Salt Stress and Ethylene Antagonistically Regulate ... · results reveal that salt stress and ethylene antagonistically regulate nucleocytoplasmic partitioning of COP1, thereby controlling

Salt Stress and Ethylene Antagonistically RegulateNucleocytoplasmic Partitioning of COP1 to ControlSeed Germination1[OPEN]

Yanwen Yu2, Juan Wang2, Hui Shi, Juntao Gu, Jingao Dong, Xing Wang Deng*, and Rongfeng Huang*

College of Life Sciences, Hebei Agricultural University, Baoding 071001, China (Y.Y., J.G., J.D.); BiotechnologyResearch Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China (Y.Y., J.W., R.H.); Schoolof Advanced Agricultural Sciences and School of Life Sciences, Peking University, Beijing 100871, China (H.S.,X.W.D.); and National Key Facility of Crop Gene Resources and Genetic Improvement, Beijing 100081, China(J.W., R.H.)

ORCID IDs: 0000-0002-5469-500X (Y.Y.); 0000-0003-1636-4450 (J.W.); 0000-0002-3039-0850 (R.H.).

Seed germination, a critical stage initiating the life cycle of a plant, is severely affected by salt stress. However, the underlyingmechanism 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 offunction of the COP1 mutant was stronger than this in the wild type. A GUS-COP1 fusion was constitutively localized to thenucleus in radicle cells. Salt treatment caused COP1 to be retained in the cytosol, but the addition of ethylene precursor1-aminocyclopropane-1-carboxylate had the reverse effect on the translocation of COP1 to the nucleus, revealing thatethylene and salt exert opposite regulatory effects on the localization of COP1 in germinating seeds. However, loss offunction of the ETHYLENE INSENSITIVE3 (EIN3) mutant impaired the ethylene-mediated rescue of the salt restriction ofCOP1 to the nucleus. Further research showed that the interaction between COP1 and LONG HYPOCOTYL5 (HY5) had arole in SSG. Correspondingly, SSG in loss of function of HY5 was suppressed. Biochemical detection showed that salt promotedthe stabilization of HY5, whereas ethylene restricted its accumulation. Furthermore, salt treatment stimulated and ethylenesuppressed transcription of ABA INSENSITIVE5 (ABI5), which was directly transcriptionally regulated by HY5. Together, ourresults reveal that salt stress and ethylene antagonistically regulate nucleocytoplasmic partitioning of COP1, thereby controllingArabidopsis seed germination via the COP1-mediated down-regulation of HY5 and ABI5. These findings enhance ourunderstanding of the stress response and have great potential for application in agricultural production.

Seed germination is a critical stage in the initiation ofplant life cycles, and it has receivedmuch attention due toits vital importance in sustainable agriculture (Weitbrechtet al., 2011). Under favorable conditions, seeds initiategermination with water uptake for imbibition, completingtheprocesswith radicle protrusionandelongation throughthe covering layers (Finch-Savage and Leubner-Metzger,2006; Holdsworth et al., 2008). Increasing evidence sug-gests that seed germination is regulated by a lot of factors,

including light, salt, and phytohormones (Finch-Savageand Leubner-Metzger, 2006; Holdsworth et al., 2008;Weitbrecht et al., 2011). For instance, light is a major envi-ronmental factor that affects seed germination (Borthwicket al., 1952; Seo et al., 2009). DE-ETIOLATED1 regulatesthe stabilization of LONG HYPOCOTYL IN FAR-RED1 (HFR1) and PHYTOCHROME INTERACTINGFACTOR1 (PIF1), and the HFR1-PIF1 complex inverselymodulates the entire genomic transcriptional net-work in phytochrome B-dependent seed germination(Shi et al., 2013, 2015). CONSTITUTIVE PHOTOMOR-PHOGENESIS1 (COP1), a central repressor of light sig-naling, is a ring finger E3 ligase (Ang et al., 1998; Toriiet al., 1998; Jang et al., 2010; Osterlund et al., 2000; Yi andDeng, 2005), which itself is regulated by multiple factors(Zhong et al., 2009; Yu et al., 2013). Nuclear COP1 levelsare attributed to dark-light transitions and are correlatedwith the repression of photomorphogenesis (von Arnimand Deng, 1994). In the dark, COP1 translocates to thenucleus and is responsible for the proteasome-mediateddegradation of photomorphogenesis-promoting factors,such as ELONGATED HYPOCOTYL5 (HY5). In con-trast, in the presence of light, COP1 is removed from thenucleus, and its activity is repressed (Torii et al., 1998).HY5 has been reported tomediate the abscisic acid (ABA)

1 This work was supported by the National Basic Research Pro-gram of China (2012CB114204) and the National Science Foundationof China (91217303). The funders had no role in the study design, datacollection and analysis, decision to publish, or preparation of themanuscript.

2 These authors contributed equally to the article.* Address correspondence to [email protected] or [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Rongfeng Huang ([email protected]).

R.H., X.W.D., and Y.Y. designed the research; Y.Y. and J.W. per-formed the research; H.S., J.G., J.D., J.W., X.W.D., and R.H. analyzedand discussed the data; R.H., X.W.D., H.S., and Y.Y. wrote the article.

