ABSTRACT

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

INTRODUCTION

Cu2+ ions are a key constituent of cytochrome c oxidase and are essential to bacteria and eukaryotes. In plants, Cu2+ ions are cofactors for many enzymes, such as plastocyanin, superoxide dismutase and polyphenol oxidase, and participate in photosynthetic electron transport, oxidative stress responses and oxidative phosphorylation. Thus, Cu2+ ions play roles in the vegetative growth, development and reproduction of plants (Marschner, 1995; Peñarrubia et al., 2010). A previous study reported that Cu2+ associated with the ethylene-binding domain is required for ETR1 (ethylene response 1) to bind the gaseous hormone ethylene. This suggests that Cu2+ ions also play a role in hormone perception (Rodríguez et al., 1999). Under copper deficiency, plants show a twisted or malformed phenotype in young leaves (Marschner, 1995). However, excess copper exhibits heavy metal toxicity and causes cell death in plants. Therefore, plants tightly balance the level of Cu2+, allowing sufficient metal co-factor to be delivered to target proteins while avoiding toxicity. Plants have evolved specific membrane transporters, such as the copper transporter (COPT) family and P-type ATPase-family copper transporters, to deliver copper intra- and intercellularly (Hirayama et al., 1999; Sancenón et al., 2003). Copper deficiency upregulates expression of the transcription factor SQUAMOSA promoter-binding protein-like 7 (SPL7) in Arabidopsis. SPL7 then targets the cis-element of the GTAC motif present in promoters of some members of the COPT gene family to increase the absorption of Cu2+ (Peñarrubia et al., 2010; Yamasaki et al., 2009). However, excess copper suppresses the expression of COPT genes to reduce the concentration of copper in plants (Sancenón et al., 2003; Yuan et al., 2011). In yeast, the CUP2 gene codes for a copper-binding transcription factor protein containing a copper fist. In elevated copper concentrations, CUP2 binds Cu2+ and activates transcription of the metallothionein genes CUP1 and CRS5 (Welch et al., 1989).

Cu2+ is also a key constituent of many bactericides and fungicides. Copper-based bactericides and fungicides are used extensively in agriculture. Bordeaux mixture, a mixture of copper sulfate (CuSO4) and slaked lime, has been widely used in vineyards, fruit farms and gardens to protect plants from infestations of downy mildew, powdery mildew and other fungi since 1854. The antimicrobial mechanisms of Cu2+ were assumed to be associated with its heavy metal toxicity. Cu2+ can suppress microbial growth by denaturing nucleic acids, inhibiting protein activity and changing plasma membrane permeabilization (Borkow and Gabbay, 2004). Interestingly, fewer microbes are reported to be resistant to copper-based bactericides and fungicides, although some microbes are tolerant to copper. For example, Pseudomonas syringae pv. tomato (Pst) isolated from tomato fields is tolerant to copper, dependent on three copper-resistant operon genes (Cha and Cooksey, 1991). However, copper compounds are still effective in controlling plant diseases. Thus, we hypothesize that Cu2+ not only inhibits bacterial and fungal growth but also elicits defense responses in plants. In our previous study, we found that Cu2+ promoted reactive oxygen species (ROS) accumulation and callose deposition, induced the expression of pathogenesis-related (PR) genes, activated MAP kinase signaling and elicited defense against Pst DC3000 in Arabidopsis (Liu et al., 2015). In addition, the Cu2+-mediated defense response is dependent on salicylic acid (SA) and ethylene signaling pathways. Thus, we assumed that Cu2+ acts as an elicitor and triggers immune responses in plants.

