Functions and Application of the AP2/ERF Transcription Factor Family in Crop Improvement


  • Zhao-Shi Xu,

    1. National Key Facility of Crop Gene Resources and Genetic Improvement (NFCRI), Key Laboratory of Crop Genetics and Breeding, Ministry of Agriculture, Institute of Crop Science, Chinese Academy of Agriculture Sciences (CAAS), Beijing 100081, China
    Search for more papers by this author
  • Ming Chen,

    1. National Key Facility of Crop Gene Resources and Genetic Improvement (NFCRI), Key Laboratory of Crop Genetics and Breeding, Ministry of Agriculture, Institute of Crop Science, Chinese Academy of Agriculture Sciences (CAAS), Beijing 100081, China
    Search for more papers by this author
  • Lian-Cheng Li,

    1. National Key Facility of Crop Gene Resources and Genetic Improvement (NFCRI), Key Laboratory of Crop Genetics and Breeding, Ministry of Agriculture, Institute of Crop Science, Chinese Academy of Agriculture Sciences (CAAS), Beijing 100081, China
    Search for more papers by this author
  • You-Zhi Ma

    Corresponding author
    1. National Key Facility of Crop Gene Resources and Genetic Improvement (NFCRI), Key Laboratory of Crop Genetics and Breeding, Ministry of Agriculture, Institute of Crop Science, Chinese Academy of Agriculture Sciences (CAAS), Beijing 100081, China
    Search for more papers by this author

Tel: +86 10 8210 9718; Fax: +86 10 8210 8798; E-mail:


inline imageYou-Zhi Ma
(Corresponding author)

Plants have acquired sophisticated stress response systems to adapt to changing environments. It is important to understand plants’ stress response mechanisms in the effort to improve crop productivity under stressful conditions. The AP2/ERF transcription factors are known to regulate diverse processes of plant development and stress responses. In this study, the molecular characteristics and biological functions of AP2/ERFs in a variety of plant species were analyzed. AP2/ERFs, especially those in DREB and ERF subfamilies, are ideal candidates for crop improvement because their overexpression enhances tolerances to drought, salt, freezing, as well as resistances to multiple diseases in the transgenic plants. The comprehensive analysis of physiological functions is useful in elucidating the biological roles of AP2/ERF family genes in gene interaction, pathway regulation, and defense response under stress environments, which should provide new opportunities for the crop tolerance engineering.


Environmental stresses, such as drought, high-salt and low-temperature, as well as pathogen and pest attacks, have adverse effects on plant growth and crop yields. These effects are caused by altered morphology and physiology, resulting from changes to processes, such as inhibition of cell division and effects on metabolism. To survive, it is hypothesized that plants have evolved a complex and elaborate signaling network that perceives signals from their surroundings and to appropriately respond to continuously changing surroundings by modulating the expression of responsive genes. It is important to investigate plants’ stress responses to improve crop productivity under stressful conditions.

Transcription factors play pivotal functions in signal transduction to activate or suppress defense gene expression, as well as in the regulation of interaction between different signaling pathways. Transcription factors, positioned at the penultimate step in signal cascade, directly regulated downstream target gene expression by virtue of DNA-binding with cis-acting elements in the promoters (Figure 1). Particular environmental conditions may affect specific mechanisms. Responses and adaptations require differential gene expression, which is regulated by specific transcription factors. More than 1,500 genes encode transcription factors in the Arabidopsis genome, e.g. NAC, b-ZIP, Myb/c, WRKY and AP2/ERF transcription factors (Riechmann et al. 2000). Among them, the AP2/ERF family has received much attention in the past decade.

Figure 1.

Abiotic and biotic stress signal transduction pathway in plants.
TFs, transcription factors.

It was hypothesized that a horizontal transfer of an HNH-AP2 endonuclease from bacteria or viruses into plants led to the origin of the AP2/ERF family (Magnani et al. 2004; Shigyo et al. 2006). AP2/ERF transcription factors are characterized by the presence of AP2/ERF DNA-binding domains that directly interact with GCC box and/or dehydration-responsive element (DRE)/C-repeat element (CRT) cis-acting elements at the promoter of downstream target genes. Sakuma et al. (2002) classified AP2/ERF transcription factors into five subfamilies: AP2 (APETALA2), RAV (related to ABI3/VP1), DREB (dehydration-responsive element binding protein), ERF (ethylene-responsive factor), and others, according to the number and similarity of the DNA-binding domains.

A variety of AP2/ERF genes were successfully identified and investigated in angiosperms (including both monocotyledonous and dicotyledonous types), gymnosperms and microorganisms (encompassing cyanobacteria, ciliates, and viruses) (Magnani et al. 2004; Shigyo et al. 2006; Xu et al. 2008a). We isolated several DRE/ERFs from wheat (Triticum aestivum), barley (Hordeum vulgare), and soybean (Glycine max) (Xu et al. 2007, 2008b; Zhang et al. 2008, 2009). The AP2/ERF transcription factors were shown to regulate diverse processes of plant development and stress responses, such as vegetative and reproductive development, cell proliferation, abiotic and biotic stress responses, and plant hormone responses (Nakano et al. 2006; Licausi et al. 2010; Sharoni et al. 2011). Therefore, it is important to elucidate the mechanisms underlying the transmission of stress signals in order to ultimately manipulate AP2/ERF regulation for improving crop stress resistance. This review paper summarizes the latest progress in the molecular functions of AP2/ERF transcription factors and their potential application in crop tolerance improvement.