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response through ABA INSENSITIVE5 (ABI5) duringseed germination and early seedling growth (Chen et al.,2008), revealing the integration of light signaling withseed germination.It is estimated that more than 6% of the world’s total

land and approximately 20% of irrigated land are greatlyaffected by salt stress, which restrains plant growth andseverely reduces crop yield (Munns and Tester, 2008).Salt has long been considered a key factor that affectsseed germination (Darwin, 1856). In the field, seed ger-mination occurs in soil. In a soil environment, salinity hasa strong impact on seed germination and impairs theestablishment of seedlings in many species (Almansouriet al., 2001; Debez et al., 2004; Zapata et al., 2004).However, the mechanism of salt inhibition of seed ger-mination (SSG) remains largely unknown. Specifically, itis unclear whether photomorphogenic factors, such asCOP1, are involved in SSG. In addition, if COP1 doesregulate SSG, its direct targets and its behavior in re-sponse to salt in germinating seed tissue remain unclear.Extensive studies have reported that ethylene signaling

components participate in salinity responses during seedgermination and seedling growth (Cao et al., 2008). Forexample, Arabidopsis (Arabidopsis thaliana) ETHYLENERECEPTOR1 (ETR1) and ETHYLENE INSENSITIVE4(EIN4) inhibit germination during salt stress,whereas ETR2stimulates it (Wilson et al., 2014). The CONSTITUTIVERESPONSE1 (CTR1) mutant ctr1-1 exhibits a high sur-vival rate under salt and osmotic stress treatments,whereas loss-of-function mutants ein2 and ein3-1 eil1-1(double mutations of EIN3 and EIN3-LIKE1 [EIL1]) exhibitremarkably reduced tolerance to salt (Achard et al., 2006;Cao et al., 2007; Lei et al., 2011). In addition, salinity-induced ethylene promotes the tolerance of Arabidopsisto soil salinity by enhancing sodium/potassium homeo-stasis (Jiang et al., 2013). Ethylene activates the expressionof salt-induced and EIN3/EIL1-dependent peroxidasegenes by increasing EIN3/EIL1 abundance to prevent theaccumulation of reactive oxygen species and enhance tol-erance to salt stress (Peng et al., 2014), which demonstratesthe essential role of ethylene in regulating the salt response.In this study, through the use of ethylene to improve

seed germination under salt stress, we discovered thatCOP1 counteracts SSG, distinctive from the regulation inphotomorphogenic development. Ethylene antagonisti-cally modulates this process through affecting nucleo-cytoplasmic partitioning of COP1, thereby controllingArabidopsis seed germination via the COP1-mediateddown-regulation of HY5 and ABI5 in the nucleus. Thisfinding is a novel and important contribution to ourunderstanding of the stress response, with great potentialfor application in agricultural production.

RESULTS

COP1 Negatively Regulates SSG

Because ethylene improves seed germination undersalt stress (Negm and Smith, 1978; Chang et al., 2010;

Lin et al., 2012; Wilson et al., 2014), we applied the phy-tohormone ethylene or its precursor 1-aminocyclopropane-1-carboxylate (ACC) to suppress SSG. Our results showedthat the addition of ACC did not obviously increase seedgermination under control conditions but significantlypromoted NaCl inhibition of seed germination comparedto the corresponding controls (Supplemental Fig. S1).Ethylene overproduction mutants (eto1/2/3) and an ethyl-ene constitutive response mutant (ctr1) promoted seedgermination under NaCl stress, but ethylene signalingmutants etr1-1 (a gain-of-function mutation) and ein2,ein3-1, and ein3 eil1 (loss-of-function mutations) showeda decrease in seed germination under NaCl stress(Supplemental Fig. S2). In addition, after treatment withaminoethoxyvinyl-Glyor silver nitrate (AgNO3), inhibitorsof ethylene biosynthesis and signaling pathway, respec-tively, eto2 displayed lower tolerance to NaCl than thecontrols (Supplemental Fig. S3). These results confirmedprevious findings that ethylene suppresses SSG (Cao et al.,2008; Lin et al., 2012; Wang et al., 2007, 2008).

Because light is a major factor controlling seed ger-mination (Borthwick et al., 1952; Seo et al., 2009) andCOP1 is the master regulator of light-associated de-velopment (Osterlund et al., 2000), we questionedwhether COP1 affects SSG. To test this hypothesis, wefirst determined that germination in Col-0 and cop1-4,which lacks the WD-40 repeat domain with a hyper-photomorphogenic phenotype (McNellis et al., 1994),showed no obvious difference under control condi-tions. The germination of cop1-4 was more sensitive toNaCl than Col-0, and ACC did not improve the ger-mination of cop1-4 under salt stress (Fig. 1A), indicatingthat COP1 is a negative regulator of SSG and is requiredfor the suppression of SSG by ethylene, distinctive fromthe regulation in photomorphogenic development.

