Regulation Mechanism Of Light Intensity On Fruit Quality And Anthocyanin Synthesis In Grape (V. Vinifera L.) Berry

Anthocyanin is a very important secondary metabolite in grapes.It is the main coloring substance and plays a decisive role in the color of red wine.At the same time,anthocyanin can also combine with other substances（such as tannin）to affect the taste of wine,thus having an important impact on the quality of wine.At present,the response mechanism of different anthocyanins to light intensity is not clear.The synthesis of anthocyanins and the regulation of gene expression patterns are not clear when the light is completely shaded.In this study,clusters of 45 days after flowering were bagged with four different light-transmissive fruit bags（A: 50%;B: 15%;C: 5%;D: 0）,the non-bagged clusters were used as control（CK）.Samples were collected at 90 days after flowering（S1）,100 days after flowering（S2）and 125 days after flowering（S3）,respectively.GC-MS,LC-MS and transcriptome sequencing techniques were used to determine and analyze the samples in different periods.The main results are as follows:1.The contents of total phenols and anthocyanins in shaded berries decreased significantly,and the synthesis of tannins and flavonoids were significantly inhibited when the transmittance of bags was less than 50%.Bagging increased the content of tartaric acid,malic acid and lactic acid in berries,and the difference was the greatest compared with control at S3 stage.Different treatments affected the synthesis and accumulation of aroma substances in berries.During ripening,with the decrease of light intensity,the types and total amount of aromatic substances synthesized by fatty acid metabolism and isoprene metabolism pathway were decreased in berries.D reduced the types of aromatic substances synthesized by amino acid metabolism,and the total amount of aromatic substances in shaded berries were significantly lower than that in other treatments.Except for heptanol,the contents of cis-7-decanal and trans-hexenal increased significantly in shaded berries,while the contents of other aromatic substances decreased after shading.2.The transcriptome results showed that the down-regulated genes increased from 502 to 1482 and the up-regulated genes increased from 721 to 2308 with the decrease of light transmittance of fruit bags at S2.Besides,at S3,the number of down-regulated genes increased from 1592 to 2429 and up-regulated genes increased from 1326 to 1599 with the decrease of light intensity.GO enrichment results showed that in the classification of biological processes,the biological processes with the largest number of differentially expressed genes were metabolic processes,cellular processes,single-organism process and response to stimulus.The number of differentially expressed genes in cell component classification is the largest in cell part,cell,membrane and organelle.Catalytic activity and binding are the most common genes in molecular functional classification.KEGG enrichment results showed that differentially expressed genes in different treatments were mainly involved in flavonoid synthesis,circadian rhythm-plant,stilbenoid,diarylheptanoid and gingerol biosynthesis and phenylalanine metabolism.qRT-PCR analysis of 14 candidate genes in flavonoid and fatty acid metabolic pathways showed that except for the variation trend of PAL（VIT₀8s0040g01710）expression level in A1 and inconsistency of transcriptome data,other genes were consistent with transcriptome sequencing data.3.The light transmittance of fruit bags decreased,which delayed the colour-changed of berries.The berries of A and CK began to turn color almost at the same time.The coloring time of B was 3 days later than CK,and the treatment of C and D was delayed for 10 days.Total anthocyanin concentration decreased with the decrease of light transmittance in fruit bags at three developmental stages.Among which,A,B,C and D decreased by 10.10%,19.23%,37.07% and 51.50% at harvest stage compared with that of CK,respectively.Twenty-four and twenty-one anthocyanins were detected in CK and D by UHPLC-MS/MS.The content of Malvidin-3-O-coumaroyl glucoside（trans）increased with the increase of light intensity,and could not be synthesized under complete shading.It belonged to light-induced anthocyanin.There was no significant correlation between the content of other anthocyanins and the change of light intensity.Malvidin-3-O-coumaroyl glucoside（cis）and Malvidin-3-O-acetylglucoside accounted for 14% and 34% of total anthocyanin content in the completely shaded berry skin,respectively.The berry skin of control accounted for 1% and 27% of the total anthocyanin content,respectively.It was a typical representative of non-light-induced anthocyanin.The synthesis of light-induced and anthocyanins is regulated by candidate functional genes and MYB,bHLH transcription factors in the flavonoid synthesis pathway.4.Bioinformatics and expression levels of bHLH gene family members in berries at different growth stages were analyzed.Ninety-six members of bHLH were divided into six subfamilies.Multiple sequence alignment revealed that the family was not conservative at both C-and N-terminal,while nearly 80 amino acids in the middle of the amino acid sequence are highly conserved.The number of exons in this family ranged from 1 to 11,and the numbers in subgroup V was larger,in subgroup III was smaller.The bHLH family of grape was distributed on all chromosomes except chromosome 9.The number of amino acids ranged from 136 to 573,the molecular weight ranged from 15249.69D to 64156.59D,and the isoelectric point ranged from 4.65 to 10.37.Except for GSVIVT01034621001,the bHLH family was hydrophobic.Most of the VvbHLH family members are mainly located in the nucleus,and the secondary structure is mainly irregular curl.Chip data analysis showed that most of the genes expressed in different growth stages and tissues were up-regulated at veraison.qRT-PCR showed that the expression level of VvbHLH family members in`Maserland’increased significantly during the color-changing period.5.VvMYB30,VvbHLH79 and VvbHLH121 were cloned and the overexpression vectors were successfully constructed.Transient expression analysis of apple peel mediated by Agrobacterium tumefaciens showed that apple peel could be colored and anthocyanin content increased significantly by injection of transformed Agrobacterium tumefaciens solution.The expression levels of VvMYB30,VvbHLH79,VvbHLH121 and functional genes（CHS,CHI,F3 H,DFR,LDOX and F3’5’H）related to anthocyanin synthesis in the skin of the pigmented part were found to be up-regulated to different degrees by qRT-PCR.Subcellular localization of onion epidermis revealed that all three genes were located in the nucleus,which was consistent with the predicted results of bioinformatics.