DELLA proteins recruit the Mediator complex subunit MED15 to coactivate transcription in land plants

Abstract

DELLA proteins are negative regulators of the gibberellin response pathway in angiosperms, acting as central hubs that interact with hundreds of transcription factors (TFs) and regulators to modulate their activities. While the mechanism of TF sequestration by DELLAs to prevent DNA binding to downstream targets has been extensively documented, the mechanism that allows them to act as coactivators remains to be understood. Here, we demonstrate that DELLAs directly recruit the Mediator complex to specific loci in Arabidopsis, facilitating transcription. This recruitment involves DELLA amino-terminal domain and the conserved MED15 KIX domain. Accordingly, partial loss of MED15 function mainly disrupted processes known to rely on DELLA coactivation capacity, including cytokinin-dependent regulation of meristem function and skotomorphogenic response, gibberellin metabolism feedback, and flavonol production. We have also found that the single DELLA protein in the liverwort Marchantia polymorpha is capable of recruiting MpMED15 subunits, contributing to transcriptional coactivation. The conservation of Mediator-dependent transcriptional coactivation by DELLA between Arabidopsis and Marchantia implies that this mechanism is intrinsic to the emergence of DELLA in the last common ancestor of land plants.

Keywords: DELLA proteins; Mediator; gibberellins; hormone signaling; transactivation.

Fig. 1.

RGA and MED15 interaction enhances gene activation. (A) Alignment of Arabidopsis DELLA selected regions (numbers above alignment refer to corresponding RGA residue) showing the presence of predicted 9aaTADs (khaki shaded residues), p53-like activation domain motif bulky residues (Φ, purple shading, or cyan shading in overlapping motif), and acidic residues (red) surrounding p53-like AD sites. “x” refers to any residue. See SI Appendix, Fig. S1. (B) Yeast one-hybrid transactivation assay of GAL4 DNA binding domain (Gal4^BD) fusions of RGA N2 region (amino acids 1 to 120) with the Φ residues shown in (A) mutated to Ala residues or not (SI Appendix, Fig. S3D). The Left side shows a drop assay of a dilution series on -His medium and the Right-side quantification of the LacZ reporter activation. (C) RT-qPCR analysis of the GAL4-responsive reporter genes in the original Y2HGold yeast strain (GAL11) or with the endogenous MED15/GAL11 gene deleted (gal11Δ) in the presence of a Gal4^BD alone or fused to RGA. S. cerevisiae ACT1 was used as reference gene. (D) Yeast two-hybrid assay using MED15a or MED15a KIX domain as bait, and RGA and RGA DELLA domain as prey. (E) Dual luciferase transactivation assay in N. benthamiana leaves using the LUC gene under the control of 5xGal operon UAS motif as the reporter, and a Gal4^BD effector fused to the RGA N-end (RGA^N) coexpressed or not with MED15a as coeffector. (F) Dual luciferase transactivation assay in N. benthamiana leaves using the LUC gene under the control of 5xGal operon UAS motif as the reporter, and either a Gal4^BD fused to the RGA N2 region with Φ residues mutated or not, coexpressed with MED15a or not. Bars represent the mean activity of three to five independent biological replicates (circles). Statistical groups in determined by Tukey’s post hoc test (P < 0.05) following ANOVA. For RT-qPCR, statistical groups were calculated independently per each gene.

Fig. 2.

MED15 is involved in DELLA-mediated transcriptional activation. (A) Venn’s diagram showing the overlap of curated genes (showing at least 1 TPM in WT and med15a^Ri, P-adj <0.01) up-regulated in PAC-treated plants (compared to PAC+GA₃-treated plants). P-value shown indicates the statistical significance of the overlap. Orange-blue circles represent the predicted DELLA mechanistic effect on EAT-Up-derived TFs found to be enriched in each subset. Orange, coactivation; blue, sequestering. (B) TPM comparison of the genes up-regulated in WT (gray dot line in A), or exclusively in med15a^Ri (blue dot line in A). Zoomed-out axes are shown in SI Appendix, Fig. S4. (C) TF numbers per family and the presence of TF binding sites in DELLA ChIP-seq experiments (2). Colors represent the predicted DELLA mechanistic effect on that TF family. Expression analyses were carried out by whole RNA sequencing of 7-d-old seedlings treated for 3 d either with 1 μM PAC, or 1 μM PAC + 100 μM GA₃.

Fig. 3.

MED15 is needed for DELLA-dependent coactivation responses. (A) Five-day-old WT, med15a^Ri, and arr1;12 seedlings grown in darkness with or without 1 μM PAC. (Scale bar, 1 mm.) (B) Rainbow plot of angle between cotyledons in 5-d-old seedlings grown in darkness with or without 1 μM PAC corresponding to (A). Measurements in mock plants are not shown (mean = 0; SD = 0). (C) DPBA staining of 8-d-old WT and med15a^Ri seedlings root tips grown in long day conditions (16L:8D), and treated for 4 d with or without 1 μM PAC. (Scale bar, 100 μm.) (D) RT-qPCR analysis of GA and IDD-responsive genes in 5 d-old WT and med15a^Ri seedlings grown in darkness with or without 1 μM PAC using PDF2.1 as reference gene. (E) ChIP-qPCR analysis of MED6 occupancy at the indicated locations of GA20ox2 promoter in 7-d-old seedlings treated with 1 μM PAC, and with or without 100 μM GA₃ for 3 d. Relative enrichment is given as a percentage of IP/Input. (F) Dual luciferase transactivation assay in N. benthamiana leaves using the LUC gene under the control of GA20ox2 promoter as the reporter, and HA-GAF1, YFP-GAI, and cMyc-MED15a coexpressed as effectors. The effector level is indicated below each bar as the agroinfiltrated OD₆₀₀. (B) shows experimental data of one representative experiment of three with at least 15 plants per genotype and treatment; statistical difference represented as Student’s t test P-value. (D–F) bars represent biological replicate means and means of technical triplicates are depicted as circles. Statistical groups in (D–F) were determined by Tukey’s post hoc test (P < 0.05) following ANOVA. For qPCR, ANOVA were performed independently per gene/genomic region.

Fig. 4.

M. polymorpha MpDELLA acts as a coactivator by recruiting MED15. (A) Yeast two-hybrid assay using MpMED15 KIX domain as bait, and MpDELLA full length (FL) or N-terminal domain (N) as prey. (B) Dual luciferase transactivation assay in N. benthamiana leaves using the LUC gene under the control of the Gal operon UAS promoter as the reporter, and a GAL4 DNA binding domain (DBD) fused to the MpDELLA N-terminal domain, and MpMED15 coexpressed as effectors. (C) Yeast two-hybrid assay using the MpDELLA GRAS domain as bait and MpMYB14 as prey. (D) Dual luciferase transactivation assay in N. benthamiana leaves using the LUC gene under the control of MpPAL (Mp1g05190) promoter as the reporter, and HA-FLAG-MpMYB14, YFP-MpDELLA, and cMyc-MpMED15 coexpressed as effectors. The effector level in (B and D) is indicated below each bar as the agroinfiltrated OD₆₀₀. Bars represent medians of three biological replicates. Biological replicate means of technical triplicates are depicted as circles. Statistical groups were determined by Tukey’s post hoc test (P < 0.05) following ANOVA.