COP1 destabilizes DELLA proteins in Arabidopsis

Abstract

DELLA transcriptional regulators are central components in the control of plant growth responses to the environment. This control is considered to be mediated by changes in the metabolism of the hormones gibberellins (GAs), which promote the degradation of DELLAs. However, here we show that warm temperature or shade reduced the stability of a GA-insensitive DELLA allele in Arabidopsis thaliana Furthermore, the degradation of DELLA induced by the warmth preceded changes in GA levels and depended on the E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1). COP1 enhanced the degradation of normal and GA-insensitive DELLA alleles when coexpressed in Nicotiana benthamiana. DELLA proteins physically interacted with COP1 in yeast, mammalian, and plant cells. This interaction was enhanced by the COP1 complex partner SUPRESSOR OF phyA-105 1 (SPA1). The level of ubiquitination of DELLA was enhanced by COP1 and COP1 ubiquitinated DELLA proteins in vitro. We propose that DELLAs are destabilized not only by the canonical GA-dependent pathway but also by COP1 and that this control is relevant for growth responses to shade and warm temperature.

Keywords: environment; gibberellin; growth; shade avoidance; thermomorphogenesis.

Fig. 1.

COP1 regulates RGA levels in Arabidopsis hypocotyls in response to shade and warmth. (A) GFP-RGA levels respond to shade and warmth in the absence of GA synthesis. We incubated the seedlings with the indicated doses of PAC for 8 h, before exposure to shade or 28 °C for 4 h. (B) GFP-(rga-∆17) is insensitive to GA-induced degradation. Seedlings of pRGA:GFP-RGA and pRGA:GFP-(rga-∆17) lines were mock treated or treated with 3 μM GA₄ for 4 h. (C) Shade or warmth reduces GFP-(rga-∆17) levels in a COP1-dependent-manner. Seedlings were exposed to the treatment for 3 d. (D and E) Dynamics of nuclear accumulation of YFP-COP1 (D) in the wild type and of GFP-RGA (E) in the wild type (circles) and cop1-4 mutants (triangles) after the transfer from darkness to light (white symbols), light to shade (green symbols), and 20 °C to 28 °C (red symbols). (F) Time course of GA₄ levels in wild-type and cop1-4 seedlings before (time = 0 h) and after transfer to 28 °C (Inset shows an extended time course). (G) Time course of nuclear levels of GFP-RGA in wild-type and cop1-4 seedlings before (time = 0 h) and after transfer to 28 °C. (H) The reduction of GFP-RGA levels in response to 28 °C is reversible, unaffected by a saturating dose of PAC and dependent on COP1. Seedlings of pRGA:GFP-RGA in the wild-type and cop1-4 backgrounds were returned to 20 °C after 3 h-treatment of 28 °C. We incubated the seedlings with PAC for 8 h before exposure to 28 °C. Confocal data (A, E, G, and H) show the normalized fluorescence intensity (NFI) in nuclei (NFI = 1 in the wild-type seedling control, and in B in the wild-type RGA control). For representative images, see D and E (8-h time point) and SI Appendix, Fig. S2 A and B. Confocal microscopy data are means and SE of 5 to 10 (A), 6 to 9 (B), 6 to 14 (C), 18 (D and E), 6 to 13 (G), and 18 (H) seedlings (a minimum of 10 and up to 50 nuclei were averaged per seedling replicate). GA₄ data are means and SE of three independent biological replicates. In A, C, and G we indicate the significance of the differences with the control condition (B also shows the difference between genotypes) in Student’s t test or ANOVA followed by Bonferroni tests. In E–G we indicate the significance of the term accounting for the interaction (Int.) between condition (light, temperature) and genotype (wild type, cop1) in multiple regression analysis. In H, we indicate the significance of the comparison with the preceding bar in ANOVA followed by Bonferroni tests. *P < 0.05, ***P < 0.005; ns, nonsignificant.

Fig. 2.

