DELLA-induced early transcriptional changes during etiolated development in Arabidopsis thaliana

Abstract

The hormones gibberellins (GAs) control a wide variety of processes in plants, including stress and developmental responses. This task largely relies on the activity of the DELLA proteins, nuclear-localized transcriptional regulators that do not seem to have DNA binding capacity. The identification of early target genes of DELLA action is key not only to understand how GAs regulate physiological responses, but also to get clues about the molecular mechanisms by which DELLAs regulate gene expression. Here, we have investigated the global, early transcriptional response triggered by the Arabidopsis DELLA protein GAI during skotomorphogenesis, a developmental program tightly regulated by GAs. Our results show that the induction of GAI activity has an almost immediate effect on gene expression. Although this transcriptional regulation is largely mediated by the PIFs and HY5 transcription factors based on target meta-analysis, additional evidence points to other transcription factors that would be directly involved in DELLA regulation of gene expression. First, we have identified cis elements recognized by Dofs and type-B ARRs among the sequences enriched in the promoters of GAI targets; and second, an enrichment in additional cis elements appeared when this analysis was extended to a dataset of early targets of the DELLA protein RGA: CArG boxes, bound by MADS-box proteins, and the E-box CACATG that links the activity of DELLAs to circadian transcriptional regulation. Finally, Gene Ontology analysis highlights the impact of DELLA regulation upon the homeostasis of the GA, auxin, and ethylene pathways, as well as upon pre-existing transcriptional networks.

Figure 1. Effect of transient gai-1 induction on known DELLA target genes.

Two-day-old, etiolated HS::gai-1 and wild type Col-0 plants grown at 22°C received a 37°C heat-shock treatment for different periods (30, 60, 120 min) and then returned to 22°C. Samples were collected at the indicated times. Expression of the transgene (A), as well as of GA20ox2 (B) and GA3ox1 (C) genes was monitored by RT-qPCR.

Figure 2. Transcriptomic analysis of early targets of DELLA proteins.

(A) Heatmap representation of the 148 best-scored genes (q-value≤8). (B) Illustration of the overlap with the datasets of DELLA target genes in two other developmental situations , (C) Heatmap representation of the differential expression of genes overlapping in the three datasets. Red and blue colors in the heatmaps represent induced and repressed genes, respectively.

Figure 3. Meta-analysis comparing microarray data from HS::gai-1, HY5 and PIF targets.

Venn diagram of microarray data from HS::gai-1, HY5 targets and quadruple pif mutant (pifQ) show common genes regulated by GAI, HY5 and PIF proteins. Heatmaps show the behavior of common GAI-HY5, GAI-HY5-PIF and GAI-PIF targets in different light conditions. Wt R/D, data are differentially expressed genes under red light compared to dark in a WT. pifQ/wt D, data are differentially expressed genes among pifQ mutant compared to wt in darkness. pifQ/wt R, data are differentially expressed genes among pifQ mutant compared to wt under red light. The heatmaps represent the differential expressions of genes overlapping in the different datasets. Red and blue colors in the heatmaps represent induced and repressed genes, respectively.

Figure 4. Over-represented cis elements among DELLA-regulated promoters.

(A) Logos of over-represented cis elements in the promoters of induced and repressed targets in the HS::gai-1 microarray experiment. (B) Logos of over-represented cis elements in the promoters of induced and repressed genes coming from the joint dataset of HS::gai-1, rga-Δ17 and GA/floral microarray targets. The logo representation was obtained at http://weblogo.berkeley.edu/ .

Figure 5. GAI directly regulates the expression of genes of the GA pathway.

Three-day-old, etiolated pGAI::gai-1-GR seedlings grown at 22°C were incubated for 5 h in water or in water (control treatment) supplemented with either 10 αM DEX (white bars), 10 αM cycloheximide (orange bars) or both (blue bars). Expression was monitored by RT-qPCR and normalized to the control treatment. Values are log ratios between the treatment and the control. Data represent mean and the standard error of the mean from three independent biological replicates. Data from each biological replicate consisted in three technical replicates that were averaged and normalized.

Figure 6. GAI directly modulates the auxin pathway and transcriptional networks.

Three-day-old, etiolated pGAI::gai-1-GR seedlings grown at 22°C were incubated for 5 h in water or in water (control treatment) supplemented with either 10 αM DEX (white bars), 10 αM cycloheximide (orange bars) or both (blue bars). Expression was monitored by RT-qPCR and normalized to the control treatment. Values are log ratios between the treatment and the control. Data represent mean and standard error of the mean from three independent biological replicates. Data from each biological replicate consisted in three technical replicates that were averaged and normalized.