The ethylene response factor AtERF98 ... - Wiley Online Library

The Plant Journal (2012) 71, 273–287

doi: 10.1111/j.1365-313X.2012.04996.x

The ethylene response factor AtERF98 enhances tolerance to salt through the transcriptional activation of ascorbic acid synthesis in Arabidopsis Zhijin Zhang1,2, Juan Wang1,2, Rongxue Zhang1,2 and Rongfeng Huang1,2,* 1 Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China, and 2 National Key Facility of Crop Gene Resources and Genetic Improvement, Beijing 100081, China Received 24 June 2011; revised 4 March 2012; accepted 5 March 2012; published online 18 June 2012. *For correspondence (e-mail [email protected]).

SUMMARY Ascorbic acid (AsA) is an important antioxidant in plants, and its biosynthesis is finely regulated through developmental and environmental cues; however, the regulatory mechanism remains unclear. In this report, the knockout and knockdown mutants of Arabidopsis AtERF98 decreased the AsA level, whereas the overexpression of AtERF98 increased it, which suggests that AtERF98 plays an important role in regulating AsA biosynthesis. AtERF98-overexpressing plants showed enhanced expression of AsA synthesis genes in the D-mannose/L-galactose (D-Man/L-Gal) pathway and the myo-inositol pathway gene MIOX4, as well as of AsA turnover genes. In contrast, AtERF98 mutants showed decreased expression of AsA synthesis genes in the D-Man/L-Gal pathway but not of the myo-inositol pathway gene or AsA turnover genes. In addition, the role of AtERF98 in regulating AsA production was significantly impaired in the D-Man/L-Gal pathway mutant vtc1-1, but the expression of the myo-inositol pathway gene or AsA turnover genes was not affected, which indicates that the regulation of AtERF98 in AsA synthesis is primarily mediated by the D-Man/L-Gal pathway. Transient expression and chromatin immunoprecipitation assays further showed that AtERF98 binds to the promoter of VTC1, which indicates that AtERF98 modulates AsA biosynthesis by directly regulating the expression of the AsA synthesis genes. Moreover, the knockout mutant aterf98-1 displayed decreased salt-induced AsA synthesis and reduced tolerance to salt. The supplementation of exogenous AsA increased the salt tolerance of aterf98-1; coincidently, the enhanced salt tolerance of AtERF98-overexpressing plants was impaired in vtc1-1. Thus, our data provide evidence that the regulation of AtERF98 in AsA biosynthesis contributes to enhanced salt tolerance in Arabidopsis. Keywords: ascorbate acid synthesis, ERF protein, D-Man/L-Gal pathway, oxidative stress, salt stress.

INTRODUCTION Ascorbic acid (AsA) plays important roles in the growth, development and stress responses of plants (Smirnoff and Wheeler, 2000; Conklin and Barth, 2004). The biosynthesis of AsA through several pathways, including the D-glucosone, D-galacturonate, myo-inositol and D-mannose/L-galactose (D-Man/L-Gal) pathways (Loewus et al., 1990; Wheeler et al., 1998; Agius et al., 2003; Lorence et al., 2004), is finely regulated in plants (Smirnoff et al., 2001; Lorence et al., 2004; Hancock and Viola, 2005; Zhang et al., 2008). Plants can synthesise AsA through the D-glucosone pathway (Loewus et al., 1990; Saito et al., 1990), but some enzymes in this pathway are not well characterised (Pallanca and Smirnoff, 1999). The uronic acids D-glucuronate and D-galacturonate ª 2012 The Authors The Plant Journal ª 2012 Blackwell Publishing Ltd

can be converted into AsA through the D-galacturonate pathway (Davey et al., 1999; Jain et al., 2000), and it has been shown that the D-galacturonate pathway may play a primary role in the specific organ or developmental stage of plants (Davey et al., 2000). Myo-inositol has also been shown to be involved in the synthesis of AsA (Lorence et al., 2004; Zhang et al., 2008); however, other studies have shown that myo-inositol is not involved in the synthesis of AsA (Endres and Tenhaken, 2009). In AsA biosynthesis pathways, the D-Man/L-Gal pathway is thought to be the most important AsA synthesis pathway in plants. In the D-Man/L-Gal pathway, AsA is sequentially synthesised by D-Man–1P, GDP–D-Man, GDP–L-Gal, L-Gal–1P, L-Gal, 273

274 Zhijin Zhang et al. and the final precursor, L-galactono-1,4-lactone (L-GalL), with catalysis through GDP–D-Man pyrophosphorylase, GDP–Man-3,5-epimerase, GDP–L-Gal phosphorylase, L-Gal dehydrogenase, and L-Gal guanyltransferase enzymes, respectively (Smirnoff et al., 2001). All of the genes involved in this pathway have been characterised in Arabidopsis (Wheeler et al., 1998; Conklin et al., 1999; Wolucka and Van Montagu, 2003; Laing et al., 2004, 2007; Dowdle et al., 2007; Linster et al., 2007). Mutations in the D-Man/L-Gal pathway result in a significantly decreased AsA content, whereas the overexpression of genes in this pathway increases the AsA levels (Conklin et al., 2000; Gatzek et al., 2002; Wolucka et al., 2003; Dowdle et al., 2007; Linster et al., 2008). For example, the GDP–D-Man pyrophosphorylase (VTC1) vtc1-1 mutant displays a decrease in the synthesis of AsA to only one-third of the amount in wild-type plants (Conklin et al., 2000). Two homologous genes, VTC2 and VTC5, encode GDP–L-Gal phosphorylase in Arabidopsis (Dowdle et al., 2007; Linster et al., 2007, 2008). The double mutant (vtc2/ vtc5), which has almost no GDP–L-Gal phosphorylase activity, displays significantly decreased AsA levels and impaired growth (Dowdle et al., 2007). The overexpression of the tomato GDP–Man-3,5-epimerase gene SIGME increases AsA content and enhances tolerance to salt and cold (Zhang et al., 2011a). In addition to de novo synthesis, AsA recycling also affects the AsA level (Smirnoff et al., 2001; Chen et al., 2003; Stevens et al., 2008). As the most abundant water-soluble antioxidant, one of the important roles of AsA is to scavenge reactive oxygen species (ROS) (Smirnoff and Wheeler, 2000) that are normally generated during photosynthesis and aerobic metabolism in plants (Conklin and Barth, 2004; Foyer and Noctor, 2009). During ROS scavenging, AsA is oxidised into monodehydroascorbate (MDHA) by AsA peroxidase (APX), and MDHA can be recycled to AsA by monodehydroascorbate reductase (MDAR) or disproportionate to dehydroascorbate (DHA) and AsA. The DHA can be recycled to AsA by dehydroascorbate reductase (DHAR) or hydrolysed to 2,3diketogulonic acid (Smirnoff and Wheeler, 2000). The overexpression of wheat DHAR significantly increased the levels of AsA in tobacco and maize, which indicates that AsA recycling also plays an important role in the regulation of AsA levels in plants (Chen et al., 2003). Thus, MDAR and DHAR play important roles in recapturing AsA and regulating the redox state of plant cells (Chen et al., 2003; Eltayeb et al., 2007; Stevens et al., 2008). Various stresses can induce the synthesis of AsA in plants (Smirnoff et al., 2001). In Arabidopsis, salt stress can induce the synthesis of AsA (Huang et al., 2005). In tomato, development and stress can induce the expression of AsA synthesis-related genes and enhance AsA synthesis (Ioannidi et al., 2009). Recent studies have shown that in Arabidopsis the F-box E3 protein AMR1 (ascorbic acid mannose pathway regulator 1) could negatively regulate the expres-

sion of D-Man/L-Gal pathway genes at the transcriptional level and inhibit AsA synthesis during leaf aging and in response to ozone (Zhang et al., 2009a). However, the specific mechanisms involved in the regulation of AsA synthesis in plants remain obscure. The ethylene response factor (ERF) proteins, which are defined by a conserved DNA-binding domain (Riechmann et al., 2000), belong to the AP2/ERF transcription factor superfamily (Nakano et al., 2006). The ERF domain was first identified in a conserved GCC-box-binding protein from tobacco (Ohme-Takagi and Shinshi, 1995), and additional studies have shown that the ERF DNA-binding domain can also be used to identify other cis-elements, such as DRE/ CRT (dehydration-responsive element/C-repeat), JERE (JA responsive element) and VWRE (vascular system-specific and wound-responsive cis-element) (Liu et al., 1998; van der Fits and Memelink, 2001; Sasaki et al., 2007). The ERF proteins have important functions in the transcriptional regulation of plant growth and development, as well as plant responses to various environmental elements (Nakano et al., 2006; Zhuang et al., 2011). The overexpression of Arabidopsis ERF1 activates the expression of genes related to pathogenesis and enhances the resistance of Arabidopsis to Botrytis cinerea and Plectosphaerella cucumerina (Berrocal-Lobo et al., 2002). The overexpression of tomato TSRF1 improves the resistance of tobacco and tomato plants to Ralstonia solanacearum-induced bacterial wilt (Zhang et al., 2004), and the overexpression of CBF1/ DREB1B enhances the tolerance of Arabidopsis, tobacco and rape to cold (Jaglo-Ottosen et al., 1998; Yang et al., 2010). Recent studies have also shown that ERF proteins could regulate the biosynthesis of plant metabolites, such as the synthesis of jasmonate, gibberellin, ethylene, lipid and wax, to enhance the tolerance to environmental stresses (van der Fits and Memelink, 2000; Aharoni et al., 2004; Taketa et al., 2008; Zhang et al., 2009b; De Boer et al., 2011; Fukao et al., 2011). In tobacco and tomato, the overexpression of LeERF2/TERF2 activated the expression of genes related to ethylene synthesis, which resulted in increased ethylene synthesis and increased tolerance to cold (Zhang et al., 2009b; Zhang and Huang, 2010). The ERF protein SHINE increased the synthesis of wax in Arabidopsis and alfalfa (Medicago sativa) and enhanced the tolerance to salt and drought (Aharoni et al., 2004). In this study, we found that the Arabidopsis ERF gene AtERF98, which was named following Nakano et al. (2006), could regulate AsA synthesis through the activation of AsA synthesis-related genes. The overexpression of AtERF98 increased the AsA content, whereas the mutants aterf98-1 and aterf98-2 displayed reduced levels of AsA. The AtERF98 mutants showed a reduced level of AsA in salt stress and decreased tolerance to salt, which indicates that AtERF98 could regulate the response to salt stress by increasing AsA synthesis.

