## 3 Discussion  
### Response of ABA and IAA Biosynthesis and Signaling  
This study deals with the responses of endogenous ABA and auxin biosynthesis and signaling, and previously identified switch genes to exogenous ABA and NAA treatments (Figures 2-4). These data provide a framework to understand numerous aspects of ABA and NAA-regulated ripening. We found that the level of endogenous ABA significantly increased after ABA treatments in 2015 (Figure 2A), which is consistent with the previous study [20]. The elevated ABA could result from both induced endogenous ABA biosynthesis and the absorb from exogenous spraying ABA. However, the uptake of exogenous  
Figure 8: Qualitative RT-PCR analysis of expression of _VviDXS_ and _VviCRTISO_ splice variants in grape berries at specific stage under NAA100 and ABA1000 treatments. The forward and reverse primers were designed from the upstream and downstream exons of the skipped exon or retention intron, respectively. Figure 7: Gene co-expressed and regulatory network. (**A**) Gene co-expressed and regulatory network related to nonisoprenoid biosynthesis. The size of the nodes corresponds to the nodes ‘betweenness centrality’, which measures how often a node in the network occurs on all shortest paths between two nodes and help identify the gene that plays a ‘bridge spanning’ role in the network. Red and blue edges indicate that the interaction between two genes is identified by WGCNA and DNA affinity purification sequencing (DAP)-seq, respectively. (**B**) Hierarchical cluster analysis of genes in the left graph. The colors of the boxes represent the intensity of the normalized gene. ABA into the grape was predicted to be an inefficient process because of the berry waxy cuticle [20; 29]. Given the increased ABA mainly came from ABA biosynthesis, a small amount of external ABA entering the berry may be enough to drive the flux of carotenoid into endogenous ABA production. This speculation is supported based on the upregulation of VviNECDs in ABA-treated berries (Figure 2B), as well as the higher correlation between ABA concentration and VviNCED1 expression (r = 0.79, \(p\) = 0.011) or VviNCED2 expression (r = 0.70, \(p\) = 0.036). However, auxin analogues NAA had both positive and negative effects on the expression of auxin biosynthetic genes (Figure 2C), and this explained why there was no significant difference in IAA concentration between NAA treatment and the control. From the aspect of interactive crosstalk, the ABA treatment inhibited the endogenous auxin production by suppressing the expression of VviTAR1 and VviYUC10, and NAA application suppressed endogenous ABA biosynthesis by synchronously down-regulating three VviNECDs at E-L 34 stage (Figure 2B). The lower expression of ABA biosynthetic genes in NAA-treated berries has been also observed in a previous study [30]. The ABA and NAA treatments also caused changes in the transcription rate of ABA and auxin signaling genes. As a whole, ABA spraying activated the ABA signaling transduction and suppressed the auxin signaling transduction, whereas the effects of NAA treatment on the two signaling pathways appeared to be complex, owing to the up-regulation or down-regulation of various genes (Figure 3). The complicated regulatory network between ABA and auxin signaling pathways has been already suggested by [PERSON] et al. [31] and [PERSON] et al. [30]. As expected, some ripening switch genes previously identified in grape berry were found to have the opposite expression behavior in response to ABA and NAA treatments, such as the DESGs in the group of a and d (Figure 4b). Indeed, VviMYBA2, VviMYBA1, VviMYBA3, and zinc finger family genes in the groups of a and d have been demonstrated to involve in berry development and ripening [32; 33]. It was considered that these TFs were up-regulated by ABA while down-regulated by NAA treatment at E-L 34 or E-L 35 stage, in turn, which monitored grape ripening.