RESEARCH ARTICLE SPECIAL ISSUE: PLANT CELL BIOLOGY

Arabidopsis thaliana ACS8 plays a crucial role in the earlybiosynthesis of ethylene elicited by Cu2+ ionsBaogang Zhang1,2,*, Haifeng Liu1,*, Xinhua Ding2,*, Jiajia Qiu1, Min Zhang3 and Zhaohui Chu1,‡

ABSTRACTCu2+ ions are required by all living organisms and play important rolesin many bactericides and fungicides. We previously reported thatCu2+ can elicit defense responses, which are dependent on theethylene signaling pathway inArabidopsis. However, themechanism bywhich Cu2+ elicits the biosynthesis of ethylene remains unclear. Here,we show that CuSO4 treatment rapidly increases the production ofethylene. In addition, it upregulates the expression of several defense-related genes and ethylene biosynthesis genes, including genesencoding S-adenosylmethionine synthase, 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) and ACC oxidase. Among thesegenes, Arabidopsis thaliana (At)ACS8 was identified as essential for thedefense response and early ethylene biosynthesis induced by Cu2+.Furthermore, Cu2+-induced AtACS8 expression depended on thecopper-response cis-element (CuRE) in the promoter of AtACS8. Ourstudy indicates that Cu2+ specifically activates the expression of AtACS8to promote the early biosynthesis of ethylene that elicits plant immunity inArabidopsis plants.

KEY WORDS: Copper fungicide, cis-element, Defense response,MAMP-triggered immunity, Phytohormone, Transcript regulation

INTRODUCTIONCu2+ ions are a key constituent of cytochrome c oxidase and areessential to bacteria and eukaryotes. In plants, Cu2+ ions arecofactors for many enzymes, such as plastocyanin, superoxidedismutase and polyphenol oxidase, and participate inphotosynthetic electron transport, oxidative stress responses andoxidative phosphorylation. Thus, Cu2+ ions play roles in thevegetative growth, development and reproduction of plants(Marschner, 1995; Peñarrubia et al., 2010). A previous studyreported that Cu2+ associated with the ethylene-binding domain isrequired for ETR1 (ethylene response 1) to bind the gaseoushormone ethylene. This suggests that Cu2+ ions also play a role inhormone perception (Rodríguez et al., 1999). Under copperdeficiency, plants show a twisted or malformed phenotype inyoung leaves (Marschner, 1995). However, excess copper exhibitsheavy metal toxicity and causes cell death in plants. Therefore,

plants tightly balance the level of Cu2+, allowing sufficient metalco-factor to be delivered to target proteins while avoiding toxicity.Plants have evolved specific membrane transporters, such as thecopper transporter (COPT) family and P-type ATPase-familycopper transporters, to deliver copper intra- and intercellularly(Hirayama et al., 1999; Sancenón et al., 2003). Copper deficiencyupregulates expression of the transcription factor SQUAMOSApromoter-binding protein-like 7 (SPL7) in Arabidopsis. SPL7 thentargets the cis-element of the GTAC motif present in promoters ofsome members of the COPT gene family to increase the absorptionof Cu2+ (Peñarrubia et al., 2010; Yamasaki et al., 2009). However,excess copper suppresses the expression of COPT genes to reducethe concentration of copper in plants (Sancenón et al., 2003; Yuanet al., 2011). In yeast, the CUP2 gene codes for a copper-bindingtranscription factor protein containing a copper fist. In elevatedcopper concentrations, CUP2 binds Cu2+ and activates transcriptionof the metallothionein genes CUP1 and CRS5 (Welch et al., 1989).

Cu2+ is also a key constituent of many bactericides andfungicides. Copper-based bactericides and fungicides are usedextensively in agriculture. Bordeaux mixture, a mixture of coppersulfate (CuSO4) and slaked lime, has been widely used in vineyards,fruit farms and gardens to protect plants from infestations of downymildew, powdery mildew and other fungi since 1854. Theantimicrobial mechanisms of Cu2+ were assumed to be associatedwith its heavy metal toxicity. Cu2+ can suppress microbial growthby denaturing nucleic acids, inhibiting protein activity and changingplasma membrane permeabilization (Borkow and Gabbay, 2004).Interestingly, fewer microbes are reported to be resistant to copper-based bactericides and fungicides, although some microbes aretolerant to copper. For example, Pseudomonas syringae pv. tomato(Pst) isolated from tomato fields is tolerant to copper, dependent onthree copper-resistant operon genes (Cha and Cooksey, 1991).However, copper compounds are still effective in controlling plantdiseases. Thus, we hypothesize that Cu2+ not only inhibits bacterialand fungal growth but also elicits defense responses in plants. In ourprevious study, we found that Cu2+ promoted reactive oxygenspecies (ROS) accumulation and callose deposition, induced theexpression of pathogenesis-related (PR) genes, activated MAPkinase signaling and elicited defense against Pst DC3000 inArabidopsis (Liu et al., 2015). In addition, the Cu2+-mediateddefense response is dependent on salicylic acid (SA) and ethylenesignaling pathways. Thus, we assumed that Cu2+ acts as an elicitorand triggers immune responses in plants.

