Root-specific CsATs enhances acylated flavonol-glycoside accumulation to promote aluminum tolerance in tea roots

Acylated flavonol glycosides can regulate plant growth, improve plant stress resistance and disease resistance. We have found that some specific acetylated flavonol glycosides accumulated in the tea roots, but their metabolic characteristics and the mechanism of biosynthetic regulation were not clear. In this paper, the flavonol glycosides in tea seedlings were qualitatively and quantitatively analyzed by UPLC-TOF-MS/MS and UPLC-QqQ-MS/MS. The results showed that acetylated kaempferol glycosides were specifically accumulated in root tips of tea plants, and these flavonol glycosides were enriched by Al treatment. Then, two acylation genes (CsAT1 and CsAT2) of the BAHD acyltransferase family that were highly expressed in the tender roots of tea plants and induced by aluminum were screened out for further function study. In vitro, the results of enzyme activity experiments indicated that the recombinant proteins CsAT1 and CsAT2 could catalyze flavonol-7-O-glucoside to the corresponding acetylated flavonol glycosides and flavonol. When CsAT1 was transiently overexpressed in Nicotiana benthamiana, the protein CsAT1 could hydrolyze kaempferol-7-O-glucoside to kaempferol. When CsAT1 was heterologous overexpressed in Arabidopsis thaliana, the substrate kaempferol-7-O-glucoside decreased significantly over time. Using hairy root experiment system, CsAT1 was stably overexpressed in the roots of tea plants. The results of metabolic analysis indicated that the content of non-acylated flavonol glycosides remained unchanged or decreased in the roots of tea plants overexpressing CsAT1, while the accumulation of acylated flavonol glycosides significantly increased. In conclusion, acylated flavonol glycosides were specifically accumulated in tea roots, its biosynthesis is catalyzed by acyltransferase CsATs and induced by aluminum. This study laid a foundation for further revealing the mechanism of aluminum tolerance of tea plants, and had certain guiding significance in the cultivation and breeding of tea plant.