To investigate the relationship between COP1 andethylene signaling in SSG, our data showed that eto23cop1-4, similar to cop1-4, had lower seed germinationunder salt stress than eto2 and Col-0 (Fig. 1B), furtherdemonstrating that COP1 is downstream of ethylene inthe regulation of SSG.We next evaluated the germinationof ein3-13COP1ox and cop1-43EIN3ox under salt stress.Transgenic lines overexpressing COP1 (COP1ox inNossen [No] background; vonArnimandDeng, 1994) andEIN3 (EIN3ox in Col-0 background; Zhong et al., 2009)were more tolerant to salt than their corresponding con-trols (Fig. 1, C and D). However, cop1-43EIN3ox, whichlacks COP1, exhibited lower germination, as did cop1-4,than Col-0 (Fig. 1C). Additionally, ein3-13COP1ox, whichlacks EIN3, exhibited higher germination, as did COP1ox,than No3Col-0 (Fig. 1D), indicating that COP1 functionsgenetically downstream of EIN3 in ethylene-promotedseed germination under salt stress.

Salt Promotes Cytosolic Partitioning of COP1

Some of the seeds completed germination within2 d after imbibition, but this process was delayed ap-proximately 1 to 2 d by the addition of 100 mM NaCl(Supplemental Fig. S4). We then used 1- to 1.5-d

Plant Physiol. Vol. 170, 2016 2341

The Retention of COP1 in Cytosol after Salt Stress

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germinating seeds with emerging radicles for gene ex-pression and protein detection in the following assays.In photomorphogenesis, COP1 activity is correlatedwith the nucleocytoplasmic partitioning of COP1. In thedark, COP1 is mainly located in the nucleus, mediatingthe degradation of its target proteins, such as HY5.However, light can inhibit this activity by reducing thenuclear abundance of COP1 and allowing the accu-mulation of HY5 to promote photomorphogenesis (vonArnim and Deng, 1994; Osterlund et al., 2000; Yu et al.,2013). To determine whether COP1 movement plays arole in SSG, we quantified the nuclear and cytoplasmiclocalization of COP1 in a transgenic line that constitu-tively expressed a GUS-COP1 fusion protein. In theprotruding radicles, GUS-COP1 was localized mainlyto the nucleus in both light and darkness (Fig. 2A),which was consistent with a previous report in Arabi-dopsis roots (von Arnim and Deng, 1994). However,NaCl treatment maintained the localization of GUS-COP1 to the cytosol in both light and darkness(Fig. 2A), revealing that NaCl prevented the translocationof GUS-COP1 from the cytosol to the nucleus.

Additionally, ACC treatment did not obviously in-crease the amount of nucleus-enriched GUS-COP1 (Fig.2A), which was similar to the effect of ACC on seedgermination under control conditions. Interestingly,this retentive effect of NaCl on the cytosolic localizationof GUS-COP1 was impaired by the addition of ACC inthe presence of NaCl, and the amount of nucleus-enriched GUS-COP1 recovered to a level that wassimilar to that in the samples without NaCl treatment(Fig. 2A). These data indicate that ACC andNaClmighthave opposite effects on the movement of COP1 be-tween nucleus and cytosol.

In addition, we performed cell fractionation experi-ments to directly detect the levels of COP1 in the nu-cleus and cytosol using COP1 antibodies. To excludethe influences of de novo protein production and deg-radation during the process, we supplemented thesamples with cycloheximide (CHX) and MG132, withor without ACC and NaCl, in light or darkness for 16 h.Consistent with the observations shown in Figure 2A,COP1 was mainly localized to the nucleus in bothlight and darkness. Additionally, the repression of

Figure 1. COP1 mutation impairs ethylene-promoted seed germination under saltstress. A, Statistical analysis of seed ger-mination with the indicated treatment(100 mM NaCl and 10 mM ACC) at 4 d ofgermination. “Control” and “NaCl” indicatethe absence or presence of salt treatment,respectively. P values were calculatedwith atwo-tailed Student’s t test assuming equalvariances (*P , 0.05). B to D, The timecourse of seed germination with treatmentwith 100mMNaCl. The data show the rate ofgerminated seeds compared to their corre-sponding controls on the same day of ger-mination. Each value shown represents themean 6 SD of three independent biologicalexperiments.

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Yu et al.



translocation to the nucleus by NaCl was reversed bythe addition of ACC (Fig. 2B). Thus, our results dem-onstrate that ethylene and salt inversely modulate thelocalization of COP1 during seed germination, inde-pendent of the transition between light and darkness.We then examined the localization of GUS-COP1 in

the ein3-1 background. With loss of function of EIN3 inthe ein3-13GUS-COP1 hybrid, althoughNaCl treatmentstill attenuated the translocation of GUS-COP1 to thenucleus, ACC treatment did not reverse this process (Fig.2C). Cell fractionation experiments using ein3-1 eil1 fur-ther confirmed the above observation, i.e. the amount ofCOP1 protein localized in the nucleus was decreased byNaCl treatment, and the addition ofACCdid not recoversalt-restrained COP1 localization in cytosol (Fig. 2D),strongly suggesting that the reversal of ethylene on salt-induced COP1 movement is inactivated by the removalof EIN3 and EIL1.

