Fig. 3
MaSPL4/5/25/42/44 positively control MIR528 transcription by binding to the GTAC motifs in the MIR528 promoter. a The subcellular localization of MaSPL4/5/25/42/44 in Arabidopsis mesophyll protoplasts, using NLS-mCherry as a nuclear localization marker. b EMSA of in vitro binding of MaSPL4/5/25/42/44 to the MIR528 promoter. At the top is the MIR528 promoter diagram, which contains eight GTAC motifs. Numbers below the diagram indicate the distance away from the pre-miR528. A biotinylated probe containing three GTAC motifs sequence were incubated with the GST-MaSPLs, while the probe incubated with GST protein was used as a negative control. Biotin-free probes containing three GTAC and AAAA motifs were used as cold competitors and mutant competitors, respectively. c Y1H assay shows that MaSPL4/5/25/42/44 bind to the promoter of MIR528 in vitro. A 210 bp fragment containing the EMSA probe sequence was inserted into the pABAi vector. The full-length CDS of MaSPL4/5/25/42/44 were cloned into the pGADT7 vector and the empty vectors were used as the negative control. Yeast with pABAi vector and pGADT7 vector were cultured on SD/-Leu/+AbA200 medium. d The activation of MIR528 by MaSPL4/5/25/42/44 as determined by DLR. The LUC/REN ratio of the empty vector plus promoter-reporter was set as 1. Values are presented as means ± standard error (SE) of six biological replicates (n = 6). The asterisks (**) indicate significant differences at 0.01 level according to two-tailed unpaired Student’s t-test