Salt Stress and Ethylene Antagonistically Regulate Nucleocytoplasmic Partitioning of COP1 to Control Seed Germination

Abstract

Seed germination, a critical stage initiating the life cycle of a plant, is severely affected by salt stress. However, the underlying mechanism of salt inhibition of seed germination (SSG) is unclear. Here, we report that the Arabidopsis (Arabidopsis thaliana) CONSTITUTIVE PHOTOMORPHOGENESIS1 (COP1) counteracts SSG Genetic assays provide evidence that SSG in loss of function of the COP1 mutant was stronger than this in the wild type. A GUS-COP1 fusion was constitutively localized to the nucleus in radicle cells. Salt treatment caused COP1 to be retained in the cytosol, but the addition of ethylene precursor 1-aminocyclopropane-1-carboxylate had the reverse effect on the translocation of COP1 to the nucleus, revealing that ethylene and salt exert opposite regulatory effects on the localization of COP1 in germinating seeds. However, loss of function of the ETHYLENE INSENSITIVE3 (EIN3) mutant impaired the ethylene-mediated rescue of the salt restriction of COP1 to the nucleus. Further research showed that the interaction between COP1 and LONG HYPOCOTYL5 (HY5) had a role in SSG Correspondingly, SSG in loss of function of HY5 was suppressed. Biochemical detection showed that salt promoted the stabilization of HY5, whereas ethylene restricted its accumulation. Furthermore, salt treatment stimulated and ethylene suppressed transcription of ABA INSENSITIVE5 (ABI5), which was directly transcriptionally regulated by HY5. Together, our results reveal that salt stress and ethylene antagonistically regulate nucleocytoplasmic partitioning of COP1, thereby controlling Arabidopsis seed germination via the COP1-mediated down-regulation of HY5 and ABI5. These findings enhance our understanding of the stress response and have great potential for application in agricultural production.

Figure 1.

COP1 mutation impairs ethylene-promoted seed germination under salt stress. A, Statistical analysis of seed germination with the indicated treatment (100 mm NaCl and 10 μm ACC) at 4 d of germination. “Control” and “NaCl” indicate the absence or presence of salt treatment, respectively. P values were calculated with a two-tailed Student’s t test assuming equal variances (*P < 0.05). B to D, The time course of seed germination with treatment with 100 mm NaCl. The data show the rate of germinated seeds compared to their corresponding controls on the same day of germination. Each value shown represents the mean ± sd of three independent biological experiments.

Figure 2.

Salt and ethylene reversely affect the translocation of COP1. A and C, GUS staining and statistical analysis of GUS-COP1 localization in the emerging radicle. P values were calculated with a two-tailed Student’s t test assuming equal variances (*P < 0.05). Germinating transgenic seeds (1 d) were treated with or without 100 mm NaCl and/or 10 μm ACC for 16 h. The cell nuclei in the radicle were stained with 0.5 μg/mL 4′,6-diamino-phenylindole (DAPI). Bars = 5 µm. B and D, Immunoblotting of COP1 using germinating seeds (1 d) pretreated with 100 μm CHX for 12 h and then treated with or without NaCl and/or 10 μm ACC for another 16 h. N, Nuclear protein; S, soluble fraction containing cytoplasmic protein. Immunoblotting was performed using anti-COP1, -histone 3 (H3), and -actin antibodies. The actin and H3 signals were used to confirm equal protein loading. “Light” and “Dark” indicate continuous white light and darkness, respectively.

Figure 3.

Interaction between COP1 and HY5 plays a role in SSG. A and B, Time course of seed germination without (Control) or with 100 mm NaCl treatment (Salt treatment). The data show the rate of germinated seeds compared to the corresponding controls on the same day of germination. Each value shown represents the mean ± sd of three independent biological experiments.

Figure 4.

HY5 acts downstream of EIN3 in ethylene-promoted seed germination under salt stress. A and B, Time course of seed germination with treatment with 100 mm NaCl. The data show the rates of germinated seeds compared to the corresponding controls on the same day of germination. Each value shown represents the mean ± sd of three independent biological experiments.

Figure 5.

Salt and ethylene oppositely modulate the stabilization of HY5 protein. A, HY5 accumulation in different phenotypes. B, HY5 stability in response to NaCl and/or ACC treatment in Col-0. C, HY5 protein levels in response to NaCl and/or ACC treatment in ein3 eil1. Germinating seeds (1 d) were pretreated with 100 μm CHX for 12 h and then treated with or without 100 mm NaCl and/or 10 μm ACC for the indicated time. Protein extracts were prepared from germinated seeds. Immunoblotting was performed with anti-HY5 and antiactin antibodies. Numbers indicate the relative levels of HY5 protein.

Figure 6.

ABI5 mutation enhances seed germination under salt stress. A and B, Time course of seed germination without (Control) and with 100 mm NaCl treatment (Salt treatment). The data show the rates of germinated seeds compared to the corresponding controls on the same day of germination. Each value shown represents the mean ± sd of three independent biological experiments.

Figure 7.

HY5 directly controls ABI5 transcription during seed germination. A, ABI5 expression in different genotypes. Col-0 and mutant seeds were kept in MS for 1.5 d and then transferred to MS medium with 100 mm NaCl and/or 10 μm ACC for 2 h. ABI5 transcripts were quantified with qPCR relative to TUB4. The transcript levels in Col-0 were set to 1. B, Schematic diagram of the promoter regions of ABI5 and RbcSA. Black lines represent the promoter regions of the two genes. Black boxes on the line represent the putative G-box (5′-CACGTG-3′); numbers above indicate the distance from the translational start site (ATG), shown as +1. Regions between the two pairs of arrowheads (red lines) indicate the DNA fragments used for ChIP-PCR. C, ChIP analysis showing the in vivo binding of HY5 to the ABI5 promoter. The ChIP analysis used 1.5-d germinating 35S:HY5-HA seedlings. IgG was used as a negative control, and the interaction between the RbcSA promoter and HY5 was used as a positive control. Each value shown represents the mean ± sd of three independent biological experiments.

Figure 8.

The COP1-HY5 complex is involved in ethylene-promoted seed germination under salt stress. COP1 was mainly localized to the nucleus in radicle cells (top). Under NaCl stress, salt retained the localization of COP1 in the cytosol, subsequently causing the accumulation of HY5 and the expression of ABI5, which inhibited seed germination (middle). Ethylene increased in response to the pressure of water uptake by the imbibed seed and the radicle protrusion; this reversed the retention of COP1 in the cytosol due to salt and promoted the localization of COP1 into the nucleus to destroy HY5, decreasing the expression of ABI5, which then facilitated seed germination (bottom).