Abscisic acid‐induced cytoplasmic translocation of constitutive photomorphogenic 1 enhances reactive oxygen species accumulation through the HY5‐ABI5 pathway to modulate seed germination

Abstract Seed germination is a physiological process regulated by multiple factors. Abscisic acid (ABA) can inhibit seed germination to improve seedling survival under conditions of abiotic stress, and this process is often regulated by light signals. Constitutive photomorphogenic 1 (COP1) is an upstream core repressor of light signals and is involved in several ABA responses. Here, we demonstrate that COP1 is a negative regulator of the ABA‐mediated inhibition of seed germination. Disruption of COP1 enhanced Arabidopsis seed sensitivity to ABA and increased reactive oxygen species (ROS) levels. In seeds, ABA induced the translocation of COP1 to the cytoplasm, resulting in enhanced ABA‐induced ROS levels. Genetic evidence indicated that HY5 and ABI5 act downstream of COP1 in the ABA‐mediated inhibition of seed germination. ABA‐induced COP1 cytoplasmic localization increased HY5 and ABI5 protein levels in the nucleus, leading to increased expression of ABI5 target genes and ROS levels in seeds. Together, our results reveal that ABA‐induced cytoplasmic translocation of COP1 activates the HY5‐ABI5 pathway to promote the expression of ABA‐responsive genes and the accumulation of ROS during ABA‐mediated inhibition of seed germination. These findings enhance the role of COP1 in the ABA signal transduction pathway.

. BBX21 interacts with HY5 interfering with the HY5-mediated induction of ABI5 transcription, while BBX21 directly interacts with ABI5 weakening ABI5 binding to its own promoter (Chen et al., 2008;Kang et al., 2018;Xu et al., 2014). Although some downstream components of the light signalling pathway have been shown to regulate ABA inhibition of seed germination, little is known about the involvement of upstream light signalling components.
Constitutive photomorphogenic 1 (COP1) is an upstream core repressor in the light signal transduction pathway and exert its role by ubiquitinating transcription factors involved in photomorphogenesis to promote skotomorphogenesis (Han et al., 2020). In plants, COP1 binds SPA (suppressor of PhyA-105) to form a COP1-SPAS protein complex, regulating many physiological and biochemical processes. COP1's nucleocytoplasmic partitioning plays an important role in controlling its function although the molecular mechanisms controlling such partitioning are still unclear (Balcerowicz et al., 2017;Podolec & Ulm, 2018).
The role of COP1 is not restricted to light signalling, and it has also been implicated in the physiological response to phytohormones, clock regulation, temperature perception, biotic stress, and so forth (Jeong et al., 2010;Martinez et al., 2018;Wang et al., 2019;Xu, Zhu, et al., 2016). Several roles for COP1 in phytohormone signalling have gradually emerged, although its involvement in ABA signalling is relatively obscure. Absence of COP1 reduces stomatal sensitivity to ABA, weakens ABAinduced microtubule depolymerization in guard cells, and reduces the activity of SLAC1 (slow anion channel-associated1) in guard cells (Khanna et al., 2014). Recent studies have shown that COP1 regulates ABA-induced stomatal response by degrading the negative ABA regulators protein phosphatase type 2Cs (PP2Cs) and activating ABA downstream signalling pathways (Chen et al., 2021). Aside from the two mentioned examples, the involvement of COP1 in ABA-mediated responses is unknown.

