Fig. 6

PbZFP1 positively mediates ABA biosynthesis in fruit flesh. a Dual-luciferase assays reveal the regulatory effects of 12 selected TFs on the promoters of ABA biosynthetic genes. The standard error bars were calculated from at least five replicates, and two independent experiments were performed. b A yeast-one-hybrid assay shows the binding of PbZFP1, PbHB1, PbHB2, and PbHB3 to the upstream region (− 500 bp to − 100 bp) of the initiation codon of PbAAO. SD/-Leu, SD medium lacking Leu; SD/Leu + AbA²⁰⁰, 200 ng/mL Aureobasidin A was added in the SD medium lacking Leu. c Electrophoretic mobility shift assays reveal the binding of the PbAAO promoter by PbZFP1, PbHB1, PbHB2, and PbHB3. “ + ” and “–” indicate the presence and absence of recombinant TF protein, biotin-labeled probe, cold probe, or biotin-labeled mutant, respectively. The concentrations of cold probe were tenfold (10 ×) and 50-fold (50 ×) of labeled probes. The expression level of PbZFP1, PbHYB1, PbSDR2, and PbAAO were tested by qRT-PCR in the transgenic calli (d) and transiently transformed fruit flesh (f, h). ABA content was measured in the transgenic calli (e) and transiently transformed fruit flesh (g, i). OE represents the over-expression of PbZFP1, while pSAK277 represents the empty vector and served as the control. RNAi represents the virus-induced gene silencing of PbZFP1, while TRV1/2 represents the empty vectors of pTRV1 and pTR2. Analysis of variance was calculated by Student’s t test. Single and double asterisks stand for the level of significance at P-value < 0.05 and < 0.01, respectively