In the past decades, many elicitors have been shown to induce thedefense response and protect plants from pathogen infection.Among these elicitors, microbe-associated molecular patterns(MAMPs) are the best studied. A number of MAMPs, such aschitin, peptidoglycan, lipopolysaccharide, flagellin and theelongation factor thermo unstable (ER-Tu), have been identifiedas inducing the immune response in plants (Felix et al., 1993, 1999;Gust et al., 2007; Kunze et al., 2004; Meziane et al., 2005). InReceived 10 February 2017; Accepted 11 July 2017

1State Key Laboratory of Crop Biology, College of Agronomy, ShandongAgricultural University, Tai an, 271018, Shandong, PR China. 2Shandong ProvincialKey Laboratory for Biology of Vegetable Diseases and Insect Pests, College of PlantProtection, Shandong Agricultural University, Tai an, 271018, Shandong, PR China.3National Engineering Laboratory for Efficient Utilization of Soil and FertilizerResources, College of Resources and Environment, Shandong AgriculturalUniversity, Tai an, Shandong 271018, China.*These authors contributed equally to this work

‡Author for correspondence (zchu@sdau.edu.cn)

B.Z., 0000-0002-5448-0092; H.L., 0000-0002-0537-930X; J.Q., 0000-0001-5302-3095; Z.C., 0000-0001-8320-7872

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addition to MAMPs, there are also many chemical compounds thatcan induce the plant defense response, including thiamine,riboflavin and rutin (Ahn et al., 2005; Dong and Beer, 2000;Yang et al., 2016). Flagellin 22 (flg22), a 22-amino-acid sequenceof the conserved N-terminal region of flagellin, is one of the mostwell-studied elicitors and can elicit a defense response after beingrecognized by FLS2, a leucine-rich repeat (LRR) receptor-likekinase (Gómez-Gómez and Boller, 2000). The flg22-inducedresponses include ion fluxes, oxidative burst, callose deposition,PR gene expression, ethylene production and seedling growthinhibition (Boller and Felix, 2009).The phytohormone ethylene plays a pivotal role in many aspects

of plant growth and development. In addition, it is also a majorregulator of plant responses to biotic and abiotic stresses (Pieterseet al., 2012). Pathogen attack and environmental stimuli such aswounding, flooding, heavy metal stress or ozone exposure caninduce ethylene production in plants (Keunen et al., 2016; Moederet al., 2002; Nie et al., 2002; Rao et al., 2002). The ethylenesynthesis pathway, which contains three steps from the precursormethionine, has been extensively researched in plants. In thefirst step, methionine is conversed to S-adenosylmethionine (SAM)by SAM synthase. Then, the conversion of SAM to1-aminocyclopropane-1-carboxylate (ACC) is catalyzed by ACCsynthase (ACS). Finally, ACC is oxidized to ethylene by ACCoxidase (ACO). The conversion of SAM to ACC by ACS is the rate-limiting step of the ethylene biosynthesis pathway. ACS genes play apivotal role in ethylene biosynthesis and their regulation has beenintensively studied. In Arabidopsis, the ACS gene family includes12 members, and only 8 of them are involved in the biosynthesis ofethylene (Yamagami et al., 2003). Tsuchisaka and Theologis (2004)reported that exogenous indole-3-acetic acid enhances theexpression of AtACS2, 4, 5, 6, 7, 8 and 11 in the root, and thatwounding of hypocotyl tissue induced the expression of AtACS2, 4,6, 7, 8 and 11. In Arabidopsis, AtACS2 and AtACS6 were stronglyupregulated upon challenge with Pst DC3000 or Botrytis cinerea(Guan et al., 2015; Han et al., 2010).In a previous report, we showed that Cu2+-elicited defense

responses in Arabidopsis were dependent on SA and ethylenesignaling pathways (Liu et al., 2015). However, whether and howCu2+ activates the production of ethylene remains unclear. In this

study, we report that Cu2+ rapidly induces the production ofethylene. In addition, Cu2+-induced ethylene production mainlyrelies on the AtACS8 gene.We demonstrate that the copper-responsecis-element (CuRE) plays an important role in Cu2+-activatedAtACS8 transcription. Together, our results suggest that AtACS8 isresponsible for the production of ethylene and Cu2+-elicited defenseresponses.

RESULTSCu2+ ions promote the production of ethylene in ArabidopsisIn a previous study, we showed that Cu2+ could elicit the defenseresponse in Arabidopsis, and that the Cu2+-activated defenserequired the SA and ethylene signaling pathways (Liu et al.,2015). Moreover, Arteca and Arteca (2007) reported that copperstress increased ethylene production in Arabidopsis. However, themolecular mechanism remains unknown. To further test therelationship between Cu2+ and ethylene production, we examinedthe production of ethylene in Arabidopsis plants treated with CuSO4

or MgSO4. We found that CuSO4-treated plants produced moreethylene than MgSO4-treated plants 24 h post treatment (hpt)(Fig. 1A). We also examined the transcription of the ethyleneresponsive factor 1 (ERF1) gene, which is a marker of the ethylenesignaling pathway (Alonso and Stepanova, 2004), to confirm theactivation of ethylene signaling by Cu2+. Compared with theMgSO4-treated plants, the transcript level of ERF1was significantlygreater in CuSO4-treated Arabidopsis (Fig. 1B). These resultsdemonstrate that Cu2+ promotes ethylene production and activatesethylene signaling in Arabidopsis.

