Effects of sun shading on anthocyanin components and the expression of genes related to anthocyanin synthesis in Marselan grape

【Objective】In order to improve the structural stability of anthocyanins in wine grapes, avoid rapid color fading during berry ripening, and enhance the color stability of grapes and wine, we selected Marselan (Vitis vinifera L.) grape as the experimental material, one of the main wine grape varieties grown in the eastern foothills of the Helan Mountains, and investigated the effects of shading at different veraison stages on anthocyanin components and the expression of synthesis- related genes in wine grape.【Methods】Using the wine grape Marselan as the test material, the top of grapevines was shaded from the beginning of veraison to the harvest stage, and this treatment was T1. The top of the grapevines was sun-shaded from the complete veraison stage to the harvest stage, which was referred to as T2 treatment. Vines that grew normally without shading served as the control. The shading net adopted a black single-layer shading net with a shading rate of 50% and a specification of 10 meters wide and 25 meters long. All three treatments were designed as a single plot with three biological replicates.High-performance liquid chromatography (HPLC) was used to qualitatively and quantitatively analyze the contents and components of anthocyanins. RNA transcriptome sequencing technology (RNA- seq) and real-time quantitative fluorescence PCR technology (qRT-PCR) were used to investigate the expression levels of genes related to anthocyanin synthesis.【Results】Sunshade can significantly reduce the soluble solids content during the ripening period of grape berry, treatments at different stages can increase the titratable acid content of harvested berries, and shading the vine during the complete veraison stage can promote the accumulation of reducing sugar content and total anthocyanin content in the berries. The 16 types of anthocyanins detected in each treatment were consistent, indicating that shading had no effect on the types of grape anthocyanins. Simultaneously, shading treatment increased the modification ratio of grape acylated anthocyanins, increased the stability of anthocyanin structure, and further improved the color stability of anthocyanins. After shading treatment, the contents of delphinidin (Dp), cyanidin (Cy), petunidin (Pt) and peonidin (Pn) anthocyanins were significantly reduced, while shading treatment during the complete veraison stage significantly increased the content of malvidin (Mv) anthocyanins, indicating that shading had different effects on anthocyanins at different stages. The synthesis of anthocyanins was influenced by both external environmental factors and the expression of key enzymes involved in the internal regulatory synthesis pathway. The study showed that the expression levels of functional genes related to anthocyanin synthesis were different after shading at various stages. During the initial stage of veraison and shading during the harvesting period, the expression levels of CHI, DFR and OMT genes were upregulated compared to other treatments, with DFR and OMT reaching significant levels during the harvesting stage. During the complete veraison stage and shading during the harvesting stage, the expression levels of PAL, CHS3, F3’H, F3’5’H, UFGT, GST4 and MYB90 genes were upregulated compared to other treatments during the harvesting stage. qRT- PCR analysis revealed that among the 15 screened genes, the expression levels of CHI, F3H, F3’5’H, F3’H, DFR, LDOX, UFGT and OMT showed inconsistent trends with transcriptome sequencing in each treatment, while the changes in other genes were consistent with transcriptome sequencing results.【Conclusion】There was no significant difference in the effect of shading during the initial veraison stage and the complete veraison stage on berry quality. The complete veraison stage shading increased the transcription level of anthocyanin synthesis related genes, promoted anthocyanin synthesis, increased the total anthocyanin content of grapes, and significantly increased the content of Mv class anthocyanins. At the same time, complete shading during the veraison stage increased the modification ratio of acylated anthocyanins, and enhanced the stability of anthocyanin color, which provided a theoretical basis for effectively alleviating the problem of rapid color fading caused by unstable anthocyanin structure in grapes and wines. F3’H and F3’5’H played a crucial role in the biosynthesis pathway of anthocyanins, and the ratio of Cy class anthocyanins catalyzed by F3’H to Dp class anthocyanins catalyzed by F3’5’ H affected the color of berry peels. This study found that during the early veraiosn stage, when shading and harvesting, the expression of F3’H was up-regulated and the expression of F3’5’H was down-regulated. During the complete veraison stage of shading, both F3’H and F3’5’H were up-regulated during harvesting. However, the Cy and Dp anthocyanin contents with the shading treatment during the initial veraison stage were significantly higher than those with the shading treatment during the complete veraison stage. Perhaps due to the significant positive correlation between CHI expression level Cy and Dp content, and the significantly higher CHI expression level with shading treatment during the early veraison stage compared to shading treatment during the complete veraison stage, the Cy and Dp content with shading treatment during the early veraison stage was significantly higher than that with shad-ing treatment during the complete veraison stage. There may be differential expression of other key genes, leading to differences in Cy and Dp contents, which requires further research.