COP1 destabilizes DELLAs. (A) The reduction of GFP-RGA levels by warm temperature or shade requires the 26S proteasome and COP1. Confocal data show the NFI in nuclei (NFI = 1 in the wild-type seedling control). NFI data are means and SE of 6 to 9 seedlings (10 to 30 nuclei were averaged per seedling replicate). Asterisks indicate that the difference is statistically significant (Student’s t test, **P < 0.01 and ***P < 0.001; ns, nonsignificant). (B and C) COP1 destabilizes RGA (B) and the GA-insensitive rga-Δ17 (C) in N. benthamiana leaves. HA-RGA and HA-(rga-Δ17) were transiently expressed alone or with Flag-COP1 in leaves of N. benthamiana. For MG132 treatments, leaves were infiltrated with a solution of 25 μM of the inhibitor 8 h before sampling. HA-GFP was used as control to demonstrate the specificity of COP1 action. Blots show data from three individual infiltrated leaves per mixture. Plots show HA-RGA and HA-(rga-Δ17) normalized against HA-GFP. Data are means and SE of three leaves from one experiment, repeated twice with similar results. Asterisks indicate that the difference is statistically significant (Student’s t test, *P < 0.05 and **P < 0.01; ns, nonsignificant).

Fig. 3.

COP1 physically interacts with DELLA proteins. (A and B) Y2H assays showing the interaction between N-terminal deleted versions of GAI and RGA with COP1 (A) and SPAs (B). L, leucine; W, tryptophan; H, histidine. Numbers indicate the dilutions used in the drop assay. (C) Coimmunoprecipitation assays showing interactions in planta. YFP-M5GAI and YFP-RGA52 were transiently expressed in leaves of N. benthamiana together with DsRED-COP1-HA, c-myc-SPA1, or both. Proteins were immunoprecipitated with anti-GFP antibody-coated paramagnetic beads. Leaves expressing DsRED-COP1-HA or c-myc-SPA1 alone were used as negative controls. The arrowhead and asterisk mark the coimmunoprecipitated c-myc-SPA1 and a nonspecific band, respectively. (D) YFP-GAI and YFP-RGA colocalize with DsRED-COP1-HA in nuclear bodies in the presence of c-myc-SPA1. Fusion proteins were transiently expressed in leaves of N. benthamiana and observed by confocal microscopy. One representative nucleus is shown. (E) BiFC assay showing that COP1 and SPA1 form a complex with M5GAI or RGA52 in nuclear bodies. The indicated proteins were expressed in leaves of N. benthamiana and observed by confocal microscopy. One representative nucleus is shown. (F) Representative HEK-293T cells cotransfected with mVenus-COP1, NLS-mCerulean-SPA1, and either GAI-mCherry or RGA-mCherry. (Scale bar, 10 μm.) The arrowheads point to two representative speckles co-occupied by DELLAs, SPA1, and COP1. (G) Fluorescence from GAI-mCherry or RGA-mCherry accumulates in the regions corresponding to speckles formed by COP1 and SPA1 in cells coexpressing mVenus-COP1 and mCerulean-SPA1-NLS. Data are mean from 10 to 13 transfected cells. The boxes extend from the first to the third quartile around the median, while whiskers go down to the smallest value and up to the largest. Asterisks indicate that the difference is statistically significant with the control condition (Student’s t test, ****P < 0.0001).

Fig. 4.

COP1 ubiquitinates GAI and RGA. (A and B) The 6xHis-M5GAI (A) and 6xHis-RGA52 (B) ubiquitination assay using recombinant MBP-COP1, rice E2, and unmodified ubiquitin. (C) The 6xHis-M5GAI ubiquitination assay using unmodified and HA-tagged ubiquitin. Modified and unmodified 6xHis-M5GAI and 6xHis-RGA52 were detected with anti-GAI and anti-6xHis antibodies, respectively.

Fig. 5.

COP1 regulates the rate of hypocotyl elongation in response to shade and warm temperature in a DELLA-dependent manner. Bars indicate the hypocotyl growth rate of seedlings of the indicated genotypes during deetiolation or after transfer to shade or 28 °C measured over a period of 9 h. Where indicated, seedlings were germinated and grown in the presence of 5 μM GA₄. Values correspond to the mean and SE of 8 (light treatments) or 24 (temperature treatments) replicate boxes; 10 seedlings were averaged per replicate box. Asterisks indicate that the difference is statistically significant with the control condition (Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.005; ns, nonsignificant).