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. 2022 Nov 20;24(2):123–141. doi: 10.1111/mpp.13280

FIGURE 4.

FIGURE 4

VqNSTS4 has unconventional transcriptional regulation activity and carries specific VqAL4 binding sites. (a) cis‐Regulatory element analysis in the promoters of VqNSTS genes. MYB and WRKY binding elements were found in the ProVqNSTS2, ProVqNSTS3, and ProVqNSTS6 promoters. The ProVqNSTS4 promoter included a G‐rich element in addition to the MYB and WRKY binding elements. (b) Yeast one‐hybrid assays were carried out to determine whether VqWRKYs, VqMYBs, and VqAL4 could bind directly to the promoters of VqNSTS genes. VqMYB154 can bind to ProVqNSTS2, VqWRKY3 and VqWRKY53 can bind to ProVqNSTS3, VqMYB14 can bind to ProVqNSTS6, and VqAL4 can bind to ProVqNSTS4. (c) Yeast one‐hybrid assays were conducted to demonstrate that VqAL4 cannot bind to ProVqNSTS4 m2 , ProVqNSTS4 m3 , ProVqNSTS4 d , ProVqNSTS4 m5 , or ProVqNSTS4 m6 . (d) Luminescence intensity. (e) Ratio of firefly luciferase (LUC) to Renilla luciferase (REN) activity. The standard deviation (SD) was calculated from three independent replicates. One‐way analysis of variance (Tukey's test) was carried out. Asterisks indicate significant differences at *p < 0.05, **p < 0.01. (f) Chromatin immunoprecipitation–quantitative PCR assays were carried out to demonstrate that VqAL4 binds to the promoter of VqNSTS4 via the CACCTC/GAGGTG element. The SD was calculated from three independent replicates. Asterisks indicate significant differences (*p < 0.05, **p < 0.01, Student's t test).