AP2/ERF Transcription Factor Regulatory Networks in Hormone and Abiotic Stress Responses in Arabidopsis

1Abiotic stresses such as water scarcity, extreme temperature and high salinity lead to arrested plant growth and ultimately result in massive agricultural losses. Plants have developed sophisticated regulatory mechanisms to respond to external stress signals in a timely manner to ensure optimal growth and stress tolerance. Among these, hormonal signaling pathways and stress responsive transcriptional factors function together to form an interconnected network. In addition, environmental changes are often multifactorial, with several stresses occurring simultaneously. Instead of the linear stress signaling pathways, these regulatory components lead to more complex responses. Under abiotic stress conditions, stress hormones such as abscisic acids (ABA) and ethylene (ET) are induced, whereas the production and distribution of growth promoting hormones, such as gibberellins (GAs), brassinosteroids (BRs), and cytokinins (CTK) are also altered to enable optimal responses. The regulation of plant hormone signaling during abiotic stresses is partially mediated by hormone- and stress-responsive transcription factors.

2APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) family transcription factors (AP2/ERFs) have emerged as key regulators of several abiotic stresses and respond to multiple hormones. Numerous AP2/ERFs mutants with altered abiotic stress responses and hormone sensitivity have been identified, positioning this family of transcription factors as promising candidates to study the interactions between abiotic stresses and hormones. Several properties of AP2/ERFs, such as induction upon specific stresses and diverse DNA binding preferences, enable these transcription factors to integrate responses of multiple stimuli and participate in different regulatory processes. In this review, we focus on Arabidopsis thaliana to summarize the regulation and function of AP2/ERFs in hormone and abiotic stress responses. The function of AP2/ERFs in crops has been extensively reviewed elsewhere. Thus, only a few examples of AP2/ERFs in other species will be discussed. Finally, we investigate AP2/ERFs regulatory networks using publicly available transcriptome data to verify known and uncover novel roles of AP2/ERFs in hormone and stress responses.

3AP2/ERFs are characterized by an APETALA2 (AP2)/Ethylene Responsive Element Binding Factor (EREB) domain, which consists of 40–70 conserved amino acids involved in DNA binding. AP2/ERFs contain the four major subfamilies: APETALA2 (AP2), RELATED TO ABSCISIC ACID INSENSITIVE 3/VIVIPAROUS 1 (RAV), DEHYDRATION-RESPONSIVE ELEMENT BINDING proteins (DREBs) (subgroup A1–A6) and ETHYLENE RESPONSIVE FACTORS (ERFs) (subgroup V-X). As transcription factors, AP2/ERFs regulate genes involved in diverse biological processes such as growth, development, hormone and stress responses through several mechanisms including transcriptional and post-translational control.

4AP2/ERFs expression is tightly regulated to enable proper stress responses. Gene expression profiling studies have shown that most AP2/ERFs are expressed at low levels under normal conditions, whereas the expression can be induced or repressed at certain growth stages, by hormones and stress stimuli. In many cases, AP2/ERFs expression is regulated by the conserved cis-elements present in their promoter regions, or a combination of multiple responsive elements (Figure 1 left). For example, Arabidopsis DREB2A is highly induced under dehydration and heat conditions. The expression of DREB2A is controlled by HEAT SHOCK FACTOR1 (HSFA1) and ABRE-BINDING PROTEINs/ABRE-BINDING FACTOR 3 (AREB1/AREB2/ABF3) through binding to Heat Shock Element (HSE) and ABA Response Element (ABRE) motifs on its promoter, respectively. Moreover, by aligning 20 Arabidopsis DREB gene promoters to the motifs related to abiotic stresses, it was found that DREB1A/CBF3, DREB1C/CBF2, DREB2C, DREB2G, and DEAR3 have the most types of these motifs including HSE motif to which heat shock factors bind, Low-Temperature Responsive element (LTR) that is important for the induction of cold regulated genes, and ABRE motif that responds to ABA. AP2/ERFs whose promoters contain ethylene-responsive (EBS) motifs are also likely to respond to ET signaling.

5Moreover, AP2/ERFs expression might be affected by histone modifications. Histone modifications such as phosphorylation, ubiquitination, methylation, and acetylation can either activate or repress transcription by creating more “open” or “closed” chromatin configurations. A study in peanut (Arachis hypogaea L.) showed that the general inhibition of histone deacetylases (HDACs) and Polyethylene Glycol (PEG) treatment induced the acetylation around peanut DREB1 (AhDREB1) promoter region and resulted in increased AhDREB1 expression. Given that chromatin features such as histone variants and post-translational histone modifications are altered by abiotic stresses, and these influences could be inherited to the next generation, the study of epigenetic regulation of AP2/ERFs will advance our understating the mechanisms controlling AP2/ERFs.

