177N. Tuteja and S. Singh Gill (eds.), Plant Acclimation to Environmental Stress,DOI 10.1007/978-1-4614-5001-6_8, Springer Science+Business Media New York 2013
Plants are affected by many unfavorable conditions including both biotic (e.g., bacteria, fungi, nematode, virus, weeds, parasitic plants, and insects) and abiotic stresses (e.g., drought, cold, salinity, freezing, heat, and water logging) that negatively affect their growth and productivity. It has been estimated that 90 % of total arable land experi-ence one or more kind of environmental stresses (Dita et al . 2006). These conditions are worsening over time because of global climatic changes and developing stress-tolerant crops are becoming more important to minimize crop loss and to increase productivity (Agarwal et al. 2006 ; Vinocur and Altman 2005 ; http://www.ipcc.ch ).
Unlike other organisms, plants are sessile, and in an attempt to overcome the imposed stresses, they trigger a cascade of molecular events when subjected to stress. These events include changes in gene expression which eventually lead to physiological and biological modi cations necessary to enhance tolerance to adverse conditions. The advent of genomics and proteomics has been helpful in understanding these stress-signal transduction regulatory networks and studies have suggested that transcription factors (TFs) play very important roles in the expres-sion of stress-responsive genes (Eulgem 2005 ; Fowler and Thomashow 2002 ; Yamaguchi-Shinozaki and Shinozaki 2006 ) .
S. Krishnaswamy Department Agricultural Food and Nutritional Science , University of Alberta , Edmonton , AB , Canada T6G 2P5
Southern Crop Protection and Food Research Centre , Agriculture and Agri-Food Canada London , ON , Canada , N5V 4T3
S. Verma M. H. Rahman N. Kav (*) Department Agricultural Food and Nutritional Science , University of Alberta , Edmonton , AB , Canada T6G 2P5 e-mail: Nat.Kav@ualberta.ca
Chapter 8 APETALA2 Gene Family: Potential for Crop Improvement Under Adverse Conditions
Sowmya Krishnaswamy , Shiv Verma , Muhammad H. Rahman, and Nat Kav
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TFs are DNA-binding proteins that interact with speci c cis -elements in the promoter regions of genes and regulate their expression by activating or repress-ing the recruitment of RNA polymerase (Karin 1990 ; Nikolov and Burley 1997 ) . A single TF regulates the expression of several other genes including TFs them-selves and are therefore considered to be important molecular targets for the genetic manipulation of cellular processes in plants (Hussain et al. 2011 ) . Indeed, transcriptional regulators, considered a dominant class of the gene fam-ily, played a major role in selection and domestication along with morphological development in plants, which led to dramatic improvement in productivity of most extensively grown crops worldwide like rice, wheat, and maize (Doebley et al. 2006 ) . Given the importance of TFs in regulation of metabolic pathways, it is not surprising that signi cant portion of plant genome encodes TFs. For example, approximately 5% of the Arabidopsis genome encodes for TFs (Riano-Pachon et al. 2007 ) .
APETALA2/ethylene response element-binding protein (AP2/EREBP) TF fam-ily is the major group among the TF families in Arabidopsis with 147 genes com-prising about 9 % of the total TFs (Feng et al. 2005 ) . In higher plants, close to 200 AP2 TF genes have been reported ( http://plntfdb.bio.uni-potsdam.de/v3.0/ ). For instance, the genomes of rice (Nakano et al. 2006 ) , grapevine (Jaillon et al. 2007 ) , and poplar (Zhuang et al. 2008 ) encode 139, 132, and 200 AP2/ERF-related pro-teins, respectively. The name AP2 arises from the protein APETALA that is involved in ower development (Jofuku et al. 1994 ) .
AP2/EREBPs are characterized by the presence of a DNA-binding domain called AP2 domain, about 68 amino acids long (Hao et al. 1998 ; Riechmann and Meyerowitz 1998 ) . Based on the presence of one or two AP2-DNA-binding domains, the family is further divided into four subfamilies, the AP2, DREB, ERF, RAV, and others (Sakuma et al. 2002 ) . AP2 subfamily encodes proteins with two AP2 domains and these proteins are implicated in various growth events like meristem determinance, organ identity, and ower development (Saleh and Pags 2003 ) . Examples of pro-teins belonging to this class include AP2, baby boom (BBM), Glossy15 (GL15), and AINTEGUMENTA (ANT) (Krizek 2009 ; Moose and Sisco 1996 ; Passarinho et al. 2008 ) . The DREB (dehydration-responsive element binding), ERF (ethylene-responsive factors), and RAV (related to ABI3/VP1) subfamily genes encode pro-teins with only one AP2 domain and members of these subfamilies have been implicated in stress signaling network (Guo et al. 2005 ; Saleh and Pags 2003 ; Gutterson and Reuber 2004 ; Shinwari et al. 1988 ) . The DREB groups were identi ed as genes encoding TFs involved in dehydration-responsive regulon (Liu et al. 1998 ; Stockinger et al. 1997 ) , whereas ERF groups were identi ed as binding factors mediating the ethylene response (Fujimoto et al. 2000 ) . The RAV groups were identi ed by Kagaya et al. ( 1999 ) as proteins with two DNA-binding domains, an AP2 and a B3 motif, and these proteins are involved in hormone and stress responses (Alonso et al. 2003 ; Hu et al . 2004 ; Sohn et al. 2006 ) . Examples of proteins belong-ing to DREB, ERF, and RAV subfamilies include C-repeat/dehydration-responsive element-binding factors (CBFs/DREBs), ERFs, LePtis, TINY, abscisic acid insensi-tive (ABI4), and RAV proteins (Riechmann 2000 ; Sakuma et al. 2002 ) .