Divergent sets of defense genes regulated by AP2/ERFs

DREBs and ERFs are two major subfamilies of the AP2/ERF family and play crucial roles in the regulation of abiotic- and biotic-stress responses. Commonly, DREB transcription factors activate multiple dehydration/cold-regulated (RD/COR) genes by interacting with DRE/CRT elements (A/GCCGAC) present in the promoters of RD/COR genes that are responsive to both water deficit and low-temperature, such as COR15A, RD29A/COR78, and COR6.6 (Stockinger et al. 1997; Liu et al. 1998; Lucas et al. 2011). ERF transcription factors directly regulate pathogenesis-related (PR) gene expression by virtue of DNA-binding with the GCC-box (GCCGCC), such as prb-1b (PR1), β-1, 3-glucanase (PR2), chitinase (PR3), and osmotin (PR5) (Ohme-Takagi and Shinshi 1995; Buttner and Singh 1997; Zarei et al. 2011). However, the flanking sequences that extend beyond the AP2/ERF domain affected binding activity to cis-acting elements (Tournier et al. 2003; Canella et al. 2010) although two conserved amino acid residues, 14th Val (V)/Ala (A) and 19th Glu (E)/Asp (D) in the β-sheet of AP2/ERF domains, play important roles in the recognition of cis-acting elements (Sakuma et al. 2002). For example, the barley DREB protein HvDRF1 bound preferably to a CT-rich element (T(T/A)ACCGCCTT), rather than DRE/CRT motif (Xue and Loveridge 2004). It was reported that changes of Gly156 to arginine and Phe62 to serine increased the GCC-box binding activity of Brassica napus ERF-B3-hy15 protein (Jin et al. 2010). Tobacco ERF factor ORC1 required the presence of both GCC-motif (TGCGCCC) and G-box (GCACGTTG) elements in the promoters of the tobacco nicotine biosynthesis genes for maximum transactivation (De Boer et al. 2011). Therefore, it is possible that different DREB/ERF transcription factors are involved in regulation of different downstream target genes.

Recently, Sun et al. (2008) reported that TINY, a DREB-like factor, was capable of binding to both DRE and GCC-box with similar affinity and could activate the expression of both the DRE- and the GCC-box-containing genes. Interestingly, several ERF proteins also bound to the DRE/CRT element besides GCC-box in vitro. Moreover, overexpression of these ERF genes resulted in an elevated expression of the DRE/CRT-containing RD/COR genes (reviewed by Xu et al. 2008a). These factors binding to the DRE/CRT element are mostly from ERF Cluster VII, except for the Tsi1 protein that belongs to Cluster V (Xu et al. 2008a). Accumulating data show that some ERFs participate in other cis-element regulations. For example, ERN (ERF for required nodulation) combined specifically to the NF-box (Andriankaja et al. 2007). Arabidopsis RAP2.6 is able to bind to the GCC-box and CE1 element (TGCCACCGG) (Zhu et al. 2010). Lee et al. (2010c) demonstrated that a group of Arabidopsis AP2/ERFs, especially those belonging to the subgroup B-3, were able to bind the CE1 element. Therefore, ERFs could be involved in the transduction of various signals and regulation of divergent sets of downstream defense genes or developmental genes in response to stresses.

AP2 subfamily has been reported to bind the element GCAC(A/G)N(A/T)TCCC(A/G)ANG(C/T) (Nole-Wilson and Krizek 2000; Gong et al. 2007). This DNA element is quite distinct from the consensus sequence CCGA/CC bound by DREB and ERF subfamilies and, surprisingly, is not composed of two similar half sites, since members of the AP2 subfamily contain a double AP2/ERF domain (Krizek 2003). Members of RAV subfamily contain an AP2/ERF domain in C-terminal regions and a B3-domain in N-terminal regions. Pepper CARAV1 could recognize and bind to CAACA and CACCTG motifs and activate the reporter gene in yeast (Sohn et al. 2006).

Microarray technologies and transcriptome analysis are powerful global analysis tools for discovering many stress-inducible genes involved in stress response and tolerance. Transcriptome analysis of transgenic plants is expected to uncover novel stress regulated genes driven by cis-elements. Our results of transcriptome analysis of transgenic wheat plants provided preliminarily evidences that AP2/ERF maybe participate in more cis-element regulations to activate more genes, including heat shock protein 70 (HSP70) and lipid transfer protein (LTP) (ZS Xu et al. unpubl. data).

Post-translational modification and interaction of AP2/ERFs with other proteins

Transcriptional and post-transcriptional regulation plays an essential role in the adaptation of cellular functions to the environmental changes (Saibo et al. 2009; Walley and Dehesh 2010). The activity of DREB-2 type transcription factors was reported to be subject to post-transcriptional modification or phosphorylation responses. For example, overexpression of Arabidopsis AtDREB2A and rice OsDREB2A did not activate downstream genes under normal growth conditions, suggesting that AtDREB2A and OsDREB2A require post-translational modification for activation (Liu et al. 1998; Dubouzet et al. 2003). Recently, Agarwal et al. (2007) showed that phosphorylated PgDREB2A could not bind to DRE/CRT elements. Sakuma et al. (2006a) found that deletion of a region between residues 136 and 165, containing a Ser/Thr-rich motif, led to a constitutively active form of AtDREB2A. We isolated a wheat DREB gene TaAIDFa, whch encodes a protein lacking a Ser/Thr-rich region (a putative phosphorylation site) relative to TaAIDFb/c. Overexpression of TaAIDFa activated transcription of RD29A, COR15A, and ERD10 in transgenic Arabidopsis plants. Therefore, the putative phosphorylation sites on these DREB-2 type proteins are possible motifs for negative regulation (Xu et al. 2008b). However, to date, no DREB-2 type factors phosphorylated by a Ser/Thr protein kinase have been isolated. Therefore, whether Ser/Thr-rich region is involved in phosphorylation suppression requires further investigation due to lack of rigorous demonstration.

