Behavior of 3-isobutyl-2-methoxypyrazine biosynthesis related to proposed precursor and intermediate in wine grape

•Incorporate the proposed precursor of IBMP biosynthesis into grapes in situ.•Incorporate the proposed intermediate of IBMP biosynthesis into grapes in situ.•IBMP content of grapes with added leucine and leucinamide increased.•IBMP content of grapes increased faster in leucinamide treatment than leucine.•Key genes expression levels of IBMP biosynthesis in berry skin increased. Although 3-alkyl-2-methoxypyrazines (MPs) are very important flavor compounds in grape and wine, the metabolic pathway of these compounds has not been fully elucidated, and only a few studies have focused on it to date. In this study, we performed in situ incorporation of the proposed precursor (l-Leu) and the key intermediate (2-amino-4-methylpentanamide; AMPA) of 3-isobutyl-2-methoxypyrazine (IBMP) biosynthesis into Cabernet Sauvignon clusters. The IBMP content and the expression levels of key related genes were monitored. IBMP content decreased shortly after l-Leu or AMPA treatment, but subsequently increased significantly, with raised levels of O-methyltransferase genes (VvOMT1 and VvOMT3) in the berries between veraison and maturation. We speculate that l-Leu may be a precursor of IBMP biosynthesis and that AMPA may be a key intermediate.