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Ampelopsis grossedentata leaves are highly rich in dihydromyricetin. They have been used to make tea in China for centuries. Dihydromyricetin has many potential applications in foods and medicine. This are because it has five phenolic hydroxyl groups. However, the hydroxylases involving the biosynthesis of dihydromyricetin have not been identified and characterized. In this study, a series of hydroxylases genes, including flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), flavonoid 3′,5′-hydroxylase (F3′5′H), and cytochrome P450 reductase (CPR), were identified after RNA sequencing. The full-length CDSs of AgF3H, AgF3′H, AgF3′5′H, and AgCPR genes were amplified from the cDNA library of leaves. The aforementioned enzymes were expressed and verified in Saccharomyces cerevisiae. Through the substrate specificity assay, the functional AgF3′H, AgF3′5′H, and AgCPR in A. grossedentata were identified. The dihydromyricetin hydroxylation process in A. grossedentata was successfully identified. We found that substantial carbon flux occurred through the Naringenin (NAR)–Eriodictyol (ERI)–Dihydroquercetin (DHQ)–Dihydromyricetin (DHM) and NAR–Dihydrokaempferol (DHK)–DHQ–DHM pathways. This study provides some reference for the development and utilization of the germplasm resources and molecular breeding of A. grossedentata. [ABSTRACT FROM AUTHOR]

Rice plants contain high basal levels of salicylic acid (SA), but some of their functions remain elusive. To elucidate the importance of SA homeostasis in rice immunity, we characterized four rice SA hydroxylase genes ( OsSAHs ) and verified their roles in SA metabolism and disease resistance. Recombinant OsSAH proteins catalyzed SA in vitro, while OsSAH3 protein showed only SA 5-hydroxylase (SA5H) activity, which was remarkably higher than that of other OsSAHs that presented both SA3H and SA5H activities. Amino acid substitutions revealed that three amino acids in the binding pocket affected SAH enzyme activity and/or specificity. Knockout OsSAH 2 and OsSAH3 ( sah KO) genes conferred enhanced resistance to both hemibiotrophic and necrotrophic pathogens, whereas overexpression of each OsSAH gene increased susceptibility to the pathogens. sah KO mutants showed increased SA and jasmonate levels compared to those of the wild type and OsSAH -overexpressing plants. Analysis of the OsSAH3 promoter indicated that its induction was mainly restricted around Magnaporthe oryzae infection sites. Taken together, our findings indicate that SA plays a vital role in immune signaling. Moreover, fine-tuning SA homeostasis through suppression of SA metabolism is an effective approach in studying broad-spectrum disease resistance in rice.