PtrWRKY75 promotes SA biosynthesis by activatingPAL1 transcription
We speculated that WRKY75 might promote SA biosynthesis during drought. Therefore, we measured the SA content in WT and OE lines under non-stress conditions and dehydration for 4 h. The free SA concentration increased under dehydration treatment, especially in the OE lines, where its peak level was approximately double that in WT (Figure 3A). Next, we explored how WRKY75 promotes SA biosynthesis. On the basis of previous research, we examined the expression level of SA INDUCTION-DEFICIENT 2 (SID2 ) (Nawrath and Métraux, 1999) andPHENYLALANINE AMMONIA LYASE 1 (PAL1 ) (Cochrane et al. , 2004) under non-stress and dehydration conditions. SID2 andPAL1 encode crucial enzymes in two SA biosynthesis pathways. ThePtrPAL1 transcript levels were markedly higher in OE lines than in WT, whereas PtrSID2 transcript levels did not differ between OE lines and WT, or between drought and non-stress conditions (Figure 3B, C). This result suggested that WRKY75 might directly or indirectly induce PAL1 expression.
To assess whether WRKY75 directly binds to the PAL1 promoter to regulate its transcription, we performed electrophoretic mobility shift assay (EMSA) experiments. WRKY TFs are known to specifically bind to the W-box (TTGACT) sequence, and we identified one putative W-box sequence in the PAL1 promoter (Figure 3D). The full-length WRKY75 protein tagged with His (His-WRKY75) was capable of binding to probes containing the W-box, but not to the probe harboring a single nucleic acid mutation (TTAACT) (Figure 3E). These results suggested that WRKY75 can directly bind to the promoter of PAL1 .
To further investigate the positive regulation of PAL1transcription by WRKY75, promoter transactivation assays were conducted in tobacco leaves by transient expression. The PAL1 promoter was fused with the β-GLUCURONIDASE (GUS ) reporter gene to generate reporter constructs, and the 35S:WRKY75 construct was used as the effector (Figure 3F). The results revealed that WRKY75 activated the promoter of PAL1 in vivo under both non-stress and dehydration conditions, especially the latter. Taken together, these results indicated that WRKY75 can promote SA production by activating PAL1 transcription.