6Another point of AP2/ERF regulation occurs at the level of splicing. Alternative splicing is required to create a functional isoform for some AP2/ERFs in rice (Oryza sativa) (OsDREB2A/2B), maize (Zea mays) (ZmDREB2A), wheat (Triticum aestivum) (WDREB2), and barley (Hordeum vulgare) (HvDRF1). In these cases, plants produce an inactive AP2/ERF form containing stop codons before the DNA binding domain during normal conditions, while under stress conditions, the exon with a premature stop codons is excluded to generate a functional transcription factor. In addition, microRNA (miRNA)-mediated RNA silencing and translation repression was shown to regulate AP2/ERFs. miRNA172 targets Arabidopsis AP2 messenger RNA to inhibit its translation. Several other miRNAs such as miRNA156 and miRNA838 have also been shown to regulate AP2/ERFs in common bean (Phaseolus vulgaris).

7In addition to transcriptional control, post-transcriptional regulation modulates the activity and abundance of AP2/ERF proteins (Figure 1 middle). One of the modifications, phosphorylation, affects AP2/ERF protein transactivity and stability. In Arabidopsis, SNF1-RELATED PROTEIN KINASES (SnRKs), a positive regulator in ABA signaling pathway, interacts with and phosphorylates RAV1 to inhibit its transcriptional repression function, while ERF104 and ERF6 are phosphorylated by mitogen-activated protein kinases (MPKs) to positively regulate pathogen responses. The stability of Arabidopsis DREB2A is also affected by phosphorylation. DREB2A is destabilized by phosphorylation on its negative regulatory domain (NRD), which is probably mediated by Casein Kinase 1 (CK1). Indeed, many kinases such as GSK3-like kinase BR-INSENSITIVE 2 (BIN2) and SnRKs have been reported to mediate abiotic stresses. However, information about how these kinases regulate AP2/ERFs is limited. Therefore, future studies such as phosphoproteomics under abiotic stresses and upon perturbation of specific kinases could help identify AP2/ERF protein modifications, leading to potential strategies to modify the protein activity and stability of AP2/ERFs to confer abiotic stress tolerance.

8The protein stability of AP2/ERFs is also regulated by ubiquitin-mediated protein degradation through the 26S proteasome pathway as revealed in Arabidopsis (Figure 1 middle). Under non-stress conditions, DREB2A, ERF53, and ERF75/RAP2.2 proteins are directly ubiquitinated by RING domain E3 ligases DREB2A-INTERACTING PROTEIN1/2 (DRIP1/2), RAlGDS-LIKE (RGLG1/2), and SEVEN IN ABSENTIA OF ARABIDOPSIS2 (SINAT2), respectively. A CUL3-based E3 ligase adaptor BTB/POZ AND MATH DOMAIN proteins (BPMs) also mediates DREB2A and ERF59/RAP2.4 degradation. Additionally, the stability of several Arabidopsis ERF-VIIs is controlled by the N-end rule pathway, where the N terminal Met (Nt-Met) of ERF71/HRE2, ERF72/RAP2.3, and ERF74/RAP2.12 is removed and the second amino acid Cys (Nt-Cys) is oxidized into cysteine sulfinic/sulfonic acid in an oxygen-dependent manner. This process triggers ubiquitination mediated by the E3 ligase PROTEOLYSIS 6 (PRT6). Particularly, RAP2.12 interactes with acyl-CoA binding protein 1/2 (ACBP1/2) and is localized on the plasma membrane under normal conditions. During the limited oxygen conditions, RAP2.12 de-associates from ACBP1/2 and moves into the nucleus by an unknown mechanism. ET also promotes RAP2.3 nuclear localization. Overall, a number of components have been identified that regulate AP2/ERF stability, localization and activity. Since many AP2/ERFs are involved in stress responses, this tight regulation of AP2/ERF protein likely ensures that plants effectively respond to environmental stimuli without ectopic activation of AP2/ERF-mediated stress responses.

9To regulate target genes, AP2/ERFs have conserved DNA binding preferences. Typically, DREBs recognize Dehydration-Responsive or C-Repeat Element (DRE/CRT) with A/GCCGAC core sequence on stress-responsive genes to confer resistance to drought, cold and heat abiotic stresses. ERFs bind to Ethylene-Response Element (ERE) with AGCCGCC core sequence (also known as GCC-box) to confer resistance to biotic stresses. However, many Arabidpsis DREBs and ERFs have been reported to bind to both DRE/CRT and ERE elements, implying their potential roles in both abiotic and biotic stress. For instance, DREBs (including TINY, CBF1, ERF53, RAP2.4, and TG/RAP2.4A) and ERFs (including ERF1, ERF4, and ERF71) bind to both DRE and ERE elements. Similarly, the conserved DNA binding preferences of AP2/ERF are also expanded to other species such as rice, wheat, maize, soybean (Glycine max), and tobacco (Nicotiana tabacum). Recently, the combination of high-throughput protein-binding microarray and relevant transcriptome data demonstrated that transcription factors with high structure identity share similar DNA binding sites, which also enables them to share some biological relevance and explains their functional redundancy. It provides a way to predict unknown AP2/ERFs function. Additionally, AP2/ERFs recognize cis-elements that diverge significantly from these motifs (Table 1). For example, Coupling Element 1 (CE1: TGCCACCG), Coupling Element 3-like (CE3-like: CGCG), Hypoxia-Responsive Promoter Element (HRPE) and non-specific sequences are recognized by AP2/ERFs. Through different partners upon binding to different sites, the diversity of AP2/ERFs DNA binding broadens the scope of target genes and might enable them to participate in different regulatory processes. To date, both in vivo (ChIP-chip and ChIP-seq) and in vitro (DAP-seq, SELEX-seq and Protein-Binding Microarrays (PBMs)) techniques are helpful to decipher the AP2/ERFs transcriptional regulatory code.