Interaction between COP1 and HY5 Plays a Role in theRegulation of SSG

COP1 is an E3 ligase that targets multiple proteins,including HY5, for degradation via protein-protein in-teractions. COP1 contains three structural modules: anN-terminal RING-finger, a predicted coiled-coil domain,and C-terminal WD-40 repeats, which are all impor-tant for interaction (Ang et al., 1998; Torii et al., 1998;Osterlund et al., 2000). In the above research, we revealedthat the transgenic line overexpressing full length ofCOP1 displayed inhibition of SSG (Fig. 1D), but theoverexpression of defective COP1 lacking the coiled-coildomain (DCoil) or lacking both the coiled-coil andRING-finger domains (DRDC) in Col-0 background didnot improve SSG, showing a similar seed germinationto Col-0 under salt stress (Fig. 3, A and B). Combinedwith the promotion of SSG in the loss-of-function cop1-4

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 mM ACC for 16 h. The cellnuclei in the radicle were stained with 0.5 mg/mL 49,6-diamino-phenylindole (DAPI). Bars = 5 mm. B and D, Immunoblotting ofCOP1 using germinating seeds (1 d) pretreated with 100 mM CHX for 12 h and then treated with or without NaCl and/or 10 mM

ACC for another 16 h. N, Nuclear protein; S, soluble fraction containing cytoplasmic protein. Immunoblotting was performedusing 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.





mutant, which lacks theWD-40 repeat domain (Fig. 1, A–C), our analyses reveal that the three HY5-interactingdomains in COP1 play important roles in the regulationof SSG. The results are consistent with their photomor-phogenic phenotypes, i.e. COP1ox shows suppression ofphotomorphogenic development, whereas the photo-morphogenic changes are very weak or absent in DCoiland DRDC (Torii et al., 1998), implying that the HY5-interacting domains of COP1 that function in light sig-naling are also functional in SSG.

It has been reported that the N-terminal 77 aminoacids of HY5 are essential for the interaction of HY5with COP1 (Ang et al., 1998; Torii et al., 1998). The seedgermination of transgenic lines containing a deletion ofthe N-terminal 77 amino acids of HY5 (HY5-DN77) ina Col-0 or loss-of function mutant hy5 backgroundshowed similar trends to Col-0 under control condi-tions but were more sensitive to salt stress than Col-0(Fig. 3, A and B), which was consistent with their phe-notype in photomorphogenic development (Ang et al.,1998; Yu et al., 2013). These results demonstrate thatHY5 takes part in the COP1-regulated SSG.

To further investigate how HY5 participates in SSG,we then showed that hy5 and eto2 mutants were moretolerant, whereas an overexpression line of HY5 (35S::HY5)wasmore sensitive to salt stress than Col-0 duringseed germination. However, the hybrid eto2335S::HY5displayed lower seed germination than eto2 and hy5,similarly to the transgenic line 35S::HY5 (Fig. 4A). Thus,HY5 is a positive regulator of SSG and participates inethylene-suppressed SSG.

To determine whether HY5 functions downstream ofEIN3 in ethylene-suppressed SSG, similarly to COP1,we next observed the seed germination of the doublemutant ein3-1 hy5 under salt stress. Our results showedthat ein3-1 displayed lower germination than Col-0,whereas the double mutant ein3-1 hy5 had a similargermination to hy5, showing higher salt tolerance thanCol-0 (Fig. 4B). These results demonstrate that bothCOP1 and HY5 function genetically downstream ofEIN3 to orchestrate ethylene-suppressed SSG.

Salt Improves the Stabilization of HY5 Protein

To determine how HY5 is involved in SSG, we firstdetermined HY5 expression in ethylene mutants withor without NaCl treatment. Although NaCl greatly in-duced HY5 expression, the transcript levels did notshow obvious differences between the ethylenemutants and Col-0, suggesting that ethylene doesnot transcriptionally modulate HY5 expression in thesalt response (Supplemental Fig. S5). Interestingly, wefound that HY5 protein was greatly decreased in theendogenous ethylene overproduction mutant eto2 andthe ethylene constitutive response mutant ctr1 but wasnot changed in ethylene signaling component mutants(Fig. 5A), providing biochemical evidence that endog-enous ethylene restrainsHY5 protein accumulation.Wethen examined the protein stability of HY5 with orwithout NaCl or ACC treatment in samples pretreatedwith the protein synthesis inhibitor CHX to exclude denovo protein synthesis. We found that HY5 proteingradually decreased with time under control condi-tions, but NaCl treatment greatly preserved HY5 ac-cumulation (Fig. 5B), revealing that salt stress stabilizesHY5 protein. The addition of ACC promoted the deg-radation of HY5 (Fig. 5B), further demonstrating thenegative regulatory function of ethylene on HY5accumulation.

To further examine the regulation of HY5 by ethyleneand salt, we next examined the accumulation of HY5protein by treating Col-0 germinating seeds with 10 mM

ACC plus 100 mM NaCl after pretreatment with 50 mM

CHX. Our data showed that ACC greatly restrained theaccumulation of HY5 induced by NaCl (Fig. 5B). Thosedata indicate that ethylene and salt oppositely regulateHY5 protein levels posttranscriptionally, supportingthe antagonistic regulation by ethylene and salt ofCOP1 translocation in the germinating radicle. Thisconclusion was further supported by the detection ofHY5 protein in the ethylene-insensitive double mutantein3 eil1. NaCl treatment still increased HY5 proteinaccumulation, whereas the inhibitory effect of ACCwas

Figure 3. Interaction between COP1 andHY5 plays a role in SSG. A and B, Timecourse of seed germination without(Control) or with 100 mM NaCl treatment(Salt treatment). The data show the rateof germinated seeds compared to thecorresponding controls on the same day ofgermination. Each value shown representsthe mean 6 SD of three independent bio-logical experiments.