On the contrary, the phosphorylation by Ser/Thr protein kinase Pto and mitogen-activated protein kinase (MAPK) BWMK1 enhanced DNA-binding activity of ERFs Pti4 and OsEREBP1, respectively (Gu et al. 2000; Cheong et al. 2003). The activity of ORC1 was post-translationally upregulated by a jasmonic acid (JA)-modulated MAPK phosphorylation cascade, and possibly other kinases (De Boer et al. 2011). Similarly, we showed that the interaction mediated by wheat MAPK protein TaMAPK1 strengthened the activation activity of TaERF1 to reporter genes (Xu et al. 2007). These ERFs commonly contain Ser/Thr-rich regions or phosphorylation motifs. Therefore, putative phosphorylation sites in these ERFs play a positive role in regulation of activation activity (Xu et al. 2008b).

Signal transduction and target gene regulation are involved in a complex signaling network. SIZ1-dependent sumoylation of ICE1 may activate and/or stabilize the protein, facilitating expression of CBF3/DREB1A and repression of MYB15, initiating many changes in gene expression, and leading to low temperature tolerance in Arabidopsis (Miura et al. 2007). At present, DREB-mediated signal transduction mechanism and pathway is not clear. Some DREBs/ERFs regulate gene expression indirectly by either interacting physically with other proteins or activating transcription factors (Table 1). DBF1 interacted with DBF1-interactor protein 1 (DIP1), a protein containing a conserved R3H single-strand DNA-binding domain, enhances maize rab17 promoter activity, and control the levels of target gene expression during stress conditions (Saleh et al. 2006). Interaction of grape VvDREB with ASR transcription factor VvMSA in the nucleus regulating the expression of a glucose transporter (Saumonneau et al. 2008). ORC1 and a basic helix-loop-helix (bHLH) factor cooperatively mediate JA-elicited nicotine biosynthesis in tobacco (De Boer et al. 2011). Schramm et al. (2008) found that overexpression of DREB2A directly regulated the expression of heat-shock transcription factor HsfA3 that activated expression of many heat-inducible genes in the transcriptional cascade. Our results showed that the ERF factor W17 interacted with HSP90 and PPR proteins in vivo (Qiu et al. 2011). Therefore, DREBs/ERFs provide signal transduction and gene modulation of adequate multiplicity and complexity to permit subtle responses to environmental stresses.

Table 1. Interaction mechanisms of DREBs/ERFs with other proteins
SubfamiyTarget proteinInteractive proteinSource organismInteraction mechanismReference
 DBF1DIP1 (containing a R3H single-strand DNA-binding domain)Maize (Zea mays)Interaction enhanced maize rab17 promoter activity and control the levels of target gene expression during stress conditionsSaleh et al. 2006
CBF3/DREB1AICE1 (MYC TF)ArabidopsisE3 ligase-mediated sumoylation of ICE1 may activate and/or stabilize the protein, facilitating expression of CBF3/DREB1AMiura et al. 2007
VvDREBVvMSA (ASR TF)Grape (Vitis vinifera)Interaction regulated the expression of a glucose transporterSaumonneau et al. 2008
DREB2AHsfA3 (HSF TF)ArabidopsisDREB2A directly regulated the expression of HsfA3 that activated heat-inducible genes in the transcriptional cascade.Schramm et al. 2008
DREB1A/CBF3, DREB2A, and DREB2CABF2 (bZIP TF)ArabidopsisPhysically interaction may control ABA-regulated gene expressionLee et al. 2010b
 EREBP2/EREBP3NLP (enzyme)Tobacco (Nicotiana tabacum)Interaction of EREBP2/EREBP3 with NLP regulated PR genesXu et al. 1998
Pti4Pto (Ser/Thr protein kinase)Tomato (Solanum lycopersicum)The phosphorylation enhanced DNA-binding activityGu et al. 2000
OsEREBP1BWMK1 (MAPK kinase)Rice (Oryza sativa)The phosphorylation enhanced DNA-binding activityCheong et al. 2003
OsEBP-89OsBP-5 (MYC TF)RiceThe interaction synergistically regulates the transcription of the waxy geneZhu et al. 2003
ERF3NtUBC2 (ubiquitin-conjugating enzyme)TobaccoThe repression activity of ERF3 was enhanced by the dominant-negative inhibition of ubiquitin-conjugation activity of NtUBC2Koyama et al. 2003
AtEBPOBF4 (bZIP TF), ACBP2 (acyl-CoA-binding protein)ArabidopsisACBP2 interacted with AtEBP via its ankyrin repeatsButtner and Singh 1997; Li and Chye 2004
AtERF7PKS3 (Ser/Thr protein kinase)ArabidopsisAtERF7 was phosphorylated by kinase PKS3 in vitro, suggesting that AtERF7 might be a substrate of PKS3Song et al. 2005
AtERFAtSAP18 (Sin3A-associated protein)ArabidopsisAssociation of AtERF with AtSAP18 and histone deacetylase HDA19Song and Galbraith 2006
TaERF1TaMAPK1 (MAPK kinase)Wheat (Triticum aestivum)The interaction strengthened the activation activity of TaERF1Xu et al. 2007
ORC1MAPK or other kinasesTobaccoA JA-modulated phosphorylation cascade upregulated the activity of ORC1De Boer et al. 2011
ORC1bHLH factorTobaccoCooperatively mediated JA-elicited nicotine biosynthesisDe Boer et al. 2011