10Through directly binding to target gene promoters, AP2/ERFs can either activate or repress target gene expression (Figure 1 right). Besides an N-terminal DNA binding domain, the C-terminal activation domain of AP2/ERFs mediates the activation of target gene expression in Arabidopsis and rice. Recently, an activation EDLL motif was identified from Arabidopsis ERF98 and AP2 subfamily. The EDLL motif is strong enough to override the repression effect mediated by ERF-associated amphiphilic repression (EAR) motif, which emphasizes the transactivation activity of AP2/ERFs. However, AP2/ERFs containing an EAR motif with the consensus sequence LxLxL or DLNxxP, or B3 repression domain (BRD) with R/KLFGV sequence exhibit a repressive effect on target genes. The EAR motif containing AP2/ERFs also interact and recruit transcription co-repressors like TOPLESS (TPL) and TOPLESS-RELATED (TPR) or histone modifiers to suppress target gene expression. In Arabidopsis, ERF7 interacts with a human global co-repressor SIN3 homolog (ATSIN3) that in turn interacts with Histone Deacetylase 19 (HDA19). Similarly, ERF3 interacts with SIN3 Associated Polypeptide P18 (SAP18) and then recruits HDA19 to repress gene expression. The BRD motif containing Arabidopsis RAV1 and RAV2 also display repressive activities. There is a report demonstrating that genes co-regulated with corresponding AP2/ERFs were enriched with the AP2/ERFs targets. Therefore, by analyzing the enrichment of cognate motifs in AP2/ERFs co-regulated genes, it is possible to identify putative target genes of transcription factors and predict their biological functions.

11AP2/ERFs regulate numerous abiotic stresses such as cold, drought, heat, salt, and freezing. Although many AP2/ERFs are proposed to form an abiotic stress-specific regulatory network, the ability of AP2/ERFs to respond to multiple stimuli and regulate different stresses enable them to form a more complex stress response network. In this network, AP2/ERFs also respond to abiotic stresses with varying dynamic patterns: some AP2/ERFs are induced quickly and continuously, whereas others are regulated by prolonged stress, which indicates they might have mutual influence on each other’s function. However, the detailed mechanisms of how different AP2/ERFs cooperate or antagonize with each other are yet to be established. Therefore, in addition to studying the function of individual transcription factors in this family, it is also necessary to study the relationship between different AP2/ERFs in abiotic stress responses. Here we summarize recent works on the DREBs and ERFs regulation in cold, drought, heat and salt stress responses particularly in Arabidopsis, and we also discuss the mutual regulation of different AP2/ERFs.

12DREBs have been extensively examined in abiotic stresses, where they respond to and positively regulate cold, drought, heat and salt tolerance by directly regulating stress-responsive genes (Figure 2). Among these, DREB1s (DREB-A1 subgroup) containing several C-Repeat-Binding Factors (CBFs) play major roles in acquisition of freezing tolerance. CBFs together with another major cold responsive transcription factor, Inducer of CBF Expression (ICE), establish a central cold response pathway to activate a majority of DRE containing Cold Responsive Genes (CORs) in Arabidopsis. CORs encode Late Embryogenesis Abundant (LEA) proteins and enzymes for sugar metabolism and fatty acid desaturation that provide the protection for plants from cold stress. The roles of Arabidopsis CBFs in stress responses have been characterized by genetics using cbf mutants generated by CRISPR/Cas9 genome editing techniques and CBFs overexpression lines, as well as transcriptome analysis. CBFs are also reported to positively regulate plant drought and salt tolerance, which might due to a common set of stress responsive genes. Despite having a clear function in cold response pathways, how CBFs regulate different stresses and the mechanisms by which they confer stress tolerance are still unknown. Environmental changes are usually multifactorial and several stresses often occur simultaneously. Therefore, the multiple roles of CBFs in abiotic stresses might be necessary for plants to overcome stresses and it will be interesting to examine how CBFs regulate genes under different stress conditions.

13In addition to CBFs, transcriptome profiling in Arabidopsis also identified several other regulators that are activated during cold acclimation, including members in the DREB-A5 group. The DREB-A5 group contains six members with EAR motifs (DEARs) acting as transcriptional repressors on DRE motif containing genes, which provides a repression effect in the DREB-regulated abiotic stress network. Among these, DEAR1 likely acts upstream of CBFs, while DEAR6/RAP2.1 acts downstream of CBFs. Overexpressed DEAR1 suppressed the cold induced expression of CBFs and displayed reduced plant freezing tolerance. RAP2.1 is induced by cold, as well as in plants constitutively overexpressing CBFs, but the induction of RAP2.1 by cold has a later onset than the induction of CBF2. RAP2.1 was first identified as a downstream CBFs subregulon and negatively modifies plants cold tolerance. The presence of the DRE motif in the RAP2.1 promoter region also suggests that RAP2.1 might be a direct target of CBFs. Although the integration of DEAR1 and RAP2.1 in the CBF pathway still needs to be examined using genetics, this negative regulation mechanism might provide checks and balances that minimize the adverse effects of prolonged stress responses. In addition to the negative role of RAP2.1 in cold stress, it also negatively regulates drought tolerance, in that overexpression of RAP2.1 resulted in sensitivity to drought. However, how RAP2.1 regulates drought response, whether it regulates other abiotic stresses and what other DEARs function in abiotic stresses are interesting questions to answer in the future. The checks and balances of DREBs’ function in stress were also revealed in Brassica napus, where two groups DREBs (Group I and Group II) regulate cold stress responsive genes sequentially. The early induced Group I DREBs activate cold stress response pathways, whereas the Group II DREBs which are expressed later competitively inhibited Group I DREBs function.