ª 2012 The Authors The Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 71, 273–287

Regulation of AtERF98 in ascorbic acid biosynthesis 275 RESULTS Identification of the ERF protein AtERF98 in the regulation of AsA biosynthesis The ERF proteins play key roles in many diverse cellular processes, such as hormone signal transduction, the response to stress and the regulation of metabolism (Aharoni et al., 2004; Taketa et al., 2008; De Boer et al., 2011; Fukao et al., 2011). Our previous studies showed that the ERF proteins TERF1 and JERF3 are involved in the regulation of stress responses (Zhang et al., 2005; Wu et al., 2008). Subsequent studies have indicated that TERF1 and JERF3 can regulate the expression of genes related to the synthesis of AsA and increase the AsA levels in tobacco (Figure S1 in Supporting Information). To study the ERF-mediated regulation of AsA biosynthesis in detail, we investigated 18 mutants from the Arabidopsis Biological Resource Center (ABRC) of TERF1 and JERF3 homologous genes. First, we applied the nitroblue tetrazolium (NBT)-staining assay (Conklin et al., 2000) to estimate the differences in the AsA content in leaves of 3-week-old wild-type Columbia-0 (WT, Col-0) and mutant plants. The NBT-staining assay showed that the two AtERF98 (At3g23230) mutants, aterf98-1 (SAIL_1142_D01) and aterf98-2 (SAIL_213_E01), had lower AsA levels than the WT plants. To further analyse AsA synthesis in the AtERF98 mutants, we measured the AsA content in 3-week-old leaves of the WT and AtERF98 mutants using an AsA oxidase-based spectrophotometric assay (Conklin et al., 2000), which showed that the two mutants had markedly decreased AsA levels (Figure 1). The AsA contents of aterf98-1 and aterf98-2 were 65% and 80%,

Figure 1. Isolation of ethylene response factor (ERF) mutants involved in the regulation of ascorbic acid (AsA) synthesis in Arabidopsis. Leaves from three 3-week-old WT (wild-type Arabidopsis), and aterf98-1 and aterf98-2 mutant plants were used to measure the AsA content as described in the Experimental procedures. The experiments were repeated five times. The bars represent () the standard error (SE), and the asterisks denote the results that were significantly different from those in the WT (**P < 0.01 and *P < 0.05). The statistical significance was evaluated using the t-test.

respectively, of that in the WT plants (Figure 1). These results suggest that AtERF98 may be involved in regulating the synthesis of AsA. The two T-DNA insertion mutations aterf98-1 and aterf982 were identified using the T-DNA left internal primer LB2 and the 3¢ and 5¢ primer pairs of AtERF98 (Table S1) via the polymerase chain reaction (PCR). The aterf98-1 mutation is located at )131 bp, whereas aterf98-2 is located )338 bp upstream of the AtERF98 transcription start site (Figure 2a). The sequence of AtERF98 contains a 420-bp open reading frame (ORF) that encodes a protein of 139 amino acids. This protein contains one typical ERF DNA-binding domain, which suggests that AtERF98 is an ERF protein with 66.9% and 76.5% identity in this ERF domain to JERF3 and TERF1, respectively (Figure 2b). This homology also suggests that AtERF98 has DNA-binding characteristics similar to those of JERF3 and TERF1. The expression of AtERF98 was not detected in aterf98-1 and was only weakly detected in aterf98-2 (Figure 2c), which indicates that aterf98-1 and aterf98-2 are knockout and knockdown mutants, respectively. More interestingly, the overexpression of AtERF98 in aterf98-1 increased the AsA content compared with the WT and aterf98-1 (Figure 2d), which further suggests that AtERF98 is involved in the regulation of AsA synthesis. The regulation of AtERF98 in AsA biosynthesis occurs through the D-Man/L-Gal pathway To further study how AtERF98 regulates the biosynthesis of AsA, we overexpressed AtERF98 in a Col-0 background (OX lines, Figure S2a) to measure the AsA content. The AsA level was approximately 1.6- to 1.7-fold higher in the OX lines than that in the WT (Figures 3a and S2b), which indicates that AtERF98 plays an important role in regulating the biosynthesis of AsA in Arabidopsis. Because the content of AsA is regulated through synthesis and recycling (Smirnoff et al., 2001; Chen et al., 2003; Stevens et al., 2008), the relationship between AtERF98 and AsA biosynthesis was further evaluated. Firstly, the expression of AsA biosynthetic genes was analysed by using quantitative real-time PCR (Q-PCR). The AtERF98-overexpressing lines OX3 and OX8 displayed increased expression of the D-Man/L-Gal pathway genes VTC1, VTC2, GalDH and GLDH, as well as the myo-inositol oxygenase gene MIOX4, which the first enzyme in the myoinositol pathway (Lorence et al., 2004). In contrast, the mutants aterf98-1 and aterf98-2 displayed reduced transcript levels for VTC1 and VTC2, and the expression of other synthesis genes was not affected (Figure 3b). Secondly, considering that MDHA and DHA can be recycled into AsA via the AsA–glutathione (AsA-GSH) cycle with the cooperation of MDAR, DHAR and glutathione reductase (GR) (Smirnoff et al., 2001; Chen et al., 2003; Stevens et al., 2008), the expression of MDARs, DHARs and GRs was detected in AtERF98 mutants and OX lines. We found that the expression of the Arabidopsis AsA recycling genes monodehydroascorbate


276 Zhijin Zhang et al.

Figure 2. Identification of AtERF98 mutants. (a) Integration positions of the T-DNA insertions in the aterf98-1 and aterf98-2 mutants. The T-DNA insertions in aterf98-1 or aterf98-2 were identified using PCR amplification. The insertion of T-DNA occurred in the promoter region of AtERF98 (At2g230233). (b) The alignment of the AtERF98 ERF domain with known ethylene response factor (ERF) proteins. The shaded boxes indicate the percentage of sequences at that position with the same amino acid identity (dark grey, 100%; light grey, 60–80%). (c) Expression of AtERF98 in aterf98-1 and aterf98-2. The expression of AtERF98 in wild type (WT), aterf98-1 and aterf98-2 plants was analysed using RT-PCR amplification with 35 cycles. The expression of Tubulin4 was used as the internal control using RT-PCR amplification with 22 cycles. (d) The ascorbic acid (AsA) levels in aterf98-1 and in the AtERF98-overexpressing plants with an aterf98-1 background. OE1 (overexpressing line 1)/aterf98-1 and OE2/aterf98-1 indicate different overexpression lines in the aterf98-1 background. The assay was repeated three times. The bars represent () SE, and the asterisk indicates results that were significantly different from those obtained in the WT (**P < 0.01 and *P < 0.05). The statistical significance was evaluated using the t-test.

reductase 3 (MDAR3), chloroplast dehydroascorbate reductase (ChlDHAR), cytosolic dehydroascorbate reductase (CytDHAR) and glutathione reductase 1 (GR1) was significantly higher in the transgenic OX lines than in the WT, but there was no obvious difference in expression between the WT and the mutants (Figure S3). Thus, these results suggest that AtERF98 is required for the activation of D-Man/L-Gal pathway genes, such as VTC1 and VTC2, and plays a nonessential role in the regulation of MIOX4 and genes involved in AsA recycling. To further investigate the regulation of AtERF98 in AsA biosynthesis, we overexpressed AtERF98 in the mutant vtc1-1 (OE lines) in which the de novo synthesis of AsA through the D-Man/L-Gal pathway was inhibited (Conklin et al., 1999) to distinguish the regulation of the OX transgenic lines. Although the expression levels of AtERF98 were significantly increased in the different OE lines (Figure S4a), the AsA content in the OE lines was slightly increased compared with that in vtc1-1 but markedly reduced compared with that in the AtERF98-overexpressing line in the Col-0 background (Figures 3c and S4b). Consistent with the finding of the previous study (Conklin et al., 2000), the level of AsA in vtc11 [1.3 lmol g)1 fresh weight (FW)] was approximately onethird of that in the WT (3.6 lmol g)1 FW). The AsA levels in OE9 and OE10 were 1.8 and 2.1 lmol g)1 FW, respectively, whereas that in OX3 was 5.9 lmol g)1 FW (Figure 3c). These

data showed that the AsA content in the AtERF98-overexpressing lines of the vtc1-1 background (OE9 and OE10) were approximately one-third of that in the AtERF98-overexpressing line with the Col-0 background (OX3), indicating that AtERF98 regulates the level of AsA predominantly through the D-Man/L-Gal pathway. In contrast, there were no obvious differences in the expression of AtERF98, AsA synthesis genes or AsA recycling genes between the transgenic OX and OE lines (Figure 3d). Thus, the mutation of VTC1 did not alter the effects of AtERF98 on the expression of AsA synthesis or recycling genes, but the overall synthesis of AsA was limited, further suggesting that AtERF98 regulates AsA levels primarily through the D-Man/L-Gal synthesis pathway. The interaction of AtERF98 with the promoter of VTC1 Ethylene response factor proteins can directly or indirectly activate the expression of downstream genes (Chakravarthy et al., 2003). To elucidate how AtERF98 regulates the transcripts of AsA synthesis genes to control AsA biosynthesis, we analysed the effect of AtERF98 on the expression of VTC1. We first cloned the 3148-bp VTC1 promoter sequence upstream of the translation start codon ATG using PCR amplification. We analysed the promoter sequence using PLACE (http://www.dna.affrc.go.jp/PLACE/signalscan.html)