Quantitative differences in gene expression in Arabidopsistreated with CuSO4To gain a better understanding of the role of Cu2+ in the plantdefense response, we generated the transcriptome profiles fromseedlings exposed to CuSO4 using RNA sequencing. There were sixsamples in total, with each treatment having two biologicalreplicates. More than 0.14 billion reads were generated, withapproximately 24 million reads from each sample (Table S3). InCuSO4-treated Arabidopsis plants, 2206 and 1009 genes showedgreater than twofold up- or down-regulation (P<0.05) at 2 hpt,whereas 2423 and 590 genes were up- or down-regulated at 24 hpt,

Fig. 1. Cu2+ ions increase ethylene levelsand upregulate the expression of ERF1 inArabidopsis. Arabidopsis seedlings weregrown on sterile agar medium. Ethylene wasmeasured with a gas chromatographequipped with a photoionization detector.(A) Cu2+ promotes ethylene production inArabidopsis. The levels of ethylene from theleaves of 20 seedlings treated with MgSO4 orCuSO4 were measured 24 hpt. Data are themean±s.d. from three independent biologicalreplicates. Asterisk indicates that a significantdifference between the mock control andCuSO4 treatment was detected at P<0.05level (t-test; P<0.05). (B) CuSO4 treatmentactivates ethylene signaling in Arabidopsis.Seedlings were treated with MgSO4 orCuSO4 for 24 h. Expression of the ERF1gene was quantified by qRT-PCR, usingArabidopsis AtActin2 as a control tonormalize expression levels. Data representthe mean±s.d. from three independentbiological replicates.

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respectively, compared with control plants (Fig. 2A; Table S4).Most of the Cu2+-regulated genes fell into very broad GO categoriessuch as metabolic process, cellular process, localization andresponse to stimulus (Fig. S1). In addition to a large group ofgenes with unknown functions (516 genes), a considerable numberof the genes upregulated at 2 hpt can be classified as being involvedin signal perception (113 genes encoding receptor-like kinases andresistant proteins), signal transduction (121 genes), transcriptionalregulation (79 genes) and ethylene signal (42 genes encodingethylene biosynthesis proteins and ethylene responsive factors)(Table S5).Among the rapidly (2 hpt) upregulated genes, 370 genes were

also identified as being upregulated by flagellin (Zipfel et al., 2004)and EF-Tu (Zipfel et al., 2006) (Fig. 2B; Table S4), such asWRKY29 (At4g23550), WRKY33 (At2g38470), WRKY53(At4g23810), ERF1 (At4g34410), ERF5 (At5g47230) and ERF6(At4g17490). To confirm Cu2+-mediated upregulated expression inthe RNA sequencing experiment, we performed quantitative real-time polymerase chain reaction (qRT-PCR) assays. The qRT-PCRresults showed that the expression patterns of all the selected genesin the plants treated with CuSO4 were similar to those in the RNAsequencing experiment (Fig. 2C; Fig. S2). GO-based functionalprofiling showed that 79.30% of the upregulated genes can be

classified into metabolic process, cellular process and response tostimulus (Fig. S1E).

Cu2+ ions activate the ethylene biosynthesis pathwayin ArabidopsisAmong the genes that are rapidly induced more than twofold at thetranscriptional level at 2 hpt, we detected the following genes:MTO3 (At3g17390), SAM1 (At1g02500), SAM2 (At4g01850),ACS2 (At1g01480), ACS6 (At4g11280), ACS7 (At4g26200), ACS8(At4g37770), ACS11 (At4g08040), ACO1 (At2g19590), ACO4(At1g05010) and ACO5 (At1g77330) (Fig. 3A). These genes havepreviously been shown to be involved in the biosynthesis ofethylene in Arabidopsis (Mao et al., 2015; Wang et al., 2002; Yangand Hoffman, 1984). We performed qRT-PCR using gene-specificprimers to confirm the expression patterns of these genes, and foundthat the expression of all the selected genes in Arabidopsis treatedwith CuSO4 was similar to that in the RNA sequencing experiment(Fig. 3B). These results suggest that Cu2+ induces the expression ofgenes in the ethylene biosynthesis pathway in Arabidopsis.

Cu2+ ions rapidly promotes ethylene productionThe genes involved in the ethylene biosynthesis pathway and ethylenesignaling pathway were upregulated at 2 hpt (Figs 2C and 3),

Fig. 2. Gene expression is regulated by Cu2+

ions in Arabidopsis. (A) Genes upregulated morethan twofold at 2 and 24 h after CuSO4 treatment.(B) The percentage of upregulated anddownregulated genes in different functionalcategories in CuSO4-treated Arabidopsis plants at2 hpt. (C) qRT-PCR profiles of six genes that areupregulated by flg22, elf26 and Cu2+, usingArabidopsis AtActin2 as a control to normalizeexpression levels (mean±s.d.; n≥3).

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suggesting that Cu2+ activates ethylene within 2 h. To confirm thishypothesis, we examined the production rate of ethylene inArabidopsis treated with CuSO4 or MgSO4 at different time points.We found that, at 0.25 hpt CuSO4- andMgSO4-treated plants showedsimilar ethylene production rates. However, CuSO4-treated plantsshowed a significantly faster rate of ethylene production thanMgSO4-treated plants at 0.5 hpt. In addition, the rate of ethyleneproduction peaked at 2 hpt (Fig. 4B). Compared with MgSO4-treated plants, the level of ethylene in CuSO4-treated plants was109% higher at 0.5 hpt. After 1- and 2-h treatments of wild-typeseedlings with CuSO4, the level of ethylene was 168% and 131%higher than that in MgSO4-treated seedlings, respectively (Fig. 4A).These results suggest that Cu2+ rapidly induces ethylene productionin Arabidopsis.To confirm that Cu2+ rapidly promotes ethylene production, we

examined the transcription of ERF1 in Arabidopsis at 2 and 24 hafter CuSO4 treatment. At 2 hpt, ERF1 gene expression wassignificantly induced by Cu2+ (Fig. 2C), suggesting that Cu2+

activates ethylene signaling within 2 h.