14DREB2s from DREB-A2 are mostly involved in plant drought and heat tolerance, which has been reviewed in detail elsewhere. Briefly, DREB2s are induced upon drought and heat, and positively regulate DRE containing drought responsive genes such as LEAs, and heat responsive genes such as heat chaperons. Moreover, members in DREB-A4 family such as HARDY (HRD) and in DREB-A6 family such as ERF53, RAP2.4, and TG/RAP2.4A also positively regulate drought and salt tolerance. Overexpression of HRD in Arabidopsis or rice remarkably improved plant drought and salt tolerance. Overexpression of TRANSLUCENT GREEN (TG) resulted in vitrified leaves with increased water content in cells, leading to increased drought tolerance. TG regulates cell water homeostasis mainly by directly activating several aquaporin genes, as well as Ascorbate Peroxidases (APx) genes encoding chloroplast peroxidases that functions to protect against photo-oxidative stresses caused by Reactive Oxygen Species (ROS).

15Beside Arabidopsis, DREBs’ improvement in stress tolerance has been extensively studied in various crops such as OsDREB1s and OsDREB2s in rice, ZmDREB2A in maize, TaDREB1 in wheat, and HvDRF1 in barley. These DREBs provide abundant candidate genes for the engineering of stress tolerance in crops. However, given that DREBs are a major family of AP2/ERF transcription factors that integrate multiple abiotic stress signals, cooperate or antagonize one another, and modulate downstream stress responsive genes, studying the DREB gene regulatory network will provide a platform for a more comprehensive understanding of abiotic stress responses and guide the genetic engineering of crops.

16Members in ERF subfamily also contribute to abiotic stress responses. Recently, two groups of ERFs have emerged as central players of abiotic stress regulation in Arabidopsis (Figure 2). CYTOKININ RESPONSE FACTORS (CRFs) in ERF-VI subfamily are induced by CTK as well as multiple abiotic stresses to positively regulate osmotic and freezing tolerance. CRF6, whose induction is dependent on the perception of CTK, alleviated the H2O2 damage on plants to positively regulate oxidative response. CRF4, one of several CRFs not transcriptionally regulated by CTK, positively regulates freezing tolerance by promoting CORs expression. However, the mechanisms by which CRFs confer stress tolerance remain to be determined.

17Members of ERF-VII subfamily in Arabidopsis as well as rice have been demonstrated to play major roles in flooding, low oxygen (hypoxia) and submergence tolerance and their redundant function in hypoxia responses has been reviewed. For example, rice SUBMERGENCE 1A (SUB1A), and SNORKEL1/2 (SK1/SK2) positively regulate flooding tolerance by two opposite mechanisms: SUB1A mediates a quiescence strategy associated with reduced growth and respiration whereas SK1/SK2 promote a deep-water escape strategy allowing rapid growth of petioles, stems, and vascular changes.

18In Arabidopsis, ERF-VIIs have conserved N-terminal domains that allow them to be degraded under anoxia conditions though oxygen-dependent N-end rule pathway. Five members including ERF71/HRE2, ERF72/RAP2.3, ERF73/HRE1, ERF74/RAP2.12, and ERF75/ RAP2.2 are induced by limited oxygen. With limited oxygen, these ERF-VIIs accumulate and positively regulate hypoxia responsive genes involved in sugar metabolism, fermentation and ET biosynthesis to achieve hypoxia tolerance. Apart from hypoxia responses, ERF-VIIs also regulate oxidative and osmotic stresses. Overexpression of RAP2.2, RAP2.3 and RAP2.12 (RAPs) results in a higher survival rate from low oxygen, oxidative and osmotic stresses, while rap2.12-2 rap2.3-1 double mutants are sensitive to these stresses.

19RESPIRATORY BURST OXIDASE HOMOLOG D (RbohD), a NADPH oxidase, helps to generate ROS burst. It is reported that RAPs regulate abiotic stresses via an RbohD-dependent mechanism. Apart from being a toxic by-product of biochemical processes, ROS serve as signaling molecules to trigger stress responses and transduce signals crossing cells according to its lower molecular weight and fast cell diffusion. First it was found that the ROS production and RbohD expression were compromised in single erf74 and double erf74 erf75 mutants at an early stage, which resulted in compromised stress responsive gene expression and stress tolerance. Given that ERF74 binds to RbohD promoter to activate its expression, the RbohD dependent ROS activation was essential for ERF74 and ERF75 mediated hypoxia resistance. However, too much ROS can cause cell injury and cell death. To overcome adverse effects of ROS, overexpression of ERF74 promoted increased ROS scavenging enzymes and stress responsive genes at later stage. Therefore, ERF74 acts as an on-off switch to control RbohD-dependent ROS burst in response to different stresses in Arabidopsis. This newly identified mechanism provides more details and divides the stress response into early and later stages, as well as ROS balance. One example that rice ERFs OsLG3 induced ROS scavenging to positively regulate stress tolerance was reported recently, suggesting the similar mechanism exists in rice.

