163N. Tuteja and S. Singh Gill (eds.), Plant Acclimation to Environmental Stress,DOI 10.1007/978-1-4614-5001-6_7, Springer Science+Business Media New York 2013
Various abiotic stresses such as drought, salinity, and extremes of temperature pose a major challenge for survival of plants and have a great impact on crop productivity. The world population is increasing at an alarming rate and on the contrary, availability of food resources is decreasing due to the abiotic stress factors. Thus, there is a great need to generate stress-tolerant crop plants with improved sustenance and better yield. Plants elicit several responses to combat the adverse effects of various abiotic stresses, including production and accumulation of osmolytes, maintenance of intracellular ion homeostasis, and scavenging of reactive oxygen species. Understanding plant responses to abiotic stresses at the molecular level provides an essential foundation for future breeding and genetic engineering programs. Abiotic stress response is a complex trait, which involves interplay of numerous regulatory molecules at the cellular level. Various approaches have provided a holistic view of the ongoing cellular activities in response to abiotic stresses.
Numerous genes which are induced in response to abiotic stresses have been identi ed and the products of these genes are supposed to enhance stress tolerance in plants. The role of several stress-inducible genes has been explored, which regulate gene expression via various signal transduction pathways. Among these genes, transcription factors represent master switches controlling several target genes and are considered most important for regulation of gene expression. Although several transcription factors have been implicated in abiotic stress responses, only a few master switches/regulons have been identi ed and characterized in detail so far. The identi cation of master switches, which control stress-inducible genes, seems to be the most challenging task. Serious endeavor needs to be made in this direction to
A. Bhattacharjee M. Jain (*) National Institute of Plant Genome Research (NIPGR) , Aruna Asaf Ali Marg , New Delhi 110 067 , India e-mail: firstname.lastname@example.org
Chapter 7 Homeobox Genes as Potential Candidates for Crop Improvement Under Abiotic Stress
Annapurna Bhattacharjee and Mukesh Jain
164 A. Bhattacharjee and M. Jain
identify promising master regulator genes and eventually gain insights into the complex gene regulatory network, which leads to abiotic stress responses.
Several excellent comprehensive reviews are available on various aspects of complex signal transduction pathways controlling abiotic stress responses, emerging trends in genomics of abiotic stress responses and gene regulatory networks involved in abiotic stress responses and tolerance (Bartels and Sunkar 2005 ; Chinnusamy et al. 2004 ; Hirayama and Shinozaki 2010 ; Mahajan and Tuteja 2005 ; Nakashima et al. 2009 ; Urano et al. 2010 ; Vij and Tyagi 2007 ; Yamaguchi-Shinozaki and Shinozaki 2006 ) . In this review, we provide a brief overview of the role of transcrip-tion factors in abiotic stress responses and majorly focus on the emerging role of homeobox transcription factors in abiotic stress responses and their potential as target genes for engineering stress tolerance.
2 Overview of Stress-Responsive Genes
Several studies in Arabidopsis and rice have revealed that quite a large proportion of the genome is involved in abiotic stress responses. The global expression pro ling in various plant species has revealed that expression of thousands of genes is altered in response to various abiotic stress conditions (Hadiarto and Tran 2011 ; Hirayama and Shinozaki 2010 ; Nakashima et al. 2009 ; Urano et al. 2010 ; Vij and Tyagi 2007 ) . These stress-responsive genes have been broadly categorized in two groups (Yamaguchi-Shinozaki and Shinozaki 2006 ) . The rst group is comprised of genes involved directly in abiotic stress tolerance, including those encoding for metabolic proteins like water channel proteins, enzymes required for synthesis of various osmoprotectants (like sugars, glycine-betaine, and proline), proteins that aid in protecting macromolecules and membranes (for example, LEA proteins, osmotin, chaperones), proteases for protein turnover and detoxi cation enzymes like glutathione-S-transferases. The second group is comprised of genes encoding for regulatory proteins like protein kinases, transcription factors, protein phosphatases, and other signaling molecules. Overall, the expression of genes involved in diverse cellular processes is altered in response to abiotic stresses. The comparative analysis has revealed that a larger fraction of stress-responsive genes are common among various plant species, indicating the conserved mechanism of abiotic stress response. Further, many of these genes have been analyzed for their exact function and ability to provide stress tolerance in transgenic plants (Vij and Tyagi 2007 ) .