The regulation of AP2/ERFs has been extensively analyzed in the past few years, but information is lacking on post-translational regulation. Therefore, it is imperative to isolate and identify interacting proteins for further investigation of the resistant mechanism of transcription factors. We preliminarily demonstrated that the DREB factor W18 may be involved in the E2 ubiquitin pathway and calcium-dependent protein kinase (CDPK)-mediated signal transduction pathways. The CDPK receives the Ca2+ signal and activates W18 which regulates the expression of the downstream stress-related genes under stress conditions. When plant does not need overmuch DREB proteins, E2-Ub/E3 interacted with W18 to form an ubiquitinated complex which will be degraded by 26S proteasome (H.T. Sun et al. unpubl. data). Interaction with different factors provides a possibility that AP2/ERFs function in many biological processes. More interacting proteins are expected to be identified to provide an overview of the biological roles of AP2/ERF family genes in signal transduction, gene regulation, and defense response under stress environments. However, knowledge of interacting proteins of AP2/ERFs is still limited.

Recent studies indicate that molecular dynamics as specific homodimerizations and heterodimerizations as well as modular flexibility and posttranslational modifications determine the functional specificity of transcription factors in environmental adaptation (reviewed by Golldack et al. 2011). Some transcription factors may be modulated by miRNA-mediated posttranscriptional silencing and reactive oxygen species signaling. In addition, DNA methylation and posttranslational modifications of histones highly influence the efficiency of stress-induced gene expression (reviewed by Golldack et al. 2011). Interactions of different factors will be essential for targeted and efficient genetic engineering of improved stress tolerance in plants. Therefore, investigation in this area is urgently required.

DREBs used as prominent candidates for enhancing crop abiotic stress tolerance

Accumulation of abscisic acid (ABA) plays an important role in abiotic stress signal transduction pathways, mediating a number of key growth and physiological processes in plants, including suppression in seed germination, induction of stomatal closure thereby minimizing transpiration to prevent water loss, and acceleration of abscission and senescence (Wasilewska et al. 2008). It is now evident that ABA production is enhanced under water limited conditions and can effectively protect plants against drought stress (Xiong et al. 2002; Ashraf 2010; Guo et al. 2011). Various transcription factors, including AP2/ERFs, have been reported to engage in ABA-mediated gene expression (Fujita et al. 2011). Recently, DREB1A/CBF3, DREB2A, and DREB2C proteins have been reported to physically interact with AREB/ABF proteins (Lee et al. 2010b), which supports the view that DREB/CBFs and AREB/ABFs may interact to control ABA-regulated gene expression.

DREB genes display responses to exogenous ABA, such as barley HvDRF1 and wheat TaDREB2 and TaAIDFa (Xue and Loveridge 2004; Egawa et al. 2006; Xu et al. 2008b). ABA was shown to be involved in the regulation of DREB activity and increased promoter activity (Kizis and Pages 2002; Xu et al. 2008b). In addition, several DREBs have been reported to be positive and negative mediators of ABA and sugar responses, mainly during germination and the early seedling stage (reviewed by Fujita et al. 2011). Recently, it was demonstrated that the CE1-like CACCG motif in the ABI5 promoter is required for ABI4 transactivation in Arabidopsis protoplasts (Bossi et al. 2009). Therefore, ABA may play an important role in regulating the transcriptional activity of DREBs.

DREBs show variation in some conserved motifs and biological functions in divergent species, and are dichotomized as DREB1- and DREB2-types, which are involved in separate signal transduction pathways under abiotic stresses (Dubouzet et al. 2003). Expression of DREB1-type genes was specifically induced by low-temperature stress in Arabidopsis and rice (Liu et al. 1998; Sakuma et al. 2002; Dubouzet et al. 2003; Lucas et al. 2011). In contrast, DREB2-type genes respond to dehydration and high-salt stresses (Liu et al. 1998; Dubouzet et al. 2003). Recently, a DREB1-type gene, Ca-DREBLP1, was found to be induced by water-deficit and salt stresses in hot pepper, more like the expression patterns of DREB2-type genes (Hong and Kim 2005). Other DREB1-type genes, such as soybean GmDREB2, moss PpDREB1, and Caragana korshinskii CkDREB, were induced by drought, high-salt and low-temperature stresses, and by ABA treatment (Chen et al. 2007; Liu et al. 2007; Wang et al. 2011). The maize DREB2-type gene, ZmDREB2A, was shown to accumulate under heat stress in seedling stage (Qin et al. 2007). Therefore, it is possible that DREBs belonging to the same type show different response patterns under environmental stresses.

Many DREB1-type genes inserted into plants by transformation were capable of improving multiple abiotic tolerances in agricultural crops including tobacco (Kasuga et al. 2004), wheat (Pellegrineschi et al. 2004), rice (Dubouzet et al. 2003; Oh et al. 2005; Ito et al. 2006), chrysanthemum (Hong et al. 2006a, b, c), potato (Pino et al. 2008), and C. korshinskii (Wang et al. 2011) (Table 2). For example, DREB1A driven by the rd29A promoter resulted in enhanced drought tolerance in wheat plants (Pellegrineschi et al. 2004). Similarly, peanut plants transformed with rd29A:DREB1A had higher transpiration efficiency and accumulated considerably higher levels of some key antioxidant enzymes and proline content than the wild type under drought stress (Bhatnagar-Mathur et al. 2007, 2009). Overexpression of DREB1A in rice plants resulted in improved tolerance to drought and salinity (Oh et al. 2005). We obtained cotton GhDREB transgenic wheat that had improved tolerance to drought, high salt, and freezing stresses through accumulating higher levels of soluble sugar and chlorophyll in leaves after stress treatments. No phenotypic differences were observed between transgenic plants and their non-transgenic controls, which suggested that GhDREB might be used to improve wheat stress tolerance through genetic engineering (Gao et al. 2009).