Regulation of AtERF98 in ascorbic acid biosynthesis 277

Figure 3. AtERF98 enhances the expression of genes involved in ascorbic acid (AsA) biosynthesis and regulates the synthesis of AsA through the D-mannose/ L-galactose pathway. (a) Levels of AsA in AtERF98-overexpressing plants with a Col-0 background. OX3 (overexpressing line 3) and OX8 indicate two independent AtERF98overexpressing lines with a Col-0 background. (b) The expression of genes related to AsA biosynthesis in AtERF98 mutants and AtERF98-overexpressing plants determined by quantitative real-time PCR (Q-PCR) amplification. (c) The AsA levels in AtERF98-overexpressing plants with a vtc1-1 background OE9 (overexpressing line 9) and OE10 indicate AtERF98-overexpressing lines in the vtc1-1 background. (d) The expression of genes related to the de novo biosynthesis and recycling of AsA in wild type (WT), mutant and overexpressing plants with the Col-0 or vtc1-1 background was determined using Q-PCR amplification. Tubulin4 was used as the internal control, and the expression of each gene in the WT was assigned a value of 1. The bars represent the SE (), and the asterisks indicate the results that were significantly different from WT (**P < 0.01 and *P < 0.05). The statistical significance of three repeated assays was evaluated using the t-test.

and found that the VTC1 promoter contains two DRE core sequences (ACCGAC) upstream of the transcription start site between )2310 and )2305 bp and )1699 and )1694 bp. Based on the sequence characteristics of the VTC1 promoter, we generated promoter segments of different lengths with or without the DRE core sequences. The full-length promoter and its segments were cloned upstream of the GUS reporter, and the resulting promoter–GUS plasmids were transformed into Agrobacterium tumefaciens. The binding of AtERF98 to the promoter of VTC1 was analysed using a transient expression assay in tobacco leaves. The interaction of AtERF98 with the P1 or P2 fragments resulted in a 15-fold enhancement of GUS activity, and a marked decrease in GUS activity was observed for the P3 and P4 fragments that lacked both of the DRE core sequences compared with the

control (Figure 4a), which indicates that AtERF98 directly activates the transcription of VTC1 by interacting with VTC1 promoter fragments containing the DRE cis-element. To further investigate the interaction of AtERF98 with DRE in vivo, a chromatin immunoprecipitation (ChIP) assay was performed. We first overexpressed the AtERF98-haemagglutinin (HA) fusion protein in Arabidopsis. Specific primers were designed for two segments of the VTC1 promoter. Both segments contained the DRE cis-element: DRE-1 contained the region from )2447 to )2107 bp, and DRE-2 contained the region from )1812 to )1565 bp. We found that DRE-1 and DRE-2 were both present in the input samples. After affinity selection, only the fragment containing DRE-2 was detectable in the eluate of AtERF98-HA, whereas DRE-1 was not detected in the eluate (Figure 4b), which indicates that



Figure 4. AtERF98 binds to the promoter of VTC1 in transient expression and chromatin immunoprecipitation assays. (a) AtERF98 binding to the promoter of VTC1 in the transient expression assay. The top panel shows schematics of the GUS reporter vectors containing different VTC1 promoters and the effector vectors. The bottom panel shows the interaction of AtERF98 with the different VTC1 promoter fragments using the transient assay. Agrobacterium tumefaciens containing the reporter vector and the effector vectors was injected into tobacco leaves to analyse the interaction of AtERF98 with the VTC1 promoter through the activity of GUS. The results shown represent data from three assays. The asterisks indicate significant differences from the leaves with no effector (**P < 0.01 and *P < 0.05), and the bars represent the SE (). (b) AtERF98 identifies the promoter fragment of VTC1 containing a dehydration-responsive element (DRE) in vivo. Arabidopsis plants expressing haemagglutinin (HA) were used as a negative control.

AtERF98 can specifically bind to the DRE-2 fragment of the VTC1 promoter to activate the expression of VTC1. The role of AtERF98 in the salt response To study the role of AtERF98 in detail, Q-PCR was used to examine the expression patterns of AtERF98 after the plants were subjected to different stresses and hormones. Ethylene was found to quickly induce the expression of AtERF98, which rapidly reached a maximum level after 1 h. Treatment with NaCl or H2O2 also quickly induced the expression of

Figure 5. The expression pattern of AtERF98 and VTC1 induced by ethylene, salt and H2O2. The expression of AtERF98 and VTC1 following exposure to ethylene, salt and H2O2 was analysed using quantitative PCR amplification. (a) The expression patterns of AtERF98. (b) The expression patterns of VTC1. The expression of Tubulin4 was used as an internal control, and the levels of each gene in the control condition (0 h) were assigned values of 1. The asterisks indicate the results of three repeated assays that showed significant differences from the control condition (**P < 0.01 and *P < 0.05). The statistical significance was evaluated with the t-test, and the bars represent the SE ().

AtERF98, which peaked after 2 h (Figure 5a). These findings suggest that AtERF98 may be involved in these stress responses. Interestingly, the expression of VTC1 was also rapidly induced by ethylene and quickly reached a maximum level after 1 h; however, VTC1 expression was slowly induced upon treatment with NaCl or H2O2 and reached its maximum level after 8 and 4 h, respectively (Figure 5b). These results suggest that VTC1 might be located downstream of AtERF98 and could be regulated by AtERF98 in response to H2O2 and NaCl stress, which further supports the idea that AtERF98 transcriptionally modulates the expression of the VTC1 gene. Next, we examined whether AtERF98 regulates the response to salt stress. To study the role of AtERF98 in the


Regulation of AtERF98 in ascorbic acid biosynthesis 279

Figure 6. AtERF98 enhances the tolerance to salt in Arabidopsis. (a) The phenotypes of the wild type (WT), AtERF98 mutants and AtERF98overexpressing lines following salt stress. Five-day-old seedlings were transferred to MS medium containing NaCl and grown for another 5 days. (b) The survival rates (%) in the AtERF98 mutants and AtERF98-overexpressing lines following salt stress. The 5-day-old seedlings of AtERF98 mutants and AtERF98-overexpressing lines were grown on MS medium containing NaCl for another 6 days, and the surviving seedlings (those possessing green leaves) were counted. The bars represent the SE () of three assays, and the asterisks represent significant differences from WT, as indicated by the t-test (**P < 0.01 and *P < 0.05).

we examined the content of osmotically active solutes. Our results showed a lack of a significant difference in the proline and soluble sugar content among WT, aterf98-1, aterf98-2 and the OX lines under normal growth conditions and with salt treatment, although the salt treatment greatly increased the proline and soluble sugar content (Figure S5a,b). Salt stress also affects the ionic and redox homeostasis of the plant cell (Munns et al., 2008; Miller et al., 2010). Therefore, we further analysed the effects of AtERF98 on ionic homeostasis in the salt response by examining the Na+/K+ concentration ratio in mutants and transgenic lines. Our results showed that the Na+/K+ concentration ratio of WT, aterf98-1, aterf98-2 and the OX lines displayed no obvious difference under either normal growth conditions or in the presence of salt treatment (Figure S5c). The effects of AtERF98 on the redox state during the salt response were also examined. Our data showed that the overexpression of AtERF98 reduced the accumulation of H2O2 in the leaf under salt stress (Figure 7a) while the knockout or knockdown mutants enhanced it, which indicates that AtERF98 enhances the tolerance to salt, possibly by controlling the increase in ROS during salt stress. Because the increase in ROS may damage the DNA, proteins and lipids in the cell and because malondialdehyde (MDA) is an important product of lipid peroxidation (Tajdoost et al., 2007), we further determined the MDA content. There was no obvious difference in the MDA content between Col-0, theAtERF98 mutants and the overexpressing plants (OX lines) during normal growth conditions, but after NaCl treatment, the MDA content was significantly higher in the AtERF98 mutants than in Col-0, and lowest in the OX lines (Figure 7b). Similarly, the chlorophyll content in Col-0, the AtERF98 mutants and the OX lines was not obviously different under normal growth conditions, but after NaCl treatment the chlorophyll content was significantly reduced in the AtERF98 mutants and increased in the OX lines (Figure 7c), which suggests that AtERF98 may protect plant cells from ROS-mediated damage by reducing the accumulation of ROS during salt stress. The function of AsA in the AtERF98-regulated response to salt

response to salt stress, 5-day-old seedlings were transferred to MS medium with 180 mM NaCl, and their phenotypes were observed after culturing for another 5 days. The different lines exhibited marked variability in their tolerance to salt (Figure 6a). The transgenic lines showed a markedly enhanced salt tolerance (Figure 6a). Further statistical analysis indicated that 60–65% of the seedlings in the OX3 and OX8 lines remained green, whereas approximately 15–30% of the plants in aterf98-1 and aterf98-2 did (Figure 6b), indicating that AtERF98 is an important positive regulator of the salt response. Salt stress affects the osmotic homeostasis of the plant cell (Munns et al., 2008); therefore,