In the past decades, many elicitors have been shown to induce the defense response and protect plants from pathogen infection. Among these elicitors, microbe-associated molecular patterns (MAMPs) are the best studied. A number of MAMPs, such as chitin, peptidoglycan, lipopolysaccharide, flagellin and the elongation factor thermo unstable (ER-Tu), have been identified as inducing the immune response in plants (Felix et al., 1993, 1999; Gust et al., 2007; Kunze et al., 2004; Meziane et al., 2005). In addition to MAMPs, there are also many chemical compounds that can 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 sequence of the conserved N-terminal region of flagellin, is one of the most well-studied elicitors and can elicit a defense response after being recognized by FLS2, a leucine-rich repeat (LRR) receptor-like kinase (Gómez-Gómez and Boller, 2000). The flg22-induced responses include ion fluxes, oxidative burst, callose deposition, PR gene expression, ethylene production and seedling growth inhibition (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 major regulator of plant responses to biotic and abiotic stresses (Pieterse et al., 2012). Pathogen attack and environmental stimuli such as wounding, flooding, heavy metal stress or ozone exposure can induce ethylene production in plants (Keunen et al., 2016; Moeder et al., 2002; Nie et al., 2002; Rao et al., 2002). The ethylene synthesis pathway, which contains three steps from the precursor methionine, has been extensively researched in plants. In the first step, methionine is conversed to S-adenosylmethionine (SAM) by SAM synthase. Then, the conversion of SAM to 1-aminocyclopropane-1-carboxylate (ACC) is catalyzed by ACC synthase (ACS). Finally, ACC is oxidized to ethylene by ACC oxidase (ACO). The conversion of SAM to ACC by ACS is the rate-limiting step of the ethylene biosynthesis pathway. ACS genes play a pivotal role in ethylene biosynthesis and their regulation has been intensively studied. In Arabidopsis, the ACS gene family includes 12 members, and only 8 of them are involved in the biosynthesis of ethylene (Yamagami et al., 2003). Tsuchisaka and Theologis (2004) reported that exogenous indole-3-acetic acid enhances the expression of AtACS2, 4, 5, 6, 7, 8 and 11 in the root, and that wounding of hypocotyl tissue induced the expression of AtACS2, 4, 6, 7, 8 and 11. In Arabidopsis, AtACS2 and AtACS6 were strongly upregulated 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 ethylene signaling pathways (Liu et al., 2015). However, whether and how Cu2+ activates the production of ethylene remains unclear. In this study, we report that Cu2+ rapidly induces the production of ethylene. In addition, Cu2+-induced ethylene production mainly relies on the AtACS8 gene. We demonstrate that the copper-response cis-element (CuRE) plays an important role in Cu2+-activated AtACS8 transcription. Together, our results suggest that AtACS8 is responsible for the production of ethylene and Cu2+-elicited defense responses.

RESULTS

Cu2+ ions promote the production of ethylene in Arabidopsis

In a previous study, we showed that Cu2+ could elicit the defense response in Arabidopsis, and that the Cu2+-activated defense required the SA and ethylene signaling pathways (Liu et al., 2015). Moreover, Arteca and Arteca (2007) reported that copper stress increased ethylene production in Arabidopsis. However, the molecular mechanism remains unknown. To further test the relationship between Cu2+ and ethylene production, we examined the production of ethylene in Arabidopsis plants treated with CuSO4 or MgSO4. We found that CuSO4-treated plants produced more ethylene than MgSO4-treated plants 24 h post treatment (hpt) (Fig. 1A). We also examined the transcription of the ethylene responsive factor 1 (ERF1) gene, which is a marker of the ethylene signaling pathway (Alonso and Stepanova, 2004), to confirm the activation of ethylene signaling by Cu2+. Compared with the MgSO4-treated plants, the transcript level of ERF1 was significantly greater in CuSO4-treated Arabidopsis (Fig. 1B). These results demonstrate that Cu2+ promotes ethylene production and activates ethylene signaling in Arabidopsis.

Fig. 1.

Cu2+ ions increase ethylene levels and upregulate the expression of ERF1 in Arabidopsis.Arabidopsis seedlings were grown on sterile agar medium. Ethylene was measured with a gas chromatograph equipped with a photoionization detector. (A) Cu2+ promotes ethylene production in Arabidopsis. The levels of ethylene from the leaves of 20 seedlings treated with MgSO4 or CuSO4 were measured 24 hpt. Data are the mean±s.d. from three independent biological replicates. Asterisk indicates that a significant difference between the mock control and CuSO4 treatment was detected at P<0.05 level (t-test; P<0.05). (B) CuSO4 treatment activates ethylene signaling in Arabidopsis. Seedlings were treated with MgSO4 or CuSO4 for 24 h. Expression of the ERF1 gene was quantified by qRT-PCR, using Arabidopsis AtActin2 as a control to normalize expression levels. Data represent the mean±s.d. from three independent biological replicates.

Fig. 1.

Cu2+ ions increase ethylene levels and upregulate the expression of ERF1 in Arabidopsis.Arabidopsis seedlings were grown on sterile agar medium. Ethylene was measured with a gas chromatograph equipped with a photoionization detector. (A) Cu2+ promotes ethylene production in Arabidopsis. The levels of ethylene from the leaves of 20 seedlings treated with MgSO4 or CuSO4 were measured 24 hpt. Data are the mean±s.d. from three independent biological replicates. Asterisk indicates that a significant difference between the mock control and CuSO4 treatment was detected at P<0.05 level (t-test; P<0.05). (B) CuSO4 treatment activates ethylene signaling in Arabidopsis. Seedlings were treated with MgSO4 or CuSO4 for 24 h. Expression of the ERF1 gene was quantified by qRT-PCR, using Arabidopsis AtActin2 as a control to normalize expression levels. Data represent the mean±s.d. from three independent biological replicates.