1798 APETALA2 Gene Family: Potential for Crop Improvement Under Adverse Conditions
2 Gene Regulation by AP2 TFs
As mentioned previously, ERF and DREB subfamily proteins are the major groups in AP2 family. For instance, in Arabidopsis out of 147 AP2 genes, 65 belong to ERF and 56 belong to DREB subfamily (Sakuma et al. 2002 ) . The ERF subfamily pro-teins interact with ethylene response elements (ERE) or GCC box and regulate the expression of ethylene-inducible pathogenesis-related genes such as prb-1b, b -1, 3-glucanase, chitinase, and osmotin (Bttner and Singh 1997 ; Ohme-Takagi and Shinshi 1995 ; Xu et al. 1998, 2006 ) . They can act as both activators and repressors of gene expression. For example, Arabidopsis AtERF1, AtERF2, and AtERF5 func-tion as activators of GCC-dependent transcription, while AtERF3, AtERF4, and AtERF7 act as repressors of GCC-dependent transcription (McGrath et al. 2005 ; Xu et al. 2006 ) .
The DREB subfamily proteins interact with C-repeat or dehydration response elements (DRE) and regulate the expression of low-temperature and/or water de cit responsive genes (Jaglo-Ottosen et al. 1998 ; Kasuga et al. 1999 ; Liu et al. 1998 ) . The DRE (5 -TACCGACAT-3 ) elements are found in the promoters of drought and cold-inducible genes like rd29A (Yamaguchi-Shinozaki and Shinozaki 1994 ) . Similar to DRE, the C-repeat 5 -TGGCCGAC-3 (containing the core 5 -CCGAC-3 ) elements are found in the COR (cold-regulated) genes like cor15a , rab18 , kin1 , and kin2 (Baker et al. 1994 ; Kurkela and Borg-Franck 1992 ; Kurkela and Franck 1990 ; Lang and Palva 1992 ) . Liu et al. ( 1998 ) using reporter genes demonstrated, for the rst time, that DREB proteins DREB1 and DREB2 act as activators of pro-moters harboring DRE elements. Overexpression studies have also demonstrated the role of DREB genes in DRE-dependent gene regulation. For example, overex-pression of DREB1 - and DREB2 -induced expression of regulatory region rd29A which is involved in abiotic stress signaling (Liu et al. 1998 ) . Moreover, DREB1A overexpression induced the expression of COR genes such as rd29A/cor78/lti78 , kin1 , cor6.6 / kin2 , cor15a , cor47/rd17 , and erd10 (Kasuga et al. 1999 ) .
Earlier, it was thought that DREB-dependent regulation is involved only in abi-otic stresses, whereas ERF genes are involved mostly in biotic stresses (Guo and Ecker 2004 ; Shinozaki and Yamaguchi-Shinozaki 2000 ) . However, recent studies have indicated that DREB and ERF-type AP2 TFs are involved in multiple path-ways activated by both kinds of stresses. For example, overexpression of tobacco ERF, Tsi1 , enhances resistance to both Pseudomonas syringae as well as osmotic stress. In addition, the Tsi1 protein was shown to be capable of binding to DRE/CRT elements in vitro (Park et al. 2001 ) . Furthermore, the overexpression of CaERFLP1 resulted in enhanced expression of salt-inducible LT145 which contains multiple DRE/CRT elements in its promoter (Lee et al. 2004 ) . In addition, a series of ERF TFs were found to interact with the DRE/CRT motif in vitro (Yi et al ., 2004; Li et al. 2005 ; Xu et al. 2007 ) . Similarly, DREB-type AP2 TFs have been shown to regulate biotic stress signaling. For instance, DREB2A , a regulator of dehydration-respon-sive pathway was found to cross talk with Adr1 (activated disease resistance 1) activated signaling network (Chini et al. 2004 ) . Furthermore, a DREB-like factor,
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TINY, demonstrated its ability to interact with both DRE and ERE elements with similar af nity and activated reporter genes containing these elements (Sun et al. 2008 ) . Moreover, the overexpression of TINY in seedlings enhanced the expres-sion of both DRE- and the ERE-containing genes in transgenic Arabidopsis (Sun et al. 2008 ) . Similarly, it was demonstrated that RAP2.4 acts as a transactivator of both DRE- and ERE-mediated genes that are responsive to light, drought, and ethylene (Lin et al. 2008 ) . Recently, DEAR1 (DREB and EAR motif protein 1) gene whose expression is elevated in response to both biotic and abiotic stresses, when overexpressed, showed constitutive expression of PR genes and tolerance to P. syringae in transgenic Arabidopsis plants (Tsutsui et al. 2009 ) . Similarly, the overexpression of PgDREB2A resulted in the upregulation of dehydrins and heat-shock protein genes as well as NtERF5 that mediate expression of PR genes (Agarwal et al. 2010 ) . Therefore it appears that some DREB and ERF transcription factors have a regulatory role in mediating cross talk between biotic and abiotic stress signaling pathways.
