VvTPS14 is a linalool synthase activated by VvbZIP3 and repressed by VvMADS4.

Linalool is a key aromatic terpene that imparts the characteristic Muscat flavor in grape berries. However, the upstream transcriptional mechanisms regulating its biosynthesis remain largely unknown. Here, integrated GC-MS and transcriptomic analyses across berry development in Muscat and non-Muscat cultivars identified VvTPS14 as a terpene synthase gene whose expression strongly correlated with the linalool accumulation pattern. Functional characterization confirmed that VvTPS14-2 encodes a chloroplast-localized enzyme that catalyzes linalool biosynthesis from GPP in vitro. Transient and stable overexpression of VvTPS14-2 in Nicotiana benthamiana leaves, grape berries, and Vitis amurensis callus consistently enhanced linalool production, establishing VvTPS14 as a key linalool synthase. Upstream regulatory mechanisms of VvTPS14 were elucidated through yeast one-hybrid screening, which identified VvbZIP3 and VvMADS4 as direct binders of the VvTPS14 promoter. Dual-luciferase and overexpression assays demonstrated that VvbZIP3-4 (a splice variant) activates, whereas VvMADS4 represses, the expression of VvTPS14. This regulatory pattern was conserved across heterologous and homologous systems. These findings reveal a complete transcriptional regulatory module controlling linalool biosynthesis and provide potential targets for molecular breeding of flavor traits in grapes.