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.