In plant cells, ROS can be controlled by enzymatic and nonenzymatic pathways. Because antioxidative enzymes, such as superoxide dismutase (SOD), catalase (CAT) and APX, play important roles in the response to oxidative stress, we first analysed the expression of ROS-related genes. The expression levels of the AsA peroxidase genes APX2, APX3, APX6, sAPX and catalase (CAT1) and the superoxide dismutase genes FSD1 and FSD2 were enhanced in the AtERF98-overexpressing plants (Figure S6a,b). This result indicates that these antioxidant enzymes may be involved in regulating the ROS levels in the OX lines. However, their expression in the aterf98-1 and aterf98-2 mutants was not


280 Zhijin Zhang et al. notably different from that in WT (Figure S6a,b), demonstrating that the transcriptional regulation by AtERF98 of genes for antioxidative stress enzymes was not essential for the function of AtERF98 in regulating the response to salt stress. As the most abundant water-soluble antioxidant, AsA plays an important role in scavenging ROS (Smirnoff and Wheeler, 2000), and increasing evidence has shown that AsA can scavenge ROS and enhance salt tolerance (Shalata and Neumann, 2001; Zhang et al., 2011a). Because AtERF98 can regulate the synthesis of AsA and enhance the tolerance to salt, we further examined whether the modulation of AsA synthesis by AtERF98 is associated with the response to salt. To analyse the role of AsA in AtERF98-mediated salt tolerance, we used transgenic OE lines to limit the AtERF98-mediated increase in the production of AsA. The OE lines were less tolerant to salt than the OX lines, as determined by 180 mM NaCl treatment for 5 days (Figure 8a). Further statistical analysis indicated that the OE lines exhibited a lower survival than the OX lines (Figure 8b). For example, after treatment with salt stress (180 mM NaCl) for 6 days, the survival rate was 62–67% in the OX lines but only 35–39% in the OE lines (Figure 8b), supporting the hypothesis that the synthesis of AsA is essential for the enhanced salt tolerance conferred by AtERF98. The modulation of AsA biosynthesis and the salt response by AtERF98

Figure 7. AtERF98 enhances the tolerance to salt in Arabidopsis by inhibiting accumulation of reactive oxygen species and decreasing the damage resulting from oxidative stress. (a) The H2O2 content in the leaves of wild type (WT), AtERF98 mutants, and AtERF98-overexpressing plants with or without salt treatment determined using histological staining. (b) The malondialdehyde (MDA) content in WT, AtERF98 mutants and overexpressing plants upon salt treatment. (c) The chlorophyll content in WT, AtERF98 mutants and AtERF98-overexpressing plants upon salt treatment. The above assays were repeated three times. The bars represent the SE (). The asterisks indicate significant differences in the MDA or chlorophyll content in the mutant or transgenic plants in comparison with WT as determined by the t-test (**P < 0.01, and *P < 0.05).

Studies have shown that salt stress can induce AsA synthesis in plants (Shalata and Neumann, 2001; Huang et al., 2005; Figure S7). To analyse the role of AsA in the salt response regulated by AtERF98, we examined whether the regulation of AtERF98 in the synthesis of AsA is affected by salt. We first examined whether AtERF98 is necessary for salt-induced production of AsA using Col-0 and the knockout mutant aterf98-1. NaCl markedly and quickly induced the production of AsA in Col-0 but not in aterf98-1 (Figure 8c), which indicates that AtERF98 is required for salt-induced synthesis of AsA. This result is consistent with the previous data describing the expression of the AtERF98 and VTC1 genes (Figure 5). In contrast, the synthesis of the antioxidant glutathione was similar in WT, aterf98-1, aterf98-2 and the OX lines both under normal growth conditions with salt treatment (Figure S8), indicating that the activity of AtERF98 in the salt response does not involve the regulation of glutathione synthesis. We further analysed whether AtERF98-mediated AsA synthesis contributes to salt tolerance by using supplemental exogenous AsA during salt treatment. As expected, the supplementation of AsA reversed the tolerance to salt in sensitive mutants and even enhanced the response in the OX lines (Figure 9a). The survival rates increased from 12–19% and 42–45% (salt treatment without AsA addition) to 60–62% and 67–68% (salt treatment with AsA


Regulation of AtERF98 in ascorbic acid biosynthesis 281 Figure 8. AtERF98 is involved in salt-induced ascorbic acid (AsA) synthesis and enhances the tolerance to salt through AsA synthesis. (a) The phenotypes of wild type (WT), vtc1-1 and AtERF98-overexpressing lines with a Col-0 and vtc1-1 background following salt stress. (b) The survival rates (%) of AtERF98-overexpressing lines in different backgrounds after salt stress treatment. The surviving plants (possessing green leaves) were counted. (c) The AsA levels in the WT, AtERF98 mutant and overexpressing plants after salt treatment. The 5day-old seedlings were grown on MS medium containing 100 mM NaCl for different times as indicated, and then the AsA level was measured. The bars represent the SE () of three repeated assays, and the asterisks indicate results that were significantly different from WT as determined by the t-test (**P < 0.01, and *P < 0.05).

supplementation) in the mutant and OX lines, respectively (Figure 9b), which strongly supports the idea that the regulation of AtERF98 in AsA synthesis causes the alteration of the salt response. In addition, we further determined the MDA and chlorophyll content of plants with AsA supplementation under salt treatment. The salt-modulated MDA levels were significantly decreased, and chlorophyll content obviously increased in the AtERF98 mutants as observed in the WT Col-0 after supplementation with exogenous AsA (Figure 9c,d), which further showed that AtERF98 may protect plant cells from ROS-mediated damage by regulating the production of AsA during salt stress. DISCUSSION Ascorbic acid plays pivotal roles in growth, development and the response to stress in plants. The pathways for the synthesis of AsA have been elucidated (Smirnoff and Wheeler, 2000; Hancock and Viola, 2005). Although it was reported that the F-box protein AMR1 negatively regulates AsA synthesis at the transcriptional level by decreasing the expression of the AsA synthesis genes in Arabidopsis (Zhang et al., 2009a), the detailed regulatory mechanism still remains unclear. In the present investigation, we provided further insights into the role of the ERF protein AtERF98 in AsA synthesis through the direct transcriptional modulation of genes related to AsA synthesis in the D-Man/L-Gal path-

way. We also showed that this regulation is critical for saltinduced AsA synthesis, which contributes to enhanced salt tolerance in Arabidopsis. The regulation of ERF proteins in plant growth and development, as well as in the responses of plants to various environmental stresses, has been well documented (Jaglo-Ottosen et al., 1998; Berrocal-Lobo et al., 2002; Zhang et al., 2004, 2011b; Nakano et al., 2006; Yamaguchi-Shinozaki and Shinozaki, 2006; Fukao et al., 2011). For example, CBF1/2/3 can improve the tolerance of Arabidopsis, tomato, tobacco and rice to drought, cold and salt by activating the expression of osmosis-related genes (Jaglo-Ottosen et al., 1998; Yamaguchi-Shinozaki and Shinozaki, 2006). Moreover, the modulation of ERF proteins in the biosynthesis of metabolites, such as ethylene, wax, jasmonate, nicotine and gibberellin, has been reported (van der Fits and Memelink, 2000; Aharoni et al., 2004; Achard et al., 2008; Taketa et al., 2008; Zhang et al., 2009b; De Boer et al., 2011; Li et al., 2011; Wan et al., 2011; Wang et al., 2012). The ERF protein SHINE and its rice homolog OsWR1 enhance the tolerance of plants to drought by influencing wax synthesis (Aharoni et al., 2004; Broun et al., 2004; Kannangara et al., 2007; Wang et al., 2012). In this investigation, we observed that the AsA level was markedly reduced in the AtERF98 knockout and knockdown mutants, whereas the AsA content was obviously increased in the AtERF98-overexpressing transgenic



Figure 9. AtERF98 scavenges reactive oxygen species and enhances tolerance to salt by increasing the level of ascorbic acid (AsA). (a) The phenotypes of wild type (WT), AtERF98 mutant and AtERF98-overexpressing plants under salt stress with or without exogenous AsA. The 7-day-old seedlings were grown on MS medium or MS medium containing salt for 7 days with or without AsA. (b) The survival rates (%) of the plants under salt treatment with or without exogenous AsA. The 7-day-old seedlings plants were treated with salt for 10 days, and then the surviving plants were counted. (c) The malondialdehyde (MDA) content of plants under salt treatment with or without exogenous AsA. (d) The chlorophyll content of plants under salt treatment with or without exogenous AsA. Control indicates seedlings were grown on MS medium; NaCl + AsA and NaCl indicates seedlings were grown on MS medium containing salt with or without 1 lM exogenous AsA, respectively. To analyse the MDA, chlorophyll and total glutathione contents under salt stress, 5-day-old seedlings were grown on MS medium containing salt for another 5 days, and the indicated content was measured. All assays were repeated three times. The bars represent the SE () of three repeated assays. The statistical significance was evaluated using the t-test (**P < 0.01, and *P < 0.05).

