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. 2019 May 17;17(11):2184–2198. doi: 10.1111/pbi.13131

Figure 3.

Figure 3

S‐RNase induces MdD1 expression via the JA‐MdMYC2 signalling pathway in pollen tubes. (a) Yeast one‐hybrid (Y1H) analysis showing that MdMYC2 binds to the MdD1 promoter fragment containing the G‐box motif and MeJA responsiveness motif. The promoter of MdD1 was divided into four fragments (S1‐S4). S1 and S2 fragment contain G‐box motif, respectively; S3 fragment contains MeJA responsiveness motif; S4 fragment without the binding motif as a negative control. X‐α‐gal was used as a screening marker. The empty vector and the MdD1 promoter were used as negative controls. These experiments were repeated three times. (b) Chromatin immunoprecipitation (ChIP)‐PCR showing the in vivo binding of MdMYC2 to the MdD1 promoter. Chromatin samples were extracted from pollen tubes and precipitated with an anti‐MdMYC2 antibody. Eluted DNA was used to amplify the sequences neighbouring the G‐box by qPCR. Four regions (P1–P4) were examined. The ChIP assay was repeated three times, and the enriched DNA fragments in each ChIP were used as one biological replicate for qPCR. Data are the mean ± SEM. *P < 0.05. (c) β‐glucuronidase (GUS) reporter activity analysis, showing that MdMYC2 activates the MdD1 promoter and transcriptional activity was significantly enhanced under MeJA treatment. The MdMYC2 effector vector, together with the reporter vector containing the MdD1 promoter, was co‐injected into tobacco leaves to analyse the GUS activity. The empty vector as control, together with the reporter vector containing the MdD1 promoter, was co‐injected into tobacco. Three independent transfection experiments were performed. Data are the mean values ± SEM. *P < 0.05, **P < 0.01. (d) Fold change of MdD1 relative expression by qRT‐PCR. The following pollen tube treatments were used: methyl jasmonate (MeJA) and the inhibitor (DIECA for MeJA); a treatment with the above inhibitors followed by S‐RNase treatment; a treatment with S‐RNase with or without MdMYC2 silencing. Pollen tubes without any treatment were used as controls. Data from three biological replicates were combined using a linear mixed‐effects model. The final data were normalized to the expression in the untreated pollen tubes (control). Data are the mean ± SEM. *P < 0.05.