Fig. 3. Genome-wide activation of PhANGs is insufficient to fully activate the PEP in pifq.

a Immunoblots showing the levels of the PEP complex and its core component rpoB in 2-d-old and 4-d-old Col-0 and pifq 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. The bar graph shows the qualification of the relative levels of the PEP complex, which were calculated using the relative level of rpoB in the PEP complex (BN-PAGE) divided by the relative level of the total rpoB (SDS-PAGE) in each sample. Different letters denote statistically significant differences in the relative PEP levels among the genotypes (ANOVA, Tukey’s HSD, P  ≤  0.05, n = 2). b qRT-PCR analysis of the steady-state transcript levels of psbA, psbB, and rbcL in 2-d-old and 4-d-old Col-0 and pifq seedlings grown in the dark. The transcript levels were calculated relative to those of PP2A. The fold changes between the transcript levels in the mutants and Col-0 are shown. Asterisks indicate statistically significant differences in the transcript levels between the mutant and Col-0 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. c Venn diagram showing PIF-repressed PhANGs in 2-d-old and 4-d-old dark-grown seedlings. d Box-and-whisker plots showing the fold changes in LHCs, CHLs, and nPEACs in 2-d-old and 4-d-old dark-grown pifq seedlings compared with the Col-0 controls. Each data point represents a gene; data points above the dotted magenta line are statistically significantly upregulated in the mutants by at least two-fold. The lower and upper bounds of the box represent the 25th and 75th percentile respectively; the center bar represents the median. The lower and upper end of the whiskers represent minimum and maximum values respectively. Dots outside of the whiskers represent outliers based on Tukey’s fences. Asterisks indicate statistically significant differences between the 2-d and 4-d samples based on two-tailed Student’s t-test (^***P ≤ 0.001, n = 6 biological replicates for 2-d-old dark-grown pifq, and n = 3 biological replicates for 4-d-old dark-grown pifq). n.s. indicates no significant change. e qRT-PCR analysis of the steady-state transcript levels of representative LHCs (LHCB1.1, LHCB1.4, and LHCA1) and nPEACs (PsaN, AtpD, and GAPA-2) in 2-d-old and 4-d-old dark-grown Col-0 and pifq seedlings. The transcript levels were calculated relative to those of PP2A. The fold changes between the mutants and Col-0 are shown. Asterisks indicate statistically significant differences between the mutant and Col-0 based on two-tailed Student’s t-test (^*P ≤ 0.05, ^**P ≤ 0.01). 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 e are provided in the Source Data file.