lines, which indicates that AtERF98 plays an important role in the regulation of AsA biosynthesis. Increasing evidence has revealed that multiple pathways regulate the biosynthesis of AsA in plants (Loewus et al., 1990; Wheeler et al., 1998; Agius et al., 2003; Lorence et al., 2004; Zhang et al., 2008), but the evidence for the role of some pathways in AsA biosynthesis is controversial. For example, the myo-inositol pathway has been shown to be involved in AsA synthesis in plants (Lorence et al., 2004), but the overexpression of MIOX4 did not affect the AsA content in Arabidopsis (Endres and Tenhaken, 2009). Consistent with reports from Endres and Tenhaken, no obvious difference in the expression of MIOX4 was observed between the AtERF98-overexpressing plants in the Col-0 and vtc1-1 backgrounds but the AsA levels were significantly different, which indicates that the AtERF98-activated expression of MIOX4 does not contribute to the regulation of AsA biosynthesis. The D-Man/L-Gal pathway is the major synthesis pathway for

plant AsA, and the transcript levels of the genes in the D-Man/ L-Gal pathway strictly regulate the synthesis of AsA (Smirnoff

and Wheeler, 2000). In agreement with the changes in the AsA content in Arabidopsis, the transcript levels of the AsA synthesis genes VTC1, VTC2 and VTC4 increased in continuous light (Yabuta et al., 2007). The F-box protein AMR1 regulates AsA synthesis by decreasing the expression of AsA synthesis genes during leaf aging and in response to ozone (Zhang et al., 2009a). The expression of the D-Man/L-Gal pathway genes was increased and decreased consistent with the increasing and decreasing AsA levels in the AtERF98overexpressing Arabidopsis lines and the AtERF98 knockout/ knockdown mutants, respectively. In addition, the transient expression and ChIP assays showed that AtERF98 directly interacts with the DRE-containing region of the VTC1 promoter. An analysis of the cis-elements showed that the promoters of other key genes in the D-Man/L-Gal pathway contain the DRE and/or the GCC box (Table S2), which is


Regulation of AtERF98 in ascorbic acid biosynthesis 283 considered to be identified by ERF proteins. Importantly, although the expression of the AtERF98-activated AsA synthesis genes was not obviously different between the OX lines (AtERF98-overexpressing lines in Col-0 backgrounds) and OE lines (AtERF98-overexpressing lines in the vtc1-1 backgrounds), the AtERF98-enhanced AsA synthesis in OX lines was significantly inhibited in OE lines. In addition to de novo synthesis, recycling could also affect the AsA content (Chen et al., 2003; Stevens et al., 2008). Although the overexpression of AtERF98 promoted the expression of genes related to the AsA-GSH cycle, such as APX3, APX6, ChlDHAR, CytDHAR and GR1, AtERF98 did not affect the expression of these AsA-GSH cycle genes in the AtERF98 mutants. In contrast, the expression of the AsA synthesis genes VTC1 and VTC2 was markedly impaired in the AtERF98 mutants, and the mutation of VTC1 inhibited the increasing AsA production in the AtERF98-overexpression transgenic plants, which indicates that AtERF98 regulates AsA synthesis through the de novo but not the recycling pathway. Taken together, our data clearly show that AtERF98-modulated AsA synthesis occurs primarily through the transcriptional activation of the D-Man/L-Gal pathway genes. Oxidative stress seriously affects the growth and development of plants during periods of abiotic stress (Conklin and Barth, 2004; Foyer and Noctor, 2009). As the most abundant antioxidant, the synthesis and recycling of AsA play vital roles in regulating plant stress responses (Huang et al., 2005; Ushimaru et al., 2006; Wang et al., 2010; Foyer and Noctor, 2011). In plant cells, accumulated ROS can be transformed into H2O2, and APX can scavenge H2O2 by converting it into H2O through AsA. During this process, the AsA is oxidised into MDHA and DHA. This MDHA and DHA can be recycled to AsA by MDAR and DHAR, respectively, through the AsA-GSH recycling pathway. Therefore, increased AsA synthesis and recycling activity can help plants maintain redox homeostasis (Ushimaru et al., 2006; Wang et al., 2010). In fact, under most physiological circumstances, such as photoprotection and the response to stress, the synthesis and recycling of AsA both play important roles in scavenging of ROS (Noctor and Foyer, 1998; Foyer and Noctor, 2011); however, AsA synthesis and recycling seem to have different roles in the plant stress response. For instance, the activity of AsA recycling enzymes did not change in vtc1-1, but the tolerance of the mutant to stresses was much lower than that in the WT plants (Mu¨ller-Moule´ et al., 2004; Huang et al., 2005). In contrast, the overexpression of DHAR did not significantly increase the AsA level, but it enhanced the plants’ tolerance to stress (Ushimaru et al., 2006). As a substrate of AsA peroxidases, AsA can prevent oxidative damage in plants through both the non-enzymatic and enzymatic pathways (Foyer and Noctor, 2011). Therefore, a higher AsA level allows plant cells to avoid damage caused by oxidative stress. High salt stress disturbs the ion balance in plant cells and also promotes the accumulation of

ROS, causing damage to proteins and organelle membranes, including chloroplasts. The recovery of the salt tolerance of aterf98-1 upon the supplementation of exogenous AsA indicates that AtERF98 enhances the salt tolerance of Arabidopsis by mediating the activation of AsA synthesis. Interestingly, increasing numbers of reports have revealed that the ERF proteins, such as Sub1A and JERF3, can scavenge ROS to enhance the stress tolerance of plants through the antioxidant enzyme pathway (Wu et al., 2008; Fukao et al., 2011). Although the overexpression of AtERF98 activated the expression of antioxidant enzyme genes, such as CAT1, the AtERF98 mutants (with decreasing tolerance to salt) did not affect the expression of those genes. In addition, the AtERF98 mutants limited salt-induced AsA synthesis, revealing that the ERF protein AtERF98 plays a key role in AsA synthesis, subsequently scavenging ROS during the salt response. EXPERIMENTAL PROCEDURES Plant materials and growth conditions The mutants and transgenic lines used in this study were derived from the WT Arabidopsis Col-0 ecotype. The homozygous aterf98-1 (SAIL_1142_D01) and aterf98-2 (SAIL_213_E01) mutants were obtained from the ABRC and identified using PCR amplification. Homozygous simple-insert T3 plants were used in this study. For the salt-tolerance assay, germinated seeds from WT, AtERF98 mutants and overexpressing lines were cultured on MS medium for 5 days under normal growth conditions (22C, 14-h light/10-h dark). When the roots grew to approximately 1 cm, the seedlings were transferred to MS medium containing 180 mM NaCl, and cultured for another 5–10 days before the phenotypes were observed. For the measurement of AsA and the extraction of total RNA, the seedlings were grown in soil for 3 weeks, and approximately 0.5 g FW of each sample was harvested. To characterise the expression of AtERF98 induced by ethylene, H2O2 and salt, 3-week-old WT seedlings were grown in soil and sprayed with 100 lM ACC (1-aminocyclopropane-1-carboxylate) or 100 lM H2O2 or soaked with water containing 100 mM NaCl. The samples were then harvested and stored in liquid nitrogen until further use.

Determination of ascorbic acid The qualitative AsA assay utilised NBT as a staining reagent for the visual determination of the level of AsA, as described previously (Conklin et al., 2000). The Arabidopsis leaves were excised, laid on a sheet of Whatman chromatography paper (Whatman, Maidstone, UK) and crushed with a heavy spatula. An aqueous NBT solution (10 ll of 1 mg ml)1; Sigma, St Louis, MO, USA) was pipetted directly onto each squashed leaf. Within 5 min, a bluish-purple formazan precipitate appeared around each sample. The quantitative assays were performed in accordance with the AsA-oxidase-based spectrophotometric assay described by Yabuta et al. (2007). Briefly, AsA extracts were generated from the tissue frozen in liquid nitrogen by grinding in 6% (v/v) perchlorate and centrifuging at 13 000 g for 10 min; 200 ll of the supernatant was added to 1800 ll of sodium succinate (0.2 M, pH 12.7). The AsA content was determined by measuring the optical density at 265 nm after the addition of 5 units of AsA oxidase (Sigma) or 10 ll of 25 mM dithiothreitol and incubation at room temperature (25C) for 30 min, respectively.


284 Zhijin Zhang et al. Reverse transcription PCR and quantitative real-time PCR Total RNA (5 lg samples) was isolated from 3-week-old seedlings from the different plant lines using TRIZOL as recommended by the manufacturer (Tiangen, Beijing, China) and subsequently used for reverse transcription in a 50 ll reaction mixture with a reverse PCR kit (Toyobo, Osaka, Japan). The RT-PCR amplifications were performed in 35 cycles for the expression of AtERF98 and 22 cycles for the internal control, Tubulin4. Quantitative real-time PCR was performed as described previously (Zhang et al., 2009b). All data were normalised to the expression levels of the WT control (internal control Tubulin4), which was standardised to a value of 1. The GenBank accession numbers of the genes and primers used are listed in Table S1.