AP2 TFs also regulate the expression of members of the same family. For instance, it was demonstrated that CBF2/DREB1C acts as a negative regulator of CBF1 / DREB1B and CBF3 / DREB1A expression during cold acclimatization (Novillo et al. 2004 ) . In addition, AP2 TFs are subjected to different temporal regu-lation to ensure transient and controlled expression of stress-related genes. For instance, in Brassica napus, it has been observed that trans- active Group I factors that bind with DREBs are expressed immediately on exposure to cold stress to turn on the DRE-mediated signaling pathway, whereas trans -inactive Group II proteins were expressed at later stages compete with the Group I to bind with the DRE and prevent the activation, and thus block the signal pathway (Zhao et al. 2006 ) . AP2 TFs can also regulate their own expression like many other TFs. For example, the protein RAP2.1 possesses an AP2 domain that binds to DREs and regulates desic-cation/cold-regulated ( RD/COR ) gene. Additionally, RAP2.1 can negatively regu-late its own expression and keep the expression of stress response genes under tight control (Dong and Liu 2010 ) .
Since AP2 TFs have been demonstrated to have important role in regulation of many genes in addition to their own expression, they have received much attention in recent time as ideal candidates for crop improvement. In addition, other proteins like inducer of CBF expression 1 (ICE1), calmodulin-binding transcription activa-tor (CAMTA), ZAT12 (a zinc nger protein) that are involved in the regulation of AP2 family proteins, may also serve as good targets for manipulation (Chinnusamy et al. 2003 ; Doherty et al. 2009 ; Vogel et al. 2005 ) .
3 Abiotic Stress Tolerance
Once the cis-regulatory elements of DREB/ERF TFs were identi ed as CRT/DRE and GCC elements, genetic and molecular approaches were used to investigate the potential utility of AP2/EREBP TFs from a wide variety of plants in order to enhance
1818 APETALA2 Gene Family: Potential for Crop Improvement Under Adverse Conditions
stress tolerance. Much of the data has come from overexpression and loss-of- function analysis and a list of characterized AP2 TFs from various species is presented in Table 8.1 . AP2/EREBP members have demonstrated their crucial role in regulating different kinds of abiotic stress response, including drought, low temperature, salin-ity, and hypoxia (Haake et al. 2002 ; Hinz et al. 2010 ; Novillo et al. 2004 ; Oh et al. 2005, 2007 ; Yang et al. 2011 ) .
DREB subfamily has been classi ed into six (A1A6) groups (Sakuma et al. 2002 ) , and DREB1A and DREB2A are the most studied genes among the DREBs. Group A1 contains CBF s and DDF (dwarf and delayed owering) genes. The DREB1/CBF cold-response pathway is well characterized in Arabidopsis and rice ( Oryza sativa ) (Yamaguchi-Shinozaki and Shinozaki 2006 ) . Three DREB1/CBF genes, namely CBF1 (also called as DREB1b ), CBF2 (also called as DREB1c ), and CBF3 (also called as DREB1a ) have been isolated from Arabidopsis (Gilmour et al. 1998 ; Liu et al. 1998 ; Stockinger et al. 1997 ) . From rice, DREB1/CBF homologs such as OsDREB1A , OsDREB1B , OsDREB1C , and OsDREB1D have been isolated (Dubouzet et al. 2003 ) . In response to low-temperature stress, these genes are quickly induced, and their products activate the CBF regulon to improve freezing tolerance (Agarwal et al. 2006 ; Nakashima and Yamaguchi-Shinozak 2006 ) . Overexpression of DREB1A with constitutive and stress-inducible promoters in Arabidopsis has resulted in multiple abiotic stress tolerance including freezing stress tolerance (Kasuga et al. 2004 ; Liu et al. 1998 ) . Additionally, overexpression of rice OsDREB1A in Arabidopsis resulted in expression of stress-related genes and consequent improved tolerance to abiotic stresses including drought, high salt, and freezing (Dubouzet et al. 2003 ) .
Metabolome analysis of DREB1A/CBF3 overexpressing Arabidopsis plants has demonstrated that monosaccharides, disaccharides, oligosaccharides, and sugar alcohol pro les were similar to the low-temperature-regulated metabolome (Cook et al. 2004 ; Maruyama et al. 2009 ) which suggests DREB1A may enhance tolerance by regulating genes involved in stress response. Indeed, the crucial role of group A1 DREBs...