SUPPRESSOR OF PHYTOCHROME B-4 #3 reduces the expression of PIF-activated genes and increases expression of growth repressors to regulate hypocotyl elongation in short days

Abstract

SUPPRESSOR OF PHYTOCHROME B-4 #3 (SOB3) is a member of the AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED (AHL) family of transcription factors that are involved in light-mediated growth in Arabidopsis thaliana, affecting processes such as hypocotyl elongation. The majority of the research on the AHLs has been conducted in continuous light. However, there are unique molecular events that promote growth in short days (SD) compared to constant light conditions. Therefore, we investigated how AHLs affect hypocotyl elongation in SD. Firstly, we observed that AHLs inhibit hypocotyl growth in SD, similar to their effect in constant light. Next, we identified AHL-regulated genes in SD-grown seedlings by performing RNA-seq in two sob3 mutants at different time points. Our transcriptomic data indicate that PHYTOCHROME INTERACTING FACTORS (PIFs) 4, 5, 7, and 8 along with PIF-target genes are repressed by SOB3 and/or other AHLs. We also identified PIF target genes that are repressed and have not been previously described as AHL-regulated, including PRE1, PIL1, HFR1, CDF5, and XTR7. Interestingly, our RNA-seq data also suggest that AHLs activate the expression of growth repressors to control hypocotyl elongation, such as HY5 and IAA17. Notably, many growth-regulating and other genes identified from the RNA-seq experiment were differentially regulated between these two sob3 mutants at the time points tested. Surprisingly, our ChIP-seq data suggest that SOB3 mostly binds to similar genes throughout the day. Collectively, these data suggest that AHLs affect gene expression in a time point-specific manner irrespective of changes in binding to DNA throughout SD.

Keywords: AHL; Arabidopsis; Hypocotyl; PIF; SD; SOB3.

Fig. 1

AHLs repress hypocotyl growth in SD. Hypocotyl measurements of sob3-6, WT Col-0, and SOB3-D five-day-old seedlings grown in SD. Error bars represent standard error of the mean. In a Welch’s t test (unpaired two-tailed t test with unequal variance) compared with the wild type: P ≤ 0.0001 = ****

Fig. 2

AHLs repress the expression of growth-promoting genes while also activating growth repressors. RNA-seq analysis of SOB3-D and sob3-6 seedlings in SD. A SD photoperiod diagram depicting the time points used to harvest seedings for RNA-seq. The diagram starts at the beginning of day five and ends at the beginning of day six. B, C AHL-repressed (B) or -induced (C) nuclear-encoded genes identified from the RNA-seq data based on having higher or lower expression in sob3-6 than SOB3-D, respectively. In (B), AHL-repressed PIFs, PIF-target genes, and BR-associated genes are named. In (C), AHL-induced growth repressors are named

Fig. 3

SOB3 binds a similar number of genes at ZT4, ZT9, and ZT24. Overlap between genes found to be bound by SOB3 in different replicates of the ChIP-seq experiment at A) ZT4, B) ZT9, or C) ZT24. ChIP 1 is the first replicate, ChIP 2 is the second replicate, and ChIP 3 is the third replicate. Yellow outlines indicate genes that were used for subsequent analysis, i.e. those bound in at least two replicates within a time point

Fig. 4

Most SOB3-bound genes are bound at ZT4, ZT9, and ZT24. Overlap between genes bound by SOB3 in at least two ChIP-seq replicates at each time point

Fig. 5

SOB3 binds the same types of genomic regions at different time points. SOB3 binding pattern distribution determined from the ChIP-seq data based on annotations obtained using HOMER software. Distribution of SOB3 binding at A ZT4, B ZT9, and C ZT24. Promoter-TSS is defined as -1,000 bp to + 100 bp in relation to a transcription start site. TTS is defined as -100 bp to + 1000 bp in relation to a transcription termination site

Fig. 6

SOB3 exhibits similar binding to target genes at ZT4, ZT9, and ZT24. Distribution of SOB3 binding, as determined from the ChIP-seq data, to genes identified as SOB3-bound at one, two, or three time points

Fig. 7

Motifs associated with TCP-binding, PIF4-binding, and AHL-binding are all enriched in regions bound by SOB3. Top 4 enriched motifs from the ChIP-seq data at each time point: A ZT4, B ZT9, and C ZT24. Enriched motifs were identified in the 300 bp regions surrounding SOB3 peaks using CentriMo and DREME software in MEME-Suite.

Fig. 8

SOB3-bound genes are more frequently repressed than activated by AHLs in SD. Overlap between AHL-repressed or AHL-induced genes identified from the RNA-seq data and SOB3-bound genes identified from ChIP-seq at each of the three time points tested: A ZT4, B ZT9, and C ZT24

Fig. 9

AHLs directly regulate the expression of somewhat different types of genes at ZT4, ZT9, and ZT24. Top 25 most enriched GO terms among genes identified as SOB3-bound and AHL-repressed (A), or SOB3-bound and AHL-induced (B) at ZT4, ZT9 or ZT24. The relative P value for each GO term is indicated by color, with darker shades representing smaller P values

Fig. 10

SOB3 exhibits similar binding to genes identified as directly repressed by AHLs at ZT4, ZT9, and ZT24. Distribution of SOB3 binding, as determined from the ChIP-seq data, to genes identified as both SOB3-bound and AHL-repressed at only one or two time points

Fig. 11

SOB3 exhibits similar binding to genes identified as directly induced by AHLs at ZT4, ZT9, and ZT24. Distribution of SOB3 binding, as determined from the ChIP-seq data, to genes identified as both SOB3-bound and AHL-induced at only one or two time points