Generation of transgenic Arabidopsis The full-length ORF of AtERF98 was cloned into the plant expression vector pCAMBIA 1307 using XbaI and SpeI sites. Subsequently, the plants (Col-0, aterf98-1 and vtc1-1 background, respectively) were transformed with an A. tumefaciens LBA4404 strain expressing the constructed vectors using the floral dip method (Li et al., 2011). The transgenic lines were selected with hygromycin and confirmed using PCR amplification. Homozygous single-insertion plants (the segregation ratio of T2 seeds to hygromycin resistance was 3:1, and the T3 seeds did not segregate) were used in this study. The AtERF98-overexpressing lines in Col-0 and vtc1-1 were named OXs and OEs, respectively (the ‘s’ indicates a number representing each specific transgenic line).

Transient expression assay and GUS activity analysis The reporter construct was generated using full-length or shortened VTC1 promoter sequences in pCAMBIA 1381 cloned into the PstI and NcoI sites upstream of the GUS reporter gene. For the transient expression assay, tobacco leaves were co-infiltrated with Agrobacterium containing the reporter vector and the effector vector containing AtERF98 in the plant expression vector pCAMBIA 1307 using the XbaI and SpeI sites. After 36 h of growth in the dark, the infected leaves were used to analyse the activity of GUS. The plant proteins were extracted, and their fluorescence was measured as described by Zhang et al. (2009b). The aliquot at time zero was used as the control, and the fluorescence was measured using a fluorometer (FL500; Kowa, Nagoya, Japan). The protein concentration was determined using a Protein Assay kit (Bio-Rad, Hercules, CA, USA).

Chromatin immunoprecipitation assay For the ChIP assay, the full-length ORF of AtERF98 was cloned into the plant expression vector pCAMBIA 1307 and fused with HA. Subsequently, AtERF98-HA was transformed into Col-0 using the floral dip method. Plants transformed with the pCAMBIA 1307HA vector and expressing the HA tag alone were used as a control. The HA and AtERF98-HA-overexpressing plants were referred to as HA and AtERF98-HA, respectively. Chromatin immunoprecipitation was conducted as described by Li et al. (2011). Briefly, the leaves (1 g) of Arabidopsis plants overexpressing AtERF98-HA and pCAMBIA1307-HA were fixed with 1% formaldehyde at room temperature (25C) for 10 min with gentle agitation. The chromatin solution was then sonicated to shear the DNA into approximately 500-bp fragments. After centrifugation (13 000g), 300 ll of the supernatant was diluted to 3 ml, and 60 ll of protein G agarose/salmon sperm DNA was added (New York, USA). After preclearing at 4C for 1 h, the chromatin was divided into two 1.5-ml aliquots, each containing 30 ll of monoclonal HA immobilised onto Sepharose fast flow beads (HA affinity matrix)

(Covance, Princeton, NJ, USA). After incubation at 4C overnight, the beads were washed with wash buffer, followed by elution with elution buffer. The eluates were subjected to digestion with proteinase K (Merck, Whitehouse, NJ, USA) and RNase. The target DNA fragments were then extracted using phenol and precipitated with ethanol. The PCR analysis was performed using equal amounts of DNA from the input fractions, washes and eluates in 42 cycles using amplimers for the DNA fragments DRE-1 and DRE-2.

Measurement of malondialdehyde and chlorophyll content Malondialdehyde and the chlorophyll content were evaluated in leaves from 5-day-old Arabidopsis plants grown in MS medium (control) or MS medium containing 180 mM NaCl for an additional 5 days. For the exogenous AsA treatment, 1 lM AsA was added to the MS medium. Lipid peroxidation was reflected by the MDA content, which was determined by the thiobarbituric acid (TBA) reaction described by Madhava Rao and Sresty (2000). Briefly, protoplast extraction was performed by homogenising 0.1 g of leaf tissue with 1 ml of 0.1% (w/v) trichloroacetic acid (TCA), and then the homogenate was centrifuged at 10 000 g for 10 min. Then, 4 ml of 20% TCA containing 0.5% TBA was added to the supernatant, and the mixture was boiled at 95C for 15 min, quickly cooled on ice, and centrifuged at 10 000 g for 5 min. Then, the MDA content was determined by measuring the optical density at 532 and 600 nm and was calculated using the extinction coefficient reported by Madhava Rao and Sresty (2000). Chlorophyll was extracted from approximately 0.1 g of leaves directly into 100% dimethyl formamide at a ratio of 5% (w/v). The absorbance of the extracts at 664 and 647 nm was measured using a spectrophotometer. The total chlorophyll (chlorophyll a + chlorophyll b) was determined as described by Aono et al. (1993).

Analysis of proline, soluble sugar, and glutathione content and Na+/K+ The seedlings growing in soil were used to measure the proline, soluble sugar and glutathione content and analyse the Na+/K+. For the salt treatment, 3-week-old seedlings growing in soil were watered, followed by treatment with 100 mM NaCl for 5 days, and then 180 mM NaCl for an additional 5 days. Subsequently, the leaves were collected and used to measure the proline, soluble sugar, glutathione, Na+ and K+ contents. For the determination of proline, a 0.5 g sample was homogenised in 2 ml of 3% aqueous sulphosalicylic acid and centrifuged at 5000 g for 5 min. The proline content was determined as described by Bates et al. (1973). To measure the soluble sugar content, a 0.1 g sample was dried and homogenised and then the soluble sugars were extracted with 80% ethanol and collected by centrifugation at 6000 g for 10 min. The soluble sugar content was estimated according to Mandre et al. (2002). To measure the total glutathione content, 0.5 g of Arabidopsis leaves was ground in liquid nitrogen. The glutathione content was determined by spectrophotometry according to Huang et al. (2005). The samples were homogenised with 0.1% TCA (pH 2.8), 1 mM EDTA, and 1% polyvinylpolypyrrolidone (PVPP) and centrifuged at 10 000 g for 10 min at 4C. The supernatant was mixed with 500 mM 2-amino2-(hydroxymethyl)-1,3-propanediol (TRIS)–HCl (pH 8.0) buffer containing 10 mM 5,50-dithio-bis (2-nitrobenzoic acid) (DTNB) or 500 mM TRIS–HCl (pH 8.0) buffer containing 1 U glutathione reductase (Sigma), 1 mM EDTA, 3 mM MgCl2 and 150 lM NADPH for 15 min, respectively. The total glutathione content was determined at 412 nm. To measure the contents of Na+ and K+, the samples were dried at 105C for 1 h, followed by 80C for 12 h, and the dry weight was determined. Then Na+ and K+ were extracted with 10 ml of 0.1 M HCl after ashing using a muffle furnace for 5 h. The Na+ and


Regulation of AtERF98 in ascorbic acid biosynthesis 285 K+ contents were determined by a Z-5000 Polarized Zeeman Atomic Absorption Spectrophotometer (Hitachi, Tokyo, Japan) according to Xu et al. (2006).

Staining of H2O2 To analyse the accumulation of H2O2 in the leaves under salt treatment, the histological staining of H2O2 was performed according to the method described by Thordal-Christensen et al. (1997) with minor modifications. Briefly, the excised leaves were incubated in 1% diaminobenzidine (DAB)-HCl, pH 3.8, in the dark at room temperature (25C) for 24 h. After boiling in ethanol (80%, v/v) for 10 min to remove the chlorophyll, the H2O2 appeared as a reddishbrown spot because of the instant polymerisation of DAB.

ACKNOWLEDGEMENTS This work was financially supported through the National Basic Research Program of China (2007CB108800 and 2012CB114204), the National Science Foundation of China (31070270) and the Grant Special Program of Transgenic Plants and Animals in China (2009ZX08009-073B and 2011ZX08001-002).

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Figure S1. Expression of oxidation-related genes and ascorbic acid levels in wild type and TERF1- and JERF3-overexpressing tobacco plants. Figure S2. AtERF98 expression and ascorbic acid content in AtERF98-overexpressing lines with a Col-0 background. Figure S3. The level of ascorbic acid recycling gene transcripts in wild type and AtERF98-overexpressing plants. Figure S4. AtERF98 expression and ascorbic acid content in AtERF98-overexpressing lines in the vtc1-1 background. Figure S5. The soluble sugar, proline and Na+/K+ contents in wild type, AtERF98-overexpressing and mutant plants. Figure S6. Expression of reactive oxygen species-scavenging genes in wild type, AtERF98-overexpressing and AtERF98 mutant plants. Figure S7. The ascorbic acid (AsA) level in Arabidopsis leaves under salt treatment. Figure S8. Total glutathione content in wild type, AtERF98 mutant and AtERF98-overexpressing plants under salt treatment. Table S1. Accession numbers and primers used in the study. Table S2. Analysis of potential cis-elements identified by the ethylene response factor proteins in the promoters of key genes in the D-mannose/L-galactose pathway. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

