Branched-chain amino acid catabolism initiates volatile synthesis in Gentiana triflora.

Abstract

Main conclusion: This study identified GeBCAT2 as a key gene in catalyzing the first step of branched chain amino acid biosynthesis in Gentiana triflora, thereby contributing to unpleasant floral odor emission. Gentians, widely cultivated as ornamental flowers in Japan, primarily originate from the endemic gentian species Gentiana triflora and G. scabra. This study analyzed volatile compounds in Japanese gentians using gas chromatography-mass spectrometry. Results showed that G. triflora flowers consistently emitted 3-methylbutanoic acid, 2-methylbutanoic acid, and isobutyric acid, which are volatile organic compounds derived from branched-chain amino acids (BCAAs) and associated with unpleasant odors. In contrast, G. scabra flowers did not emit these compounds. Although the BCAA metabolism has been widely studied, its catabolic pathways in gentians remain unclear. Therefore, we performed precursor feeding experiments to quantitatively verify the role of BCAAs and their corresponding keto acids in producing odorous volatiles. We also cloned and functionally analyzed two Gentiana BCAAs transferase genes (GeBCAT1 and GeBCAT2). Both genes were more highly expressed in flowers than in leaves, with expression levels higher in G. triflora than in G. scabra. Enzymatic assays with recombinant proteins demonstrated that GeBCAT1 and GeBCAT2 participate in BCAA-related catabolic reactions. Notably, GeBCAT2's substrate specificity for BCAAs correlated with unpleasant odor intensity in G. triflora, suggesting that it serves as the primary enzyme initiating unpleasant odor biosynthesis in gentians. These findings provide valuable insights into volatile biosynthesis in gentians and offer a foundation for breeding cultivars with reduced unpleasant odors.