The emerging role of various plant hormones has also been explored in the context of abiotic stress responses. Abscisic acid (ABA) is found to be the key hormone produced in plants under stress conditions and is crucial for abiotic stress responses (Hirayama and Shinozaki 2007 ) . The application of exogenous ABA often mimics the abiotic stress responses. The expression of several stress- responsive genes is also induced by ABA (Shinozaki et al. 2003 ; Zhu 2002 ) . However, several studies report that many stress-responsive genes do not show response to ABA, suggesting the existence of two signal transduction pathways,
1657 Homeobox Genes as Potential Candidates for Crop Improvement
namely ABA-dependent and ABA-independent pathways, in abiotic stress responses (Nakashima et al. 2009 ; Zhu 2002 ) . Other phytohormones, such as sali-cylic acid, ethylene, and jasmonic acid have also been shown to play important roles in abiotic stress responses directly or via interplay with ABA (Fujita et al. 2006 ; Grant and Jones 2009 ; Pieterse et al. 2009 ) . Recently, auxin has also been implicated in abiotic stress responses. Quite a large number of auxin-responsive genes have been shown to be differentially expressed under various abiotic stress conditions (Jain and Khurana 2009 ) .
Although a comprehensive knowledge has accumulated about the stress- responsive genes, the biggest challenge is to decipher their functions and logically integrate the available knowledge to understand the mechanism underlying abiotic stress response and selection of most suitable target gene(s) for improving stress tolerance.
3 Overview of Gene Regulatory Network Involved in Abiotic Stress Response
The differential expression of a large number of genes indicates that the gene regulatory network operative during abiotic stress is very complex in plants. The expression of several transcription factor encoding genes is also induced in response to various abiotic stresses. The transcription factors (TFs) are considered as master regulators of the gene expression. An individual TF can govern the expression of numerous target genes by binding to speci c cis -regulatory motifs present in their promoters either independently or in coordination with other proteins constituting gene regulatory network (Nakashima et al. 2009 ; Urano et al. 2010 ) . Some of the stress-associated TFs are themselves regulated at the transcriptional level. This type of transcriptional regulatory system is called regulon and is required for ne-tuned gene expression in response to abiotic stresses.
TFs and their regulons involved in both ABA-dependent and ABA-independent pathways have been identi ed and characterized in plants (Nakashima et al. 2009 ) . The regulons involving dehydration-responsive element (DRE) binding protein 1 (DREB1)/C-repeat binding factor (CBF) and DREB2 TFs regulate stress response via ABA-independent pathway. These TFs bind to the conserved DRE motif A/GCCGACNT sequence in the promoter region of their target genes. The regulons involving ABA-responsive element (ABRE) binding protein (AREB)/ABRE bind-ing factor (ABF) TFs act via ABA-dependent pathway (Nakashima et al. 2009 ) . AREB/ABF TFs harboring a bZIP type DNA-binding domain binds to the ABRE (PyACGTGG/TC) and plays a pivotal role in ABA-dependent gene activation (Choi et al. 2000 ) . Other regulons comprised of NAC and MYB/MYC TFs are also sup-posed to regulate the abiotic stress response via ABA-independent pathway. NAC proteins recognize a MYC-like target sequence and activate downstream gene expression (Tran et al. 2006 ) . The components of these regulons and their functions have been found to be conserved in dicots and monocots indicating the common regulatory mechanisms of gene expression among them in response to stress
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(Nakashima et al. 2009 ) . Recently, another regulon involving the transcriptional regulator, multiprotein bridging factor 1 (MBF1), has been identi ed in Arabidopsis , which regulates heat-response (Suzuki et al. 2011 ) .
Although most of the TF regulons are functional and have overlapping roles in response to multiple stresses, some of them are speci c to particular abiotic stress condition(s) only. DREB1/CBF regulon responds to cold stress and controls the expression of several downstream genes. The overexpression of DREB1/CBF of Arabidopsis and homologous genes from other plants in transgenics resulted in strong tolerance to abiotic stresses, including drought, high salinity, and freezing (Dubouzet et al. 2003 ; Kasuga et al. 1999 ; Qin et al. 2004 ) . DREB2 regulon func-tions in both osmotic and heat stress responses in Arabidopsis , whereas in dehydra-tion and high salinity in grasses (Nakashima et al. 2009 ) . Arabidopsis AREB/ABFs are induced in response to ABA, dehydration, and high salinity. The overexpression of AREB1 resulted in ABA hypersensitivity and drought tolerance (Fujita et al. 2005 ) . It has also been suggested that phosphorylation may be responsible for the activation of AREB/ABFs (Fujii et al. 2007 ; Furihata et al. 2006 ; Uno et al. 2000 ) . The response of AREB/ARF regulon to dehydration and high salinity has been found to be conserved in rice also (Kagaya et al. 2002 ; Kobayashi et al. 2005 ) . Further, the overexpression of NAC genes conferred tolerance to drought stress in transgenic Arabidopsis plants and up-regulated several stress-inducible genes (Fujita et al. 2004 ; Tran et al. 2004 ) . It has also been demonstrated that NAC TFs act along with ZF-HD proteins to activate the expression of downstream target gene, EARLY RESPONSE TO DEHYDRATION 1 ( ERD1 ) (Tran et al. 2006 ) . In rice, one of the NAC TFs has been found to be responsive to ABA, abiotic stresses and biotic stresses, and its overexpression imparted enhanced stress tolerance in trans-genic plants (Nakashima et al. 2007 ) .
