D299T Mutation in CYP76F14 Led to a Decrease in Wine Bouquet Precursor Production in Wine Grape.

Abstract

Background: Bouquet is a crucial characteristic indicative of wine quality that develops during the aging stage. The cytochrome P450 VvCYP76F14 multi-functionally catalyzes linalool into (E)-8-hydroxylinalool, (E)-8-oxolinalool, and (E)-8-carboxylinalool, which are direct precursors for wine bouquet. Wine bouquet was closely related to VvCYP76F14 activities.

Method: The VvCYP76F14 genes were cloned from five wine grape varieties using a homologous cloning method. The variation in residues of VvCYP76F14s were assessed by multiple alignment of amino acid sequences. Functional studies were implemented by in vitro enzyme activity and transient over-expression systems.

Results: D299T variation was observed in VvCYP76F14s of 'Yantai 2-2-08', 'Yantai 2-2-19', and 'Yantai 2-3-37' offspring lines, which was correlated with the decreased content of wine bouquet precursors of (E)-8-hydroxylinalool, (E)-8-oxolinalool, and (E)-8-carboxylinalool, respectively. Notably, the key amino acid residue D299 was located at the phase 0 intron positions of VvCYP76F14 genes isolated from distinct wine grape varieties or offspring lines, respectively. Notably, VvCYP76F14s of the 'Yantai2-2-08', 'Yantai 2-2-19', and 'Yantai 2-3-37' mutant lines exhibited lower in vitro enzymatic activities than those of 'L35' and 'Merlot'. In addition, the transient expression of VvCYP76F14 cloned from 'L35' and 'Merlot' restored the levels of wine bouquet precursors in berries of three D299T mutant lines, respectively, whereas VvCYP76F14 cloned from D299T mutant lines failed.

Conclusions: D299T variation was observed in three offspring lines and D299T mutation in VvCYP76F14 led to the decrease in wine bouquet precursor contents. VvCYP76F14 was implicated in the regulation of wine bouquet precursors in wine grapes.