Roles of enzymes VvNCED1 and VvCCD1 in β-Damascenone biosynthesis: insights into aroma development in grapes.

Grape (Vitis vinifera L.) is a widely cultivated fruit with significant economic value. β-Damascenone, a key norisoprenoid compound, contributes to the distinctive floral and fruity aroma of grape berries. However, the biosynthesis of this compound remains unresolved. Integrated transcriptomic and metabolomic analyses across 17 developmental stages of V. vinifera cv. Cabernet Sauvignon revealed stage-dependent regulation of aroma metabolism. Notably, norisoprenoid accumulation peaked at veraison, with the expression of both 9-cis-EPOXYCAROTENOID DIOXYGENASE 1 (VvNCED1) and CAROTENOID CLEAVAGE DIOXYGENASE 1 (VvCCD1) exhibiting a synchronized temporal pattern. Exogenous ABA treatment enhanced norisoprenoid accumulation and the expression of VvNCED1, NEOXANTHIN SYNTHASE (VvNXS), and CAROTENOID CLEAVAGE DIOXYGENASE 4b (VvCCD4b). Enzymatic assays using recombinant VvNCED1 demonstrated its capacity to cleave 9'-cis-neoxanthin in vitro, generating putative C25-allenic-apo-aldehyde and xanthoxin (an ABA precursor). Subsequent in vitro reactions with recombinant VvCCD1 converted C25-allenic-apo-aldehyde into grasshopper ketone, which undergoes conversion to β-damascenone. We propose two complementary pathways: 1) ABA-mediated regulation: VvNCED1-driven ABA biosynthesis upregulates norisoprenoid biosynthetic genes; 2) Direct precursor channeling: VvNCED1 and VvCCD1 sequentially process 9'-cis-neoxanthin into β-damascenone precursors, with grape-specific acidic conditions or uncharacterized enzymes facilitating final conversion. This collaborative VvNCED1-VvCCD1 model bridges carotenoid metabolism, ABA metabolism, and aroma development, providing insights into the spatiotemporal coordination of berry maturation and flavor compound accumulation.