Climate Change and Plant Abiotic Stress Tolerance || Coping with Drought and Salinity Stresses: Role of Transcription Factors in Crop Improvement

24

Coping with Drought and Salinity Stresses: Role of Transcription

Factors in Crop Improvement

Karina F. Ribichich, Agustı́n L. Arce, and Raquel Lı́a Chan

Abstract

Knowledge about the role of transcription factors in the eukaryotic domain, mainlyin yeast, Drosophila, human, and later in plants, has significantly advanced sincethe first discovery of these regulatory proteins in prokaryotes 50 years ago.Transcription factors are involved in a variety of biological processes, includingresponses to environmental changes. In this chapter, we focus on planttranscription factors involved in the responses to abiotic stresses, mainly droughtand salinity. We analyze the modular structure that characterizes common andkingdom-specific groups or families and the main environmental stimuli thatmodulate the responses through different signaling pathways. Thereafter, wereview examples of the roles played by transcription factors in classical breedingapproaches for crop improvement. Finally, we revise the discovery, characteriza-tion, and use of transcription factors as biotechnological tools. With regard to thelast point, we discuss the experimental methods and parameters adopted toevaluate tolerant phenotypes, and the apparent dilemma between crop yield andstress tolerance.

24.1

Transcription Factors: A Historical Perspective

Around 50 years ago, RNA polymerase activity was discovered in rat liver [1]. A fewmonths later, Stevens [2] and, independently, Hurwitz et al. [3] reported a similaractivity in Escherichia coli, showing that prokaryotes and eukaryotes shared thesame kind of enzyme. At that time, there was no idea of how gene expression wasregulated or if, in fact, gene expression had any kind of regulation. Thereafter, itwas an earlier growth in bacterial transcription research than in eukaryotes that putthe regulation of gene expression into focus.In 1961, Jacob and Monod [4] published a milestone paper that laid the basis for

genetic regulation research. They declared that protein synthesis in bacteria wasnot just regulated by “the DNA molecule whose specific self-replicating structure,

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Climate Change and Plant Abiotic Stress Tolerance, First Edition. Edited by Narendra Tuteja and Sarvajeet S. Gill.� 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.

through mechanisms unknown, becomes translated,” but by other regulator genethat controlled the rate of protein synthesis through an intermediary – the“repressor”. This idea provided the foundation of the transcription factor concept.Four years later, in 1965, Englesberg et al. [5] postulated the positive control of geneexpression by “activators” after the results they obtained studying gene expressionregulation in the arabinose operon.In the same year (1969) in which Burgess et al. [6] identified the sigma factor as a

subunit required for the core polymerase to recognize a promoter and to initiatetranscription, Roeder and Rutter [7] isolated three distinct DNA-dependent nuclearRNA activities from developing sea urchin embryos. In those days, proteins wereknown to be expressed in a regulated way in response to specific signals, butthe knowledge about how this regulation was achieved was at an early stage. Overthe next decade, research revealed that the eukaryotic RNA polymerases werecomposed of several subunits and showed no capacity of initiating transcriptionselectively at purified promoters [8]. Curiously, no eukaryotic sigma-like factorscould be identified, strengthening the idea that the transcriptional machineryidentified in bacteria could be different to that present in eukaryotes.Progress in eukaryotic transcription knowledge came at the end of the 1970s,

when Weil et al. [9] developed a cell-free system capable of RNA polymerase II-dependent viral promoter transcription. Analysis of mammalian cell extracts usedin the experiment showed that multiple factors were required for accurate initiationof transcription by a purified RNA polymerase II [10]. They were named basal orgeneral transcription factors, because it was assumed that they were involved in thetranscription of all genes; they would thereafter be named TFIIA, B, D, E, F and H.Soon, it became evident that purified RNA polymerase II and general transcriptionfactors were not sufficient to explain the variations of transcriptional regulationobserved in vivo, suggesting that other factors should exist. Also during the 1970s,several independent experiments explored the specificity of the transcriptionfactors and the cis-acting sequences recognized by them. Their modular nature wasestablished in the mid-1980s when Brent and Ptashne [11] showed that a eukaryotictranscriptional activator bore the DNA specificity of a prokaryotic repressor in ayeast system.Additional proteins forming complexes of multiple subunits, termed coactivators

or corepressors, generally work as adaptors between RNA polymerase II and generaltranscription factors. One of the groups of these additional proteins was collectivelynamed the TATA binding protein (TBP)-associated factors (TAFs) [12,13], whichconstitutes, in association with TBP, the TFIID complex. They are necessary actors inregulated transcription and different pools of TAFs in association with distinct TBP-type factors suggested a new kind of regulation at the core transcription level [14].The discovery of a second group of coregulator proteins (known as Mediator) was

one of the main contributions of Kornberg’s research in the comprehension ofeukaryotic transcriptional mechanisms [15,16]. This huge protein complex (around30 subunits in Metazoa), apparently ubiquitous from yeast, Drosophila, and humanto plants, links the initial transcriptional activators to the basal transcriptionmachinery and makes the access of transcriptional factors to covalently modified

642 24 Coping with Drought and Salinity Stresses: Role of Transcription Factors in Crop Improvement

chromatin easier. In addition, Mediator acts as a global coactivator and corepressoras was revealed by studies with Mediator-subunit mutants in vivo in which theselective effects on transcription were abolished. There is clear evidence topostulate Mediator as an expanded bridge between components of the transcrip-tional machinery [17] and it appears that Mediator is essential for transcription atnearly every eukaryotic promoter. Its modular nature, attained by three differentgroups of subunits, accomplishes the distribution of functional activities [18,19]. Ina similar way, it is supposed that the modular structure of specific transcriptionfactors, composed of different domains, reflects how they act in regulating theexpression of specific gene targets following spatial and temporal patterns.Over the two following decades, with the development of high-throughput

technologies, knowledge of transcriptional regulation of gene expression hasincreased and gained new dimensions. Some of the most relevant mechanisms thatare now beginning to be better understood include promoter methylation, dynamicalterations of the chromatin structure that modulate DNA accessibility (e.g. covalentmodifications of histones, ATP-dependent chromatin remodeling, histone variantsinterchange and reorganization of higher-order chromatin structure by the cohesin–CCTC-binding factor (CTCF) complex), covalent modifications of RNA polymerases,transcriptional roadblock proteins, transcriptional interference, and homo- andheterodimerization of transcription factors (in the last case, transcription factorsfrom the same or different families) (Figure 24.1).