20Additionally, many other Arabidopsis ERFs also regulate abiotic stresses. ERF1 and Ethylene- and Salt-inducible ERF genes (ESEs) in ERF-IX group positively regulate plant salinity tolerance by promoting salt responsive gene expression. ERF6, another member in ERF-IX group, triggers growth inhibition to confer long-term osmotic stress tolerance. RAP2.6L from ERF-X subgroup improves drought and salt tolerance. Additionally, Arabidopsis RAVs, especially AP2s, play central roles in developmental processes, such as organ number and size control, shoot and root meristem maintenance, flower initiation and growth. Members in these subfamilies are also reported to mediate diverse abiotic stress responses. AINTEGUMENTA (ANT) controls organ cell number and size throughout shoot development. ANT also negatively regulates salt tolerance by repressing SOS3-LIKE CALCIUM BINDING PROTEIN 8 (SCABP8/CBL10), a putative Ca2+ sensor that protects Arabidopsis shoots against salt stress and maintains ion homeostasis. Overexpression of Arabidopsis RAV1 and RAV2 in cotton increased fiber length and even obtained the same yield under drought stress compared with control conditions.

21Similar as DREBs, ERFs in other plant species like rice, wheat and tomato are also involved in a broad range of abiotic stresses. Overall, these findings provide the potential of engineering high-efficiency crops under stress conditions. In summary, ERFs function to receive multiple stress signals and control a diverse set of stress responsive genes, where many ERFs have cooperative or antagonistic regulation on stress responses. Therefore, constructing ERF-specific gene regulatory networks would be interesting to provide insight as to how ERFs function as a unit to regulate common downstream genes.

22In addition to directly regulating abiotic stresses, AP2/ERFs are also involved in hormone signaling and hormone mediated-stress responses. Plant hormones affect abiotic stresses by triggering a wide range of physiological processes. ABA and ET are major stress hormones that are induced under abiotic stress conditions and regulate stress responses associated with AP2/ERFs. GAs, CTK, and BRs are growth-related hormones that promote cell growth, proliferation and differentiation. It is becoming increasingly evident that these growth-related hormones also have direct and/or indirect effects on abiotic stresses. Because stress tolerance is often associated with trade-offs between growth and stress, abiotic stress-tolerant plants usually have lower growth rates and productivity. Numerous studies in Arabidopsis have shown AP2/ERFs confer plant stresses tolerance associated with growth losses (Figure 2), such as CBFs, DREB2A, HARDY, TG, ERF6, and RAP2.6. However, the growth loss can be minimized by driving expression via the stress-induced RD29A promoter. Thus, in addition to understanding the basis of AP2/ERFs in abiotic stresses, it is also important to explore their roles in the hormone-regulated stresses responses. AP2/ERFs are involved in plant hormone-mediated abiotic stresses through the following aspects: (1) AP2/ERFs transcripts are regulated by hormones; (2) AP2/ERFs alter hormone sensitivity and gene expression by cooperating or antagonizing various hormone signaling components; (3) AP2/ERFs regulate hormone biosynthesis or metabolism via feedback regulation (Figure 3). In the following sections we emphasize how abiotic stress-induced (ABA and ET) and growth-promoting (GAs, CTK, and BRs) hormones are regulated by AP2/ERFs in Arabidopsis through these mechanisms.

23The plant hormone ABA is a pivotal hormone that regulates abiotic stress responses including drought, salinity, cold and heat stresses. ABA exerts a protective function through inducing stomata closure, modulating root architecture, and promoting the synthesis of osmolytes. During stress conditions such as water deprivation and osmotic pressure, the rate-limiting ABA biosynthetic enzyme Nine-cis-Epoxycarotenoid Dioxygenase (NCED) is rapidly up-regulated to promote ABA biosynthesis. ABA is then sensed by a large family of PYRABACTIN RESISTANCE1 /PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYR/PLY/RCAR) receptors that interact with Protein Phosphatase 2C (PP2Cs) as a ternary complex to release PP2Cs’ inhibition on SnRK2 kinases (SnRK2s). The active SnRK2s phosphorylate downstream substrate proteins, including AREBs/ABFs, ion channels, and enzymes such as NADPH oxidases, thereby inducing ABA responses. Generally, transcription factors regulate abiotic stresses through ABA-dependent or ABA-independent pathways.