Copper ion-induced defense response and ethyleneproduction rely on AtACS8 geneThe heavy metal cadmium was recently shown to induce theproduction of ethylene in Arabidopsis, mainly via increasedtranscription of AtACS2 and AtACS6 (Schellingen et al., 2014).The transcription levels of AtACS2 and AtACS6 were also elevatedby CuSO4 treatment (Fig. 3). To test the role of AtACS2 and AtACS6in Cu2+-induced ethylene production, we studied the effects of Cu2+

on acs2-1/acs6-1 double knockout mutant plants. Compared withMgSO4-treated plants, CuSO4-treated wild-type and acs2-1/acs6-1mutant plants exhibited significantly increased ethylene emission at

2 hpt. In addition, no significant difference was observed in theethylene levels of wild-type and acs2-1/acs6-1 mutant plants.However, exposure to CuSO4 for 24 h led to significantly lowerethylene levels in the mutants than in the wild-type plants (Fig. 5A).Consistent with the increased ethylene emission, the transcriptionsof ERF1 and ERF5 could be induced with CuSO4 treatment in acs2-1/acs6-1 mutant plants at 2 hpt (Fig. 5B). The induction expressionlevel was attenuated in acs2-1/acs6-1 mutants compared with thewild-type plants (Fig. 5B). This result shows that the AtACS2 andAtACS6 genes are required for later (24 h) ethylene production, butnot early (2 h) ethylene production.

Our RNA sequencing and qRT-PCR results suggest that Cu2+

rapidly induces expression of the AtACS8 gene (Fig. 3). To verifythe role of the AtACS8 gene in Cu2+-induced ethylene production,ethylene emission was measured in wild-type and acs8 knockoutmutant plants after MgSO4 or CuSO4 treatments. At 2 hpt, nosignificant differences in ethylene level were observed betweenacs8 knockout mutant plants treated with MgSO4 and CuSO4

(Fig. 5C). By contrast, 24-h exposure to CuSO4 resulted in asignificantly higher level of ethylene than MgSO4 exposure in acs8mutant plants (Fig. 5C). We also examined the transcription ofERF1 andERF5 in acs8mutant plants. As shown in Fig. 5D, CuSO4

treatment completely lost the induction of ERF1 and ERF5 at 2 hpt.This result suggests that the rapid ethylene production induced byCu2+ relies on the AtACS8 gene. To confirm this result, weexamined ethylene production in acs1-1 acs2-1 acs6-1 acs4-1 acs5-2 acs9-1 acs7-1 acs11-1 octuple mutant plants after MgSO4 orCuSO4 treatments. The octuple mutant plants could produce moreethylene and expressed a higher level of the AtACS8 gene than wild-type plants. However, CuSO4 treatment resulted in similar ethylenelevels in the wild-type and octuple mutant plants (Fig. S3). The

Fig. 3. Expression of genes in the ethylene biosynthesis pathway after CuSO4 treatment. (A) Heatmap of the expression of genes in the ethylenebiosynthesis pathway in response to Cu2+. (B) qRT-PCR profiles of five upregulated ethylene biosynthesis genes induced by 2 h of CuSO4 treatment (mean±s.d.;n≥3). AtActin2 gene was used as a control to normalize expression levels.

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results suggest that AtACS8 is required for Cu2+-activated earlyethylene signaling.To determine whether the AtACS8 gene is required for the

Cu2+-mediated defense response, we assessed the effects of Cu2+ onprotection against Pst DC3000 in wild-type, acs2-1/acs6-1 doubleknockout mutant and acs8 knockout mutant plants. Comparedwith MgSO4 treatment, CuSO4 treatment resulted in a significant(P<0.01) decrease in the Pst DC3000 population in wild-typeand acs2-1/acs6-1 double knockout mutant plants. Meanwhile, inacs8 mutant plants, there were no significant (P<0.05) differencesin Pst DC3000 infection between acs8 knockout mutantplants treated with MgSO4 and CuSO4 (Fig. 5E). Taken together,our results strongly suggest that the AtACS8 gene plays an essentialrole in Cu2+-mediated early ethylene production and defenseresponses.

Identification of CuRE in the copper ion-inducible expressionof AtACS8To identify the mechanisms underlying Cu2+-induced AtACS8expression, we used the GFP reporter gene to analyze the AtACS8promoter. The GFP open reading frame (ORF) was inserteddownstream of the AtACS2, AtACS6 or AtACS8 promoter. Theconstructs were transiently expressed in Nicotiana benthamiana(NB) and analyzed for their responsiveness to CuSO4 treatment.GFP under the AtACS6 promoter showed a strong fluorescencesignal in leaves treated with MgSO4 and CuSO4. However, incontrast to MgSO4 treatment, CuSO4 treatment could activate GFPexpression driven by the AtACS2 and AtACS8 promoters. In

