调控酿酒葡萄果实C13-降异戊二烯香气物质合成的分子生理机制

C13-Norisoprenoids, a group of volatile compounds that arise from the cleavage of carotenoids, make a vital contribution to wine aroma due to their extremely low threshold and significantly varietial and geographical characteristics. However, so far studies on C13-norisoprenoids in grapes are mostly concerned about analysis of metabolites, and there has been lack of systemic research as to the biosynthesis and regulation of C13-norisoprenoids. Therefore it is essential to understand the mechanism controlling the accumulation of C13-norisoprenoids in wine grape berries grown under China's continental monsoon climate which will contribute to the promotion of our wine quality. The present project will focus on two key reactions in norisoprenoid biosynthetic pathway of wine grape berries: one dealing with the reaction from geranylgeranyl pyrophosphate to carotenoids by the catalysis of phytoene synthase (PSY), and the other with the reaction from carotenoids to C13-norisoprenoids that is catalyzed by carotenoid cleavage dioxygenase (CCD). The effects of grape variety, vine training system and bunch exposure on the accumulation of carotenoids and free/bound C13-norisoprenoids and on the expression of the genes encoding PSY and CCD are assessed, which will discover critical factors affecting the production of C13-norisoprenoids. The regulation of transcription factors (PIFs andRAP2s) on gene expression and its mechanism are further studied, which aims to explore the molecular physiological mechanism regulating the accumulation of C13-norisoprenoids in wine grape berries. The findings obtained will provide a scientific basis for the production of high quality grapes.

C13-降异戊二烯是一类由类胡萝卜素裂解产生的香气物质，由于其极低的阈值和明显的品种与地域特性，在葡萄酒特征香气构成中有着举足轻重作用，但迄今对葡萄果实C13-降异戊二烯的研究主要在产物分析的层面，关于其合成调控机制仍不明晰；探明大陆季风性气候区酿酒葡萄C13-降异戊二烯的积累与调控机制，对提升我国葡萄酒质量具有积极贡献。本项目拟以酿酒葡萄为试材，针对类胡萝卜素合成与裂解产生C13-降异戊二烯两个代谢环节，研究品种、整形方式与疏叶处理对果实类胡萝卜素和游离态/糖苷结合态C13-降异戊二烯的积累及关键酶（八氢番茄红素合成酶PSY和类胡萝卜素裂解双氧合酶CCD）基因表达的影响，明确调控果实C13-降异戊二烯物质积累的主要因素；研究转录因子PIFs和RAP2s对PSY和CCD基因表达的调节及其机制，揭示调控葡萄果实C13-降异戊二烯产生的分子生理机制，为优质酿酒葡萄的生产提供科学依据。

C13-降异戊二烯是一类由类胡萝卜素裂解产生的香气物质，具有很低的感官阈值，是非芳香型葡萄酒花果香气的重要组分，然而，迄今对葡萄果实中C13-降异戊二烯物质积累与调控机制仍知之甚少。本项目重点关注异戊二烯代谢中类胡萝卜素合成以及裂解产生C13-降异戊二烯两个关键环节，研究了基因型和微环境对相关基因表达和代谢物积累的影响及其生理机制。主要研究成果如下：（1）利用真核表达系统证实了VvCCD1和VvCCD4b均可裂解番茄红素产生 6-甲基-5-庚烯-2-酮、裂解β-胡萝卜素产生β-紫罗兰酮和β-环柠檬醛。VvCCD1还可裂解番茄红素生成香叶醛，VvCCD4a裂解β-胡萝卜素只生成β-环柠檬醛。（2）品种之间比较发现，萜烯含量较高的葡萄品种，其降异戊二烯含量及VvCCDs基因表达也相对较高，在果实发育过程中VvCCD1表达与降异戊二烯含量变化趋势一致，该基因上有4个SNP位点，可能与降异戊二烯含量关联。（3）产区间、年份间和避雨与露地栽培间的比较发现，无论是麝香型‘小白玫瑰’还是非芳香型 ‘赤霞珠’，强光干旱气候条件会导致降异戊二烯合成减少，而使类胡萝卜素更多走向于ABA合成支路；与西部产区相比，我国东部产区同品种葡萄果实可积累较多的降异戊二烯物质。（4）在新疆地区开展整形、疏果、果际摘/挪叶和果穗全套袋处理试验，结果均表明，光照强度是影响降异戊二烯物质积累的主要非生物因素，在以强光干旱为主要气候特点的新疆地区，单蔓整形和摘叶等均提高了转色前果实中叶黄素和β-胡萝卜素含量，但减少果实成熟期降异戊二烯合成；疏果（保持1-1.5穗/梢）则显著提高了成熟果实中各种降异戊二烯组分的含量。（5）利用烟草瞬时表达系统和分别转VvPSY、VvCCD1、VvCCD4a和VvCCD4b启动子介导的GUS基因拟南芥研究证实，这些基因表达均受光和温度的调控；本研究还首次鉴定到了正调控VvCCD4a基因表达的转录因子（VvREVEILLE6，属Myb-like家族）。该项目研究成果为我国不同生态产区酿酒葡萄香气品质提升提供了重要的科学依据，有效地解决了国产葡萄酒果香较弱的问题。以本项目成果作为理论依据之一研发了我国酿酒葡萄栽培管理技术，并已经在主产区推广示范，相关技术成果也在2016年获得了中国食品工业协会科学技术奖特等奖。