Fig 2: Immune analysis of TBS proteins and other plastid-localized proteins (as references) in light-exposed WT (Col-0), ntrc, trxf1, and ntrc/trxf1 plants under different growth conditions. (A,B): The plants were initially grown for 3 weeks under SD (8 h light/16 h dark, normal light (NL, 160 µmol photons m-2 s-1). (A) Then, the SD-grown seedlings were exposed for an additional week to high light (HL 500 µmol photons m-2 s-1). Samples were harvested after 4 h of light and 16 h of darkness. (B) The SD-grown three-week-old seedlings were exposed to low (LL, 40 µmol photons m-2 s-1) or high light (HL) for a week and then, samples were harvested 4 h after the start of the light phase. (C): Expression of selected TBS genes in WT (Col-0) grown under different light conditions. SD-grown three-week-old plants under NL were either left in NL or transferred to HL or LL for another week. The gene expression of the different growth conditions is given relative to the expression under NL (dashed line), with ACT2 (AT3G18780) as the reference gene. The leaf samples were harvested in the middle of the light period (4 h after the start of light exposure) or at the end of the dark period (16 h) of NL-exposed plants. The relative transcription was calculated using the 2-??Ct method. Means and standard deviations refer to four biological replicates.

Fig 3: Complementation of the spl7-1 mutant by the SPL7prom:HA-SPL7-HA:SPL7term transgene. A, Photographs of the WT, the spl7-1 mutant and a selected transgenic homozygous spl7-1 SPL7prom:HA-SPL7-HA:SPL7term (HA-SPL7-HA) line. Shown are 21-day-old seedlings cultivated in low-Cu (no CuSO4 added) or control (0.5 µM CuSO4) medium supplemented with 1% (w/v) sucrose and solidified with EDTA-washed agar in vertically oriented glass petri plates. B, Relative SPL7 transcript levels according to RT–qPCR, in the shoots of seedlings cultivated as in (A). Data are means ± sd (n = 3 technical replicates, that is independent PCR runs, each with three replicate wells per transcript). Data from two additional transgenic lines are shown in Supplemental Figure S9. C, Immunoblot detection of HA-SPL7-HA (expected at ~90 kDa) in shoots of WT, spl7-1, and HA-SPL7-HA seedlings cultivated as in (A). PVDF membranes were stripped and reprobed with a-ACTIN (ACT) antibody (shown as an LC). The full image and results from an independent experiment are shown in Supplemental Figure S10. D, Validation of ChIP prior to sequencing using ChIP-qPCR of previously implicated direct SPL7 target genes. Data are mean ± SD (n = 3 technical replicates on a PCR plate) of relative DNA enrichment of the promoter regions of FSD1, MIR408 as a positive control gene, and ACT7 (negative control gene), quantified by ChIP-qPCR on immunoprecipitation (IP) and input samples. Chromatin was isolated from shoot tissues of HA-SPL7-HA and WT seedlings (WT, negative control) cultivated in R 1 (see Figure 7A). Input samples represent aliquots taken after chromatin shearing and before the addition of a-HA for the IP.

Fig 4: Global identification of SPL7 DNA-binding sites. A, Venn diagrams showing the reproducibility of results. Given are the numbers of SPL7-binding segments (peaks) identified by ChIP-seq in each of four independent experiments (each with independent plant cultivation, denoted as R 1 to R 4) across the genome. ChIP-seq was conducted on shoots of 21-day-old seedlings cultivated on control (0.5 µM Cu) and low-Cu (no Cu added) medium supplemented with 1% (w/v) sucrose and solidified with EDTA-washed agar in vertically oriented glass petri plates. B, Number of SPL7-binding segments identified by ChIP-seq (shown on a dark background in A) and associated loci. C, Validation of ChIP-seq results by ChIP-qPCR. Data are mean ± sd (n = 3 technical replicates on a PCR plate) of relative DNA enrichment of the promoter regions of novel candidates FSD1, CITF1, CITF2 (bHLH23), with ACTIN7 (ACT7) as a negative control. Chromatin was independently isolated from a different replicate pool of shoots of HA-SPL7-HA and WT seedlings (WT, negative control) cultivated in R 1 (see A), generating independent immunoprecipitate (IP) and input samples. Input samples represent aliquots taken after chromatin shearing and before the addition of a-HA for the IP. See Supplemental Figure S12, J and K for two additional independent experiments. D–I, The GTACTRC motif (D–F, E-value = 8.20 × 10-3, 21 motif sites) and the TCTTCTST motif (G–I, E-value = 3.40 × 10-2, 19 motif sites) identified by MEME motif analysis. Motif frequency positional distribution (200-bp bin size), with conservation logo generated by MEME as an inset (D and G). Density plot summarizing the distances of motif from the center of peaks from ChIP-seq (75-bp bin size) (E and H). Number of motif copies within single peaks (F and I). The gene body (gray box) of all corresponding genes was normalized to 2,000 bp (D and G). The curved line indicates the shape of the distribution (E and H). TSS: transcriptional start site, TTS: transcriptional termination site (positions from Arabidopsis TAIR10 genome annotation).