addition, the fluorescence in AtACS8-driven GFP was stronger thanAtACS2-driven GFP (Fig. S4). The promoter of AtACS8was chosenfor further analysis. Site-directed deletions of the AtACS8 promoterat -1155, -901 and -661 were generated (Fig. 6A). Compared withMgSO4 treatment, CuSO4 treatment activated GFP driven by the-1655 and -1155 promoters. However, when driven by the -901 and-661 promoters, theGFP gene was not induced by CuSO4 treatment(Fig. 6A and B). The above result suggests that the DNA fragmentfrom -902 to -1155 is required for Cu2+-induced expression of theAtACS8 gene. We performed in silico scanning for Cu2+-inducedelements using Jaspar (Mathelier et al., 2015) with a profile scorethreshold of 80%. The analysis revealed one CuRE located at -967to -957 in the promoter region (Fig. S5), indicating the candidatecis-element controlling Cu2+-induced AtACS8 expression. Then, weused site-directed promoter deletions at -967 and -956 to drive theGFP reporter gene and analyzed these constructs for theirresponsiveness to Cu2+ (Fig. 6C). Cu2+ induced GFP expressionwhen GFP was driven by the -967 promoter but not when GFP wasdriven by the -956 promoter, which only lacks the CuRE (Fig. 6Cand D). Using in silico scanning, we found that the promoters ofthose ethylene biosynthesis genes induced by CuSO4 treatmentcontain 3–9 (7.10±1.79) CuREs, which is significantly (P<0.05)more than that of 100 random genes (Fig. S6). We alsoconstructed CuRE-35S mini and the 35S mini to drive the GFPreporter gene. In addition, Cu2+ induced a GFP signal in leavestransiently expressing CuRE-35S mini: GFP, but not in leavesexpressing 35S mini: GFP (Fig. 6C,D). The function of CuRE inthe Cu2+-induced expression of AtACS8 was further confirmed in

Fig. 4. Cu2+ ions rapidly promote the biosynthesis ofethylene in Arabidopsis. Arabidopsis seedlings weregrown on sterile agar medium. Ethylene was measured in agas chromatograph equipped with a photoionizationdetector. (A) The accumulation of ethylene from the leaves ofArabidopsis seedlings treated with MgSO4 or CuSO4 at theindicated times. Data represent the mean±s.d. from threeindependent biological replicates. Asterisks indicate that asignificant difference between the mock control and CuSO4

treatment was detected at P<0.05 level (t-test; P<0.05).(B) Replot of the data in (A) as the average rates of ethyleneproduction. Error bars indicate s.d. (n=3).

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transgenic Arabidopsis plants. The constructs containing CuREsshowed strong GFP signal after CuSO4 treatment, whereasconstructs lacking CuREs showed weak GFP signal (Fig. 6E,F).The results suggest that CuREs are required for Cu2+ to regulatethe expression of gene AtACS8.

DISCUSSIONCu2+ ions act as an elicitor, inducing defense responsesin ArabidopsisPreviously, we have demonstrated that Cu2+ elicited defenseresponses and protected plants against pathogens (Liu et al.,

Fig. 5. See next page for legend.

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2015). CuSO4 treatment promotes ROS accumulation and callosedeposition, activates MAP kinase signaling and upregulates theexpression of PR genes (Liu et al., 2015). All these responsereactions elicited by Cu2+ ions imply that they act in a similar mannerto other MAMP molecules, such as flg22 and elf18 (Zipfel, 2008).About 9.5% of the genes upregulated by Cu2+, flg22 and elf18, wereassociated with a response to stimulus (Fig. S1). In this study, wehave demonstrated that Cu2+ also elicited early ethylene productionand activated ethylene signaling in Arabidopsis. Ethylene emissionis one of the early events in the Arabidopsis response to MAMPssuch as flg22 and elf18 (Zipfel et al., 2006). CuSO4 treatment rapidlyinduced the production of ethylene 30 min after treatment (Fig. 4).This is the second study to identify similarities between Cu2+ andMAMP molecules. Furthermore, 2 h after CuSO4 treatment,WRKY22, WRKY29, WRKY33, WRKY53 and WRKY55 wereupregulated (Fig. 2 and Table S4); the expression of these genes isalso induced by MAMPs such as flg22, elf18, Atpep1 and chitin(Wan et al., 2008; Yamaguchi et al., 2010; Zipfel et al., 2004, 2006).These five WRKY genes are involved in plant responses to bothbacterial and fungal pathogens (Asai et al., 2002; Deslandes et al.,2002; Zheng et al., 2006). This is especially true for WRKY22 andWRKY29, which are considered early defense markers of flg22 andfunction downstream of MPK3 and MPK6 (Asai et al., 2002). Weconclude that Cu2+ acts as an elicitor, inducing a MAMP-triggeredimmunity (MTI)-like response in Arabidopsis. This can explain, atleast partially, why Cu2+ has broad bactericide and fungicide effects.Although Cu2+ is similar to flg22 and elicits a defense response in

Arabidopsis, there are also many differences between the responseselicited by Cu2+ and flg22. Flg22 treatment rapidly induced theexpression of FLS2,MEKK1,MKK4 andMPK3, which are upstreamof WRKY22 and WRKY29 (Asai et al., 2002; Zipfel et al., 2004).These genes were demonstrated to be part of the MAP kinasesignaling cascade in the immune response induced by flg22 (Asaiet al., 2002). However, Cu2+ did not upregulate the expression of thesereceptors or MAP kinase genes (Table S4), suggesting thatArabidopsis perceives Cu2+ with different MAP kinase signalingpathways. The following genes are some of theMAPkinase genes thatare rapidly induced at the transcriptional level by Cu2+ but not flg22:MAPKKK5 (At5g66850), MEKK3 (At4g08470) and MAPKKK19(At5g67080). These genes are members of the MEKK family, andfew studies have reported their functions in response to abiotic andbiotic stresses. The functions of these threeMEKK familymembers inthe Cu2+-mediated defense response require further research.