Quantitative differences in gene expression in Arabidopsis treated with CuSO4

To gain a better understanding of the role of Cu2+ in the plant defense response, we generated the transcriptome profiles from seedlings exposed to CuSO4 using RNA sequencing. There were six samples in total, with each treatment having two biological replicates. More than 0.14 billion reads were generated, with approximately 24 million reads from each sample (Table S3). In CuSO4-treated Arabidopsis plants, 2206 and 1009 genes showed greater 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, respectively, compared with control plants (Fig. 2A; Table S4). Most of the Cu2+-regulated genes fell into very broad GO categories such as metabolic process, cellular process, localization and response to stimulus (Fig. S1). In addition to a large group of genes with unknown functions (516 genes), a considerable number of the genes upregulated at 2 hpt can be classified as being involved in signal perception (113 genes encoding receptor-like kinases and resistant proteins), signal transduction (121 genes), transcriptional regulation (79 genes) and ethylene signal (42 genes encoding ethylene biosynthesis proteins and ethylene responsive factors) (Table S5).

Fig. 2.

Gene expression is regulated by Cu2+ ions in Arabidopsis. (A) Genes upregulated more than twofold at 2 and 24 h after CuSO4 treatment. (B) The percentage of upregulated and downregulated genes in different functional categories in CuSO4-treated Arabidopsis plants at 2 hpt. (C) qRT-PCR profiles of six genes that are upregulated by flg22, elf26 and Cu2+, using Arabidopsis AtActin2 as a control to normalize expression levels (mean±s.d.; n≥3).

Fig. 2.

Gene expression is regulated by Cu2+ ions in Arabidopsis. (A) Genes upregulated more than twofold at 2 and 24 h after CuSO4 treatment. (B) The percentage of upregulated and downregulated genes in different functional categories in CuSO4-treated Arabidopsis plants at 2 hpt. (C) qRT-PCR profiles of six genes that are upregulated by flg22, elf26 and Cu2+, using Arabidopsis AtActin2 as a control to normalize expression levels (mean±s.d.; n≥3).

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 as WRKY29 (At4g23550), WRKY33 (At2g38470), WRKY53 (At4g23810), ERF1 (At4g34410), ERF5 (At5g47230) and ERF6 (At4g17490). To confirm Cu2+-mediated upregulated expression in the RNA sequencing experiment, we performed quantitative real-time polymerase chain reaction (qRT-PCR) assays. The qRT-PCR results showed that the expression patterns of all the selected genes in the plants treated with CuSO4 were similar to those in the RNA sequencing experiment (Fig. 2C; Fig. S2). GO-based functional profiling showed that 79.30% of the upregulated genes can be classified into metabolic process, cellular process and response to stimulus (Fig. S1E).

Cu2+ ions activate the ethylene biosynthesis pathway in Arabidopsis

Among the genes that are rapidly induced more than twofold at the transcriptional 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 have previously been shown to be involved in the biosynthesis of ethylene in Arabidopsis (Mao et al., 2015; Wang et al., 2002; Yang and Hoffman, 1984). We performed qRT-PCR using gene-specific primers to confirm the expression patterns of these genes, and found that the expression of all the selected genes in Arabidopsis treated with CuSO4 was similar to that in the RNA sequencing experiment (Fig. 3B). These results suggest that Cu2+ induces the expression of genes in the ethylene biosynthesis pathway in Arabidopsis.

Fig. 3.

Expression of genes in the ethylene biosynthesis pathway after CuSO4 treatment. (A) Heatmap of the expression of genes in the ethylene biosynthesis 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.

Fig. 3.

Expression of genes in the ethylene biosynthesis pathway after CuSO4 treatment. (A) Heatmap of the expression of genes in the ethylene biosynthesis 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.

Cu2+ ions rapidly promotes ethylene production

The genes involved in the ethylene biosynthesis pathway and ethylene signaling pathway were upregulated at 2 hpt (Figs 2C and 3), suggesting that Cu2+ activates ethylene within 2 h. To confirm this hypothesis, we examined the production rate of ethylene in Arabidopsis treated with CuSO4 or MgSO4 at different time points. We found that, at 0.25 hpt CuSO4- and MgSO4-treated plants showed similar ethylene production rates. However, CuSO4-treated plants showed a significantly faster rate of ethylene production than MgSO4-treated plants at 0.5 hpt. In addition, the rate of ethylene production peaked at 2 hpt (Fig. 4B). Compared with MgSO4-treated plants, the level of ethylene in CuSO4-treated plants was 109% higher at 0.5 hpt. After 1- and 2-h treatments of wild-type seedlings 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 production in Arabidopsis.

