Effects of grafting on metabolites and flavonoid metabolic pathways of tea plant based on widely targeted metabolomics analysis

Objective This study aimed to analyze changes in metabolites and flavonoid metabolic pathways in leaves of Lingyun Baihao tea before and after grafting， so as to provide theoretical references for the replanting and transformation and industrial high quality development of Lingyun Baihao tea in old tea gardens in the future.Method Taking Guire 2 as the scion and Lingyun Baihao tea as the rootstock， the grafted tea plants were used as research subjects. One bud and two leaves samples of Guire 2 were collected before and after grafting and labeled as C and B samples， respectively. Widely targeted metabolomics technology was used to detect the metabolite content in tea before and after grafting to perform principal component analysis （PCA） and orthogonal partial least squares discriminant analysis （OPLS-DA）. The metabolite content was normalized using the range method. Cluster analysis was performed on differential metabolites， which were screened based on the criteria of log₂ Fold Change≥2 or log₂ Fold Change≤0.5 and variable importance in projection （VIP）≥1. Pathways related to differential metabolites of flavonoids were analyzed based on the KEGG database.Result Both PCA and OPLS-DA could completely separate sample B and sample C. The contribution rate of the first principal component （PC1） was 89.80%， and the contribution rate of the second principal component （PC2） was 3.04%. Cluster analysis results showed significant differences in metabolites between the two samples， with the greatest changes observed in flavonoids after grafting. The metabolites in tea showed significant differences. A total of 3495 metabolites were detected in two tea plant samples of Guire 2 before and after grafting， of which 2919 metabolites showed significant differences. Compared with sample C， sample B had 2042 metabolites with significantly up-regulated VIPs and 877 metabolites with significantly down-regulated VIPs. The analysis of the top 20 significantly differential metabolites in tea plants of Guire 2 before and after grafting showed that sample B had 17 differential metabolites whose fold changes were significantly higher than those in sample C； the differential metabolites were mainly concentrated in KEGG metabolic pathways such as flavonoid glycoside Ⅲ biosynthesis， unsaturated fatty acid biosynthesis， anthocyanin synthesis， starch and sucrose metabolism， and caffeine metabolism. Moreover， 665 flavonoids showed significant differences between the two samples before and after grafting. The main pathways related to significantly differential metabolites of flavonoids were primarily concentrated in the flavonoid biosynthesis pathway， anthocyanin biosynthesis pathway， isoflavone biosynthesis pathway， and flavonoid and flavonol biosynthesis pathway， and these four pathways had 30， 10， 10， and 23 significantly differential metabolites （mainly flavonoids） whose contents were mostly up-regulated.Conclusion Grafting of tea plant Guire 2 can significantly alter the metabolites in tea leaves， promote the accumulation of flavonoids in tea leaves， and thereby improve tea quality.