ABA-induced increase in ROS levels in seeds is closely linked to the enhanced expression of the NADPH oxidases RbohD and
RbohF, which are key components in the production of ROS (Choudhury et al., 2017;Torres & Dangl, 2005). However, the regulatory mechanisms controlling ABA-induced ROS accumulation during seed germination remain elusive, and the roles of light signalling components in this process are largely unknown.
In this article, we demonstrate that COP1 negatively regulates ABAmediated inhibition of seed germination. We found that disruption of COP1 enhanced seed sensitivity to ABA. In seeds, ABA-induced COP1 accumulation in the cytoplasm. In turn, ABA-induced COP1 cytoplasmic localization increased HY5 and ABI5 protein levels in the nucleus, leading COP1 MODULATES ABA-MEDIATED INHIBITION OF SEED GERMINATION to increased expression of ABI5 target genes and raised ROS levels in seeds.

| Seed germination
After surface disinfection, Arabidopsis thaliana seeds were sown on MS medium containing 0, 0.1 or 0.5 μM ABA, vernalized at 4°C for 2 days, and placed in an incubator at 22°C for germination experiments. The time at which seeds were placed in the culture room was considered as 0 h, germination was determined by protrusion of the radicle from the seed coat, and recorded every 12 h. More than 50 seeds per genotype were used for each experimental condition, and every experiment repeated three times. Germination rate was calculated as the proportion of germinated seeds from the total in each experiment. Seeds were germinated in an incubator at 22°C with 12-h light/12-h dark cycle (100 μmol photons m −2 sec −1 ).

| Nitroblue tetrazolium (NBT) staining assay
NBT staining assay of germinated seeds was performed as described by Wang et al. (2020) with slight modifications. Five milligrams of NBT were dissolved in 6 ml of 20 mM potassium phosphate buffer (pH 6.7) con-

| Cell fractionation assay
Nuclear and cytoplasmic proteins were extracted as described by . Briefly, 0.5 g of germinated seeds were ground in liquid nitrogen and mixed with 2 ml of fractionation lysis solution the initial low-speed centrifugation (×1500g) was centrifuged at ×10 000g at 4°C for 10 min, and the precipitate collected as cytoplasmic fraction. Western blot analysis was performed using anti-GFP antibody. Actin was used as an internal marker for cytoplasmic proteins and proliferating cell nuclear antigen (PCNA) was used as an internal marker for nuclear proteins. Three biological replicates were performed.

| Quantitative Real-Time PCR
An RNA purification kit (Tiangen) was used to extract RNA from germinating seeds, and DNA contamination removed by DNaseI (Invitrogen) treatment. cDNA was synthesized from 2 μg RNA using oligo dT primers (TaKaRa) and MMLV Reverse Transcriptase (Promega). Quantitative real-time PCR was performed using the SYBR Taq Premix system (TaKaRa) on a LightCycler480 detection system (Roche) according to the manufacturer's instructions. The Actin2 gene was used as an internal control for normalization purposes. Three biological replicates and three technical replicates were performed for each treatment. The primer sequences are listed in Table S1.

| RESULTS
3.1 | COP1 acts as a negative regulator of the ABA-mediated inhibition of seed germination in Arabidopsis In a previous study on the role of COP1 in stomatal movement (Chen et al., 2021), we obtained anecdotal evidence that seed germination under ABA treatment is different in COP1-deficient mutants compared with WT. To follow on those observations, we performed germination assays in WT, two independent cop1 mutants (cop1-4 and cop1-6) and a previously characterized functional complementation line (GUS-COP1) (Yu et al., 2016) in the absence or presence of ABA ( Figure 1). In the absence of ABA all genotypes showed similar germination rates (Figure 1a,b), but the germination rates of cop1-4, cop1-6 mutants were significantly lower than those of WT and GUS-COP1 under the two ABA concentrations assayed (0.1 μM and 0.5 μM ABA) (Figure 1a,b). Germination rates of cop1-4, cop1-6 seeds in media containing 0.1 μM ABA (58.6% and 47.3%) were significantly lower than those of WT (83.3%) and GUS-COP1 (84.6%) seeds after 48 h (Figure 1a,b). Similarly, germination rates of cop1-4 and cop1-6 seeds sawn on media containing 0.5 μm ABA (5.3% and 2%) were also significantly lower than those of WT (26%) and GUS-COP1 (45.3%) (Figure 1a,b). In addition, we examined the germination dynamics of WT, cop1-4, cop1-6 and GUS-COP1 seeds in the presence or absence of 0.5 μM ABA for 96 h. Differences in germination rates in 0.5 μM ABA between cop1-4 and cop1-6 lines and WT and GUS-COP1 lines was initially detected 36 h after sowing and remained for the rest of the time examined (Figure 1c, Figure S1).
These results suggest that the absence of COP1 enhances the inhibitory effect of ABA on seed germination of A. thaliana.

