Ethylene promotes hypocotyl growth and HY5 degradation by enhancing the movement of COP1 to the nucleus in the light

Abstract

In the dark, etiolated seedlings display a long hypocotyl, the growth of which is rapidly inhibited when the seedlings are exposed to light. In contrast, the phytohormone ethylene prevents hypocotyl elongation in the dark but enhances its growth in the light. However, the mechanism by which light and ethylene signalling oppositely affect this process at the protein level is unclear. Here, we report that ethylene enhances the movement of constitutive photomorphogenesis 1 (COP1) to the nucleus where it mediates the degradation of long hypocotyl 5 (HY5), contributing to hypocotyl growth in the light. Our results indicate that HY5 is required for ethylene-promoted hypocotyl growth in the light, but not in the dark. Using genetic and biochemical analyses, we found that HY5 functions downstream of ethylene insensitive 3 (EIN3) for ethylene-promoted hypocotyl growth. Furthermore, the upstream regulation of HY5 stability by ethylene is COP1-dependent, and COP1 is genetically located downstream of EIN3, indicating that the COP1-HY5 complex integrates light and ethylene signalling downstream of EIN3. Importantly, the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) enriched the nuclear localisation of COP1; however, this effect was dependent on EIN3 only in the presence of light, strongly suggesting that ethylene promotes the effects of light on the movement of COP1 from the cytoplasm to the nucleus. Thus, our investigation demonstrates that the COP1-HY5 complex is a novel integrator that plays an essential role in ethylene-promoted hypocotyl growth in the light.

Figure 1. HY5 is required for ethylene-promoted hypocotyl growth in the light.

(A, C) Morphological observations and (B, D) statistical analyses of hypocotyl length. Images were taken after 5 days of incubation in MS medium supplemented with or without 10 µM ACC. The data indicate the mean values plus the standard deviation (SD) from three independent experiments with approximately 30 seedlings. P-values (ACC treatment vs. non-treatment) were calculated by a two-tailed Student's t-test assuming equal variances (*P<0.05). (E) HY5 accumulation in Col-0 and hy5. Protein extracts were prepared from 5-day-old seedlings after treatment with or without 25 µM ACC for 15 h. (F) The effects of different ACC concentrations on HY5 stability in 35S::HY5 seedlings. (G) A time course of HY5 stability in response to ACC treatment in 35S::HY5 seedlings. Five-day-old seedlings were treated either with the indicated concentrations of ACC treatment for 16 h or with 25 µM ACC for the indicated times. The arrow in (F) and (G) indicates endogenous HY5, whereas the upper band corresponds to transgenic HY5. (H) Effect of the 26S proteolysis inhibitor MG132 on HY5 protein accumulation in Col-0 under normal growth conditions. Five-day-old seedlings were treated with 25 µM ACC plus 0.1% DMSO or 5 µM MG132 for 15 h. Immunoblotting was performed with anti-HY5 and -TUB4 antibodies. The TUB4 signals confirm equal protein loading. Numbers indicate the relative protein levels of HY5.

Figure 2. Ethylene signalling positively promotes hypocotyl growth and HY5 degradation.

(A) Morphological observations and (B) statistical analyses of hypocotyl length after 5 days of incubation in MS medium supplemented with or without 10 µM ACC. The data indicate the mean values plus the SD from three independent experiments with approximately 30 seedlings. P-values (ACC treatment vs. non-treatment) were determined with a two-tailed Student's t-test assuming equal variances (*P<0.05). (C) HY5 accumulation after treatment with or without ACC treatment. Protein extracts were prepared from 5-day-old seedlings after treatment with or without 25 µM ACC for 15 h. Immunoblotting was performed with anti-HY5 and -TUB4 antibodies. The TUB4 signals indicate equal protein loading. Numbers indicate the relative protein levels of HY5.

Figure 3. HY5 operates downstream of EIN3 to modulate ethylene-promoted hypocotyl growth.