Fig. 4.

Cu2+ ions rapidly promote the biosynthesis of ethylene in Arabidopsis.Arabidopsis seedlings were grown on sterile agar medium. Ethylene was measured in a gas chromatograph equipped with a photoionization detector. (A) The accumulation of ethylene from the leaves of Arabidopsis seedlings treated with MgSO4 or CuSO4 at the indicated times. Data represent the mean±s.d. from three independent biological replicates. Asterisks indicate that a significant 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 ethylene production. Error bars indicate s.d. (n=3).

Fig. 4.

Cu2+ ions rapidly promote the biosynthesis of ethylene in Arabidopsis.Arabidopsis seedlings were grown on sterile agar medium. Ethylene was measured in a gas chromatograph equipped with a photoionization detector. (A) The accumulation of ethylene from the leaves of Arabidopsis seedlings treated with MgSO4 or CuSO4 at the indicated times. Data represent the mean±s.d. from three independent biological replicates. Asterisks indicate that a significant 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 ethylene production. Error bars indicate s.d. (n=3).

To confirm that Cu2+ rapidly promotes ethylene production, we examined the transcription of ERF1 in Arabidopsis at 2 and 24 h after CuSO4 treatment. At 2 hpt, ERF1 gene expression was significantly induced by Cu2+ (Fig. 2C), suggesting that Cu2+ activates ethylene signaling within 2 h.

Copper ion-induced defense response and ethylene production rely on AtACS8 gene

The heavy metal cadmium was recently shown to induce the production of ethylene in Arabidopsis, mainly via increased transcription of AtACS2 and AtACS6 (Schellingen et al., 2014). The transcription levels of AtACS2 and AtACS6 were also elevated by CuSO4 treatment (Fig. 3). To test the role of AtACS2 and AtACS6 in Cu2+-induced ethylene production, we studied the effects of Cu2+ on acs2-1/acs6-1 double knockout mutant plants. Compared with MgSO4-treated plants, CuSO4-treated wild-type and acs2-1/acs6-1 mutant plants exhibited significantly increased ethylene emission at 2 hpt. In addition, no significant difference was observed in the ethylene levels of wild-type and acs2-1/acs6-1 mutant plants. However, exposure to CuSO4 for 24 h led to significantly lower ethylene levels in the mutants than in the wild-type plants (Fig. 5A). Consistent with the increased ethylene emission, the transcriptions of ERF1 and ERF5 could be induced with CuSO4 treatment in acs2-1/acs6-1 mutant plants at 2 hpt (Fig. 5B). The induction expression level was attenuated in acs2-1/acs6-1 mutants compared with the wild-type plants (Fig. 5B). This result shows that the AtACS2 and AtACS6 genes are required for later (24 h) ethylene production, but not early (2 h) ethylene production.

Fig. 5.

Ethylene emission and the defense response to Pst DC3000 in acs8 and acs2-1/acs6-1 mutant plants. (A) Comparison of the ethylene production rates in 2-week-old wild-type or acs2-1/acs6-1 mutant plants exposed 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 the transcriptions of ERF1 and ERF5 in 4-week-old wild-type or acs2-1/acs6-1 mutant plants exposed to CuSO4 or MgSO4 for 2 or 24 h. Wild-type Col-0 and acs2-1/acs6-1 mutants were treated with MgSO4 or CuSO4. Expression of the ERF1 and ERF5 genes were quantified by qRT-PCR, using Arabidopsis AtActin2 as a control to normalize expression levels. Data represent the mean±s.d. from three independent biological replicates. (C) Comparison of the ethylene production rates in 2-week-old wild-type or acs8 mutant plants exposed 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 ERF1 and ERF5 in wild-type and acs8 mutant plants exposed to CuSO4 or MgSO4 for 2 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 before infiltration of Pst DC3000, and the bacterial population in the leaf was measured 3 days after inoculation. Data represent the mean±s.d. from six independent biological replicates.

Fig. 5.

