Genome Wide Binding Site Analysis Reveals Transcriptional Coactivation of Cytokinin-Responsive Genes by DELLA Proteins

Abstract

The ability of plants to provide a plastic response to environmental cues relies on the connectivity between signaling pathways. DELLA proteins act as hubs that relay environmental information to the multiple transcriptional circuits that control growth and development through physical interaction with transcription factors from different families. We have analyzed the presence of one DELLA protein at the Arabidopsis genome by chromatin immunoprecipitation coupled to large-scale sequencing and we find that it binds at the promoters of multiple genes. Enrichment analysis shows a strong preference for cis elements recognized by specific transcription factor families. In particular, we demonstrate that DELLA proteins are recruited by type-B ARABIDOPSIS RESPONSE REGULATORS (ARR) to the promoters of cytokinin-regulated genes, where they act as transcriptional co-activators. The biological relevance of this mechanism is underpinned by the necessity of simultaneous presence of DELLAs and ARRs to restrict root meristem growth and to promote photomorphogenesis.

Fig 1. Genome-wide occupancy of RGA at target loci.

(A) Genomic location of the statistically significant peaks of GFP-RGA along its target genes. (B) Gene ontology analysis of RGA targets, using ReviGO. (C) Statistically significant over-representation of cis elements for different transcription factor families. The p value for each element is indicated. Bars represent the number of genes with at least one copy of the corresponding cis element in the ChIP peak. Colours indicate induction (red), repression (blue), both (yellow) or no effect (gray) by DELLAs across all published transcriptomic datasets. Please note that each ChIP peak may contain more than one cis element, therefore the sum of all genes in the graph is much larger than the 421 genes associated to ChIP peaks.

Fig 2. ARR1 and GAI interact physically in plants.

(A) Y2H assay of the interaction between ARR1 and truncated versions of GAI. (H, Histidine; 3-AT, 5mM 3-aminotriazol). (B) Y2H assay of the interaction between M5-GAI and truncated versions of ARR1. (H, Histidine; 3-AT, 5mM 3-aminotriazol). (C) Bimolecular Fluorescence Complementation assay of the interaction between GAI and ARR1 in agroinfiltrated N. benthamiana leaves. Size bars, 10μm. (D) Analysis of the interaction between HA-ARR1 or HA-ARR1ΔDDK with YFP-GAI by co-immunoprecipitation (co-IP) with anti-GFP in agroinfiltrated leaves of N. benthamiana. (E) Co-IP assay of the interaction between myc-GAI and HA-ARR1 in Arabidopsis protoplasts. The arrowhead indicates the size of the expected HA-ARR1 band. (F) Co-IP showing the interaction between RGA and ARR1-YFP-HA in Arabidopsis seedlings. Proteins were immunoblotted and consecutively detected with anti-GAI and anti-HA-peroxidase conjugate antibodies. The anti-GAI polyclonal antibody recognizes both GAI and RGA. Forty micrograms of soluble proteins were loaded as input and as unbound control. Soluble proteins from the null mutants gai-t6 and rga-24 grown for 7 days in 0.5 μM PAC plates in continuous light were used as controls. The blue lines in the marker lane indicate the position of the 64 kDa and 98 kDa bands in the upper and middle panels, respectively. Stained bands in the marker lane in the lower panel correspond to 64 kDa and 50 kDa. WT, wild-type Col-0; ARR1, 35S::ARR1-YFP-HA.

Fig 3. DELLAs promote ARR1 activity.

(A) Expression in Arabidopsis roots of GFP under the control of the CK- and ARR1-responsive TCS element, after treatments with 0.5 μM trans-zeatin and 1 μM GA₄. (B) Luciferase assays in N. benthamiana leaves agroinfiltrated with HA-ARR1, YFP-GAI, and myc-M5GAI, using the LUC gene under the control of the wild-type and mutant versions of the TCS element, and the constitutively expressed Renilla luciferase (REN) for normalization. The values represent the ratio between both luciferase activities and are the average of three biological replicates. Error bars are the standard deviation. One and two asterisks denote statistical significance (p<0.05 and p<0.005 respectively). The lower panel contains the western-blot analysis of the protein samples corresponding to equal mixtures from the three leaves used for the LUC assays with the wild-type TCS elements.

Fig 4. ARR1 and DELLA act as transcriptional co-regulators in Arabidopsis.

(A) Heat map representation of the Arabidopsis gene set whose regulation by ARR1ΔDDK:GR or CKs depends on DELLA proteins. The colour scale represents Z-scores. (B) Enrichment of GO categories of all ARR1 target genes in the presence of DELLAs, visualized with ReviGO. (C) Gene expression analysis by RT-qPCR in response to short-term ARR1ΔDDK:GR induction with or without PAC. (D) Gene expression analysis by RT-qPCR in response to short-term induction of gai-1 with or without BA. (E) ChIP analysis of RGA::GFP-RGA at the promoters of six representative common targets for ARR1 and DELLAs. (F) Fold enrichment of GFP-RGA at selected target promoters in the presence (+DEX) vs the absence (-DEX) of ARR1ΔDDK:GR, in F1 seedlings of a cross between RGA::GFP-RGA and 35S::ARR1ΔDDK:GR plants. In this experiment, qPCR values of ChIP samples were normalized per input in each condition (-DEX, and +DEX), and here we show the ratio between those two conditions. (G) ChIP analysis of endogenous RGA at the promoters of six representative common targets for ARR1 and DELLAs, in the wild type and in arr1 arr12 mutants. ChIP was performed with anti-RGA antibodies. For (C-G), data correspond to single biological samples analyzed in triplicates. A second biological sample showed equivalent results.

Fig 5. Physical interaction between ARR1 and DELLAs regulates division at the root meristem and photomorphogenesis.

(A) Root meristem size of 35S::ARR1ΔDDK:GR seedlings grown for 4 days with and without GAs. (n = 20; data are mean ± SD, ***p<0.001 in a Student’s t-test with respect to control plants). Arrowheads mark the extension of the meristem. (B) Angle between cotyledons of 35S::ARR1ΔDDK:GR seedlings grown for 4 days with and without GAs in darkness. (n = 18; data are mean ± SD; ***p<0.001 in a Student’s t-test with respect to seedlings treated with DEX without GAs). (C) Angle between cotyledons of 35S::ARR1ΔDDK:GR seedlings in gai rga double mutant or in an otherwise wild-type background. Seedlings were grown for 4 days in darkness. (n = 15; data are mean ± SD; ***p<0.001 in a Student’s t-test with respect to 35S::ARR1ΔDDK:GR GAI RGA seedlings treated with DEX). (D) Angle between cotyledons of wild-type and arr1 arr12 seedlings grown for 4 days with and without PAC in darkness. (n = 18; data are mean ± SD; ***p<0.001 in a Student’s t-test with respect to PAC-treated wild-type seedlings). Experiments were performed as indicated in Materials and Methods. Equivalent treatments of wild-type seedlings with DEX did not cause any change in root meristem size and or the angle between cotyledons.