| COP1 regulates ABA-induced ROS accumulation during seed germination
External stimuli such as H 2 S, high salt stress, etc. and the phytohormone ABA can enhance ROS levels in seeds, inhibiting germination (Penfield, 2017). NBT staining of germinating seeds revealed stronger staining in cop1-4 and cop1-6 mutant seeds compared WT and GUS-COP1 under ABA treatment (Figure 2a To obtain additional evidence that the link between COP1 and ABA during seed germination is related to ROS, we measured the effect of adding the ROS scavenger glutathione (GSH) to germination medium containing 0.5 μM ABA. GSH is an important antioxidant in plants that can effectively reduce ROS accumulation in vivo. As observed earlier (Figure 1), germination rates on 0.5 μM ABA were significantly lower in cop1 mutants compared to WT and GUS-COP1 lines (Figure 2j, Figure S2). Addition of 100 μM GSH to the ABA-containing media partially decreased the hypersensitivity shown by cop1 mutants while addition of 300 μM GSH to the media recovered germination rates in cop1 mutants to WT levels ( Figure 2j, Figure S2).
These results suggest that GSH can alleviate the increased inhibitory effect of ABA on germination observed in cop1 mutants.

| ABA induces cytoplasmic accumulation of COP1 and increased ROS production
The nucleocytoplasmic partitioning of COP1 plays an important role in its function (Podolec & Ulm, 2018). Accumulation of COP1  showing COP1 enrichment in the nucleus ( Figure S3a,b). These results suggest that ABA promotes the localization of COP1 to the cytoplasm during seed germination.
To complement our initial observations, we measured the relative amounts of CFP-COP1 in nuclear and cytoplasmic protein fractions in transgenic CFP-COP1 seeds. Our results show that, in the absence of ABA, the majority of CFP-COP1 is detected in the nuclear fraction of germinating seeds, while seeds germinated in the presence of 0.5 μM ABA contain most of the CFP-COP1 protein in the cytoplasm (Figure 3b). These results suggest that ABA promotes COP1 accumulation in the cytoplasm during seed germination.
While among these three genes, only SOD2 expression levels in  Figure S3a,b). Therefore, these results indicate that ABAinduced ROS production is not the cause for the cytoplasmic localization of COP1 during ABA treatment. Overall, our results indicate that ABA induces the accumulation of COP1 in the cytoplasm during seed germination, which results in higher ROS levels in the seed.
3.4 | The HY5-ABI5 pathway regulates the effect of COP1 in the ABA-mediated inhibition of seed germination and ROS accumulation Previous studies have shown that HY5 regulates ABA-mediated inhibition of seed germination by enhancing ABI5 expression through binding to the ABI5 promoter or direct interaction with ABI5 (Chen et al., 2008;Wang et al., 2021). HY5 and ABI5 have also been linked to ROS production (Bi et al., 2017;Chen et al., 2013;, raising the posibility that the HY5-ABI5 pathway could regulate ROS accumulation during ABA inhibition of seed germination. To explore whether the role of COP1 in the ABA-induced ROS accumulation in seeds is mediated by the HY5-ABI5 pathway, we obtained and analysed cop1-4/hy5-ks50 and cop1-4/ abi5-1 double mutants (Figure 4, Figures S7 and S8). Arabidopsis seeds from Col-0, Ws, cop1-4, hy5-ks50 and cop1-4/hy5-ks50 had similar germination rates on MS medium, but differences were apparent in medium containing 0.5 μM ABA (Figure 4a,b). Single cop1-4 mutant seeds showed the strongest ABA inhibition while hy5-ks50 mutants showed the lowest ABA inhibition. The two WT ecotypes, Col-0 and Ws, were identical to F I G U R E 2 Loss of constitutive photomorphogenic 1 (COP1) enhances reactive oxygen species (ROS) accumulation in germinating seeds under abscisic acid (ABA) treatment. (a) Germinated seeds of cop1 mutants showed deeper nitroblue tetrazolium (NBT) staining under ABA treatment than wild type (WT) seeds. WT, cop1-4, cop1-6 and GUS-COP1 seeds were vernalized and germinated for 48 h on MS medium containing 0 or 0.5 μM ABA, before staining with NBT (scale = 0.5 mm). (b) Relative staining intensity was measured on the seeds shown in (a). Statistical analyses were performed using a one-way ANOVA. Data are means ± SD (n > 30 seeds per genotype), **p < 0.01. Transgenic CFP-COP1 seeds were treated