Ethylene emission and the defense response to Pst DC3000 in acs8 and acs2-1/acs6-1 mutant plants. (A) Comparison of the ethylene production rates in 2-week-old wild-type or acs2-1/acs6-1 mutant plants exposed 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 the transcriptions of ERF1 and ERF5 in 4-week-old wild-type or acs2-1/acs6-1 mutant plants exposed to CuSO4 or MgSO4 for 2 or 24 h. Wild-type Col-0 and acs2-1/acs6-1 mutants were treated with MgSO4 or CuSO4. Expression of the ERF1 and ERF5 genes were quantified by qRT-PCR, using Arabidopsis AtActin2 as a control to normalize expression levels. Data represent the mean±s.d. from three independent biological replicates. (C) Comparison of the ethylene production rates in 2-week-old wild-type or acs8 mutant plants exposed 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 ERF1 and ERF5 in wild-type and acs8 mutant plants exposed to CuSO4 or MgSO4 for 2 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 before infiltration of Pst DC3000, and the bacterial population in the leaf was measured 3 days after inoculation. Data represent the mean±s.d. from six independent biological replicates.

Our RNA sequencing and qRT-PCR results suggest that Cu2+ rapidly induces expression of the AtACS8 gene (Fig. 3). To verify the role of the AtACS8 gene in Cu2+-induced ethylene production, ethylene emission was measured in wild-type and acs8 knockout mutant plants after MgSO4 or CuSO4 treatments. At 2 hpt, no significant differences in ethylene level were observed between acs8 knockout mutant plants treated with MgSO4 and CuSO4 (Fig. 5C). By contrast, 24-h exposure to CuSO4 resulted in a significantly higher level of ethylene than MgSO4 exposure in acs8 mutant plants (Fig. 5C). We also examined the transcription of ERF1 and ERF5 in acs8 mutant 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 by Cu2+ relies on the AtACS8 gene. To confirm this result, we examined ethylene production in acs1-1 acs2-1 acs6-1 acs4-1 acs5-2 acs9-1 acs7-1 acs11-1 octuple mutant plants after MgSO4 or CuSO4 treatments. The octuple mutant plants could produce more ethylene and expressed a higher level of the AtACS8 gene than wild-type plants. However, CuSO4 treatment resulted in similar ethylene levels in the wild-type and octuple mutant plants (Fig. S3). The results suggest that AtACS8 is required for Cu2+-activated early ethylene signaling.

To determine whether the AtACS8 gene is required for the Cu2+-mediated defense response, we assessed the effects of Cu2+ on protection against Pst DC3000 in wild-type, acs2-1/acs6-1 double knockout mutant and acs8 knockout mutant plants. Compared with MgSO4 treatment, CuSO4 treatment resulted in a significant (P<0.01) decrease in the Pst DC3000 population in wild-type and acs2-1/acs6-1 double knockout mutant plants. Meanwhile, in acs8 mutant plants, there were no significant (P<0.05) differences in Pst DC3000 infection between acs8 knockout mutant plants treated with MgSO4 and CuSO4 (Fig. 5E). Taken together, our results strongly suggest that the AtACS8 gene plays an essential role in Cu2+-mediated early ethylene production and defense responses.

Identification of CuRE in the copper ion-inducible expression of AtACS8

To identify the mechanisms underlying Cu2+-induced AtACS8 expression, we used the GFP reporter gene to analyze the AtACS8 promoter. The GFP open reading frame (ORF) was inserted downstream of the AtACS2, AtACS6 or AtACS8 promoter. The constructs were transiently expressed in Nicotiana benthamiana (NB) and analyzed for their responsiveness to CuSO4 treatment. GFP under the AtACS6 promoter showed a strong fluorescence signal in leaves treated with MgSO4 and CuSO4. However, in contrast to MgSO4 treatment, CuSO4 treatment could activate GFP expression driven by the AtACS2 and AtACS8 promoters. In addition, the fluorescence in AtACS8-driven GFP was stronger than AtACS2-driven GFP (Fig. S4). The promoter of AtACS8 was chosen for further analysis. Site-directed deletions of the AtACS8 promoter at -1155, -901 and -661 were generated (Fig. 6A). Compared with MgSO4 treatment, CuSO4 treatment activated GFP driven by the -1655 and -1155 promoters. However, when driven by the -901 and -661 promoters, the GFP gene was not induced by CuSO4 treatment (Fig. 6A and B). The above result suggests that the DNA fragment from -902 to -1155 is required for Cu2+-induced expression of the AtACS8 gene. We performed in silico scanning for Cu2+-induced elements using Jaspar (Mathelier et al., 2015) with a profile score threshold of 80%. The analysis revealed one CuRE located at -967 to -957 in the promoter region (Fig. S5), indicating the candidate cis-element controlling Cu2+-induced AtACS8 expression. Then, we used site-directed promoter deletions at -967 and -956 to drive the GFP reporter gene and analyzed these constructs for their responsiveness to Cu2+ (Fig. 6C). Cu2+ induced GFP expression when GFP was driven by the -967 promoter but not when GFP was driven by the -956 promoter, which only lacks the CuRE (Fig. 6C and D). Using in silico scanning, we found that the promoters of those ethylene biosynthesis genes induced by CuSO4 treatment contain 3–9 (7.10±1.79) CuREs, which is significantly (P<0.05) more than that of 100 random genes (Fig. S6). We also constructed CuRE-35S mini and the 35S mini to drive the GFP reporter gene. In addition, Cu2+ induced a GFP signal in leaves transiently expressing CuRE-35S mini: GFP, but not in leaves expressing 35S mini: GFP (Fig. 6C,D). The function of CuRE in the Cu2+-induced expression of AtACS8 was further confirmed in transgenic Arabidopsis plants. The constructs containing CuREs showed strong GFP signal after CuSO4 treatment, whereas constructs lacking CuREs showed weak GFP signal (Fig. 6E,F). The results suggest that CuREs are required for Cu2+ to regulate the expression of gene AtACS8.