Themechanism of copper ion-mediated ethylene productionThe phytohormone ethylene plays roles in plant growth,development, and the response to biotic and abiotic stresses.Various stress stimuli, such as pathogen infection, exposure to aheavy metal, wounding and ozone, have been reported to increaseethylene production in plants (Boller, 1991; O’Donnell et al., 1996;Keunen et al., 2016; Rao et al., 2002). Exposure to 5, 10, 25 or100 µM cadmium increased the level of ethylene in Arabidopsisafter 24 and 72 h (Schellingen et al., 2014). Copper-inducedincreases in ethylene production were also found in Arabidopsis(Arteca and Arteca, 2007) and wheat (Groppa et al., 2003).However, in another study, exposing Arabidopsis to 5 µM cadmiumfor 16 days resulted in decreased ethylene production (Carrió-Seguíet al., 2015). In addition, no significant changes in ethylene level weredetected for Arabidopsis seedlings grown in the presence of 25 or50 µMcopper for 9 days (Lequeux et al., 2010). These results suggestthat heavy metal-mediated ethylene production is dependent on theconcentration of metal, plant age and exposure time.

The amino acid methionine is the biological precursor ofethylene, and the conversion of methionine to SAM by SAMsynthase is the first step in ethylene biosynthesis (Yang andHoffman, 1984). The sam1/sam2 double mutants showedsignificantly lower ethylene levels than wild-type plants (Maoet al., 2015). In addition, SAM1 overexpression lines producedmore SAM and ethylene than Col-0 (Mao et al., 2015). These datasuggest that upregulation of SAM synthase genes increases the levelof ethylene. However, very few studies have reported the biotic orabiotic stress-mediated regulation of the transcription of SAMsynthase genes (Broekaert et al., 2006). Interestingly, CuSO4

treatment induced the expression of SAM1 (At1G02500), SAM2(At4G01850) and MTO3 (At3G17390) by more than twofold at2 hpt in our study (Fig. 2; Table S4).

ACOs catalyze the last step in ethylene biosynthesis, andmembers of the ACO gene families are induced by different bioticand abiotic stress stimuli. For example, wounding, flooding, heavymetal stress and ozone exposure were shown to enhance ACO geneexpression in potato (Nie et al., 2002), tomato (Moeder et al., 2002;Nakatsuka et al., 1998) and Arabidopsis (Herbette et al., 2006;Schellingen et al., 2014). However, in a microarray experiment,none of the ACO genes were induced by copper treatment, althoughenhanced production of ethylene was found (Weber et al., 2006). Inour study, AtACO1, 2, 4 and 5 were induced by Cu2+ (Fig. 3),suggesting that these ACO genes are also regulated in Cu2+-inducedethylene production.

In addition to SAM synthase genes and ACO genes, we alsofound that ACS genes were upregulated by CuSO4 treatment (Fig. 2;Table S4). The ACS-catalyzed conversion of SAM to ACC is therate-limiting step in the ethylene biosynthesis pathway. Thetranscriptional regulation of ACS genes to promote ethyleneproduction has been thoroughly studied. Using beta-glucuronidaseand GFP as reporters, Tsuchisaka and Theologis (2004) examinedthe spatial and temporal expression patterns of ACS genes inArabidopsis and showed that different abiotic stress stimuli result indifferent patterns of expression among the various ACS gene familymembers. Peng et al. (2005) found that hypoxia stress specificallyinduced the expression ofAtACS2,AtACS6,AtACS7 andAtACS9. In arecent study, Schellingen et al. (2014) noticed that cadmium-inducedethylene production relied on AtACS2 and AtACS6. Meanwhile, atransgenic Arabidopsis line with enhanced glutathione contentshowed higher levels of ACC and ethylene than wild-type plants.In addition, glutathione induced the transcription of AtACS2 andAtACS6, which is dependent on WRKY33 (Datta et al., 2015). Guan

Fig. 5. Ethylene emission and the defense response to Pst DC3000 inacs8 and acs2-1/acs6-1 mutant plants. (A) Comparison of the ethyleneproduction rates in 2-week-old wild-type or acs2-1/acs6-1 mutant plantsexposed to CuSO4 or MgSO4 for 2 or 24 h. Data are shown as the mean±s.d.from three independent biological replicates. (B) Comparison of thetranscriptions of ERF1 and ERF5 in 4-week-old wild-type or acs2-1/acs6-1mutant plants exposed to CuSO4 or MgSO4 for 2 or 24 h. Wild-type Col-0 andacs2-1/acs6-1mutants were treated with MgSO4 or CuSO4. Expression of theERF1 and ERF5 genes were quantified by qRT-PCR, using ArabidopsisAtActin2 as a control to normalize expression levels. Data represent the mean±s.d. from three independent biological replicates. (C) Comparison of theethylene production rates in 2-week-old wild-type or acs8 mutant plantsexposed to CuSO4 or MgSO4 for 2 or 24 h. Data are shown as the mean±s.d.from three independent biological replicates. (D) The transcriptions of ERF1andERF5 in wild-type and acs8mutant plants exposed to CuSO4 orMgSO4 for2 or 24 h. (E) AtACS8 is required in the Cu2+-elicited defense response. Wild-type Col-0 and acs8 mutants were treated with MgSO4 or CuSO4 4 h beforeinfiltration of Pst DC3000, and the bacterial population in the leaf wasmeasured 3 days after inoculation. Data represent the mean±s.d. from sixindependent biological replicates.