Yu et al.




disabled in ein3 eil1 (Fig. 5C). Taken together, theseresults indicate that ethylene and salt antagonisticallyregulate HY5 protein levels, thus affecting the salt re-sponse during seed germination.

The Expression of ABI5, Transcriptionally Activated byHY5, Participates in SSG

ABI5, a well-known transcription factor that affectsseed germination, is transcriptionally activated by thephotomorphogenic factor HY5 (Finkelstein and Lynch,2000; Chen et al., 2008). To address whether the ex-pression of ABI5 was associated with SSG, we firstdemonstrated that seed germination in different geno-types of ABI5 exhibited no obvious differences undercontrol conditions (Fig. 6A). However, loss of functionof ABI5 (abi5-8) displayed better germination, identicalto the function of hy5 in SSG, than Col-0 under salt

stress. Furthermore, the overexpression lines of ABI5 inCol-0 or hy5 background showed worse germinationthan Col-0 under salt stress (Fig. 6B), demonstratingthat ABI5 acts as a positive regulator for SSG.

We then investigated whether ABI5 expression wascontrolled by ethylene. Our analyses showed that ABI5expression was inhibited in ethylene overproductionmutants (eto1/2/3) and was upregulated in loss-of-function mutants (ein2/3) under control conditions.Although salt greatly increased ABI5 expression in allof the tested genotypes, ABI5 transcripts were signifi-cantly reduced in etomutants compared with Col-0 butwere enhanced in ein mutants (Supplemental Fig. S6),revealing the transcriptional regulation of ABI5 tran-scripts by endogenous ethylene. In addition, the ex-pression ofABI5 induced by NaCl was repressed by thetreatment of ACC in Col-0 (Fig. 7A), consistent with theeffects of ethylene and salt on HY5 protein levels.

Figure 5. Salt and ethylene oppositelymodulate the stabilization of HY5 protein.A, HY5 accumulation in different pheno-types. B, HY5 stability in response to NaCland/or ACC treatment in Col-0. C, HY5protein levels in response to NaCl and/orACC treatment in ein3 eil1. Germinatingseeds (1 d) were pretreated with 100 mM

CHX for 12 h and then treated with orwithout 100 mM NaCl and/or 10 mM ACCfor the indicated time. Protein extractswere prepared from germinated seeds.Immunoblotting was performed with anti-HY5 and antiactin antibodies. Numbersindicate the relative levels of HY5 protein.

Figure 4. HY5 acts downstream of EIN3 inethylene-promoted seed germination undersalt stress. A and B, Time course of seedgermination with treatment with 100 mM

NaCl. The data show the rates of germinatedseeds compared to the corresponding con-trols on the same day of germination. Eachvalue shown represents the mean 6 SD ofthree independent biological experiments.






Furthermore, the inhibitory effect of ACC on ABI5 ex-pression was disabled in the ein3 eil1mutant, indicatingthat ABI5 participates in ethylene-suppressed SSG.

We next conducted the investigation to detectwhether ABI5 expression was modulated by COP1 andHY5 in ethylene-promoted seed germination under saltstress. Our results showed that the expression of ABI5in cop1-4 was higher than in Col-0, and ACC treatmentfailed to repress the ABI5 transcripts induced by salttreatment, similarly to these in ein3 eil1 (Fig. 7A). Cor-respondingly, the expression of ABI5 in the hy5mutantwas maintained at a lower level than in Col-0 no matterif it was with or without the treatment of ACC and/orNaCl (Fig. 7A), providing evidence that the expressionofABI5might be controlled byHY5, consistent with thetranscriptional activation of HY5 on ABI5 in seed ger-mination and early seedling growth (Chen et al., 2008).Thus, our analyses demonstrate that COP1 and HY5oppositely regulate ABI5 transcription in ethylene-suppressed SSG.

It has been reported that HY5 binds to the G-boxCACGTG in the ABI5 and RbcSA promoters(Chattopadhyay et al., 1998; Chen et al., 2008). Wenext conducted chromatin immunoprecipitation (ChIP)assays using 35S:HY5-HA plants to verify the binding ofHY5 to theABI5promoter in germinating seeds. Possiblebinding regions in the promoters of ABI5 and RbcSA (asa positive control) were selected for PCR amplification(Fig. 7B). HY5 was directly associated with the ABI5promoter (Fig. 7C), indicating thatHY5 directly activatesABI5 expression during seed germination.

