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.