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et al., (2015) reported that acs2-1/acs6-1 double mutant plantsproduce less ethylene and were more susceptible to pathogens. In ourstudy, RNA sequencing and qRT-PCR revealed that Cu2+ induced

the expression of AtACS2, AtACS6, AtACS7, AtACS8 and AtACS11but suppressed the expression of AtACS4 (Fig. 3). CuSO4 treatmentenhanced the fluorescence signal in leaves expressing GFP driven by

Fig. 6. Induction of GFP driven by AtACS8 promoter deletion constructs. (A) Scheme of the different promoter deletions. The different deleted promoterfragments were cloned upstream of the GFP gene. (B) Agrobacterium GV3101 containing the GFP gene driven by AtACS8-deleted promoters were inoculatedinto N. benthamiana leaves. Three days later, the plants were treated with CuSO4 or MgSO4. Plants were photographed at 12 hpt. (C) Schematic diagram ofsite-directed promoter deletions at -967 and -956, CuRE: 35S mini and 35S mini. The blue box indicates the CuRE element. (D) Agrobacterium GV3101containing the GFP gene driven by the AtACS8 promoter with deletions at -967 and -956, CuRE: 35S mini or 35S mini were inoculated into N. benthamianaleaves. The effects of Cu2+ were measured as in (B). (E) Induction of GFP driven by AtACS8-deleted promoters were identified in transgenic Arabidopsis.The plants were treated with CuSO4 or MgSO4 and photographed at 12 hpt. (F) Induction of GFP driven by CuRE: 35S mini or 35S mini were identified intransgenic Arabidopsis. The plants were treated with CuSO4 or MgSO4 and photographed at 12 hpt. RLU, relative light unit.

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AtACS2, AtACS6 or AtACS8 promoters (Fig. S4). However, the acs2-1/acs6-1 double mutant showed similar levels of ethylene to wild-type plants 2 h after MgSO4 or CuSO4 treatment (Fig. 5A).Consistent with this, Cu2+ ions were still able to protect acs2-1/acs6-1 mutants from Pst DC3000 (Fig. 5C). At the same time, thelevels of ethylene and the immune response in acs8 mutant plantswere significantly lower than those of wild-type plants after CuSO4

treatment (Fig. 5B and C). Therefore, it is possible that AtACS8 is themain gene regulating Cu2+-induced early ethylene production anddefense responses. Interestingly, the expression of AtACS8 iscontrolled by the circadian clock, which is tightly correlated toethylene production (Thain et al., 2004; Vandenbussche et al., 2003).It was further concluded that the enhanced transcription level ofAtACS8 is crucial for the production of ethylene in Arabidopsis.The Cu2+-induced expression of AtACS8 is dependent on the

copper-response cis-element, termed CuRE (Fig. 6). This cis-elementwas first found in the promoter of the CUP1 gene, which codes for ametallothionein protein that protects yeast against heavy metaltoxicity. Cu2+ activates the expression of CUP1 through the CUP2transcription factor protein (Welch et al., 1989). The CUP2 protein isa copper sensor that contains a copper fist, which has the function ofbinding Cu2+ and DNA (Buchman et al., 1989). The copper sensor inplants is still unknown, and no similar CUP2 protein had been foundin plants. It is possible that Cu2+ binds to a novel transcription factorcontaining a copper fist domain to regulate expression of the AtACS8gene. Completion of a screen to identify the CuRE-binding proteinwill answer this question, which needs further research.

MATERIALS AND METHODSBacterial strains and plasmidsThe bacterial strains and plasmids used in this study are described inTable S1. The Escherichia coli strains were cultured on Luria-Bertani (LB)medium at 37°C. The Pseudomonas syringae pv. tomato strain DC3000 wascultured on King’s B medium containing 50 μg ml−1 rifampicin at 28°C.The Agrobacterium tumefaciens strain GV3101 was cultured on LBmedium containing 50 μg ml−1 rifampicin at 28°C.

For promoter deletion analysis, 1655-bp, 1155-bp, 901-bp, 967-bp, 956-bp and 661-bp promoter fragments located immediately upstream of theAtACS8 start codon were cloned upstream of theGFP gene in the pCXGFP-P binary vector (Chen et al., 2009). The CuRE-35S mini promoter and 35Smini promoter were also inserted into the pCXGFP-P plasmid. Thesefragments were amplified using primers as described in the supplementarymaterial (Table S2). All plasmids were validated by sequencing.

Plant materials and chemical treatmentsSeeds of Arabidopsis (Arabidopsis thaliana ecotype Col-0), acs2-1/acs6-1(CS16581) and acs8 (SALK_006628) were obtained from The ArabidopsisInformation Resource (TAIR). Arabidopsis plants were grown in nutrientsubstrates in a growth chamber with 12-h days (at 23°C and 60–75% relativehumidity) and 12-h nights (at 21°C and 60–75% relative humidity).According to previous reports, 100 μM CuSO4 can trigger the defenseresponse but is not toxic to Pst DC3000 (Liu et al., 2015). Four- to five-week-old plants were sprayed with 100 μM (or other concentrations) CuSO4

or MgSO4 supplemented with 0.05% (v/v) Tween 20 for 4 h and thenpathogen inoculation (Liu et al., 2015), unless stated otherwise. Two-week-old plants were used for ethylene measurement after being sprayed with100 μM CuSO4 or MgSO4 supplemented with 0.05% (v/v) Tween 20 fordifferent time periods.

