## 3 Discussion
### Response of Norisoprenoid Biosynthesis  
Our study is the first to reveal the effect of exogenous ABA on both free and total norisoprenoids in grape berries at both transcriptional and post-transcriptional levels. As is known, the biosynthetic pathways of ABA and norisoprenoid share the same part of the substrate of carotenoids. The present study indicated that though ABA biosynthesis was enhanced by ABA treatment, there is no visible decrease of total norisoprenoids in ABA-treated harvested berries. It was found the total norisoprenoid levels are low in both ABA1000 and ABA800-treated berries only at E-L 34 stage (Figure 5A,B) when the concentration of ABA largely increased (Figure 2A). The previous researchers have confirmed the increase of carotenoids after ABA treatment in many fruits such as in grapes [34] and tomato [35] and seeds of bean, tobacco, beet, and corn [36]. Hence, we speculated that ABA treatment elevated the concentration of carotenoids or ABA precursors during berry ripening, thus supporting the biosynthesis of both norisoprenoids and ABA. Alternatively, since vitispirane A, vitispirane B, and MHO exhibited lower levels in ABA-treated berries in consecutive two seasons (Table S2), ABA treatments or the increased endogenous ABA may negatively regulate the accumulation of these compounds by down-regulating VviCCD4a (Figure 2B). The pre-veraison NAA treatment was found to increase total norisoprenoids at E-L 35 and E-L 36 stages in grape berries [21] and \(\beta\)-damascenone level in wine [3]. Different from ABA treatment, NAA effect on norisoprenoid biosynthesis,by comparison to ABA biosynthesis, appeared to be more apparent. NAA up-regulated a series of norisoprenoid-associated genes including VviPSY1, VviPSY2, VviPSY3, VviLECY, VviLBCY, VviZEPs, VviCCD4a, and VviCCD4b expression at a certain stage (Figure 2B), ultimately elevating the level of most norisoprenoid components at harvest, except for geranylacetone and 6-methyl-5-hepten-2-one. In tomato fruit, silencing of PSY1 can significantly reduce carotenoid accumulation, while the silencing of PSY2 or PSY3 was less efficient in controlling carotenoid biosynthesis [37], suggesting the three PSY genes played different roles in the carotenoid biosynthetic pathway. Similarly, since carotenoids are synthesized mostly from fruit formation until veraison in grape berry [1], the higher expression of VviPSY1 at E-L 34 and E-L 35 stages (Figure 7B) indicated its important role in carotenoid metabolism. In contrast, the elevated expression levels of VviPSY2 and VviPSY3 were observed at E-L 36 and E-L 38 stages, which could be responsible for the carotenoid biosynthesis after veraison. It may explain the different responses of VviPSYs to NAA treatments (Figure 2B). WGCNA further expounded that VviCCD4a expression had a high correlation with (E)-\(\beta\)-damascenone, a norisoprenoid component with the highest concentration, and (Z)-\(\beta\)-damascenone, riesling acetal, TPB, as well as total norisoprenoid concentration (Figure 6), while VviCCD4b was correlated only with the concentration of 6-methyl-5-hepten-2-one. This result is different from our previous study in which we found that total norisoprenoids and \(\beta\)-damascenone were positively correlated with the expression of VviCCD4b, rather than VviCCD4a during Cabernet Sauvignon grape berry development [12]. This difference is inferred to mainly relate to the ripening process retarding and transcriptional alteration induced by NAA. In the present study, the expression of VviCCD4a was remarkedly up-regulated by NAA treatment at E-L 34, E-L 35, and E-L 36 stages, but was down-regulated by ABA spraying at E-L 35 and E-L 36 stages (Figure 2B). Moreover, VviCCD4a co-expressed with the upstream VviPSYs and VviZEPs of norisoprenoid biosynthesis pathway (Table S5).