## 3 Results
### Effect of SA and MeJA on Carotenoid Content in Citrus Juice Sacs In Vitro
Columns and bars represent the means and SE (\(n=3\)), respectively. Different letters indicate significant differences at the 5% level by [PERSON]’s HSD test. CHS1, chalcone synthase1; CHS2, chalcone synthase2; CHI, chalcone isomerase; UFGT2, flavanone 7-O-glucosamine2; F3’H, flavonoid-3’-hydroxylase. Identification of Transcription Factor in Response to Salicylic Acid and Methyl Jasmonate Treatments  
In the present study, differentially expressed genes were investigated in the SA and MeJA treatments by using microarray analysis. As compared with the control, the expressions of 1616 genes were found to be significantly changed after 24-, 48-, and 72-h treatments of SA (Figure 5a and Supplementary Materials, Table S2). In particular, the expression of 1286 genes was upregulated, while the expression of 330 genes was downregulated by the SA treatment. In the MeJA treatment, there were 4914 genes that were differentially expressed as compared with the control (Figure 5b and Supplementary Materials, Table S3). Among them, the expression of 4404 genes was upregulated, while the expression of 510 genes was downregulated by the MeJA treatment. In this study, we found that SA treatment upregulated the key genes related to flavonoid and carotenoid biosynthesis (e.g., _CitCHS2_, _CitF3'H_, and _CitHYb_) in the juice sacs in vitro. In contrast, the expression of these key genes was downregulated by the MeJA treatment in the juice sacs in vitro. To identify the transcriptional factors that might be involved in the regulation of flavonoid and carotenoid accumulation, we further screened the genes which were upregulated in the SA treatment while downregulated in the MeJA. As shown in Figure 6, we found that there were three genes that were upregulated by SA treatment while downregulated by the MeJA treatment (Cicev10012055m, Cicev10022863m, and Cicev10025958m; Table 1). Among them, a WRKY DNA-binding protein 70 was identified (Cicev10012055m), which exhibited 45.6% homology to Arabidopsis _WRKY70_. Expression analysis of _CitWRKY70_ by using real-time PCR confirmed that it was a 9.4-fold upregulated by the SA treatment, and a 14.4-fold downregulated by the MeJA treatment as compared with the control (Figure 7). Figure 5: Hierarchical cluster analysis of 1616 salicylic acid-responsive genes and 4914 methyl jasmonate-responsive genes with more than 2-fold expression changes between salicylic acid or methyl jasmonate and control treatments. (**a**) Salicylic acid (SA)/control (CNT) signal intensity ratio; (**b**) Methyl jasmonate (MeJA)/control (CNT) signal intensity ratio. The color scale indicates a signal intensity of each gene. Tree at the left side of the matrix represents gene relationship. _Appl. Sci._**2020**, _10_, 8916 \begin{table}
\begin{tabular}{c c c c c c c c c c c} \hline \hline \multirow{2}{*}{**ID**} & \multirow{2}{*}{**mRNA**} & \multirow{2}{*}{**JGL_AGI**} & \multirow{2}{*}{**Abbreviations**} & \multirow{2}{*}{**Enzyme**} & \multicolumn{4}{c}{**SA/CNT**} & \multicolumn{4}{c}{**MeJ/A/CNT**} \\ \cline{5-10}  & & & & **24 h** & **48 h** & **72 h** & **24 h** & **48 h** & **72 h** \\ \hline C22820 & Cidev10022863m & & & & PROTEIN & & & & & \\  & & & NIM-INTERACTING 1 & 2.53 & 2.26 & 4.18 & 0.31 & 0.42 & 0.35 \\ C25898 & Cidev10025958m & AT4G10490.1 & DMR-6 & 2-oxyglutamate (20G) & & & & & & \\  & & and Fe (IJ-dependent & & & & & & \\ C12031 & Cidev10012055m & AT3G56400.1 & WRKY70 & WRKY70 & 6.52 & 5.51 & 10.30 & 0.30 & 0.16 & 0.28 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Details of three genes that were more than 2-fold upregulated in salicylic acid (SA) treatment and 2-fold downregulated in methyl jasmonate (MeJA) treatment. CNT, control. Figure 6: Two-set Venn diagram analysis of salicylic acid (SA) and methyl jasmonate (MeJA)-responsive genes in the juice sacs in vitro.