Table 2. Improving industrial and food crop abiotic stress tolerance through engineering genes for DREB transcription factors
Gene engineeredTransgenic hostSource organismTrait improvedReference
DREB1ATomato (Solanum lycopersicum)ArabidopsisConferred elevated tolerance to chilling and oxidative stressesHsieh et al. 2002
DREB1ATobacco (Nicotiana tabacum)ArabidopsisImproved drought and low-temperature stress tolerance (driven by RD29A promoter)Kasuga et al. 2004
DREB1AWheat (Triticum aestivum)ArabidopsisDelayed water stress symptoms under greenhouse conditions (driven by stress-inducible RD29A promoter)Pellegrineschi et al. 2004
DREB1ARice (Oryza sativa)ArabidopsisIncreased tolerance to abiotic stress without stunting growthOh et al. 2005
DREB1AChrysanthemum (Dendranthema vestitum)ArabidopsisEnhanced drought, salt and low temperature toleranceHong et al. 2006a, b, c
DREB1AChrysanthemumArabidopsisEnhanced heat stress toleranceHong et al. 2009
DREB1ATall fescue (Festuca arundinacea Schreb.)ArabidopsisIncreased resistance to drought and accumulation of high prolineZhao et al. 2007
DREB1APeanut (Arachis hypogaea)ArabidopsisEnhanced activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase and enhanced proline level in the transgenic plants under drought stressBhatnagar-Mathur et al. 2009
DREB1CMedicago truncatula and China RoseM. truncatulaEnhanced freezing toleranceChen et al. 2010
AtCBF1Potato (Solanum tuberosum)ArabidopsisEnhanced freezing tolerance and induces cold acclimation-associated physiological modificationsPino et al. 2008
CkDREBTobaccoCaragana korshinskiiEnhanced tolerance to high salinity and osmotic stressesWang et al. 2011
OsDREB1RiceRiceImproved tolerance to drought, high-salt and low-temperature stresses, but showed growth retardation under normal growth conditionsIto et al. 2006
HRDRiceArabidopsisShowed drought tolerance and improved water use efficiency, the ratio of biomass by enhancing photosynthetic assimilation and reducing transpiration under normal greenhouse conditionsKaraba et al. 2007
HRDTrifolium alexandrinumArabidopsisImproved drought and salt tolerance by reducing transpiration and sodium uptakeAbogadallah et al. 2011
GmDREBWheatSoybean (Glycine max)Improved drought and salt toleranceGao et al. 2005
GhDREBWheatCotton (Gossypium hirsutum)Conferred enhanced tolerance to drought, high salt, and freezing stressesGao et al. 2009
GmDREB1Alfalfa (Medicago sativa)SoybeanConferred salt toleranceJin et al. 2010

Similarly, DREB2-type genes were also shown to improve abiotic tolerance in plants. In maize, ZmDREB2A under the control of constitutive or stress-inducible promoter resulted in enhanced drought tolerance in plants (Qin et al. 2007). Overexpression of a constitutively active form of AtDREB2A, like the wheat TaAIDFa from DREB2-type with a deletion at a Ser/Thr-rich motif, activated transcription of downstream genes and improved drought and osmotic stress tolerance in transgenic Arabidopsis plants (Sakuma et al. 2006a; Xu et al. 2008b) although AtDREB2A and OsDREB2A did not activate downstream genes under normal growth conditions (Liu et al. 1998; Dubouzet et al. 2003). However, DREB2-type genes have been studied in only a limited number of plant species (reviewed by Agarwal et al. 2006). Therefore, additional DREB2-type genes are necessary to gain a more comprehensive picture of DREB regulations.

Overexpression of the Arabidopsis HRD, a DREB IIIb group (Nakano et al. 2006), showed drought tolerance and improved water usage efficiency, the ratio of biomass produced to the water used, by enhancing photosynthetic assimilation and reducing transpiration in rice under normal greenhouse conditions (Karaba et al. 2007). Furthermore, these HRD rice lines exhibited increased shoot biomass under well irrigated conditions and an adaptive increase in root biomass under drought stress (Karaba et al. 2007). An increase in leaf biomass and bundle sheath cells probably contributes to the enhanced photosynthesis assimilation and efficiency in the transgenic plants. This agrees with the fact that the efficiency of the PSII reaction centre (Fv′/Fm′) was higher in the HRD plants than in the wild-type plants (Karaba et al. 2007). Likewise, overexpression of HARDY (HRD) improved drought and salt tolerance in the field by reducing transpiration and protecting photosynthesis in clover (Trifolium alexandrinum) (Abogadallah et al. 2011).