24A great deal of studies have shown that DREBs regulate ABA-independent abiotic stresses by directly binding to DRE/CRT motifs on stress responsive genes. However, AP2/ERFs are indispensable for ABA-dependent stress responses as well. ANT, ERF53, ERF-VIIs, RAP2.6L, and RAP2.6 in Arabidopsis are induced by ABA to up-regulate DRE- and ABRE- element containing genes. Rice OsERF71 positively regulates ABA signaling to alter root architecture and confer drought tolerance. The combination of abiotic stresses and ABA also led to the further activation of DREB2s and stress inducible genes. ABA INSENSITIVE 4 (ABI4), a unique one in the DREB-A3 group is a key component of the ABA signaling pathway. Upon ABA and ROS accumulation under stress conditions, ABI4 represses CCAAT Binding Factor A (CBFA). CBFA is a subunit of the trimeric transcription complex of Heme Activator Proteins (HAPs). Repression of CBFA then allows other transcription factors to enter the transcription complex and improves the efficiency of stress responsive gene transcription.

25Apart from the positive effect of AP2/ERFs in ABA-mediated stress responses, many Arabidopsis studies have shown that AP2/ERFs also interrupt ABA signaling, resulting in reduced sensitivity on root growth inhibition and stomata closure (Figure 3). ERF18/ ORA47 activated the PP2C family phosphatase gene ABI2. At the same time ABI1 acted upstream of ORA47 to activate ORA47, leading to an ABI1-ORA47-ABI2 regulation loop that inhibits ABA signaling as well as drought tolerance. RAV1 inhibites ABA sensitivity on root growth by repressing ABI3, ABI4 and ABI5 expression. SnRK2.2, SnRK2.3, SnRK2.6 also interact with and phosphorylate RAV1 to inhibit RAV1’s transcriptional repression of targets genes.

26ET is also reported to regulate abiotic stress responses including salt, cold, and flooding. ET is synthesized from the rate limiting enzymes ACC Synthases (ACSs), a major target for the regulation for ET production under stresses. The binding of ET with its receptor ETHYLENE INSENSITIVE 1 (ETR1) deactivates CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1) kinase activity to release CTR1’s inhibition on ETHYLENE INSENSITIVE 2 (EIN2). Then the C-terminal of EIN2 translocates into the nucleus to activate ETHYLENE INSENSITIVE 3 (EIN3) as well as the transcriptional cascade of ethylene-regulated genes.

27In stress responses, AP2/ERFs, especially the ERF subfamily, are the major downstream regulatory factors of the ET signaling pathway. ET regulates several aspects of Arabidopsis abiotic stress responses, including inhibiting CBFs to negatively regulate cold stress (Figure 3). Conversely, ET positively regulates flooding and submergence mediated by ERF-VIIs, and improves salt tolerance by activating ERF1 and ESEs.

28For ET-regulated cold response, the production of ET is inhibited after exposure to cold, which results in compromised cold tolerance. Consistently, ET insensitive mutants etr1-1, ein2-5, and ein3-1 displayed increased freezing tolerance. EIN3 inhibits the expression of CBFs by directly binding to their promoters. In addition, ET plays crucial roles on plant survival and recovery from flooding, especially in rice. Flooding causes oxygen deficiency, which promotes ET production and activates the expression of a set of ERF-VIIs, whose function in hypoxia is discussed above. ET also promotes RAP2.3 nuclear localization and advances ORA59 mediated ethylene responses which is dependent on RAP2.3. However, ERF-VIIs regulate hypoxia response partially through ET-independent pathways. The induction of ERF73/HRE1 under hypoxia was not completely abolished in ethylene-insensitive mutants or in the presence of ethylene biosynthesis inhibitors. ERF-VIIs also negatively regulate ET signaling and homeostasis probably via feedback regulations. For instance, HRE1-RNAi seedlings displayed exaggerated triple responses; ACSs was decreased in RAP2.2 overexpression plants, but up-regulated in rap2.2-2 knockout mutant. However, how ERF-VIIs control ET homeostasis via negative feedback mechanism under stresses needs further investigation.

29ET also has complex regulation in salt stress, which has been extensively discussed. In Arabidopsis, ET signaling is required for plant tolerance to salinity stress as EIN3 activates ERF1 and ESEs to activate downstream stress-related genes and promote salinity tolerance. However, knockout mutants of ACSs also led to salt tolerance, leading to an opposite conclusion in terms of ET signaling and salt tolerance. These different conclusions might be due to the different mutants, growth conditions or experimental setups used. Future investigation is necessary to further our understanding of the role of ET in plant salinity response.

30The plant hormone GAs is known to promote plant growth and development. GAs have also been shown to regulate abiotic stresses, as reduced GA content slows down plant growth upon exposure to several abiotic stresses including cold, salt, and osmotic stresses. GAs are synthesized through several key oxidases including GA 20-oxidases (GA20oxs) and GA 3-oxidases (GA3oxs), and catabolized by GA 2-oxidase (GA2ox) that depletes pools of GA precursors to maintain GA homeostasis. In the absence of GAs, a group of DELLA proteins (DELLAs) inhibit GA response, and this inhibition can be released by the degradation of DELLAs in the presence of GAs. Generally, abiotic stresses cause reduction of GA content and signaling through the inhibition of GA biosynthesis enzymes mediated by Arabidopsis AP2/ERFs (Figure 3). DREB1E and DREB1F confer salt stress-induced growth retardation mostly through the repression of GA20oxs. CBF1 and ERF6 overexpression plants were sensitive to stress-induced growth retardation because of increased GA2oxs expression as well as the accumulation of DELLAs. Conversely, ERF11 promotes plant internode elongation by activating GA biosynthesis, and expression of GA3ox1 and GA20oxs are increased in ERF11 overexpression plants. Nevertheless, ERF11 and ERF6 show antagonistic regulation on stress-induced growth inhibition. ERF11 suppresses the extreme dwarf phenotype of ERF6 overexpression plants and represses ERF6-induced gene expression. The opposite regulation by ERF6 and ERF11 reveals that dynamic mechanisms must exist in plants to fine-tune and maintain the balance between plant growth and stress responses.