DISCUSSION

In agricultural production, the rate of seed germina-tion determines the planting density per unit area andsubsequently affects crop yield. In the soil environment,salinity exhibits a strong impact on seed germination,impairing the establishment of seedlings. It is important

to improve seed germination to maintain the sustain-able development of agriculture (Bewley, 1997; Finch-Savage and Leubner-Metzger, 2006; Holdsworth et al.,2008; Weitbrecht et al., 2011). Moreover, photomor-phogenic regulators are involved in light-initiatedseed germination in Arabidopsis (Shi et al., 2013, 2015)and participate in phytohormone-controlled seedlinggrowth (Chen et al., 2008; Seo et al., 2009; Yu et al.,2013). In addition, increasing numbers of studies haverevealed that ethylene suppresses SSG (Negm andSmith, 1978; Chang et al., 2010; Lin et al., 2012); how-ever, it remains unclear whether the light signalingfactor COP1 participates in SSG. In this study, we foundthat the COP1 negatively regulates SSG. Moreover,salt stress and ethylene antagonistically regulate thenucleocytoplasmic partitioning of COP1, which sup-presses the accumulation of HY5 protein, subsequentlyresulting in the transcriptional repression of ABI5 andthe increase of seed germination. Thus, our data revealthe molecular mechanism bywhich photomorphogenicfactors are involved in SSG. This finding is novel andimportant for our understanding of the stress response,and it has high potential for application in agriculturalproduction.

Light mediates plant development, in part, by or-chestrating multiple signaling cascades. In Arabidopsishypocotyl cells, nuclear GUS-COP1 levels change inresponse to dark-light transitions and are quantitativelycorrelated with the extent of the repression of photo-morphogenic development (von Arnim and Deng,1994). Ethylene suppresses hypocotyl growth by im-proving GUS-COP1 movement into the nucleus, evenunder light conditions (Yu et al., 2013). Increasing evi-dence has revealed that light is a major environmentalfactor affecting seed germination (Borthwick et al.,1952; Seo et al., 2009; Shi et al., 2013, 2015). In this study,we discovered that COP1 counteracts SSG. Impor-tantly, we reveal that the retention of COP1 in the cy-tosol following NaCl treatment is fine-tuned by theaddition of ACC, which enhances the movement of

Figure 6. ABI5 mutation enhances seedgermination 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 ofgerminated seeds compared to the corre-sponding controls on the same day of ger-mination. Each value shown represents themean6 SD of three independent biologicalexperiments.


Yu et al.



COP1 into the nucleus. Correspondingly, HY5, a down-stream regulator of COP1, participates in SSG; saltmaintains the stability of HY5,whereas ethylene reducesit. Thus, our results demonstrate that the antagonisticregulation of ethylene and salt in seed germination ismediated by the control of COP1 translocation, whichsubsequently results in dynamic changes in HY5 proteinand ABI5 transcripts, providing novel evidence that im-proves our understanding of SSG.Soil cover is associated with the increased evolution

of ethylene, which improves seed germination andseedling establishment (Gomez-Jimenez et al., 2001;Achard et al., 2006; Zhong et al., 2014). Components ofthe ethylene signaling pathway, including the receptorsCTR1, EIN2, and EIN3, play critical roles in early plantdevelopment under salt stress (Achard et al., 2006;Cao et al., 2007, 2008; Lei et al., 2011;Wilson et al., 2014).In this study, we found a connection between the

photomorphogenic factor HY5 and SSG. Our researchshowed that ethylene determines whether seeds willinitiate germination under salt stress. This fine regula-tion occurred in ethylene-insensitive mutants (etr1-1,ein2, and ein3-1), in which HY5 protein accumulatedand germination was restrained; however, in ethyleneoverproduction and constitutive expression mutants

Figure 8. The COP1-HY5 complex is involved in ethylene-promotedseed germination under salt stress. COP1 was mainly localized to thenucleus in radicle cells (top). Under NaCl stress, salt retained the lo-calization of COP1 in the cytosol, subsequently causing the accumu-lation of HY5 and the expression of ABI5, which inhibited seedgermination (middle). Ethylene increased in response to the pressure ofwater uptake by the imbibed seed and the radicle protrusion; this re-versed the retention of COP1 in the cytosol due to salt and promoted thelocalization of COP1 into the nucleus to destroy HY5, decreasing theexpression of ABI5, which then facilitated seed germination (bottom).

Figure 7. HY5 directly controls ABI5 transcription during seed germi-nation. A, ABI5 expression in different genotypes. Col-0 and mutantseedswere kept inMS for 1.5 d and then transferred toMSmediumwith100 mM NaCl and/or 10 mM ACC for 2 h. ABI5 transcripts were quan-tifiedwith qPCR relative to TUB4. The transcript levels in Col-0 were setto 1. B, Schematic diagram of the promoter regions of ABI5 and RbcSA.Black lines represent the promoter regions of the two genes. Black boxeson the line represent the putative G-box (59-CACGTG-39); numbersabove 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 analysisshowing the in vivo binding of HY5 to the ABI5 promoter. The ChIPanalysis used 1.5-d germinating 35S:HY5-HA seedlings. IgG was usedas a negative control, and the interaction between the RbcSA promoterand HY5 was used as a positive control. Each value shown representsthe mean 6 SD of three independent biological experiments.