Fig. 6.

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

Fig. 6.

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

DISCUSSION

Cu2+ ions act as an elicitor, inducing defense responses in Arabidopsis

Previously, we have demonstrated that Cu2+ elicited defense responses and protected plants against pathogens (Liu et al., 2015). CuSO4 treatment promotes ROS accumulation and callose deposition, activates MAP kinase signaling and upregulates the expression of PR genes (Liu et al., 2015). All these response reactions elicited by Cu2+ ions imply that they act in a similar manner to other MAMP molecules, such as flg22 and elf18 (Zipfel, 2008). About 9.5% of the genes upregulated by Cu2+, flg22 and elf18, were associated with a response to stimulus (Fig. S1). In this study, we have demonstrated that Cu2+ also elicited early ethylene production and activated ethylene signaling in Arabidopsis. Ethylene emission is one of the early events in the Arabidopsis response to MAMPs such as flg22 and elf18 (Zipfel et al., 2006). CuSO4 treatment rapidly induced the production of ethylene 30 min after treatment (Fig. 4). This is the second study to identify similarities between Cu2+ and MAMP molecules. Furthermore, 2 h after CuSO4 treatment, WRKY22, WRKY29, WRKY33, WRKY53 and WRKY55 were upregulated (Fig. 2 and Table S4); the expression of these genes is also 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 both bacterial and fungal pathogens (Asai et al., 2002; Deslandes et al., 2002; Zheng et al., 2006). This is especially true for WRKY22 and WRKY29, which are considered early defense markers of flg22 and function downstream of MPK3 and MPK6 (Asai et al., 2002). We conclude that Cu2+ acts as an elicitor, inducing a MAMP-triggered immunity (MTI)-like response in Arabidopsis. This can explain, at least 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 responses elicited by Cu2+ and flg22. Flg22 treatment rapidly induced the expression of FLS2, MEKK1, MKK4 and MPK3, which are upstream of WRKY22 and WRKY29 (Asai et al., 2002; Zipfel et al., 2004). These genes were demonstrated to be part of the MAP kinase signaling cascade in the immune response induced by flg22 (Asai et al., 2002). However, Cu2+ did not upregulate the expression of these receptors or MAP kinase genes (Table S4), suggesting that Arabidopsis perceives Cu2+ with different MAP kinase signaling pathways. The following genes are some of the MAP kinase genes that are 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, and few studies have reported their functions in response to abiotic and biotic stresses. The functions of these three MEKK family members in the Cu2+-mediated defense response require further research.

The mechanism of copper ion-mediated ethylene production

The 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 a heavy metal, wounding and ozone, have been reported to increase ethylene production in plants (Boller, 1991; O'Donnell et al., 1996; Keunen et al., 2016; Rao et al., 2002). Exposure to 5, 10, 25 or 100 µM cadmium increased the level of ethylene in Arabidopsis after 24 and 72 h (Schellingen et al., 2014). Copper-induced increases 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 cadmium for 16 days resulted in decreased ethylene production (Carrió-Seguí et al., 2015). In addition, no significant changes in ethylene level were detected for Arabidopsis seedlings grown in the presence of 25 or 50 µM copper for 9 days (Lequeux et al., 2010). These results suggest that heavy metal-mediated ethylene production is dependent on the concentration of metal, plant age and exposure time.

