3.4 NtMYB12a directly targets two LOX and two lipase genes
The 35S:NtMYB12a-GR seedlings were treated with DEX, CYC, and DEX+CYC, respectively. The NtCHS , NtCHI , NtF3H , andNtFLS genes, which are known direct targets of MYB12, were selected as the marker genes to show the effects of treatments. The transcriptions of these four genes were significantly induces by DEX treatment owing to the release of NtMYB12a-GR fusion protein from the nuclear membrane, but significantly repressed by CYC, which can inhibit protein synthesis in plants (Supplemental Figure S3). Moreover, the transcriptions of these four marker genes were also significantly induced under DEX+CYC treatment, indicating the regulation of NtMYB12a on the expression of these four genes requires no intermediate protein synthesis. Similarly, when compared to those in the mock seedlings, the expression levels of NtSFAR4 , NtLOX5 , NtLOX6 , andNtGDSL2 genes were significantly lower in the seedlings treated with CYC, but significantly higher in the seedlings treated with DEX or DEX+CYC at all the processing time points (Figure 7a). Meanwhile, the transcription of NtFAH1 , NtGDSL1 , NtGDSL3 , andNtGDSL4 genes were also induced by DEX, and repressed by CYC, but showed no significant difference between the mock and DEX+CYC treatment (Supplemental Figure S4a).
The putative binding sites of MYB12 were searched in the promoter regions of the eight lipid-related DEGs (Figure 7b, Supplemental Figure S4b), and ChIP-qPCR was then performed to verify the direct targets of NtMYB12a in tobacco. As shown in Figure 7c, three fragments containing the binding site of MYB12 in the promoter regions of NtSFAR4 andNtGDSL2 genes were significantly enriched after immunoprecipitation, while two fragments in the promoter regions ofNtLOX5 and NtLOX6 genes were also significantly enriched, respectively. However, the fragments with the binding site of MYB12 in the promoter regions of NtGDSL1 , NtGDSL3 , NtGDSL4 , and NtFAH1 genes were not significantly enriched (Supplemental Figure S4c). These results indicate that NtSFAR4 , NtLOX5 ,NtLOX6 , and NtGDSL2 are the direct targets of NtMYB12a.