DREBs are also shown to confer heat tolerance to transgenic plants. The constitutive active form of AtDREB2A was reported to significantly increase the tolerance to heat stress through regulating its downstream genes in Arabidopsis and maize (Sakuma et al. 2006b; Qin et al. 2007). Schramm et al. (2008) found that overexpression of DREB2A directly regulated the expression of heat-shock transcription factor HsfA3 that activated expression of many heat-inducible genes in the transcriptional cascade. However, until now, little information was available concerning the effect of DREBs on heat stress. Recently, it was reported that chrysanthemums overexpressing AtDREB1A, with significantly lower leaf electrolyte leakage, showed a higher survival rate than wild-type plants under heat stress, and displayed greatly enhanced expression of the genes including signal transduction, transcription and HSP70 in the early time, and the genes including photosynthesis and metabolism in the late time of the heat treatment (Hong et al. 2009). AtDREB1A plants maintained significantly higher photosynthetic capacity, and elevated activity of Rubisco and sucrose-phosphate synthase under heat stress, which suggests that improvement of heat stress tolerance in transgenic chrysanthemum may be associated with enhanced tolerance of photosynthesis (Hong et al. 2009).

Various studies have demonstrated that improved stress tolerance by overexpression of DREBs is associated with sustained photochemical efficiency and photosynthetic capacity (Savitch et al. 2005; Oh et al. 2007; Hong et al. 2009). However, knowledge about the regulation of photosynthesis-related genes (stomatal and non-stomatal responses) is still limited. Several transcription factors showed improved abiotic stress tolerance related to enhanced photosynthetic parameters in transgenic plants. However, few photosynthetic parameters are usually evaluated under natural field conditions (Saibo et al. 2009). Caution must be exercised when evaluating abiotic stress tolerance in transgenic plants showing a dwarf phenotype, as the improvement may be mainly due to reduced size rather than metabolic changes leading to intrinsic tolerance (Saibo et al. 2009).

ERFs, excellent candidates for improving crop abiotic and biotic stress resistance

ERF transcription factors were shown to be involved in responses to environmental stress and exhibited multiple, complex and flexible expression patterns. To better understand ERF-mediating plant defense responses, it is necessary to elucidate the signaling pathways involved in their regulation. Several important defense-signaling pathways were regulated by low-molecular-weight signal molecules, such as ethylene (ET), JA, salicylic acid (SA), and abscisic acid (ABA) (McGrath et al. 2005). There is a complex cross-talk among these signal molecules, resulting in antagonistic interactions, e.g. the SA and JA pathways, as well as the JA/ET and ABA pathways, to fine-tune defense responses (Anderson et al. 2004; Takahashi et al. 2004; Kariola et al. 2005). Accordingly, some plant defense genes coordinate induction patterns utilizing multiple signaling pathways during defense responses.

ERF genes integrate different pathogen and disease-related stimulus (i.e. ET, JA, and SA) signal pathways, such as ET and JA pathways (reviewed by Gutterson and Reuber 2004). SA signal transduction pathway can act antagonistically with the ET/JA pathway (Leon-Reyes et al. 2010; An and Mou 2011), whereas several ERFs are induced by SA, JA, or ET (Gu et al. 2000; Oñate-Sánchez and Singh 2002; Zhang et al. 2004b; Zhang et al. 2010a; Seo et al. 2010; Zarei et al. 2011). These results indicate that ERFs can coordinately integrate the SA and the ET/JA pathways, but not antagonize them, to finely modulate defense response during pathogen challenge. Overexpression of some ERFs conferred the resistance of transgenic plants to fungal and bacterial pathogens. Furthermore, multiple pathogen resistance was observed in ERF-transgenic plants (reviewed by Xu et al. 2008a).

In addition, some ERFs were activated by ABA (Qin et al. 2004; Lee et al. 2005; Wang et al. 2004; Zhang et al. 2004a; Zhang et al. 2010a), indicating a cross-talk pathway between abiotic and biotic stress responses, although antagonistic interactions between the ABA and the JA/ET signaling pathways. Therefore, ERF genes encode multifunctional factors that respond to multiple stresses, integrate various signal transductions, and thus potentially play dual roles in abiotic and biotic stresses in plants. Abiotic stresses, such as drought, salt, and freezing, activate the expression of some ERF genes from Clusters II, Va, VIb, and VII (Xu et al. 2008a; Zhu et al. 2010). Correspondingly, overexpression of these ERFs improved tolerance of transgenic plants to drought, salt, and freezing (Table 3). For example, transgenic plants overexpressing the SHN1/2/3 genes displayed significant drought tolerance, probably due to reduced stomatal density (Aharoni et al. 2004). Furthermore, overexpression of pepper CaPF1 improved tolerance to heavy metals and heat, and also contributed to organ growth by increasing organ size and cell numbers in transgenic Virginia pine (Pinus virginiana) (Tang et al. 2005). We transformed TaERF1 into Arabidopsis plants and observed that overexpression of TaERF1 improved tolerance to salt and freezing stress (Xu et al. 2007). Mantiri et al. (2008) provided evidence that MtSERF1 appeared to be essential for somatic embryogenesis and might enable a connection between stress and development.