The amino acid methionine is the biological precursor of ethylene, and the conversion of methionine to SAM by SAM synthase is the first step in ethylene biosynthesis (Yang and Hoffman, 1984). The sam1/sam2 double mutants showed significantly lower ethylene levels than wild-type plants (Mao et al., 2015). In addition, SAM1 overexpression lines produced more SAM and ethylene than Col-0 (Mao et al., 2015). These data suggest that upregulation of SAM synthase genes increases the level of ethylene. However, very few studies have reported the biotic or abiotic stress-mediated regulation of the transcription of SAM synthase genes (Broekaert et al., 2006). Interestingly, CuSO4 treatment induced the expression of SAM1 (At1G02500), SAM2 (At4G01850) and MTO3 (At3G17390) by more than twofold at 2 hpt in our study (Fig. 2; Table S4).

ACOs catalyze the last step in ethylene biosynthesis, and members of the ACO gene families are induced by different biotic and abiotic stress stimuli. For example, wounding, flooding, heavy metal stress and ozone exposure were shown to enhance ACO gene expression 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, although enhanced production of ethylene was found (Weber et al., 2006). In our study, AtACO1, 2, 4 and 5 were induced by Cu2+ (Fig. 3), suggesting that these ACO genes are also regulated in Cu2+-induced ethylene production.

In addition to SAM synthase genes and ACO genes, we also found that ACS genes were upregulated by CuSO4 treatment (Fig. 2; Table S4). The ACS-catalyzed conversion of SAM to ACC is the rate-limiting step in the ethylene biosynthesis pathway. The transcriptional regulation of ACS genes to promote ethylene production has been thoroughly studied. Using beta-glucuronidase and GFP as reporters, Tsuchisaka and Theologis (2004) examined the spatial and temporal expression patterns of ACS genes in Arabidopsis and showed that different abiotic stress stimuli result in different patterns of expression among the various ACS gene family members. Peng et al. (2005) found that hypoxia stress specifically induced the expression of AtACS2, AtACS6, AtACS7 and AtACS9. In a recent study, Schellingen et al. (2014) noticed that cadmium-induced ethylene production relied on AtACS2 and AtACS6. Meanwhile, a transgenic Arabidopsis line with enhanced glutathione content showed higher levels of ACC and ethylene than wild-type plants. In addition, glutathione induced the transcription of AtACS2 and AtACS6, which is dependent on WRKY33 (Datta et al., 2015). Guan et al., (2015) reported that acs2-1/acs6-1 double mutant plants produce less ethylene and were more susceptible to pathogens. In our study, RNA sequencing and qRT-PCR revealed that Cu2+ induced the expression of AtACS2, AtACS6, AtACS7, AtACS8 and AtACS11 but suppressed the expression of AtACS4 (Fig. 3). CuSO4 treatment enhanced the fluorescence signal in leaves expressing GFP driven by 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, the levels of ethylene and the immune response in acs8 mutant plants were significantly lower than those of wild-type plants after CuSO4 treatment (Fig. 5B and C). Therefore, it is possible that AtACS8 is the main gene regulating Cu2+-induced early ethylene production and defense responses. Interestingly, the expression of AtACS8 is controlled by the circadian clock, which is tightly correlated to ethylene production (Thain et al., 2004; Vandenbussche et al., 2003). It was further concluded that the enhanced transcription level of AtACS8 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-element was first found in the promoter of the CUP1 gene, which codes for a metallothionein protein that protects yeast against heavy metal toxicity. Cu2+ activates the expression of CUP1 through the CUP2 transcription factor protein (Welch et al., 1989). The CUP2 protein is a copper sensor that contains a copper fist, which has the function of binding Cu2+ and DNA (Buchman et al., 1989). The copper sensor in plants is still unknown, and no similar CUP2 protein had been found in plants. It is possible that Cu2+ binds to a novel transcription factor containing a copper fist domain to regulate expression of the AtACS8 gene. Completion of a screen to identify the CuRE-binding protein will answer this question, which needs further research.

MATERIALS AND METHODS

Bacterial strains and plasmids

The bacterial strains and plasmids used in this study are described in Table S1. The Escherichia coli strains were cultured on Luria-Bertani (LB) medium at 37°C. The Pseudomonas syringae pv. tomato strain DC3000 was cultured on King's B medium containing 50 μg ml−1 rifampicin at 28°C. The Agrobacterium tumefaciens strain GV3101 was cultured on LB medium 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 the AtACS8 start codon were cloned upstream of the GFP gene in the pCXGFP-P binary vector (Chen et al., 2009). The CuRE-35S mini promoter and 35S mini promoter were also inserted into the pCXGFP-P plasmid. These fragments were amplified using primers as described in the supplementary material (Table S2). All plasmids were validated by sequencing.