REFERENCES Achard, P., Gong, F., Cheminant, S., Alioua, M., Hedden, P. and Genschik, P. (2008) The cold-inducible CBF1 factor-dependent signaling pathway modulates the accumulation of the growth-repressing DELLA proteins via its effect on gibberellin metabolism. Plant Cell, 20, 2117–2129. Agius, F., Gonza¢lez-Lamonthe, R., Caballero, J.L., Mun~oz-Blanco, J., Botella, M.A. and Valpuesta, V. (2003) Engineering increased vitamin C levels in plants by over-expression of a D-galacturonic acid reductase. Nat. Biotechnol. 21, 177–181. Aharoni, A., Dixit, S., Jetter, R., Thoenes, E., van Arkel, G. and Pereira, A. (2004) The SHINE clade of AP2 domain transcription factors activates wax

biosynthesis, alters cuticle properties, and confers drought tolerance when overexpressed in Arabidopsis. Plant Cell, 19, 2866–2885. Aono, M., Kubo, A., Saji, H., Tanaka, K. and Kondo, N. (1993) Enhanced tolerance to photooxidative stress of transgenic Nicotiana tabacum with high chloroplastic glutathione reductase activity. Plant Cell Physiol. 34, 129–136. Bates, L.S., Waldren, R.P. and Teare, I.D. (1973) Rapid determination of free proline for water-stress studies. Plant Soil, 39, 205–207. Berrocal-Lobo, M., Molina, A. and Solano, R. (2002) Constitutive expression of ETHYLENE-RESPONSE-FACTOR1 in Arabidopsis confers resistance to several necrotrophic fungi. Plant J. 29, 23–32. Broun, P., Poindexter, P., Osborne, E., Jiang, C.Z. and Riechmann, J.L. (2004) WIN1, a transcriptional activator of epidermal wax accumulation in Arabidopsis. Proc. Natl Acad. Sci. USA, 101, 4706–4711. Chakravarthy, S., Tuori, R.P., D’Ascenzo, M.D., Fobert, P.R., Despres, C. and Martin, G.B. (2003) The tomato transcription factor Pti4 regulates defenserelated gene expression via GCC box and non-GCC box cis elements. Plant Cell, 15, 3033–3050. Chen, Z., Young, T.E., Ling, J., Chang, S.C. and Gallie, D.R. (2003) Increasing vitamin C content of plants through enhanced ascorbate recycling. Proc. Natl Acad. Sci. USA, 100, 3525–3530. Conklin, P.L. and Barth, C. (2004) Ascorbic acid, a familiar small molecule intertwined in the response of plants to ozone, pathogens, and the onset of senescence. Plant, Cell Environ. 27, 959–970. Conklin, P.L., Norris, S.R., Wheeler, G.L., Williams, E.H., Smirnoff, N. and Last, R.L. (1999) Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis. Proc. Natl Acad. Sci. USA, 96, 4198–4203. Conklin, P.L., Saracco, S.A., Norris, S.R. and Last, R.L. (2000) Identification of acid-deficient Arabidopsis thaliana mutants. Genetics, 154, 847–856. Davey, M.W., Gilot, C., Persiau, G., Ostergaard, J., Han, Y., Bauw, G.C. and Van Montagu, M.C. (1999) Ascorbate biosynthesis in Arabidopsis cell suspension culture. Plant Physiol. 121, 535–543. Davey, M.W., Van Montagu, M., Inze´, D., Sanmartin, M., Kanellis, A., Smirnoff, N., Benzie, I.J.J., Strain, J.J., Favell, D. and Fletcher, J. (2000) Plant L-ascorbic acid: chemistry, function, metabolism, bioavailability and effects of processing. J. Sci. Food Agric. 80, 825–860. De Boer, K., Tilleman, S., Pauwels, L., Vanden Bossche, R., De Sutter, V., Vanderhaeghen, R., Hilson, P., Hamill, J.D. and Goossens, A. (2011) APETALA2/ETHYLENE RESPONSE FACTOR and basic helix-loop-helix tobacco transcription factors cooperatively mediate jasmonate-elicited nicotine biosynthesis. Plant J. 66, 1053–1065. Dowdle, J., Ishikawa, T., Gatzek, S., Rolinski, S. and Smirnoff, N. (2007) Two genes in Arabidopsis thaliana encoding GDP-L-galactose phosphorylase are required for ascorbate biosynthesis and seedling viability. Plant J. 52, 673–689. Eltayeb, A.E., Kawano, N., Badawi, G.H., Kaminaka, H., Sanekata, T., Shibahara, T., Inanaga, S. and Tanaka, K. (2007) Overexpression of monodehydroascorbate reductase in transgenic tobacco confers enhanced tolerance to ozone, salt and polyethylene glycol stresses. Planta, 225, 1255–1264. Endres, S. and Tenhaken, R. (2009) Myoinositol oxygenase controls the level of myoinositol in Arabidopsis, but does not increase ascorbic acid. Plant Physiol. 149, 1042–1049. van der Fits, L. and Memelink, J. (2000) ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism. Science, 289, 295–297. van der Fits, L. and Memelink, J. (2001) The jasmonate-inducible AP2/ERFdomain transcription factor ORCA3 activates gene expression via interaction with a jasmonate-responsive promoter element. Plant J. 25, 43–53. Foyer, C.H. and Noctor, G. (2009) Redox regulation in photosynthetic organisms: signaling, acclimation, and practical implications. Antioxid. Redox Signal. 11, 861–905. Foyer, C.H. and Noctor, G. (2011) Ascorbate and glutathione: the heart of the redox hub. Plant Physiol. 155, 2–18. Fukao, T., Yeung, E. and Bailey-Serres, J. (2011) The submergence tolerance regulator SUB1A mediates crosstalk between submergence and drought tolerance in rice. Plant Cell, 23, 412–427. Gatzek, S., Wheeler, G.L. and Smirnoff, N. (2002) Antisense suppression of l-galactose dehydrogenase in Arabidopsis thaliana provides evidence for its role in ascorbate synthesis and reveals light modulated l-galactose synthesis. Plant J. 30, 541–553.


286 Zhijin Zhang et al. Hancock, R.D. and Viola, R. (2005) Biosynthesis and catabolism of L-ascorbic acid in plants. Crit. Rev. Plant Sci. 24, 167–188. Huang, C., He, W., Guo, J., Chang, X., Su, P. and Zhang, L. (2005) Increased sensitivity to salt stress in an ascorbate-deficient Arabidopsis mutant. J. Exp. Bot. 56, 3041–3049. Ioannidi, E., Kalamaki, M.S., Engineer, C., Pateraki, I., Alexandrou, D., Mellidou, I., Giovannonni, J. and Kanellis, A.K. (2009) Expression profiling of ascorbic acid-related genes during tomato fruit development and ripening and in response to stress conditions. J. Exp. Bot. 60, 663–678. Jaglo-Ottosen, K.R., Gilmour, S.J., Zarka, D.G., Schabenberger, O. and Thomashow, M.F. (1998) Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance. Science, 280, 104–106. Jain, A.K. and Nessler, C.L. (2000) Metabolic engineering of an alternative pathway for ascorbic acid biosynthesis in plants. Mol. Breed. 6, 73–78. Kannangara, R., Branigan, C., Liu, Y., Penfield, T., Rao, V., Mouille, G., Ho¨fte, H., Pauly, M., Riechmann, J.L. and Broun, P. (2007) The transcription factor WIN1/SHN1 regulates Cutin biosynthesis in Arabidopsis thaliana. Plant Cell, 19, 1278–1294. Laing, W.A., Bulley, S., Wright, M., Cooney, J., Jensen, D., Barraclough, D. and MacRae, E. (2004) A highly specific L-galactose-1-phosphate phosphatase on the path to ascorbate biosynthesis. Proc. Natl Acad. Sci. USA, 101, 16976–16981. Laing, W.A., Wright, M.A., Cooney, J. and Bulley, S.M. (2007) The missing step if the L-galactose pathway of ascorbate biosynthesis in plants, an L-galactose guanyltransferase, increases leaf ascorbate content. Proc. Natl Acad. Sci. USA, 104, 9534–9539. Li, Z., Zhang, L., Yu, Y., Quan, R., Zhang, Z., Zhang, H. and Huang, R. (2011) The ethylene response factor AtERF11 that is transcriptionally modulated by the bZIP transcription factor HY5 is a crucial repressor for ethylene biosynthesis in Arabidopsis. Plant J. 68, 88–99. Linster, C.L., Gomez, T.A., Christensen, K.C., Adler, L.N., Young, B.D., Brenner, C. and Clarke, S.G. (2007) Arabidopsis VTC2 encodes a GDP-L-galactose phosphorylase, the last unknown enzyme in the Smirnoff-Wheeler pathway to ascorbic acid in plants. J. Biol. Chem. 282, 18879–18885. Linster, C.L., Adler, L.N., Webb, K., Christensen, K.C., Brenner, C. and Clarke, S.G. (2008) A second GDP-L-galactose phosphorylase in Arabidopsis enroute to vitamin C. J. Biol. Chem. 283, 18483–18492. Liu, Q., Kasuga, M., Sakuma, Y., Abe, H., Miura, S., Yamaguchi-Shinozaki, K. and Shinozaki, K. (1998) Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell, 10, 1391–1406. Loewus, M.W., Bedgar, D.L., Saito, K. and Loewus, F.A. (1990) Conversion of L-sorbosone to L-ascorbic acid by a NADP-dependent dehydrogenase in bean and spinach leaf. Plant Physiol. 94, 1492–1495. Lorence, A., Chevone, B.I., Mendes, P. and Nessler, C. (2004) myo-Inositol oxygenase offers a possible entry point into plant AsA biosynthesis. Plant Physiol. 134, 1200–1205. Madhava Rao, K.V. and Sresty, T.V. (2000) Antioxidative parameters in the seedlings of pigeonpea (Cajanus cajan L. Millspaugh) in response to Zn and Ni stresses. Plant Sci. 157, 113–128. Mandre, M., Tullus, H. and Klo˜seiko, J. (2002) Partitioning of carbohydrates and biomass of needles in Scots pine canopy. Z. Naturforsch. C. 57, 296–302. Miller, G.A.D., Suzuki, N., Ciftci-Yilmaz, S. and Mittler, R.O.N. (2010) Reactive oxygen species homeostasis and signalling during drought and salinity stresses. Plant, Cell Environ. 33, 453–467. Mu¨ller-Moule´, P., Golan, T. and Niyogi, K.K. (2004) Ascorbatedeficient mutants of Arabidopsis grow in high light despite chronic photoxidative stress. Plant Physiol. 134, 1163–1172. Munns, R. and Tester, M. (2008) Mechanisms of salinity tolerance. Annu. Rev. Plant Biol. 59, 651–681. Nakano, T., Suzuki, K., Fujimura, T. and Shinshi, H. (2006) Genome-wide analysis of the ERF gene family in Arabidopsis and rice. Plant Physiol. 140, 411–432. Noctor, G. and Foyer, C.H. (1998) Ascorbate and glutathione: keeping oxygen under control. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 249–279. Ohme-Takagi, M. and Shinshi, H. (1995) Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element. Plant Cell, 7, 173–182. Pallanca, J.E. and Smirnoff, N. (1999) Ascorbic acid metabolism in pea seedlings. A comparison of D-glucosone, L-sorbosone, and L-galactono1,4-lactone as ascorbate precursors. Plant Physiol. 20, 453–462.

