Multiple regulatory roles of AP2/ERF transcription factor in angiosperm

APETALA2/ethylene response factor (AP2/ERF) transcription factor (TF) is a superfamily in plant kingdom, which has been reported to be involved in regulation of plant growth and development, fruit ripening, defense response, and metabolism. As the final response gene in ethylene signaling pathway, AP2/ERF TF could feedback modulate phytohormone biosynthesis, including ethylene, cytokinin, gibberellin, and abscisic acid. Moreover, AP2/ERF TF also participates in response to the signals of auxin, cytokinin, abscisic acid, and jasmonate. Thus, this superfamily is key regulator for connecting the phytohormonal signals. In this review, based on the evidence of structural and functional studies, we discussed the multiple regulator roles of AP2/ERF TF in angiosperm, and then constructed the network model of AP2/ERF TF in response to various phytohormonal signals and regulatory mechanism of the cross-talk.


Background
The surperfamily APELATA 2/ethylene response factor (AP2/ERF) has been studies in many plants, which have a range of 119-200 members (Du et al. 2014;Nakano et al. 2006;Rao et al. 2015;Zhuang et al. 2008), and have been reported in responses to ethylene, stress, metabolic, fruit ripening and senescence (Han et al. 2016;Koyama et al. 2013;Lee et al. 2012;Li et al. 2007;Fits and Memelink 2000;Trujillo et al. 2008;Zhu et al. 2014). All the time, regulatory mechanism of AP2/ERF TF in these fields were wide-spread studies by many scientists and their research teams, and increasing experimental evidence was exploited to elucidate the detailed roles in each field (Guo and Ecker 2004;Liu et al. 2014;Pré et al. 2008;Taketa et al. 2008;Tang et al. 2016;Xiao et al. 2013;Yin et al. 2016). Herein, research advance of AP2/ERF TF was reviewed in plant, and the doubtful viewpoints were also discussed.

Classification and DNA-binding elements
According to previous reports, the superfamily AP2/ERF members contain a common DNA binding domain, AP2 domain. Based on the difference of this domain in copy numbers, AP2/ERF TF could usually be divided into four families, AP2, ERF, RAV, and Soloist (Nakano et al. 2006;Licausi et al. 2010a). AP2 members constitute by one or additionally taking a tandem repeated AP2 domain (Kagaya et al. 1999;Licausi et al. 2013). ERF members characterize by a single AP2 domain (Nakano et al. 2006;Licausi et al. 2013). RAV members comprise by a consensus sequence elements for both AP2 domain and B3 domains (Kagaya et al. 1999;Swaminathan et al. 2008). Soloist family have little members (one or two) that also contain a single AP2 domain in all sequenced plant genome, but they strongly diverged in gene sequence from other AP2/ERF members (Du et al. 2013;Licausi et al. 2010a;Rao et al. 2015;Zhuang et al. 2008). Because of ERF family members could bind to two mainly DNAbinding elements (Hao et al. 2002;Sakuma et al. 2002), resulting in a novel DREB family is separated from ERF family (Du et al. 2013;Rao et al. 2015;Sakuma et al. 2002;Zhuang et al. 2008). Of the DREB and ERF families, all the members are further classified into six groups, A1 to A6 and B1 to B6, respectively (Sakuma et al. 2002). However, these twelve groups are re-designated with group I to X, VI-L, and Xb-L or group A to J (Nakano et al. 2006). The re-designated classification is employed in horticultural plants, such as Vitis vinifera, Prunus mume, and Solanum lycopersicon (Licausi et al. 2010a;Du et al. 2013;Pirrello et al. 2012), whereas the traditionally classification is used in other plant species, including Salix arbutifolia, Nicotiana tabacum, and Populus trichocarpa (Rao et al. 2015;Sasaki et al. 2007;Zhuang et al. 2008).
AP2/ERF proteins have strongly capacity to bind a wide range of cis-regulatory elements in promoter of target genes (Sasaki et al. 2007). Of these cis-regulatory elements, GCC-box (AGCCGCC element) and DRE/CRT (dehydrationresponsive element/C-repeat, RCCGCC element) are the mainly two DNA-binding elements (De Boer et al. 2011;Fujimoto et al. 2000;Hao et al. 1998Hao et al. , 2002Oñate-Sánchez et al. 2007;Wang et al. 2012). Noteworthy, most AP2/ERF proteins can bind GCC-box containing promoter, but the activation degree is different among members in various groups. For instance, the members are weak activators in group A, B and E, neutral in class G and H, and strong in group C, whereas that are as repressor in group F (Pirrello et al. 2012). Besides GCC-box and DRE/CRT, the elements diverged from these two also belong to cis-regulatory elements, which may be in response to different stimuli underlying various stresses (Mizoi et al. 2012;Shaikhali et al. 2008;Welsch et al. 2007). Moreover, ERF protein can also bind to VWRE (vascular wounding responsive element, GAAAAGAAAATTTC) and CE1 (coupling element, CACCG) in tobacco (Sasaki et al. 2007;Wu et al. 2008). In addition, few reports reveal that ERF proteins could interact directly with a non-GCC element containing promoters (Chakravarthy et al. 2003).