as described in (a), collected and proteins extracted. The relative abundance of CFP-COP1 in cytoplasmic and nuclear fractions was detected using anti-GFP antibodies. ACTIN was used as an internal reference for the presence of cytoplasmic proteins and proliferating cell nuclear antigen (PCNA) was used as an internal reference for the presence of nuclear proteins. N, nuclear fraction; S, soluble fraction containing cytoplasmic proteins. (c) Wild type (WT), cop1-4 mutants, COP1 nuclear-localized mutants (COP1 nu −1, COP1 nu −2), and COP1 cytoplasm-localized mutants (COP1 cyt −1, COP1 cyt −2) seeds were germinated on MS medium in the absence or presence of 0.5 μM ABA for 60 h (scale = 2 mm). (d) Germination rates for the seeds shown in (c). The time at which seeds were placed in the culture room was designated as 0 h, and the germination rates were measured every 12 h until 108 h. The values are means ± SD, (n > 50 seeds per genotype) of three replicates. cop1-4 seeds. These observations support the germination results ( Figure 4a,b). Similarly, the O 2 − and H 2 O 2 levels in cop1-4/abi5-1 seeds were significantly lower than those of cop1-4 seeds under ABA treatment (Figure 4f), suggesting that the ABI5 mutation also reduces ROS accumulation in cop1-4 seeds. In conclusion, these results indicate COP1 can regulate ABA-induced ROS accumulation through the HY5-ABI5 pathway during ABA-mediated inhibition of seed germination.
3.5 | ABA-induced cytoplasmic localization of COP1 enhances activation of the nuclear HY5-ABI5 pathway Our results so far have shown that ABA induces COP1 accumulation in cytoplasm during seed germination while genetic evidence suggests that the role of COP1 in the ABA-mediated inhibition of germination is exerted through the HY5-ABI5 pathway. We therefore hypothesized that, during seed germination, the ABA-induced cytoplasmic accumulation of COP1 could increase HY5 and/or ABI5 levels, thus activating the HY5-ABI5 pathway. To test this hypothesis, we first examined the effects of the absence of COP1 on HY5 and ABI5 protein levels in response to ABA during seed germination.
Western blot analysis of germinating WT and cop1-4 seeds in the absence or presence of ABA was performed and the HY5 protein levels visualized. As expected, the presence of ABA increased HY5 levels in WT seeds (Figure 5a). Notably, the ABA-induced HY5 levels in cop1-4 seeds were always higher than those observed in WT seeds. When the same experiment was performed using antibodies against ABI5 we observed that ABA increased ABI5 levels in WT seeds, but the increase was more pronounced in cop1-4 seeds suggesting that loss of COP1 enhances the ABA-induced increase in ABI5 levels ( Figure 5b). qPCR showed that ABA induction of the ABI5 expression levels was far more pronounced in cop1-4 and cop1-6 mutants than in WT seeds ( Figure S9a).
To investigate the effect of COP1 localization on the HY5 and ABI5 protein levels in response to ABA treatment, we analysed the cytoplasm-and nuclear-localized mutants, COP1 cyt −1 and COP1 nu −1 respectively. Western blot analysis of germinating seeds in the presence or absence of ABA revealed that the enhanced HY5 and ABI5 levels observed in cop1-4 mutants versus WT seeds was also observed in cytoplasmic localized COP1 cyt −1 lines but was not present in nuclear-localized COP1 nu −1 lines (Figure 5c,d). In addition, ABI5 transcript levels in response to ABA were higher in cop1-4 and COP1 cyt −1 seeds than in WT while COP1 nu −1 seeds showed WT levels ( Figure S9b). These results suggest that accumulation of COP1 in the cytoplasm enhances the ABA-induced increase of HY5 and ABI5 protein levels.
To further examine the effect of COP1 localization on the ABA activation of the HY5-ABI5 pathway, we examined the expression levels of several ABI5 target genes (RD29A, RAB18, EM1 and EM6) in WT, cop1-4, COP1 cyt −1, and COP1 nu −1 seeds under ABA treatment.
The results showed that, for all analysed genes, ABA consistently induced higher expression levels in cop1-4 and COP1 cyt −1 seeds compared to WT seeds, while COP1 nu −1 seeds showed similar levels of induction than WT (Figure 5e-h). This suggests that the ABAinduced accumulation of COP1 in the cytoplasm enhances the activity of the HY5-ABI5 pathway during ABA-mediated inhibition of seed germination.