31In addition to GA regulation in Arabidopsis abiotic stresses, rice regulates flooding coping submergence tolerance by two opposite GA regulations. The first quiescence strategy was that SUB1A increased the accumulation of SLENDER RICE1 (SLR1) and SLENDER RICE1 LIKE1 (SLRL1) (DELLA like proteins in rice) to restrict GA signaling and sensitivity, which resulted in inhibition of plant internode elongation and reduced carbohydrate consumption. The second deep-water escape strategy involves SK1/SK2, which lead to up-regulated GA20oxs and promoted internode elongation to escape submergence in water.

32The plant hormone CTK not only plays diverse roles in plant growth and development, but also has been reported to regulate plant abiotic stresses, one of which is mediate by CRFs (Figure 3). CRFs are essential for CTK-mediated embryo, cotyledon, and leaf development, as both single and multiple CRF1/2/3/5/6 mutants displayed cell proliferation deficient phenotypes. The roles of CRF’s regulation on CTK-mediated development were further confirmed by the transcriptome analysis of crf 1,2,5 and crf2,3,6 mutants, with or without CTK treatment in Arabidopsis. About 60% of the CTK responsive genes were regulated by both CRFs and type-B ARRs (the typical cytokinin-responsive transcription factors), suggesting a model that CRFs acted tandemly with type-B ARRs to mediate CTK response. CRF6 also cooperated with CTK signaling to inhibit stress-induced leaf senescence through a common subset of CTK-regulated genes. Apart from the CRFs positive effect in the CTK pathway, CRF6 also represses CTK-associated target genes involved in CTK biosynthesis, signaling and transport, to alleviate the adverse effect of CTK on abiotic stress. The opposite regulation between CRF6 and CTK on stresses and similar regulation on senescence suggest that CRF6 regulates CTK signaling through two subsets of genes: one set of genes alleviate the negative effect of CTK on abiotic stresses, while the other set helps CTK to promote plant development. The detailed mechanisms of CRF regulation in these processes remain to be determined. Identification of CRF target genes and the upstream signaling could allow for a better understanding about ERF-VIs function and how CTK regulates abiotic stresses.

33The plant hormone BRs play important roles throughout plant development, such as cell elongation, leaf development, pollen tube growth, xylem differentiation, senescence, and photomorphogenesis as well as stress response. BRs are sensed by plasma membrane located receptor kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1) to inhibit negative regulator BRASSINOSTEROID INSENSITIVE 2 (BIN2), leading to accumulation of transcription factors BRASSINAZOLE-RESISTANT 2/BRI1-EMS-SUPPRESSOR 1 (BES1/BZR1) to regulate 1000s of BR responsive genes involved in plant growth and stresses responses.

34BR regulates cold and drought responses through several pathways. For example, BR positively regulates cold tolerance partially though CBF-mediated cold response pathway, where BZR1 binds and promotes the expression of CBF1/CBF2 in response to cold. Cold stress also promotes accumulation of the unphosphorylated and active form of BZR1 by unknown mechanisms (; Figure 3). However, BR negatively regulates drought tolerance via antagonizing with drought induced transcription factor RD26 on drought responsive genes. BR also antagonizes with ABA pathway from receptors to transcription factors and regulates the trade-off of plants growth under stress conditions. Although there are no reports of AP2/ERFs in BR mediated drought response, BES1 and BZR1 target genes include numerous AP2/ERFs, implying that AP2/ERFs have a potential function to integrate the BR pathway with abiotic stresses. Future studies in this area will shed light on the mechanisms and roles of AP2/ERFs in BR and stress responses.

35In addition, AP2/ERFs regulate the BR pathway through different mechanisms. ERF72/RAP2.3 antagonizes BZR1 and AUXIN RESPONSIVE FACTOR 6 (ARF6) to inhibit hypocotyl elongation, while its role in BR regulated stresses response is unknown. The role of ERF72 in controlling growth implies that ERF72 might be a candidate for the study of the integration of BR and stresses. In fact, rice SUB1A mediates GA and BR cross-talk to control submergence tolerance. SUB1A activates BR biosynthesis and signaling, which in turn induces GA catabolic gene GA2ox7 to lower GAs content, and ultimately promotes rice DELLA protein accumulation.