Ethylene response
Ethylene is an important phytohorome for plant growth, development, senescence, and stress tolerance. Ethylene is synthesized by ACS (1-aminocyclopropane-1-carboxyla synthase) catalyzing substrate of SAM (S-adenosyl methionine) to form ACC (1-aminocyclopropane-1-carboxyla acid), and then impel by ACO (1-aminocyclopropane-1-carboxyla oxidase). Sequentially, how much ethylene produced in plant tissues are positive correlated to ACS and ACO activities. The produced ethylene in plant tissues is combined with ETR (Ethylene receptor) to activate constitutive triple response (CTR), and then induce expression of a set of ethylene insensitive (EIN) and Ethylene insensitive-like (EIL). The EIN/EIL proteins bind to upstream regions of ERF TFs to promote it expressed in tissues (Alexander and Grierson 2002;Guo and Ecker 2003;Solano et al. 1998). However, due to GCC-box usually presented in the promoter of ACS and ACO in many plants, the expressed ERF genes will enhance the activities of the two genes, thereby accelerate ethylene biosynthesis and signal transduction, such as LeERF1, AtERF73/HRE1, TERF2/LeERF2, and MaERF9 (Li et al. 2007;Xiao et al. 2013;Yang et al. 2011;Zhang et al. 2009). Besides the positive feedback genes, few ERF TFs also represent as repressor of ACS and ACO activities to prevent ethylene biosynthesis, including AtERF4, AtERF11, SlERF6, and MaERF11 (Lee et al. 2012;Li et al. 2011;Xiao et al. 2013;Yang et al. 2005). In addition, ERF. B3 has the ability to modulate the transcription levels of a subset of other ERF TFs (Liu et al. 2013). Noteworthy, this subset contains the aforementioned activators and repressors of ethylene biosynthesis and signal pathway genes. Thus, AP2/ERF TF is not only in response to ethylene signal transduction, but also can feedback regulate ethylene synthesis in plant tissues.

