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

<p>(<i>A</i>) Root meristem size of <i>35S</i>::<i>ARR1ΔDDK</i>:<i>GR</i> 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. (<i>B</i>) Angle between cotyledons of <i>35S</i>::<i>ARR1ΔDDK</i>:<i>GR</i> 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). (<i>C</i>) Angle between cotyledons of <i>35S</i>::<i>ARR1ΔDDK</i>:<i>GR</i> seedlings in <i>gai rga</i> 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 <i>35S</i>::<i>ARR1ΔDDK</i>:<i>GR GAI RGA</i> seedlings treated with DEX). (<i>D</i>) Angle between cotyledons of wild-type and <i>arr1 arr12</i> 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.</p

Genome-wide occupancy of RGA at target loci.

<p>(<i>A</i>) Genomic location of the statistically significant peaks of GFP-RGA along its target genes. (<i>B</i>) Gene ontology analysis of RGA targets, using ReviGO. (<i>C</i>) Statistically significant over-representation of <i>cis</i> elements for different transcription factor families. The <i>p</i> value for each element is indicated. Bars represent the number of genes with at least one copy of the corresponding <i>cis</i> 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 <i>cis</i> element, therefore the sum of all genes in the graph is much larger than the 421 genes associated to ChIP peaks.</p

DELLAs promote ARR1 activity.

<p>(<i>A</i>) Expression in <i>Arabidopsis</i> roots of GFP under the control of the CK- and ARR1-responsive TCS element, after treatments with 0.5 μM <i>trans</i>-zeatin and 1 μM GA₄. (<i>B</i>) Luciferase assays in <i>N</i>. <i>benthamiana</i> leaves agroinfiltrated with HA-ARR1, YFP-GAI, and <i>myc</i>-M5GAI, using the <i>LUC</i> gene under the control of the wild-type and mutant versions of the TCS element, and the constitutively expressed <i>Renilla</i> luciferase (<i>REN</i>) 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.</p

ARR1 and GAI interact physically in plants.

<p>(<i>A</i>) Y2H assay of the interaction between ARR1 and truncated versions of GAI. (H, Histidine; 3-AT, 5mM 3-aminotriazol). (<i>B</i>) Y2H assay of the interaction between M5-GAI and truncated versions of ARR1. (H, Histidine; 3-AT, 5mM 3-aminotriazol). (<i>C</i>) Bimolecular Fluorescence Complementation assay of the interaction between GAI and ARR1 in agroinfiltrated <i>N</i>. <i>benthamiana</i> leaves. Size bars, 10μm. (<i>D</i>) 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 <i>N</i>. <i>benthamiana</i>. (<i>E</i>) Co-IP assay of the interaction between <i>myc-</i>GAI and HA-ARR1 in <i>Arabidopsis</i> protoplasts. The arrowhead indicates the size of the expected HA-ARR1 band. (<i>F</i>) Co-IP showing the interaction between RGA and ARR1-YFP-HA in <i>Arabidopsis</i> 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 <i>gai-t6</i> and <i>rga-24</i> 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, <i>35S</i>::<i>ARR1-YFP-HA</i>.</p

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

<p>(<i>A</i>) Heat map representation of the <i>Arabidopsis</i> gene set whose regulation by ARR1ΔDDK:GR or CKs depends on DELLA proteins. The colour scale represents Z-scores. (<i>B</i>) Enrichment of GO categories of all ARR1 target genes in the presence of DELLAs, visualized with ReviGO. (<i>C</i>) Gene expression analysis by RT-qPCR in response to short-term ARR1ΔDDK:GR induction with or without PAC. (<i>D</i>) Gene expression analysis by RT-qPCR in response to short-term induction of gai-1 with or without BA. (<i>E</i>) ChIP analysis of RGA::GFP-RGA at the promoters of six representative common targets for ARR1 and DELLAs. (<i>F</i>) 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 <i>RGA</i>::<i>GFP-RGA</i> and <i>35S</i>::<i>ARR1ΔDDK</i>:<i>GR</i> 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. (<i>G</i>) ChIP analysis of endogenous RGA at the promoters of six representative common targets for ARR1 and DELLAs, in the wild type and in <i>arr1 arr12</i> mutants. ChIP was performed with anti-RGA antibodies. For (<i>C-G</i>), data correspond to single biological samples analyzed in triplicates. A second biological sample showed equivalent results.</p