TPS Expression, Not Copy Number, Explains Aroma Diversity among Grapevine (Vitis vinifera L.) Cultivars

Terpenoids are major contributors to grapevine berry and wine aroma, with mono- and sesquiterpenoids exhibiting substantial variation among cultivars. However, the genetic basis of cultivar-specific terpenoid profiles remains only partially understood due to gene duplications that expanded the terpenoid synthase (TPS) gene family across different cultivars. Here, we compared haplotype-resolved genome assemblies for seven cultivars to analyze copy number variation and expression profiles of terpenoid biosynthesis genes and terpenoid accumulation throughout flower and berry development. These assemblies resolved duplicated TPS loci at the haplotype level, revealing extensive copy number variation across cultivars and between haplotypes within a cultivar, with up to 44 mono-TPS genes on a single haplotype. We identified eight specific monoterpenoids as key drivers of cultivar-specific profiles in ripe berries. Total TPS transcript abundance correlated strongly with total terpenoid accumulation over time. In contrast, TPS copy number was largely decoupled from both expression and terpenoid accumulation, indicating that expression regulation, rather than gene dosage, underlies aroma differences among cultivars. Transcript-metabolite network analysis revealed both expected correlations between specific TPS genes and putative products as well as novel associations. We functionally validated selected candidate TPS in Nicotiana benthamiana, characterizing -farnesene, {beta}-ocimene, and multi-product -terpineol synthases. The in vivo characterization demonstrated that minor sequence variations can alter catalytic activity and product profile. Together, these findings show that the extensive duplication of TPS genes in grapevine does not itself determine terpenoid output; instead, cultivar-specific aroma arises from how these genes are expressed and from sequence differences that shape enzyme activity.