Riechmann, J.L., Heard, J., Martin, G. et al. (2000) Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Science, 290, 2105–2110. Saito, K., Nick, J.A. and Loewus, F.A. (1990) D-Glucosone and L-sorbosone, putative intermediates of l-ascorbic acid biosynthesis in detached bean and spinach leaves. Plant Physiol. 94, 1496–1500. Sasaki, K., Mitsuhara, I., Seo, S., Ito, H., Matsui, H. and Ohashi, Y. (2007) Two novel AP2/ERF domain proteins interact with cis-element VWRE for wound-induced expression of the Tobacco tpoxN1 gene. Plant J. 50, 1079– 1092. Shalata, A. and Neumann, P.M. (2001) Exogenous ascorbic acid (vitamin C) increases resistance to salt stress and reduces lipid peroxidation. J. Exp. Bot. 52, 2207–2211. Smirnoff, N. and Wheeler, G.L. (2000) Ascorbic acid in plants: biosynthesis and function. Crit. Rev. Biochem. Mol. Biol. 35, 291–314. Smirnoff, N., Conklin, P.L. and Loewus, F.A. (2001) Biosynthesis of ascorbic acid in plants: a renaissance. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 437–467. Stevens, R., Page, D., Gouble, B., Garchery, C., Zamir, D. and Causse, M. (2008) Tomato fruit ascorbic acid content is linked with monodehydroascorbate reductase activity and tolerance to chilling stress. Plant, Cell Environ. 31, 1086–1096. Tajdoost, S., Farboodnia, T. and Heidari, R. (2007) Salt pretreatment enhance salt tolerance in Zea mays L. seedlings. Pak. J. Biol. Sci. 10, 2086– 2090. Taketa, S., Amano, S., Tsujino, Y. et al. (2008) Barley grain with adhering hulls is controlled by an ERF family transcription factor gene regulating a lipid biosynthesis pathway. Proc. Natl Acad. Sci. USA, 105, 4062–4067. Thordal-Christensen, H., Zhang, Z., Wei, Y. and Collinge, D.B. (1997) Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley-powdery mildew interaction. Plant J. 11, 1187–1194. Ushimaru, T., Nakagawa, T., Fujioka, Y., Daicho, K., Naito, M., Yamauchi, Y., Nonaka, H., Amako, K., Yamawaki, K. and Murata, N. (2006) Transgenic Arabidopsis plants expressing the rice dehydroascorbate reductase gene are resistant to salt stress. J. Plant Physiol. 163, 1179–1184. Wan, L., Zhang, J., Zhang, H., Zhang, Z., Quan, R., Zhou, S. and Huang, R. (2011) The transcriptional regulation of OsDERF1 in OsERF3 and OsAP2-39 suppresses ethylene synthesis and decreases drought tolerance in rice. PLoS ONE, 6, e25216. Wang, Z., Xiao, Y., Chen, W., Tang, K. and Zhang, L. (2010) Increased vitamin C content accompanied by an enhanced recycling pathway confers oxidative stress tolerance in Arabidopsis. J. Integr. Plant Biol. 52, 400–409. Wang, Y., Wan, L., Zhang, L., Zhang, Z., Zhang, H., Quan, R., Zhou, S. and Huang, R. (2012) An ethylene response factor OsWR1 responsible to drought stress transcriptionally activates wax synthesis related genes and increases wax production in rice. Plant Mol. Biol. 8, 275–288. Wheeler, G.L., Jones, M.A. and Smirnoff, N. (1998) The biosynthetic pathway of vitamin C in higher plants. Nature, 393, 365–369. Wolucka, B.A. and Van Montagu, M. (2003) GDP-mannose 3’,5’-epimerase forms GDP-L-gulose, a putative intermediate for the de novo biosynthesis of vitamin C in plants. J. Biol. Chem. 278, 47483–47490. Wu, L., Zhang, Z., Zhang, H., Wang, X.C. and Huang, R. (2008) Transcriptional modulation of ethylene response factor protein JERF3 in the oxidative stress response enhances tolerance of tobacco seedlings to salt, drought, and freezing. Plant Physiol. 148, 1953–1963. Xu, J., Li, H.D., Chen, L.Q., Wang, Y., Liu, L.L., He, L. and Wu, W.H. (2006) A protein kinase, interacting with two calcineurin B-like proteins, regulates K+ transporter AKT1 in Arabidopsis. Cell, 125, 1347–1360. Yabuta, Y., Mieda, T., Rapolu, M., Nakamura, A., Motoki, T., Maruta, T., Yoshimura, K., Ishikawa, T. and Shigeoka, S. (2007) Light regulation of ascorbate biosynthesis is dependent on the photosynthetic electron transport chain but independent of sugars in Arabidopsis. J. Exp. Bot. 58, 2661–2671. Yamaguchi-Shinozaki, K. and Shinozaki, K. (2006) Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annu. Rev. Plant Biol. 57, 781–803. Yang, J.S., Wang, R., Meng, J.J., Bi, Y.P., Xu, P.L., Guo, F., Wan, S.B., He, Q.W. and Li, X.G. (2010) Overexpression of Arabidopsis CBF1 gene in transgenic tobacco alleviates photoinhibition of PSII and PSI during chilling stress under low irradiance. J. Plant Physiol. 167, 534–539.


Regulation of AtERF98 in ascorbic acid biosynthesis 287 Zhang, Z. and Huang, R. (2010) Enhanced tolerance to freezing in tobacco and tomato overexpressing transcription factor TERF2/LeERF2 is modulated by ethylene biosynthesis. Plant Mol. Biol. 73, 241–249. Zhang, H., Zhang, D., Chen, J., Yang, Y., Huang, Z., Huang, D., Wang, X.C. and Huang, R. (2004) Tomato stress-responsive factor TSRF1 interacts with ethylene responsive element GCC box and regulates pathogen resistance to Ralstonia solanacearum. Plant Mol. Biol. 55, 825–834. Zhang, X., Zhang, Z., Chen, J., Chen, Q., Wang, X.C. and Huang, R. (2005) Expressing TERF1 in tobacco enhances drought tolerance and abscisic acid sensitivity during seedling development. Planta, 222, 494–501. Zhang, W., Gruszewski, H.A., Chevone, B.I. and Nessler, C.L. (2008) An Arabidopsis purple acid phosphatase with phytase activity increases foliar ascorbate. Plant Physiol. 146, 431–440. Zhang, W., Lorence, A., Gruszewski, H.A., Chevone, B.I. and Nessler, C.L. (2009a) AMR1, an Arabidopsis gene that coordinately and negatively regulates the mannose/l-galactose ascorbic acid biosynthetic pathway. Plant Physiol. 150, 942–950.

Zhang, Z., Zhang, H., Quan, R., Wang, X.C. and Huang, R. (2009b) Transcriptional regulation of the ethylene response factor LeERF2 in the expression of ethylene biosynthesis genes controls ethylene production in tomato and tobacco. Plant Physiol. 150, 365–377. Zhang, C., Liu, J., Zhang, Y., Cai, X., Gong, P., Zhang, J., Wang, T., Li, H. and Ye, Z. (2011a) Overexpression of SlGMEs leads to ascorbate accumulation with enhanced oxidative stress, cold, and salt tolerance in tomato. Plant Cell Rep. 30, 389–398. Zhang, L.X., Li, Z.F., Quan, R.D., Li, G.J., Wang, R.G. and Huang, R. (2011b) An AP2 domain-containing gene ESE1 targeted by ethylene signaling component EIN3 is important for salt response in Arabidopsis thaliana. Plant Physiol. 157, 854–865. Zhuang, J., Chen, J.M., Yao, Q.H., Xiong, F., Sun, C.C., Zhou, X.R., Zhang, J. and Xiong, A.S. (2011) Discovery and expression profile analysis of AP2/ERF family genes from Triticum aestivum. Mol. Biol. Rep. 38, 745– 753.