| The higher ABA-induced ROS levels in cytoplasmic COP1 lines is dependent on the HY5-ABI5 pathway
To study whether the increased ABA-induced ROS levels observed in cytoplasmic COP1 localized lines is dependent on the HY5-ABI5 pathway, we produced and analysed double COP1 nu −1/abi5-1 and COP1 cyt −1/abi5-1 mutants ( Figure 6, Figure S10). In the absence of ABA, Col-0, Ws, cop1-4, COP1 nu −1, COP1 cyt −1, abi5-1, COP1 nu −1/ abi5-1, COP1 cyt −1/abi5-1 seeds showed similar germination rates (Figure 6a,b). In agreement with our previous results, cop1-4 and COP1 cyt −1 seeds show enhanced ABA sensitivity compared to WT (Col-0 and Ws) (Figure 6a,b) while COP1 nu −1 seeds show slightly lower sensitivity than WT. Importantly, the enhanced ABA sensitivity shown by COP1 cyt −1 seeds disappeared in the double COP1 cyt −1/ abi5-1 mutants (Figure 6a,b) indicating that the ABA hypersensitivity of COP1 cyt −1 seeds is mediated by ABI5. F I G U R E 5 Abscisic acid (ABA)-induced accumulation of constitutive photomorphogenic 1 (COP1) in the cytoplasm enhances the activity of the HY5-ABI5 pathway during ABA-mediated inhibition of seed germination. (a) Western blot analysis of HY5 protein levels in seeds of wild type (WT) and cop1-4 germinated on 0, 0.1 or 0.5 μM ABA medium for 36 h using HY5 antibodies (AS12 1867; Agriser Corp.). hy5-ks50 seeds were used as negative control. Proliferating cell nuclear antigen (PCNA) protein was used as an internal reference. The numbers represent the relative intensity of the HY5 protein band normalized versus the respective PCNA band. Three independent experiments were performed with similar results. (b) Western blot analysis of ABI5 protein levels in seeds of WT and cop1-4 germinated on 0, 0.1 or 0.5 μM ABA medium for 36 h using ABI5 antibodies (AS12 1863; Agrisera Corp.). abi5-1 seeds were used as negative control. PCNA protein was used as an internal reference. The numbers represent the relative intensity of the ABI5 protein band normalized versus the respective PCNA band. Three independent experiments were performed with similar results. (c) Western blot analysis of HY5 protein levels in seeds of WT, cop1-4, COP1 cytoplasmlocalized mutant COP1 cyt −1 and COP1 nuclear-localized mutant COP1 nu −1 germinated on 0 and 0.5 μM ABA medium for 36 h. hy5-ks50 seeds were used as negative control. PCNA protein was used as an internal reference. The numbers represent the relative intensity of the HY5 protein band normalized versus the respective PCNA band. Three independent experiments were performed with similar results. (d) Western blot analysis of ABI5 protein levels in seeds of WT, cop1-4, COP1 cyt −1 and COP1 nu −1 seeds under the same treatment conditions as in (c). abi5-1 seeds were used as negative control. PCNA protein was used as an internal reference. The numbers represent the relative intensity of the ABI5 protein band normalized versus the respective PCNA band. Three independent experiments were performed with similar results.  (Bahin et al., 2011;Bailly, 2019;Oracz & Karpinski, 2016), while light acts as an environmental signal by balancing the ABA/GA ratio to regulate seed germination (Seo et al., 2009). In addition, the light signalling component COP1 can directly participate in ABA-mediated developmental processes such as stomatal movement (Chen et al., 2021), but its role in ABA inhibition of seed germination is unclear.