Bacterial growth assayA bacterial growth assay was performed in Arabidopsis plants at 0 and3 days after inoculation. The concentration of Pst DC3000 suspension inbuffer containing 10 mM MgCl2 was adjusted to 106 CFU ml−1 andinfiltrated into 4- to 5-week-old Arabidopsis plants using a needlelesssyringe. Leaves were harvested, surface sterilized in 70% ethanol solution

for 1 min and then rinsed in sterile distilled water for 1 min. Leaf disks wereexcised from the leaves of three independent plants with a 0.5 cm2 corkborer and were ground in 1.5-ml microfuge tubes with 100 μl sterile distilledwater. The samples were serially diluted (1:10) and plated on King’s Bmedium containing 50 μg ml−1 rifampicin at 28°C for colony counting.The experiment was repeated three times. The means were compared using at-test. The standard error and t-test results were recorded.

RNA extraction, reverse transcription-PCR, quantitative real-time PCRArabidopsis plants were treated with CuSO4 or MgSO4 and whole plantswere collected to extract total RNAwith TRIzol Reagent (Invitrogen, USA).Using a HiFi Script cDNA Synthesis Kit (CWBIO, China), 1 μg of totalRNA was used to synthesize first-strand cDNA as described in themanufacturer’s instructions. qRT-PCRwas performed with a QuantStudio 6Flex Real-Time PCR System (Life Technologies, USA) using UltraSYBRMixture (CWBIO, China) as described in the manufacturer’s instructions.The primers used in qRT-PCR are described in the supplementary material(Table S2). The expression levels of genes were analyzed using the 2−⊿⊿Ct

analysis method or normalized with AtActin2 (At3g18780).

RNA-seq and data analysisTwo biological replicates of RNA samples were collected from two-week-old wild-type Arabidopsis plants treated with 100 μM CuSO4 for 0, 2 or24 h. As previously reported (Ju et al., 2017), six libraries were constructedand sequencing performed with a BGISEQ-500 by the Beijing GenomicInstitution (www.genomics.org.cn, BGI, Shenzhen, China). Clean tags weremapped to the reference genome and genes that are available in theArabidopsis TAIR10.2 reference genome. For gene expression analysis, thematched reads were calculated and then normalized to RPKM (reads perkilobase of transcript per million mapped reads) using RESM software. Thesignificance of differential gene expression was confirmed with the BGIbioinformatics service using the combination of the absolute value of log2-Ratio≥1 and P≤0.05 in this research.

Quantitation of ethylene biosynthesis ratesEthylene biosynthesis rates were measured by gas chromatography asdescribed previously (Spollen et al., 2000). Briefly, 2-week-old seedlingswere treated with CuSO4 or MgSO4, and leaves from 20 plants weredetached and placed in a 20-ml penicillin bottle. At various times afterCuSO4 or MgSO4 treatment, a 1-ml gas sample was injected into a cold trap.Ethylene was quantified using a gas chromatograph equipped with aphotoionization detector.

Promoter analysis in N. benthamianaAgrobacterium GV3101 containing the pCXGFP-P binary vector with theGFP gene driven by different promoter fragments were resuspended withAgro-infiltration buffer (10 mM MgCl2, 10 mM MES, pH 5.6, 200 mMacetosyringone), and the OD600 adjusted to 0.5. Bacterial suspensions wereinfiltrated into 4- to 6-week-old N. benthamiana leaves with a needlelesssyringe. Two or three days later, whole plants were sprayed with 100 μMCuSO4 orMgSO4 supplemented with 0.05% (v/v) Tween 20 for 12 h. Plantswere then observed using an M165 FC fluorescence stereomicroscope(Leica, Germany).

Production of transgenic Arabidopsis and promoter analysisStable transgenic Arabidopsis plants expressing the GFP gene driven bydifferent promoter fragments were generated by floral dipping of Col-0plants. Multiple positive T1 lines were selected for production of the T2

generation for experiments.The transgenic plants were sprayed with 100 μMCuSO4 or MgSO4 supplemented with 0.05% (v/v) Tween 20 for 12 h andthen observed using a LSM880NLO confocal microscope (Zeiss, Germany).

AcknowledgementsWe are grateful to TAIR for providing seeds of Arabidopsis mutants.

Competing interestsThe authors declare no competing or financial interests.

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Author contributionsConceptualization: H.L., X.D., Z.C.; Methodology: B.Z., H.L., J.Q., M.Z., Z.C.;Validation: B.Z.; Formal analysis: H.L.; Investigation: B.Z., H.L.; Resources: J.Q.,M.Z.; Data curation: B.Z.; Writing - original draft: H.L.; Writing - review & editing:Z.C.; Supervision: X.D., Z.C.; Project administration: X.D., Z.C.; Funding acquisition:X.D., Z.C., H.L.

FundingThis study was supported by the Major Application Technology Innovation Project ofShandong Province (2016) and the Natural Science Foundation of ShandongProvince, China (ZR2014CQ044, ZR2015CM004). Z.C. and X.D. were supported bythe Funds of Shandong “Double Tops” Program (2016). H.L. was funded by theChina Postdoctoral Science Foundation (2017M612310) and X.D. was funded bythe Taishan Industrial Experts Programme (20150621) of Shandong Province.

Data availabilityRNA-seq original sequence data were submitted to the database of the NCBISequence Read Archive (https://trace.ncbi.nlm.nih.gov/Traces/sra_sub/sub.cgi?acc=SRP092377&focus=SRP092377&from=list&action=show:STUDY) under theaccession number SRP092377.

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.202424.supplemental

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