(A) Morphological observations and (B) statistical analyses of hypocotyl length after 5 days of incubation in MS medium supplemented with or without 10 µM ACC. The data indicate the mean values plus SD from three independent experiments with approximately 30 seedlings. P-values (ACC treatment vs. non-treatment) were determined with a two-tailed Student's t-test assuming equal variances (*P<0.05).

Figure 4. COP1 is required for ethylene-promoted hypocotyl growth.

(A) Morphological observations and (B) statistical analyses of hypocotyl length after 5 days of incubation in MS medium supplemented with or without 10 µM ACC. The data indicate the mean values plus the SD from three independent experiments with approximately 30 seedlings. P-values (★: ACC treatment vs. non-treatment, ☆: transgenic lines vs. wild type) were determined with a two-tailed Student's t-test assuming equal variances (*P<0.05). (C, D) HY5 accumulation with or without ACC treatment. Protein extracts were prepared from 5-day-old seedlings after treatment with or without 25 µM ACC for 15 h. Immunoblotting was performed using anti-HY5 and -TUB4 antibodies. The TUB4 signals indicate equal protein loading. Numbers indicate the relative protein levels of HY5.

Figure 5. COP1-HY5 interaction is required for HY5-mediated hypocotyl growth.

(A) Morphological observations and (B) statistical analyses of hypocotyl length in transgenic lines of HY5-ΔN77 driven by the CaMV35S promoter. The images were taken after 5 days of incubation in MS medium supplemented with or without 10 µM ACC. The data indicate the mean values plus the SD from three independent experiments with approximately 30 seedlings. P-values (ACC treatment vs. non-treatment) were determined with a two-tailed Student's t-test assuming equal variances (*P<0.05).

Figure 6. COP1 acts downstream of EIN3 to modulate ethylene-promoted hypocotyl growth.

(A) Morphological observations and (B) statistical analyses of hypocotyl length after 5 days of incubation in MS medium supplemented with or without 10 µM ACC. The data indicate the mean values plus the SD from three independent experiments with approximately 30 seedlings. P-values (ACC treatment vs. non-treatment) were determined with a two-tailed Student's t-test assuming equal variances (*P<0.05). (C) HY5 accumulation with or without ACC treatment. Protein extracts were prepared from 5-day-old seedlings after treatment with or without 25 µM ACC for 15 h. Immunoblotting was performed with anti-HY5 and -TUB4 antibodies. The TUB4 signals indicate equal protein loading. Because the ein3-1 and GUS-COP1 were produced in different backgrounds, an F1 hybrid of No-0×Col-0 was used as the control. The numbers below the gel indicate the protein levels of HY5.

Figure 7. The ethylene precursor ACC improves COP1 nucleus-enriched localisation in the light.

Images (left panel) and statistical summaries (right panel) of GUS-COP1 localisation in the hypocotyl in Col-0 (A) and ein3-1 (B) under different growth conditions. The degree of nuclear enrichment of GUS staining is shown as the percentage of cells with nucleus-enriched GUS relative to the total number of GUS-stained hypocotyl cells. At least 100 cells were counted for each sample. GUS-COP1 transgenic seedlings were first grown on MS for 5 days and then grown in the dark (labelled “Dark”) or under continuous white light (50 µmol/m²s; labelled “Light”) for another 24 h with or without 25 µM ACC. The cell nuclei were stained with 0.5 µg/mL DAPI. Scale bar: 5 µm.

Figure 8. Biochemical detection of COP1 in the cytoplasm and nucleus.

Seedlings of five-day-old (A) Col-0 and (B) ein2 and ein3-1 Arabidopsis plants grown under long-day conditions (16-h light/8-h dark) were treated with 50 µM CHX and 5 µM MG132, supplemented with or without 25 µM ACC for 15 h, and then the nuclear proteins were extracted as described in the Materials and methods (N: nuclear protein, S: soluble fraction, cytoplasmic protein). Immunoblotting was performed using anti-COP1, -histone 3 (H3), and -TUB4 antibodies. The TUB4 and H3 signals confirm equal protein loading. All of the seedlings were illuminated with white light (50 µmol/m²s).