Table 3. Improving industrial and food crop abiotic and biotic stress tolerance through engineering genes for ERF transcription factors
Gene engineeredTransgenic hostSource organismTrait improvedReference
Tsi1Tobacco (Nicotiana tabacum)TobaccoEnhanced resistance against pathogen attack and osmotic stressPark et al. 2001
Tsi1Hot pepper (Capsicum annuumTobaccoEnhanced host resistance to viral, bacterial, and oomycete pathogensShin et al. 2002
NtERF5TobaccoTobaccoEnhanced resistance to tobacco mosaic virusFischer and Droge-Laser 2004
OPBP1TobaccoTomato (Solanum lycopersicum)Enhanced disease resistance and salt toleranceGuo et al. 2004
TSRF1TomatoTomatoRegulated pathogen resistance to Ralstonia solanacearumZhang et al. 2004b
JERF1/3TobaccoTomatoEnhanced salt toleranceZhang et al. 2004a; Wang et al. 2004
NtERF5TobaccoTobaccoEnhanced resistance to tobacco mosaic virus (TMV)Fischer and Droge-Laser 2004
CaERFLP1Hot pepperTobaccoImproved resistance to pathogen and high salinityLee et al. 2004
TERF1TobaccoTomatoEnhanced drought tolerance and abscisic acid sensitivity during seedling development.Zhang et al. 2005
GmERF3TobaccoSoybean (Glycine max)Increased tolerances to salt, drought and diseasesZhang et al. 2009
TERF2/LeERF2Tobacco and tomatoTobacco and tomatoEnhanced tolerance to freezing overexpressing transcription factorZhang and Huang 2010
CaPF1Virginia pine (Pinus virginiana Mill.)PepperObserved multiple pathogen resistance and improved tolerance to heavy metals and heat, and also contributed to organ growth by increasing organ size and cell numbersTang et al. 2005
Sub1A-1Rice (Oryza sativa)RiceConferred enhanced submergence toleranceXu et al. 2006; Perata and Voesenek 2007
TERF1RiceTomatoEnhanced tolerance to drought and high-salinityGao et al. 2008
AP37RiceRiceImproved grain filling rate and grain yield (16–57%) and drought tolerance without undesirable growth phenotypesOh et al. 2009
SNORKEL1 and SNORKEL2RiceRiceTriggered remarkable internode elongation via gibberellin to allow rice to adapt to deep waterHattori et al. 2009
JERF3RiceTomatoImproved tolerance to drought and osmotic stressZhang et al. 2010b
JERF1RiceTomatoImproved tolerance to droughtZhang et al. 2010c

Several ERFs were incorporated into crops, showing improvement of abiotic stress tolerance (Table 2). Overexpression of these ERFs have been recently reported in rice (Gao et al. 2008; Zhang et al. 2010b, c), tomato and tobacco (Zhang et al. 2009; Zhang and Huang 2010), and medics (Chen et al. 2010; Jin et al. 2010) leading to improvement of drought and/or salt tolerance in transgenic plants. Rice plants overexpressing AP37 showed enhanced resistance to drought and saline conditions at the reproductive growth stage. AP37 plants, without undesirable growth phenotypes, showed considerably higher tolerance to drought by producing 16–57% more grain yield over controls under severe field drought conditions (Oh et al. 2009). At present, we obtained TaERF1-overexpressing transgenic wheat plants that showed drought tolerance and exhibited increased biomass under drought and well irrigated conditions. Our results suggest that the TaERF1 gene has the potential to improve drought tolerance in wheat without causing undesirable growth phenotypes (ZS Xu et al. unpubl. data). These findings suggest the potential of these ERFs to improve abiotic tolerance in crops without causing undesirable growth phenotypes.

Recent studies have shown that a number of ERFs are involved in deepwater response. The activation of SNORKEL1 and SNORKEL2 trigger remarkable internode elongation via gibberellin to allow rice to adapt to deep water (Hattori et al. 2009). Overexpression of RAP2.2 resulted in the improvement of plant survival under hypoxic stress although it was not induced during hypoxia (Hinz et al. 2010). It was reported that AtERF73/HRE1 was involved in modulating ethylene responses under hypoxia (Licausi et al. 2010; Yang et al. 2011). Overexpression of AtERF73/HRE1 improved the tolerance of Arabidopsis plants to anoxia (Licausi et al. 2010). It might be possible that AtERF73/HRE1 plays a role in maintaining the homeostasis of H2O2 by regulating the expression of genes involved in antioxidation (Yang et al. 2011). Overexpression of Sub1A-1 enhanced submergence tolerance to rice plants (Xu et al. 2006; Perata and Voesenek 2007). Jung et al. (2010) confirmed that the presence of Sub1A-1 impacted multiple pathways of response to submergence. These results suggest that ERFs might regulate the expression of a set of genes whose products are involved in the response to hypoxic stress.

It was reported that some ERFs were involved in control of metabolite biosynthesis and trait development. ORC1 was sufficient to stimulate alkaloid biosynthesis in tobacco plants and tree tobacco (Nicotiana glauca) root cultures (De Boer et al. 2011). LeERF-1, down-regulated by light signals, might be a crucial positive regulator in regulating biosynthesis of secondary metabolites in Lithospermum erythrorhizon (Zhang et al. 2011). ERN regulated in root hairs combines specifically with the nodulin promoters and functions in early Nod factor signaling in Medicago truncatula (Middleton et al. 2007). EFD is involved in the control of nodule number and differentiation in Medicago truncatula (Vernié et al. 2008). Nud controls the covered/naked caryopsis phenotype by regulating a lipid biosynthesis pathway in barley grain (Taketa et al. 2008). Interestingly, Iwase et al. (2011) firstly found that ERF protein was involved in the control of cell dedifferentiation in response to wounding in Arabidopsis. WIND1 promotes cell dedifferentiation and subsequent cell proliferation to form a mass of pluripotent cells termed callus at the wound site. Overexpression of WIND1 is sufficient to establish and maintain the dedifferentiated status of somatic cells without exogenous auxin and cytokinin. Therefore, it is necessary to characterize the isolated homologous genes in a model plant before practical engineering into crop plants.