Stress tolerance
Stresses are the negative environment factors around plant growth and development. Both abiotic and biotic stresses are mediated by multiple transcriptional factors, such as NAC, WRKY, MYB, bHLH, bZIP, and ERF (Abe et al. 2003;Li et al. 2013;Puranik et al. 2012;Rushton et al. 2010;Singh et al. 2002;Zhang et al. 2012a). Most studies have found the importance of AP2/ERF TF in defense of various stresses. In general, the AP2/ERF TFs in response to abiotic stresses are the members of DREB family (Licausi et al. 2013;Sakuma et al. 2002). Such as AtERF98, MsERF8, JcERF011, and CaERFLP1 that enhance tolerance to salt Lee et al. 2004;Tang et al. 2016;Zhang et al. 2004Zhang et al. , 2012b. TERF2/ LeERF2, CBF1, and CBF3 exalt cold and freezing tolerances (Novillo et al. 2007;Tian et al. 2011;Zhang et al. 2010b). Sub1A, SNORKEL1 and SNORKEL2 allow rice to adapt to deep water (Fukao et al. 2006(Fukao et al. , 2011Hattori et al. 2009;Xu et al. 2006). HRE1 and HRE2 improve the tolerance of the plant to the hypoxia stress (Licausi et al. 2010b). OsWR1, JERF1, TERF1, and SHINE are positive regulators of resistance to drought (Aharoni et al. 2004;Wang et al. 2012;Zhang et al. 2005Zhang et al. , 2010a. Moreover, few of AP2/ERF TFs are involved to modulate at least two different abiotic stresses in defense response. For example, over-expression of SlERF5 in transgenic tomato plants result in high tolerance to drought and salt stress (Pan et al. 2012). Over-expression of JERF3 and SodERF3 improve resistance to drought, osmotic, salt, and freezing stresses in transgenic rice and tobacco (Trujillo et al. 2008;Wu et al. 2008;Zhang et al. 2010c). Ectopic expression of DREB2A in Arabidopsis increase endurance to drought, stress, and heat stresses (Sakuma et al. 2006a, b).
Unless enhanced tolerance to abiotic stresses, AP2/ ERF TF also are reported to be concerned in raising resistance to biotic stresses. Over-expression of NtERF5 contributes to high tolerance to Tobacco mosaic virus in Nicotiana tabacum (Fischer and Droge-Laser 2004). Silence-expression of ORA59 or RAP2.2 results in low tolerance to Botrytis cinerea in Arabidopsis thaliana (Pré et al. 2008;Zhao et al. 2012). Loss-of-function mutants of AtERF2 or AtERF14 are more susceptible against Fusarium oxysporum in Arabidopsis thaliana (McGrath et al. 2005;Oñate-Sánchez et al. 2007). Exceptionally, AtERF4 is the negatively genes in regulating Fusarium oxysporum resistance (McGrath et al. 2005). Similar to that in abiotic stress defense, few of AP2/ERF TFs have the ability to coordinate two or more biotic stresses in defense response. For instance, Over-expression of MtERF1-1 improves tolerance to Rhizoctonia solani and Phytophthora medicaginis in Medicago roots (Anderson et al. 2010). Over-expression of ERF1 in Arabidopsis conferred resistance to necrotrophic fungi including B. cinerea and Plectosphaerella cucumerina (Berrocal-Lobo et al. 2002). The tomato Transcription Factor Pti4 Regulates Defense-Related Gene Expression for Pseudomonas syringae and Erysiphe orontii by combined to GCC Box and Non-GCC Box cis Elements (Chakravarthy et al. 2003).
In addition, few AP2/ERF TFs had been reported responsible for biotic and abiotic stress, simultaneously. A typical example is the positively regulator TaPIE1 that raise the defense responses to R. cerealis and freezing stresses by activating defense-and stress-related genes (Zhu et al. 2014). Taken together, AP2/ERF TF plays very important roles in regulating defense response to all kinds of biotic and abiotic stresses.

Plant growth, development, and senescence
The life of plant is cycled through seed germination, seedling growth, organ development, and senescence. In this cycle, AP2/ERF TF also displays their regulatory roles for shaping many architectural traits. In the process of seed germination, SlERF2 positively improve transcription level of marker gene, mannanase 2, resulting in a stimulation of premature germination, and enhance hook formation of darkgrown (Pirrello et al. 2006). In the progression of plant growth and development, AIN-TEGUMENTA and AINTEGUMENTA-LIKE6 are related to flower organ growth and ovule development in Arabidopsis (Elliott et al. 1996;Jofuku et al. 1994;Klucher et al. 1996;Krizek 2009;Mizukami and Fischer 2000). Rice ethylene-response AP2/ERF factor OsEATB restricts internode elongation by down-regulating ent-kaurene synthase A, leading to a reduction of rice plant height and panicle length at maturity (Qi et al. 2011). In contrast, AtERF1, AtDREB1, and TINYT present their ability in dwarfing plant height (Liu et al. 1998;Solano et al. 1998;Wilson et al. 1996). Moreover, NtERF3, AtERF4 and AtERF8 had been found to be associated with plant aging (Koyama et al. 2013). Of these three genes, AtERF4 and AtERF8 belonged to class II ERFs in Arabidopsis, which can accelerate precocious leaf senescence by targeting the EPITHIOSPECIFIER PROTEIN/EPITHIOSPECIFYING SENESCENCE REGULATOR gene and regulating the expression of many genes related to senescence (Koyama et al. 2013). In addition, AP2/ERF TF is involved in regulating metabolite productions, such as chlorophyll, wax and cutin. The present evidences show that CitERF13 is negative regulator for chlorophyll degradation during Citrus fruit degreening by directly binding to the Cit-PPH promoter and enhancing the activity of a metabolite of pheophorbide hydrolase (Yin et al. 2016). AtWIN1, AtSHN, and HvNUD could increase an accumulation of wax and cutin on the epidermis by regulating a lipid biosynthesis pathway (Aharoni et al. 2004;Broun et al. 2004;Taketa et al. 2008). Obviously, the functions of these AP2/ERF TFs are distinctly elucidated in these reported traits, but the regulatory roles of other members should be further explored in unknown properties in future.