Analysis of O 2
This study provides several lines of evidence strongly supporting a pivotal role for COP1 in the ABA-mediated inhibition of seed germination. Firstly, disruption of COP1 enhances sensitivity to ABA and raises ABA-induced ROS levels in seeds (Figures 1 and 2). Second, COP1 substrates HY5 and ABI5, positively regulate ABA-mediated inhibition of seed germination, and mutations in HY5 and ABI5 effectively suppress cop1-4 hypersensitivity to ABA during seed germination and the enhanced ABA-induced ROS levels observed in cop1-4 seeds ( Figure 4). Overall, the evidence suggests that COP1 is a negative modulator of the ABA-mediated inhibition of seed germination via the HY5-ABI5 pathway.
COP1, as an E3 ligase, marks for degradation a number of regulators involved in light and other signalling pathways, and its function is always affected by its nucleocytoplasmic partitioning (Podolec & Ulm, 2018;Ponnu & Hoecker, 2021). Enhanced nuclear localization of COP1 by elevated temperatures facilitates hypocotyl growth by increasing HY5 degradation (Park et al., 2017). In contrast, jasmonic acid reduces the amount of nuclear-localized COP1 promoting the suppression of hypocotyl elongation (Zheng et al., 2017). Under some abiotic stress conditions, such as high salt and high temperature, COP1 is induced to accumulate in the cytoplasm suggesting that the cytoplasmic localization of COP1 promotes inhibition of seed germination Yu et al., 2016). We observed that treatment of seeds with ABA enhances COP1 localization in the cytoplasm. The hypersensitivity to ABA shown by cytoplasmic COP1 cyt lines suggests that the cytoplasmic localization of COP1 enhances the inhibitory effect of ABA on seed germination. COP1's E3 ligase function requires the participation of SPA proteins (Podolec & Ulm, 2018). Similar to cop1 mutants, spa1 mutants were also hypersensitive to ABA during seed germination (Gangappa et al., 2010), suggesting that the COP1/SPAs complex play a regulatory role in ABA-mediated inhibition of seed germination. Therefore, the cytoplasmic accumulation of COP1, induced by ABA, may result in increased degradation of cytoplasmic proteins enhancing the ABA-mediated inhibition of seed germination. Although SPAs and FIN219 have been identified as factors modulating the nucleocytoplasmic partitioning of COP1 (Balcerowicz et al., 2017;Swain et al., 2017), the mechanism that regulates this partitioning is still unclear.
ROS are key signalling intermediates involved in a wide range of plant responses and developmental processes (Bailly, 2019; Ros Barcelo & Gomez Ros, 2009). HY5 acts as the bridge between light and ROS signalling to regulate protochlorophyllide synthesis and cell death in the light during seeding establishment (Bellegarde et al., 2019;Chai et al., 2015;Chen et al., 2013) and can bind to the promoter of several ROS-responsive genes modulating their expression . ABI5 plays an important role regulating ROS balance during germination, with abi5 mutants exhibiting altered expression of genes involved in ROS metabolism and response, and impaired ROS signalling during ABA-mediated inhibition of seed germination (Bi et al., 2017). Although high ROS levels had been previously reported in cop1 mutants , the role of  These results suggest that COP1 has different regulatory roles in these two ABA-mediated