）,and the most significant difference was found at post-veraison period.Berries at veraison were selected for transcriptome analysis.Compared with C3,C1 had 174 up-regulated genes and 106 down-regulated genes,while C2 had 89 up-regulated genes and 260down-regulated genes.By KEGG,GO,and COG analysis,34 differently expressed genes（DEGs）related to carbon transport and metabolism were enriched in carbon metabolism,pentose and glucuronate interversions,starch and sucrose metabolism and glycolysis pathways.In order to further understand the molecular mechanism of carbon metabolism in grape berries,the genes of VvPUP1（VIT＿18s0001g06910）and VvFBA2（VIT＿03s0038g00670）in carbon metabolism pathway were selected as candidate genes for subsequent transformation.3.VvPUP1 was induced by reducing the fruit load.The total length of VvPUP1sequence was 1056 bp,and this gene encodes 351 amino acids.The promoter sequence of VvPUP1 with the length of 2000 bp was cloned and a transient expression vector containing GUS reporter gene（p BI121-VvPUP1-GUS）was constructed and detected its activity after injection into tobacco.The full-length sequence of VvPUP1 was cloned,and the overexpression vector p CAM-VvPUP1-FLAG and silencing vector p TRV2-VvPUP1 was constructed.These two vectors were transformed into the grape callus（cv.Pinot Noir）,which found that overexpression of VvPUP1 significantly increased the glucose,fructose,sucrose,and starch content by about 33%,39%,160%,and 150%,and increased SS,SPS,NI,andα-Amy activity by about 2.02-fold,0.53-fold,0.57-fold,and 1.27-fold,respectively.In the meanwhile,the silent expression of VvPUP1 in the grape callus decreased above indicators.Overexpression of VvPUP1 increased the expression levels of VvSUS,VvSPS1,VvHT5,VvSWEET14 and VvSUC27 in the grape callus,while silencing of VvPUP1 reduced the expression levels of these genes.When overexpressed VvPUP1 in tomato,it was found that the fruit size,fruit weight and leaf area increased compared with the wild type.The soluble sugar,starch contents,sugar-related enzyme activity in mature transgenic tomato was higher than those in the wild type.Overexpression of VvPUP1 in tomato increased the expression level of Sl BGLU13,Sl SPS1 and SLSUS-1,but decreased the expression level of Sl BMY1.These results indicated that VvPUP1 was beneficial to the development of source strength,so as to improve the sink strength,and its promoter was active.4.The gene of VvFBA2 was also induced by limiting the fruit load.The FBA family in grape was identified and five FBA genes were found.The full-length sequence of VvFBA2 was cloned,and the overexpression vector p CAMBIA1300-VvFBA2-EGFP and silencing vector TRV2-VvFBA2 were constructed.The overexpression vector p CAMBIA1300-VvFBA2-EGFP was transformed into tobacco,and discovered that VvFBA2 located in the cell membrane and chloroplast.Then the vector of p CAMBIA1300-VvFBA2-EGFP and TRV2-VvFBA2 were transformed into the grape callus,which found that the overexpression of VvFBA2 significantly improved the contents of glucose,fructose,sucrose,and starch by about 46%,90%,116%,and 471%,respectively,and increased the SS,SPS,NI,andα-Amy activity by about 0.56-fold,4.79-fold,1.07-fold,and1.39-fold,respectively,compared with the wild-type callus.However,the silent expression of VvFBA2 in the grape callus decreased the above indexes.Overexpression of VvFBA2 increased the expression levels of VvSUS,VvSPS1,VvHT5,VvCWINV1,VvSWEET14 and VvSUC27 in the grape callus,while silencing of VvFBA2 reduced the expression levels of these genes.When overexpressed VvFBA2 in tomato,it was found that the fruit size had no significant changes compared with the wild type in different periods.The soluble sugar and starch content,and sugar metabolism-related enzyme activity in the fruit were improved when overexpressed VvFBA2 in tomato.Overexpression of VvFBA2 in the tomato plant also significantly increased the expression levels of Sl BGLU13,Sl SPS1and Sl SUS-1,while decreased the expression level of Sl BMY1.These results indicated that VvFBA2 affects sugar metabolism in fruit,but does not affect the source and sink relationship.5.By comparing the functions of VvPUP1 and VvFBA2,it was found that compared with the wild type,the increase fold of fructose and sucrose content was higher by about 65%and 48%in the callus of overexpressing VvFBA2 than that in the callus of overexpressing VvPUP1.Compared with the callus of overexpressed VvPUP1,the activity of SS and NI increased 3.97-fold and 0.45-fold in the callus of overexpressed VvFBA2,respectively.The overexpression of VvPUP1 increased the starch content and fruit weight compared with the overexpression of VvFBA2 in tomato fruit.It can be concluded that overexpression of VvFBA2 can better improve the soluble sugar content and the activities of sugar-related metabolic enzymes in transgenic fruits or callus,while overexpression of VvPUP1 could better improve the starch content and fruit size.