Other subfamilies of AP2/ERFs

Some studies showed that AP2 subfamily was responsive to ABA. ADAP is considered to be a positive regulator of ABA responses, because knockout mutant plants show partial insensitivity to ABA and decreased drought tolerance (Lee et al. 2009). CHOTTO1 is probably involved in the ABA-dependent repression of GA biosynthesis (Yano et al. 2009) as well as sugar and nitrate responses in an ABI4-dependent manner during seed germination in Arabidopsis (Yamagishi et al. 2009). In addition, a member of AP2 subfamily, Rice CRL5, promotes crown root initiation. The expression of rice CRL5 was observed in the stem region where crown root initiation occurs. The crl5 mutant produced fewer crown roots and displayed impaired initiation of crown root primordial (Kitomi et al. 2011).

ABI3/VP1 mediates ABA responses and is involved in regulation of seed maturation and ABA-regulated gene expression in plant seeds (Zeng et al. 200; Sakata et al. 2010). RAV subfamily, related to ABI3/VP1 transcription factors, might be also involved in stress responses. B. napus RAV-1-HY15 is induced by cold, NaCl, and PEG treatments (Zhuang et al. 2011). Overexpression of the CARAV1 gene in transgenic Arabidopsis plants induced some PR genes and enhanced resistance against infection by Pseudomonas syringae and osmotic stresses by high salinity and dehydration (Sohn et al. 2006). In addition, overexpression of SlRAV2 in transgenic tomato plants induced the expression of SlERF5 and PR5 genes and increased bacterial wilt tolerance (Li et al. 2011). Lee et al. (2010a) identified that pepper CaRAV1 protein physically interacted with the oxidoreductase CaOXR1. Overexpression of CaRAV1 and CaOXR1/CaRAV1 conferred resistance to the biotrophic oomycete Hyaloperonospora arabidopsidis infection, and high tolerance to high salinity and osmotic stress in Arabidopsis. Together, these results suggest that RAV subfamily mediates plant defense during abiotic and biotic stresses.


A major challenge for current agricultural biotechnology is to satisfy an ever increasing demand in food production facing a constantly increasing world population that will reach more than 9 billion in 2050 (Godfray et al. 2010; Tester and Langridge 2010). This growing demand for food is paralleled by dramatic losses of arable land due to increasing severity of soil destruction by environmental conditions (Golldack et al. 2011). Plant engineering strategies for cellular and metabolic reprogramming seems to increase the efficiency of plant adaptive processes. In contrast to classical breeding, engineered crops for enhancing tolerance incorporate specifically desired genes into an organism by limiting the transfer of undesirable genes from the donor organism. Therefore, transgenic technologies are deemed efficient and precise ways of improving a desired trait. Rapid advances in transgenic technology have resulted in efficient transformation and generation of transgenic lines in a number of crops to improve traits including tolerance to abiotic and biotic stresses (Ashraf 2010). The prospects of improving abiotic stress tolerance in crops seem to be not very bright due to the involvement of many genes with additive effects (Ashraf 2010). DREB genes could be used as candidates to improve abiotic stresses in plants, including drought, salt, and cold (reviewed by Saibo et al. 2009; Hussain et al. 2011). Recently, several DREB genes were reported to enhance heat tolerance in transgenic plants (Sakuma et al. 2006b; Qin et al. 2007; Hong et al. 2009). Overexpression of some ERF genes from Clusters Ia, II, Va, and VII resulted in broad-spectrum of resistance to pathogens and improves drought, salt, and/or freezing tolerances in transgenic plants (reviewed by Xu et al. 2008a). It seems that DREBs/ERFs can be used as the candidates in improving crop stress tolerance. However, the functions of most DREBs/ERFs are still unknown. Therefore, to obtain additonal insight into this area of potentially large agronomic impact, it is essential to identify and functionally characterize new DREB/ERF genes. Since not all potential biological functions can be deduced using bioinformatics techniques, more DREB/ERF genes from more species, especially wild species, should be isolated and shown to be useful for efforts in breeding crop resistance.

Rice plants overexpressing HRD did not show reduced growth, seed yield, or germination rate; instead, they had a larger leaf canopy with more tillers under normal greenhouse conditions, and had more root biomass under drought stress (Karaba et al. 2007). However, the growth and development of transgenic plants is often affected by overexpression of extrinsic genes. In fact, uncontrolled expression of target genes in certain plants may lead to dwarf phenotypes and reduced yields. To overcome growth retardation, it seems that a stress-inducible promoter is more efficient in driving the expression of stress-related target genes in transgenic plants. Plants expressing DREB genes driven by the Arabidopsis stress-inducible promoter, RD29A have shown enhanced abiotic stress tolerance without compromising the yield (Kasuga et al. 2004; Pino et al. 2007). However, the RD29A promoter is more efficient in driving gene expression in dicots rather than in monocots (Ito et al. 2006; Saibo et al. 2009). Therefore, it is essential to more efficiently identify stress-inducible promoters to apply to different crop engineering purposes.

Considerable progress can be made in improving crop tolerance by incorporating AP2/ERF genes. However, most experiments were investigated under controlled stress conditions in laboratory or greenhouse, not in natural field conditions (Ashraf et al. 2010). To produce stress-resistant crop cultivars/lines, we will have to perform functional identification in natural field conditions other than a single simulated stress. We believe that an ideal plant can eventually be obtained from a large number of transgenic lines.

(Co-Editor: Weicai Yang)


This research was financially supported by the National Transgenic Key Project of MOA (2011ZX08002–002 and 2009ZX08009-083B). We are grateful to Drs. Long Mao and Hong-Jie Li for critical review of the manuscript.