(ctr1-1 and eto2),HY5was reduced, and seed germinationwas promoted.Moreover, the opposite regulation of seedgermination by ethylene and salt was attenuated in theein3 eil1 mutant. HY5 has also been reported to regulateABA responses by directly activating the expression oftranscription factorABI5 (Chen et al., 2008). Antagonisticinteractions between ABA and ethylene during germi-nation have been demonstrated in numerous species(Beaudoin et al., 2000; Ghassemian et al., 2000). There-fore, HY5 might integrate the ethylene and ABA signal-ing pathways during seed germination under salt stress.As stress hormones, ABA and ethylene increase undersaline conditions and regulate seed germination in dis-tinct ways. ABA specifically promotes the binding ofHY5 to the ABI5 promoter, whereas ethylene enhancesthe degradation of HY5 protein by promoting the COP1enriched in nucleus. Both the level and the activity ofHY5 protein determine the expression of ABI5, whoseaccumulation inhibits seed germination. Thus, our re-search demonstrates that HY5, as an integrator, partici-pates in the interaction between ethylene and ABA inseed germination under salt stress.

Previous study has showed that loss-of function ofCOP1 enhances photomorphogenic development (Denget al., 1991). In this research, we found that COP1 coun-teracts SSG, indicating that COP1 might have differentmechanisms responsible for seed germination and pho-tomorphogenesis. Moreover, the functional activity ofCOP1 is attributed to dark-light transitions in photo-morphogenesis (von Arnim and Deng, 1994; Torii et al.,1998). In germinating seeds, COP1 is constitutively lo-calized to the nucleus, which is not affected by light-darktransitions, but affected by salt stress, demonstrating anovel function of COP1 in SSG. In addition, the E3 ligaseCOP1 directly combines with HY5 to cause the degra-dation of HY5, and the translocation of COP1 from thenucleus to the cytoplasm has been shown to be suffi-ciently rapid to participate in light-induced responses(Osterlund et al., 2000; Pacín et al., 2013, 2014). In thisstudy, we found that COP1 also functions through itsinteraction with HY5, revealing that the COP1-HY5complex is a key regulon of SSG. Moreover, we foundthat salt reduced the translocation of COP1 into the nu-cleus. ACC addition reversed the localization of COP1 tothe nucleus, and this regulation was dependent on EIN3,showing that ethylene and salt inversely regulate thelocalization of COP1. Thus, the evidence presented heredemonstrates that COP1 localization directly results influctuations in the HY5 protein and ABI5 expressionlevels in SSG. Although it has been shown that CSN1contributes to the shuttling of COP1 between the nucleusand cytoplasm (Wang et al., 2009), the mechanism bywhich the localization of COP1 is regulated by ethyleneand salt requires further investigation.

CONCLUSIONS

Based on previous reports (Chen et al., 2008; Zhonget al., 2014) and the results of this study, we proposethat the cascade of COP1-HY5-ABI5 might play a

central role in SSG (Fig. 8). Once seeds have imbibed,salt stress prevents the entry of COP1 into the nucleus,subsequently causing HY5 protein accumulation, pro-moting ABI5 expression, and restraining seed germi-nation. In response to the mechanical pressure of wateruptake, radicle protrusion, and/or soil cover, imbibedseeds are stimulated to increase ethylene production,which promotes the localization of COP1 to the nucleusto destroy HY5. Finally, the expression of ABI5 is re-duced to facilitate seed germination.

MATERIALS AND METHODS

Plant Materials and Chemicals

All Arabidopsis (Arabidopsis thaliana) transgenic lines and mutants weregenerated in Col-0 background, with the exception of the line overexpressingGUS-COP1 in Nossen (No) background. Homozygous lines of abi5-8(SALK_013163), ctr1-1 (CS8057), etr1-1 (CS237), ein2 (CS8844), ein3-1 (CS8052),eto1 (CS3072), eto2 (CS8059), eto3 (CS8060), and hy5 (SALK_096651) wereobtained from the Arabidopsis Biological Resource Center.

The following transgenic plants and mutants were generated previously:cop1-4 (McNellis et al., 1994); ein3-1 eil1, EIN3ox, and EIN3ox3cop1-4 (Zhonget al., 2009); 35S::HY5 (Lee et al., 2007);DCoil andDRDC (Torii et al., 1998); HY5-DN77/Col-0 and HY5-DN77/hy5 (Ang et al., 1998); GUS-COP1/COP1ox (vonArnim andDeng, 1994), 35S::ABI5/Col-0 and 35S::ABI5/hy5 (Chen et al., 2008);and eto2335S::HY5, ein33hy5, and ein3-13COP1ox (Yu et al., 2013). Plantswere grown under a 16-h white light (50 mmol/m2/s)/8-h dark cycle at 22°C.All chemicals used were obtained from Sigma-Aldrich.

Seed Germination

All seeds used for the assays were planted and harvested at the same time anddried at room temperature for 3 months to break dormancy. Seeds were surface-sterilized and sown onMurashige and Skoog (MS)medium containing 3%Suc and0.5% Phytagel with the indicated supplementation (10 mM ACC, 100 mM NaCl,5mM aminoethoxyvinyl-Gly, and 5mMAgNO3). The plateswere then chilled at 4°Cin darkness for 3 d to stratify and improve the uniformity of germination beforebeingmoved to 22°Cunder a 16-hwhite light (50mmol/m2/s)/8-h dark cycle. Thepercentage of seed germinationwas scored every day in three replicates, andmorethan 50 seeds each were grown in each plate. In our assays, the tested genotypesdid not display obvious differences in seed germination under control conditions.Thus, previously reported data on ethylene-promoted seed germination are notshown in the text, whereas seed germination data on genotypes that had not beenpreviously investigated are presented as control.