Fruit ripening
Fruit is one of important tissues in fruited plants, which harbors seed formation, development, and maturity. According to respiratory intensity during ripening, fruit is divided into climacteric and non-climacteric phenotypes. The climacteric fruit must release massive ethylene at ripening, also called ethylene-dependent fruit. On the contrary, the non-climacteric fruit is ethylene-independent. To date, ethylene-dependent fleshy-fruits are the primary materials for studying fruit ripening, such as tomato, apple, and banana. In ethylene-dependent fruits, ERF, as the final response gene in ethylene signaling pathway, directly regulate fruit ripening by binding to the promoters of their downstream genes, includ- . Therefore, the regulatory role of AP2/ERF TF is multiple, and their regulated mechanism is very complex during fruit ripening.

Integration of phytohormonal signals
Phytohormones are a group of naturally occurring, organic substances which affected plant growth, development, and senescence at low concentrations. Of these phytohormones, auxin, cytokinin, and gibberellin are reported to be involved in regulation of seed germination and plant growth (Pacifici et al. 2015;Urbanova and Leubner-Metzger 2016;Werner et al. 2001). Ethylene plays extremely important roles in climacteric fleshy fruit ripening and senescence (Hayama et al. 2006;Xiao et al. 2013;Yin et al. 2008), and together with jasmonate and abscisic acid, participate in defense response to biotic and abiotic stresses (Li et al. 2011;Lorenzo et al. 2003;Pré et al. 2008). Obviously, cross-talk among these phytohormones must be carried out in plant tissues. This crosstalk is always surveyed by many scientists, and increasing evidences are emerged to elucidate the talk mechanism. Ethylene signal transduction is a general pathway during the life cycle of plant. As the final response gene in ethylene signaling pathway, AP2/ERF are also documented to be involved in response to other hormones. In rice, an AP2/ERF TF OsCRL5 is induced by treating with exogenous auxin, and inhibits cytokinin signal transduction by enhancing the activities of two repressors (Kitomi et al. 2011). Interestingly, several AP2/ERF TFs in subgroup B-5 are responsible for exogenous cytokinin, thereby designated as cytokinin response factor (Rashotte et al. 2006). Also in rice, OsEATB, which is restrained by ethylene and abscisic acid in expression level, negative regulate gibberellin biosynthesis by down-regulating a pathway gene (Qi et al. 2011). In Arabidopsis, tobacco and tomato, however, few AP2/ERF TFs are shown to modulate abscisic acid responses, such as AtERF11 and TSRF1 (Li et al. 2011;Zhang et al. 2008). The ethylene-, jasmonate-, and abscisic acid-responsive JERF1 regulates abscisic acid biosynthesis-related gene in expression level (Zhang et al. 2004;Wu et al. 2007). Moreover, NIC2 participate in mediating jasmonate-elicited nicotine biosynthesis (De Boer et al. 2011). ORA59, which was induced by jasmonate and ethylene in expression level, is the key regulator of jasmonate-and ethylene-responsive PLANT DEFENSIN 1.2 expression by binding to GCC-box element in the promoter (Pré et al. 2008;Zarei et al. 2011). AtERF2 is a positive regulator of jasmonate-responsive defense genes, while AtERF4 negative adjust jasmonateresponsive defense gene expression (McGrath et al. 2005). Overall, AP2/ERF TF is the key regulator to integrate all kinds of phytohormonal signals.

Conclusions
The AP2/ERF superfamily has hundreds of members in various plants, which contains at least one AP2 domain in all designated families. Generally, AP2/ERF TF mediates downstream responsible genes by binding to the GCC-box and/or DREB element in the promoter. Unless responses to ethylene signal, a large number of AP2/ERF members are stimulated by auxin, cytokinin, abscisic acid, and jasmonate signals. Meanwhile, several members also modulate gibberellin, cytokinin, and abscisic acid contents by directly regulating biosynthesis pathway genes of these phytohormones. Moreover, the stimulated genes would further regulate downstream effectors, resulting in changes of agronomic traits, including plant growth, defense responses, and fruit ripening (Fig. 1). In summary, AP2/ERF TF presents multiple regulatory roles in angiosperm.