processes. Under ABA treatment, cop1 mutants exhibited hypersensitivity to ABA during seed germination, but hyposensitivity to ABA in seeding establishment. Our results show that disruption of COP1 enhances seed sensitivity to ABA, probably caused by increased accumulation ABI5 protein ( Figure 5b), whereas the insensitivity of cop1 mutants to ABAmediated inhibition of post-germination was caused by reduced ABI5 protein levels (Yadukrishnan et al., 2020). Overall, it seems F I G U R E 6 ABI5 is involved in the regulation of reactive oxygen species (ROS) accumulation promoted by abscisic acid (ABA)-induced constitutive photomorphogenic 1 (COP1) cytoplasmic localization. (a) Col-0, Ws, cop1-4, COP1 nu −1, COP1 cyt −1, abi5-1, COP1 nu −1/abi5-1, COP1 cyt −1/abi5-1 seeds germinated for 48 h on MS plates containing 0 μM ABA or 0.5 μM ABA. (b) Germination rates of the genotypes shown in (a). The time at which seeds were placed in the culture room was designated as 0 h, and the germination rates were measured every 12 h until 96 h. Data are means ± SD, (n > 50 seeds per genotype) of three replicates. that COP1 involvement in these two ABA-mediated processes is exerted by regulating ABI5 protein levels. However, the detailed regulatory mechanism remains to be elucidated.
We propose a mechanistic model in which, during seed germination, ABA induces COP1 export to cytoplasm (Figure 7). The reduced nuclear levels of COP1 result in decreased degradation of HY5, increasing the levels of HY5 and, as a consequence, the levels of ABI5 in the nucleus. For exploring whether COP1 can directly degrade ABI5, the ubiquitination experiment of COP1 on ABI5 needs to be designed in the future research. Here, the enhanced ABI5 levels promote the transcription of multiple ABA-responsive genes as well as the levels of ABA-induced ROS, both of which have a detrimental effect on seed germination (Figure 7). The elucidation of this mechanism explains the role of COP1 in ABA-mediated inhibition of seed germination, and places COP1 as an integrator of the crosstalk between light and ABA signalling.

SUMMARY STATEMENT
COP1, the core repressor of light signals, functions in ABA-mediated inhibition of Arabidopsis seed germination. The ABA-induced COP1 cytoplasmic translocation increases both HY5 and ABI5 protein levels in the nucleus, leading to elevated expression of ABI5 target genes and enhanced ROS levels.
F I G U R E 7 Model for the involvement of constitutive photomorphogenic 1 (COP1) in abscisic acid (ABA)-mediated inhibition of seed germination in Arabidopsis. During Arabidopsis seed germination, ABA promotes COP1 export from the nucleus and accumulation in the cytoplasm. The reduction of COP1 levels in the nucleus enhances the HY5 stability increasing HY5 levels in the nucleus. Increase nuclear HY5 levels promote ABI5 gene expression leading to the accumulation of ABI5. Accumulation of ABI5 enhances ABA-induced reactive oxygen species (ROS) levels and promotes the expression of ABA-related genes, thus inhibiting seed germination of Arabidopsis thaliana. The red arrow line represents the transfer direction of COP1. The green blocking dotted line represents the weakened ubiquitination-mediated degradation of HY5 by reduced levels of nuclear COP1. The upward black arrows represent increased protein levels or gene expression.