Fig. 5. PEP activation is repressed redundantly by multiple PIFs.

a Immunoblots showing the levels of the PEP complex and its core component rpoB in 4-d-old Col-0, pif1, pif3, pif4, pif5, pifq, pif345, pif145, pif135, and pif134 seedlings grown in the dark. Total protein was isolated under either native or denaturing conditions and resolved via BN-PAGE or SDS-PAGE to assess the fraction of rpoB in the PEP complex or the amount of total rpoB by immunoblots, respectively. RPN6 was used as a loading control. The graph below the immunoblots shows the relative PEP levels, which were estimated using the relative level of rpoB in the PEP complex (BN-PAGE) divided by the relative level of denatured rpoB (SDS-PAGE) in each sample. Different letters denote statistically significant differences in relative PEP level either between the pif single mutants and Col-0 or between the pif triple mutants and pifq (ANOVA, Tukey’s HSD, P  ≤  0.05, n = 2). b qRT-PCR analysis of the steady-state transcript levels of select PhAPGs, including psbA, psbB, and rbcL, in the 4-d-old dark-grown seedlings shown in a. c qRT-PCR analysis of the steady-state transcript levels of select PhANGs in the 4-d-old dark-grown seedlings described in a. For b and c, asterisks indicate statistically significant differences in the transcript levels either between the single pif mutants and Col-0 or between the pif triple mutants and pifq based on two-tailed Student’s t-test (^*P ≤ 0.05, ^**P ≤ 0.01, ^***P ≤ 0.001). If the change was less than two-fold or not statistically significant, it is labeled as n.s. (not significant). Error bars represent the s.e. of three biological replicates, and the centers of the error bars represent the mean values. The source data underlying the immunoblots in a and the qRT-PCR analysis in b and c are provided in the Source Data file.