CsHSFA1b and CsHSFA2 regulate CsJAZ6 expression
Distinct from the down-regulation of JA-responsive genes, expression ofCsJAZ6 , an inhibitor of the JA signaling pathway, was significantly induced by HT treatment (Figure 3a). As indicated in Figure 4a, three HSEs were identified in the CsJAZ6 promoter region (~2 kb upstream of the start codon). The presence of these sequence elements encouraged us to investigate whether CsHSFA1a and CsHSFA2 could directly bind to the CsJAZ6 promoter. Firstly, yeast one-hybrid assays demonstrated that CsHSFA1b could recognize all three HSE elements, whereas CsHSFA2 only bound to the segment containing HSE1 (Figure 4a and Figure S1). Similarly, EMSA using a 50-bp fragment containing the HSE1 (TTCNNGAA) motif as a probe suggested that both CsHSFA1b and CsHSFA2 could bind directly to the HSE1 element in theCsJAZ6 promoter (Figure 4b, c). Subsequent competitive experiments showed that the addition of unlabeled wild-type probes at 1X concentration could partly prevent the binding of CsHSFA1b and CsHSFA2 to labelled probes; adding 10X enhanced the effect of this competition (Figure 4b, c). Based on the in vitro observation that both CsHSFA1b and CsHSFA2 bind directly to HSE1 in the CsJAZ6promoter, we subsequently explored their capacity for transcriptional activation of CsJAZ6 in tobacco leaves. Dual-Luc assays revealed that compared with the individual expression of corresponding negative controls, CsHSFA1b and CsHSFA2 activated the CsJAZ6 promoter by 3.3-fold and 2.1-fold, respectively (Figure 4d, e). This finding indicates that HT promotes CsHSFA1b and CsHSFA2 and thereby activatesCsJAZ6 transcription in planta . Together, our data suggest that CsHSFA1b and CsHSFA2 could repress JA signaling mainly through their binding to the CsJAZ6 promoter and activating its transcription.