RNA Extraction and Quantitative Real-Time PCR

Total RNAwas extracted from germinating seeds using the Plant Total RNAPurification Kit (GeneMark), and 2 mg of total RNA was used for the reversetranscription of cDNA with the FastQuant RT kit (Tiangen) according to themanufacturer’s instructions. Gene expression was measured by qPCR analysisusing SYBR Premix (Takara) in the IQ-5 real-time system (Bio-Rad). The ex-pression levels were normalized to TUB4. The primers used for qPCR are listedin Supplemental Table S1. Each qPCR was repeated independently three timesand the average was calculated.

Western-Blot Analysis

Protein was extracted from germinating seedswith 100mL of extraction buffer(150 mM NaCl, 1 mM EDTA, 50 mM Tris, pH 7.5, 2 mM sodium orthovanadate,10mMNaF, 25mM b-glycerophosphate, 10% glycerol, 1mM phenylmethylsulfonylfluoride, and 0.1% Tween 20). Ten micrograms of total protein was separated on12%PAGEgels containing SDS, andwestern blottingwas performed as previouslydescribed (Osterlund et al., 2000) using anti-HY5 or anti-COP1 antibodies (1:1,000).Actin (1:5,000) was used as a loading control. The relative HY5 protein levels wereevaluated by Image Gauge V3.12 (Fujifilm). The values of Actin signal in Col-0without treatment were normalized to 1.0.


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Subcellular Localization of GUS-COP1

Transgenic GUS-COP1 seeds were germinated onMSmedium for 1 d. Aftertreatment with or without 10 mM ACC and 100 mM NaCl for 16 h in darkness orin light, the germinating seeds were fixed using cold 90% acetone followed bythe GUS staining assay as previously described (Yu et al., 2013). To quantify thenuclear-cytoplasmic localization of GUS-COP1, the percentages of cells inwhich GUS-COP1 staining was stronger in the nucleus than in the cytoplasmwere calculated. At least 30 germinating seeds with 20 cells per seed were an-alyzed for each GUS fusion.

Microscopy

ForGUS activity assays, imageswere acquired using aZeiss Axio ImagerM1microscope and a Zeiss AxioCam HRc camera.

Nuclear Protein Extraction

Nuclear proteins were extracted at 4°C using a CelLytic PN extraction kit(Sigma-Aldrich) as previously described (Jang et al., 2010; Yu et al., 2013). Thesamples grown on MS medium for 1 d were pretreated with 100 mM CHX and5 mM MG132 (to exclude de novo protein production and degradation duringthe process) for 12 h and then treated with or without supplementation with10 mM ACC and 100 mM NaCl in the light for 16 h. The nuclear and cytosolprotein fractionwere obtained according to themanufacturer’s instructions andanalyzed by western blot.

ChIP-qPCR Analysis

The EpiQuik Plant ChIP kit (Epigentek) was used to perform the ChIPassays. IgG was used as a negative control, and the interaction between theRbcSA promoter and HY5 was used as a positive control. The primers used forqPCR are listed in Supplemental Table S1.

Accession Numbers

The sequence data from this study can be found in the Arabidopsis GenomeInitiative or GenBank/EMBL databases under the following accessionnumbers: COP1 (At2g32950), HY5 (At5g11260), ETR1 (Ag1g66340), CTR1(At5g03730), EIN2 (At5g03280), EIN3 (At3g20770), EIL1 (At2g27050), ABI5(AT2g36270), and RbcSA (At1g67090).

Supplemental Data

The following supplemental materials are available.

Supplemental Table S1. The primers used in this study.

Supplemental Figure S1. Application of ACC improves seed germinationunder salt stress.

Supplemental Figure S2. Endogenous ethylene enhances seed germinationunder salt stress.

Supplemental Figure S3. Blockage of ethylene biosynthesis or signalingreduced ethylene-promoted seed germination under salt stress.

Supplemental Figure S4. Salt stress inhibits seed germination.

Supplemental Figure S5. Ethylene does not transcriptionally activate theexpression of HY5.

Supplemental Figure S6. Ethylene transcriptionally activates the expres-sion of ABI5.

ACKNOWLEDGMENTS

We greatly appreciate the efforts of the editors and anonymous reviewers toimprove the text. We thank Dr. Ligeng Ma (Capital Normal University, Beijing,China) and Dr. Jianping Yang (Chinese Academy of Agricultural Sciences,Beijing, China) for their helpful comments and suggestions regarding this

study. This article is dedicated to the memory of Dr. Zhuofu Li, who died inJune 2012 of acute leukaemia and who contributed at the start of this study.

Received November 5, 2015; accepted February 3, 2016; published February 5,2016.

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Salt Stress and Ethylene Antagonistically Regulate ... · results reveal that salt stress and ethylene antagonistically regulate nucleocytoplasmic partitioning of COP1, thereby controlling