Using the Chou's 5-steps rule, transient overexpression technique, subcellular location, and bioinformatic analysis to verify the function of Vitis vinifera O-methyltranferase 3 (VvOMT3) protein.

3-Isobutyl-2-methoxypyrazine (IBMP) is an important odor compound that revives unripe grapes or poor-quality wine. The biosynthesis of IBMP in grape berries is under the catalysis of Vitis vinifera O-methyltranferase 3 (VvOMT3). The homologous verification in this paper was carried out with the transient overexpression technique. The results showed that both the expression levels of the VvOMT3 gene and the IBMP concentration in 'Red globe' grapes increased significantly, which suggested that VvOMT3 could function in the biosynthesis of IBMP. Based on β-glucuronidase (GUS) staining results, blue color was only observed in grape pulp, not in grape skin, which indicated that VvOMT3 was expressed in grape pulp. The outcomes of the subcellular location examination performed on the protoplasts of Arabidopsis thaliana showed that the VvOMT3 protein was located on the inner surface of the cytoplasmic membrane. In summary, the VvOMT3 enzyme may function at the inner surface of the cytoplasmic membrane of pulp cells during grape development. These results will provide a background for future research on the catalytic mechanisms of VvOMT3.