Fig. 3

Generation of independent alleles of pdx1.2 by CRISPR-Cas9. a and (b) Schematic representations of alleles of pdx1.2 generated using the CRISPR-Cas9 technology are shown on the left. The red box represents the heat shock element (HSE), which is from − 165 to − 153 bp upstream of the ATG translational start codon (+ 1). The black arrows indicate the annealing positions of the primers used for qPCR. The numbers refer to the site of insertion of a nucleotide as depicted. DNA sequencing chromatograms around the mutated sites are shown on the right. The DNA sequences of wild type (Col-0) and CRISPR mutations (1 (pdx1.2–3), A-1.8 (pdx1.2–4), B-11.11 (pdx1.2–5)) are given below each chromatogram. In each case, CRISPR resulted in the addition of a nucleotide, as depicted (in red), and is highlighted by a blue bar in the respective chromatogram. c Quantitative analysis of PDX1.2 transcript expression in wild type (Col-0) and the pdx1.2 CRISPR mutants characterized (pdx1.2–3, pdx1.2–4, pdx1.2–5). The expression relative to GAPDH in the respective lines is depicted in the absence of heat stress (-HS) or the presence of heat stress (+HS). Heat stress was induced by exposure to 37 °C for 1 h, at which time-point samples were harvested. In each case, 8-days-old Arabidopsis seedlings pre-cultivated in sterile culture at 22 °C under a 16-h photoperiod (120 μmol photons m^− 2 s^− 1) and 8 h of darkness at 18 °C were used. The data are the average of three biological and three technical replicates. Statistical differences from the wild type under the same conditions were calculated by a two-tailed Student’s t test and indicated by an asterisk for P < 0.001. In all cases, error bars represent SE