Coordinated regulation of Arabidopsis thaliana development by light and gibberellins

Abstract

Light and gibberellins (GAs) mediate many essential and partially overlapping plant developmental processes. DELLA proteins are GA-signalling repressors that block GA-induced development. GA induces degradation of DELLA proteins via the ubiquitin/proteasome pathway, but light promotes accumulation of DELLA proteins by reducing GA levels. It was proposed that DELLA proteins restrain plant growth largely through their effect on gene expression. However, the precise mechanism of their function in coordinating GA signalling and gene expression remains unknown. Here we characterize a nuclear protein interaction cascade mediating transduction of GA signals to the activity regulation of a light-responsive transcription factor. In the absence of GA, nuclear-localized DELLA proteins accumulate to higher levels, interact with phytochrome-interacting factor 3 (PIF3, a bHLH-type transcription factor) and prevent PIF3 from binding to its target gene promoters and regulating gene expression, and therefore abrogate PIF3-mediated light control of hypocotyl elongation. In the presence of GA, GID1 proteins (GA receptors) elevate their direct interaction with DELLA proteins in the nucleus, trigger DELLA protein's ubiquitination and proteasome-mediated degradation, and thus release PIF3 from the negative effect of DELLA proteins.

Figure 1. Effect of GA₃, MG132 and PAC on DELLA protein abundance

Immunoblot analysis of RGA (by anti-RGA antibody) and TAP-DELLA proteins (by anti-MYC antibody) in various light-grown Arabidopsis seedlings (genotypes labelled to the left of each panel) treated with different combinations of GA₃, MG132 and PAC. Panels on the left (four lanes) and panels on the right (two lanes) are from two independent experiments using different protein gel systems. RPN6 immunoblotting (by anti-RPN6 antibody) is used as a loading control. WT, wild type.

Figure 2. DELLA proteins and PIF3 have opposite roles in regulating Arabidopsis hypocotyl elongation

a, Images of red-light-grown seedlings. b, Hypocotyl length measurement (mean ± s.d.) of untreated seedlings (red), or seedlings treated with 10 μM GA₃ (blue) or 1 μM PAC (yellow).c, Hypocotyl length measurement (mean ± s.d.) of red-light-grown seedlings treated with increasing amounts of GA₃ or PAC (see Methods). The concentrations of GA₃ used are 0, 0.5 μM, 1 μM, 2 μM and 5 μM (from left to right). The concentrations of PAC were 0, 0.01 μM, 0.02 μM, 0.05 μM, 0.1 μM, 0.2 μM and 0.5 μM (from left to right). In b and c, hypocotyl length of untreated wild-type seedlings is set to 100%. d, Simplified diagram depicting the genetic interaction of light and GA in the control of hypocotyl elongation by PIF3 and DELLA proteins. della, rga-t2 gai-t6 rgl1–1 rgl2–1 rgl3–1.

Figure 3. DELLA proteins bind PIF3 and inhibit PIF3 activity towards its target genes

a, β-galactosidase activities from yeast two-hybrid assays (mean ± s.d.). b, BiFC analysis of RGA and PIF3. The positions of nuclei are indicated by arrows. c, Co-immunoprecipitation of RGA with PIF3 in 35S-PIF3–His–MYC seedlings. ‘RGA’ and ‘Pre’ indicate immunoprecipitation by anti-RGA antibody and pre-immune sera, respectively. d, Pull-down assays between His–PIF3 and MBP–RGA. The precipitated His–PIF3 was detected by anti-His antibody. MBP–RGA and MBP inputs were stained by Coomassie blue. e, ChIP–PCR analyses in dark-grown seedlings. f, Semi-quantitative RT–PCR analyses in dark-grown seedlings. Total, total protein extracts; IP, immunoprecipitation.

Figure 4. GA-dependent interaction between GID1s and DELLA proteins

a, b, β-galactosidase activities from yeast two-hybrid assays (mean ± s.d.). In b, β-galactosidase activities from GAI–GID1 interactions are set to 100%. c, BiFC analysis of GID1c and RGA. The positions of nuclei are indicated by arrows. d, TAP-DELLA proteins interact with GID1a–YFP. e, Detection of multi-ubiquitinated TAP-RGA. In d, e, ‘IgG’ indicates immunoprecipitation by IgG-conjugated beads. f–h, RGA interacts with GID1a–MYC (f), GID1b–Flag (g), and GID1c–HA(haemagglutinin) (h). ‘MYC’, ‘Flag’ and ‘HA’ indicate immunoprecipitation by respective antibodies. i, A working model of the nuclear protein interaction cascade in GA signalling.