## 2 Results
### Regulation of ABA and NAA on the Biosynthesis and Accumulation of Norisoprenoids
#### 2.2.2 Acquisition of the Candidate Genes Regulating Norisoprenoid Accumulation  
To identify the ABA and NAA-responsive transcription factors regulating norisoprenoid biosynthesis, an effective system biology method called weighted gene co-expression network analysis (WGCNA), was performed to find the modules of highly correlated genes and relate these modules to traits. For each module, eigengene is defined as the first principal component of the expression matrix of the module and considered to be representative of the module gene expression profiles. It is then used to correlate with traits and look for the significant association. Based on the genes differentially expressed between the treatment and control at least one phenological stage, twelve modules were established (Figure 6A). The top intramodular hub genes of each module were defined by using the intramodular connectivity measure. Among the 12 modules, the modules of \"turquoise\", \"cyan\", \"black\", and \"magenta\" were found to be associated with norisoprenoid accumulation. The module turquoise showed a strong correlation with the concentration of riseling acetal (r = 0.72, \(p\) = 7 \(\times\) 10\({}^{-7}\)), (E)-\(\beta\)-damascenone (r = 0.76, \(p\) = 7 \(\times\) 10\({}^{-8}\)), (Z)-\(\beta\)-damascenone (r = 0.75, \(p\) = 2 \(\times\) 10\({}^{-7}\)) and total norisoprenoids (r = 0.75, \(p\) = 2 \(\times\) 10\({}^{-7}\)), and moderately correlated with 6-methyl-5-hepten-2-one (r = -0.60, \(p\) = 1 \(\times\) 10\({}^{-4}\)), cis-theaspirane (r = -0.56, \(p\) = 3 \(\times\) 10\({}^{-4}\)), vitispirane B (r = 0.55, \(p\) = 5 \(\times\) 10\({}^{-4}\)), trans-theaspirane (r = -0.54, \(p\) = 8 \(\times\) 10\({}^{-4}\)) and TPB(r = 0.65, \(p\) = 2 \(\times\) 10\({}^{-5}\)) contents (Figure 6B). The module-trait relationship analysis also identified module cyan as most highly related to (E)-\(\beta\)-damascenone (r = -0.86, \(p\) = 1 \(\times\) 10\({}^{-11}\)), TPB (r = -0.70, \(p\) = 2 \(\times\) 10\({}^{-6}\)), (Z)-\(\beta\)-damascenone (r = -0.84, \(p\) = 1 \(\times\) 10\({}^{-10}\)) and total norisoprenoids (r = -0.85, \(p\) = 4 \(\times\) 10\({}^{-11}\)). The module black exhibited a moderate correlation with cis-theaspirane (r = 0.61, \(p\) = 7 \(\times\) 10\({}^{-5}\)), (E)-\(\beta\)-damascenone (r = -0.66, \(p\) = 1 \(\times\) 10\({}^{-5}\)), trans-theaspirane (r = 0.60, \(p\) = 1 \(\times\) 10\({}^{-4}\)), (Z)-\(\beta\)-damascenone (r = -0.66, \(p\) = 1 \(\times\) 10\({}^{-5}\)) and total norisoprenoids (r = -0.62, \(p\) = 5 \(\times\) 10\({}^{-5}\)). Moreover, the moderate correlation of \(\beta\)-cyclodextrin (r = 0.56, \(p\) = 4 \(\times\) 10\({}^{-4}\)) and high correlation of \(\beta\)-ionone (r = 0.78, \(p\) = 1 \(\times\) 10\({}^{-8}\)) were observed in magenta module. Interestingly, the VviCCD4a and VviCCD4b which have been reported to tightly associate with norisoprenoid accumulation [7], were presented in this module turquoise and cyan, respectively. Furthermore, it was found that VviCCD4a co-expressed with the upstream genes of norisoprenoid biosynthesis including VviPSY2, VviPSY3 and two VviZEPs in module turquoise, while VviCCD4b co-expressed with VviNCED1, VviNCED2, and VviNCED3 in module cyan. The VviPSY1, VviZDS  
Figure 5: Concentrations of free, total and individual norisoprenoids. (**A**) Concentrations of free norisoprenoids in 2015. (**B**) Concentrations of total norisoprenoids in 2015. (**C**) Concentrations of free norisoprenoids in 2016. (**D**) Concentrations of total norisoprenoids in 2016. Statistical analysis involved the comparison of treatments at each E-L stage, and different letters represent significant differences (\(p\) = 0.05). (VIT_214s0030g01740) and VviLECY were clustered in the module black. However, in module magenta, there were no known genes related to norisoprenoid biosynthesis. Therefore, to investigate the regulation of norisoprenoid biosynthesis, the top 30 hub genes in modules of turquoise, cyan, and black were further analyzed.