Taken together, TFs play important roles in abiotic stress responses and are powerful targets for engineering stress tolerance in transgenic plants because the overexpression of a single TF may lead to induction of diverse stress-responsive genes. Further, the role of many other stress-responsive TFs and their regulons need to be identi ed for understanding the molecular mechanisms of abiotic stress responses.
4 Homeobox Genes
Homeobox genes represent a class of TFs containing a conserved 180 bp long DNA sequence, which encodes for a 60 amino acid long DNA-binding domain termed as homeodomain (HD). The HD consists of three alpha helices forming a helix- turn-helix, which binds to speci c DNA sequence and regulates the expression of target genes. The rst homeobox genes were identi ed in Drosophila melanogaster and thereafter in all the eukaryotes. Homeobox genes are known to be the key regu-lators of various aspects of development, including cell fate determination and body plan speci cation.
1677 Homeobox Genes as Potential Candidates for Crop Improvement
Homeobox genes are represented by a large multigene family in plants, which have been classi ed into several distinct classes based on the amino acid sequence of HD and presence of other conserved domains. Initially, homeobox genes were classi ed into seven classes, including KNOX, BEL, ZM-HOX, HAT1, HAT2, ATHB8, and GL2 (Bharathan et al. 1997 ) . Later on, Chan et al. ( 1998 ) classi ed homeobox genes into ve groups (HD-ZIP, GLABRA, KNOTTED, PHD, and BEL). However, based on genome-wide analysis, 107 homeobox genes were classi ed into ten distinct subfamilies, including HD-ZIP I, HD-ZIP II, HD-ZIP III, HD-ZIP IV, ZF-HD, PHD, BLH, KNOXI, KNOXII, and WOX, in rice (Jain et al. 2008 ) . Among these, HD-ZIP represented the largest family comprising of at least 48 members. The expansion of homeobox gene family has been attributed due to the chromosomal segmental duplications in rice, which might be responsible for the diversi cation of their function (Jain et al. 2008 ) . Most recently, a comprehensive classi cation of plant homeobox genes based on the characterization of new motifs has been accomplished from the analysis of ten complete genomes of owering plants, mosses, Selaginella , unicellular green algae, and red algae (Mukherjee et al. 2009 ) . A total of 14 classes were identi ed across various plant species, namely HD-ZIP I-IV, PLINC, WOX, KNOX, BEL, PHD, DDT, NDX, LD, SAWADEE, and PINTOX. The conservation of homeobox genes across lineages emphasized their functional signi cance. In a recent study, it has also been shown that unchar-acterized conserved motifs outside the HD-ZIP domain of HD-ZIP I subfamily con-fers functional diversity to members of this group of homeobox genes (Arce et al. 2011 ) . Further, a greater number of homeobox genes in owering plants have been related to their higher developmental and organizational complexity (Mukherjee et al. 2009 ) .
5 Role of Homeobox Genes in Plant Development
The role of homeobox genes in plant developmental patterns has been extensively explored. The homeobox genes belonging to different subfamilies exhibit distinct expression patterns indicating their speci c regulatory roles in tissue/organ differentiation and development (Chan et al. 1998 ) . The molecular genetic analy-ses of several mutants have revealed that the KNOX family homeobox genes ( SHOOTMERISTEMLESS , BREVIPEDICELLUS , KNAT2 and KNAT6 in Arabidopsis ) are the key determinants in the maintenance of shoot apical meristem (Hake et al. 2004 ) . The role of KNOX genes as versatile regulators of plant develop-ment and diversity have been comprehensively reviewed recently (Hay and Tsiantis 2010 ) . KNOX proteins interact with HD proteins of BELL family...