36Based on AP2/ERFs roles in abiotic stresses and hormone signaling, AP2/ERFs function through complicated regulatory networks. These networks are influenced by diverse environment stimuli and plant hormones. Regulatory mechanisms such as protein–protein interactions and cooperative or antagonistic regulation of target genes are involved in dictating the output of AP2/ERF networks for plant growth, development and abiotic stresses. The large number of the AP2/ERF transcription factors coupled with functional redundancy and their diverse roles have made it difficult to fully understand AP2/ERFs networks. A promising approach is to apply computational tools to dissect AP2/ERFs function. These include analyzing promoter sequences for abiotic stress-related motifs, and constructing stress response gene regulatory networks under different stress conditions. For instance, analysis of 20 Arabidopsis DREB genes promoters demonstrated that they contained most types of HSE LTR and ABRE promoter elements. The multiple stress responsive motifs on one hand help explain the induction of DREBs under various environment stimuli, and on the other hand, they imply that these DREBs might form a central response network to control diverse abiotic stresses.

37In addition to promoter analysis, Van den Broeck et al. examined a complex, highly interconnected network of 20 Arabidopsis transcription factors (more than half are AP2/ERFs) to illustrate how stress inhibits plant growth. Under stresses condition, plants limit growth to promote survival. In this case, members from ERF-VIIIs (ERF8, ERF9, ERF11), ERF-IXs (ERF-1, ERF2, ERF5, ERF6, ERF59, ERF98) and ERF-X (RAP2.6L) were significantly up-regulated in proliferating and expanding tissues upon short-term mannitol exposure. Among these, ERF6 and RAP2.6L have already been shown to positively regulate stress tolerance and inhibit growth, implying they have potential role in growth and stress trade-offs. This work also illustrated that the activation of these ERFs was sequential. ERF5, ERF6, ERF11, and ERF98 showed a fast, strong and continuous induction, while other ERFs were regulated mildly and slowly. Transcriptomic profiling of each ERF using inducible overexpression plants demonstrated that they formed a highly interconnected gene regulation network consisting of redundant regulation mediated via interaction, activation, repression, and internal regulation. By combining osmotic stress and each individual ERFs transcriptiomic data it was possible to generate a network that simplifies the signaling cascades into linear pathways, although further genetic confirmation is needed.

38In order to gain additional insight into AP2/ERF function in abiotic stresses and hormone response pathways, we investigated Arabidopsis AP2/ERFs in gene co-expression networks that were generated by processing and classifying 1000s of public microarray datasets into tissue and process specific categories. If AP2/ERFs are involved in hormone- and stress- responsive gene regulation, then these transcription factors would be expected to connect to hormone/stress regulated genes in the network. We used transcriptome data from various stress and hormone treatments to test this idea and found that many AP2/ERFs connect to more hormone/stress regulated genes in the network than would be expected by chance (Figure 4A, Fisher’s exact test). We further divided AP2/ERFs into four clusters based on enrichment for hormone or stress responsive genes in the network. The role of CBFs in stress and hormone responses was investigated as an example. CBFs are distributed into four clusters that include AP2/ERFs enriched for cold, drought, salt and heat responsive genes. These clusters also include AP2/ERFs enriched for ABA, ET, and BR responsive genes. In line with these network predictions, the function of CBFs in ET and cold stress (including those mediated by BRs) has been confirmed by genetics or transcriptomic studies. We further compared genes differentially expressed in cbf123 triple mutants after cold treatment, BR and ET responsive genes (Figure 4B;;;; ). The comparison indicates that CBFs share a significant amount of genes with cold, ET and BR. For example, cold regulates ∼4000 genes, ∼68% of which are regulated by CBFs, supporting CBFs’ major contribution in cold tolerance. Among these, ∼20% of CBF regulated cold responsive genes are regulated by ET and BR, indicating that ET and BR regulated cold stress are dependent on the CBF pathway. ET and BR negatively and positively regulated CBFs through their specific transcription factors EIN3 and BES1/BZR1, respectively (Figure 4C). Taken together, these examples of network and transcriptome comparisons can (1) predict AP2/ERFs functions, (2) study AP2/ERFs function in specific stress/hormone responses; (3) examine the cross-talk of hormones in controlling growth and stress tolerance; (4) help clarify their upstream regulators and downstream targets.

39In summary, AP2/ERFs are regulated by stresses and control multiple plant stress responses to coordinate plant growth under stress conditions. AP2/ERFs are both regulated by plant hormone pathways and modulate plant hormone biosynthesis and signaling. Considering the complex roles of AP2/ERFs in abiotic stress and hormonal responses, future studies are required to fully understand this unique family of transcription factors. The regulation of AP2/ERFs by various hormone signaling pathways should be examined at transcriptional and post-transcriptional levels. The genome-wide identification of AP2/ERF target genes should help understand their functions as well as gauge the scope of their actions. Establishing gene regulatory networks using gene expression data and computational tools will be crucial to understand the large family of genes with 100s–1000s of target genes. Considering the complexity, the functions of AP2/ERFs should be examined in different tissues/cell types and in a temporal manner. Many AP2/ERFs are potential candidates for crops stress tolerance engineering. Thus, a full understanding of AP2/ERFs can guide crop engineering to achieve optimal plant growth and crop production under changing environmental conditions.

40Publicly available datasets were analyzed in this study. This data can be found here: http://www.plantphysiol.org/content/171/4/2744/tab-figures-data.

41ZX, TN, HJ, and YY conceived the topic. TN and HJ collected the gene regulation data. TN performed the network analysis. ZX wrote the manuscript with edits from other co-authors.

42The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.