Plant materials and chemical treatments

Seeds of Arabidopsis (Arabidopsis thaliana ecotype Col-0), acs2-1/acs6-1 (CS16581) and acs8 (SALK_006628) were obtained from The Arabidopsis Information Resource (TAIR). Arabidopsis plants were grown in nutrient substrates in a growth chamber with 12-h days (at 23°C and 60–75% relative humidity) and 12-h nights (at 21°C and 60–75% relative humidity). According to previous reports, 100 μM CuSO4 can trigger the defense response 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 then pathogen inoculation (Liu et al., 2015), unless stated otherwise. Two-week-old plants were used for ethylene measurement after being sprayed with 100 μM CuSO4 or MgSO4 supplemented with 0.05% (v/v) Tween 20 for different time periods.

Bacterial growth assay

A bacterial growth assay was performed in Arabidopsis plants at 0 and 3 days after inoculation. The concentration of Pst DC3000 suspension in buffer containing 10 mM MgCl2 was adjusted to 106 CFU ml−1 and infiltrated into 4- to 5-week-old Arabidopsis plants using a needleless syringe. Leaves were harvested, surface sterilized in 70% ethanol solution for 1 min and then rinsed in sterile distilled water for 1 min. Leaf disks were excised from the leaves of three independent plants with a 0.5 cm2 cork borer and were ground in 1.5-ml microfuge tubes with 100 μl sterile distilled water. The samples were serially diluted (1:10) and plated on King's B medium containing 50 μg ml−1 rifampicin at 28°C for colony counting. The experiment was repeated three times. The means were compared using a t-test. The standard error and t-test results were recorded.

RNA extraction, reverse transcription-PCR, quantitative real-time PCR

Arabidopsis plants were treated with CuSO4 or MgSO4 and whole plants were collected to extract total RNA with TRIzol Reagent (Invitrogen, USA). Using a HiFi Script cDNA Synthesis Kit (CWBIO, China), 1 μg of total RNA was used to synthesize first-strand cDNA as described in the manufacturer's instructions. qRT-PCR was performed with a QuantStudio 6 Flex Real-Time PCR System (Life Technologies, USA) using UltraSYBR Mixture (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 analysis

Two biological replicates of RNA samples were collected from two-week-old wild-type Arabidopsis plants treated with 100 μM CuSO4 for 0, 2 or 24 h. As previously reported (Ju et al., 2017), six libraries were constructed and sequencing performed with a BGISEQ-500 by the Beijing Genomic Institution (www.genomics.org.cn, BGI, Shenzhen, China). Clean tags were mapped to the reference genome and genes that are available in the Arabidopsis TAIR10.2 reference genome. For gene expression analysis, the matched reads were calculated and then normalized to RPKM (reads per kilobase of transcript per million mapped reads) using RESM software. The significance of differential gene expression was confirmed with the BGI bioinformatics service using the combination of the absolute value of log2-Ratio≥1 and P≤0.05 in this research.

Quantitation of ethylene biosynthesis rates

Ethylene biosynthesis rates were measured by gas chromatography as described previously (Spollen et al., 2000). Briefly, 2-week-old seedlings were treated with CuSO4 or MgSO4, and leaves from 20 plants were detached and placed in a 20-ml penicillin bottle. At various times after CuSO4 or MgSO4 treatment, a 1-ml gas sample was injected into a cold trap. Ethylene was quantified using a gas chromatograph equipped with a photoionization detector.

Promoter analysis in N. benthamiana

Agrobacterium GV3101 containing the pCXGFP-P binary vector with the GFP gene driven by different promoter fragments were resuspended with Agro-infiltration buffer (10 mM MgCl2, 10 mM MES, pH 5.6, 200 mM acetosyringone), and the OD600 adjusted to 0.5. Bacterial suspensions were infiltrated into 4- to 6-week-old N. benthamiana leaves with a needleless syringe. Two or three days later, whole plants were sprayed with 100 μM CuSO4 or MgSO4 supplemented with 0.05% (v/v) Tween 20 for 12 h. Plants were then observed using an M165 FC fluorescence stereomicroscope (Leica, Germany).

Production of transgenic Arabidopsis and promoter analysis

Stable transgenic Arabidopsis plants expressing the GFP gene driven by different promoter fragments were generated by floral dipping of Col-0 plants. Multiple positive T1 lines were selected for production of the T2 generation for experiments.The transgenic plants were sprayed with 100 μM CuSO4 or MgSO4 supplemented with 0.05% (v/v) Tween 20 for 12 h and then observed using a LSM880NLO confocal microscope (Zeiss, Germany).

Acknowledgements

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

Footnotes

Author contributions

Conceptualization: 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.

Funding

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

Data availability

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

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Competing interests

The authors declare no competing or financial interests.

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