Figure 24.1 Diagram of transcriptional

regulation of eukaryotic promoters. Major

mechanisms of transcriptional regulation are

represented: heterodimerization of specific

transcription factors, targeting of Mediator by

one or more activation domains (ADs),

alterations of chromatin structure (histone

acetylation (red triangle), DNA looping

mediated by cohesin (green ring)---CTCF

complex, phosphorylation of RNApolymerase II

C-terminal domain (red circle)), and

transcriptional interference. DBD, DNA-binding

domain;MED?, aMediator subunit, probably an

activation domain target-specific subunit.

24.1 Transcription Factors: A Historical Perspective 643

Numerous novel transcription factors were identified and associated to adiversity of biological processes in complex networks. According to structureand function, families of transcription factors were defined to accomplish abetter comprehension of the biological processes in which they are involved.Many of these families share certain biological domains, while others arefamily specific [20].Transcription factors are the main actors in the responses of organisms to

environmental variations, with consequences in growth and differentiation. Owingto the sessile nature of plants, the role of transcription factors gains particularsignificance when they respond to stress factors. Some transcription factors areinduced by a group of abiotic stress factors, like cold, heat, drought, and salinity,which suggests cross-talk in the pathways in which they are involved.Hereafter, we will discuss the characteristics used to group transcription factors

in families and that facilitate our comprehension of their role in plant stressresponses, with a special focus on drought and high salinity.

24.2

Plant Transcription Factor Families Implicated in Drought and Salinity

A large part of the genome capacity is reserved for transcription, includingtranscription factor encoding genes. Around 1500 transcription factors wereidentified in the Arabidopsis (Arabidopsis thaliana) genome [20] and 1600 in thatof rice (Oryza sativa) [21], which represents 6% and 3% of the total genes,respectively – a percentage comparable with the estimations in animals.However, differences in transcription factor families can be found among theeukaryotic kingdom. Roughly 45% of Arabidopsis transcription factors areshared with Drosophila melanogaster, Saccharomyces cerevisiae, and Caenorhabditiselegans, but transcription factor family sizes can vary more than 20-fold betweenspecies [20]. Moreover, some of these transcription factor families (e.g., MADS-box, MYB families) are much larger in Arabidopsis, which suggests that theycould be involved in plant-specific regulatory functions [20]. Considering thishypothesis, Shiu et al. [22] reported that among 19 families shared betweenanimals and plants, more than 14 are larger in plants than in animals. Theseelevated expansion rates in plants are not simply due to the higher duplicationrates of plant genomes, but to a higher degree of transcription factor geneexpansion compared to other plant genes. The high rate of expansion amongplant transcription factor genes and their parallel expansion, at least inArabidopsis and rice, suggests frequent adaptive responses to selectionpressures common among higher plants [22].In addition, there are at least two more sources of diversification. One is the de

novo creation of kingdom-specific transcription factor families, which representabout 30% of the transcription factor gene complement in Arabidopsis. The otheris domain shuffling, which has contributed to find certain domain combinations


more frequently, like the homeodomain (HD) families: HD-ZIP in plants versusHD-POU in animals [22, 72].Large gene families of plant transcription factors, which in some cases are

unique to plants, are mainly involved in stress responses. These families aregrouped according to the structure of the DNA-binding domains, the structure ofthe genes, the signaling pathways to which they can be associated and, in somecases, the presence of additional protein domains. Key stress-responsive transcrip-tion factors belong to MYB (myeloblastosis oncogene), MYC (myelocytomatosisoncogene) (in the basic helix–loop–helix (bHLH) family), AP2/ERF (apetala 2/ethylene-responsive element-binding factor), basic leucine zipper (bZIP), NAC(NAM, ATAF, and CUC), HD, and WRKY families. Plants share MYB, bHLH,bZIP, HD, and WRKY transcription factors with other kingdoms, in which they arenot necessarily involved in stress responses, while the AP2/ERF and NAC familiesare plant-specific.

24.2.1

MYB Family

MYB transcription factors are characterized by the presence of imperfect MYBrepeats (called R) of 52 amino acids involved in DNA binding and protein–protein interaction. Each MYB repeat contains three regularly spaced tryptophan(or aliphatic) residues that together form a hydrophobic core [23]. The threerepeats of the prototypic MYB protein c-Myb are referred to as R1, R2, and R3,and repeats from other MYB proteins are named according to their similarity toR1, R2, or R3 of c-Myb [24]. In plants, MYB proteins can be classified into threesubfamilies, R-MYB, R2R3-MYB, and R1R2R3-MYB (MYB3R) depending on thepresence of one, two, or three tandem MYB repeats, respectively. The first MYBsubfamily has more structural diversity, with the number of MYB repeatsranging from one to four, but with several subgroups of single MYB (R-MYB)proteins [23].DNA binding by most MYB proteins (R2R3-MYBs and MYB3Rs) involves

dimerization. It has been difficult to identify specific DNA-contacting residuesthat define distinct DNA-binding specificities. Consensus DNA sequences CC(T/A)ACC and ANNC(G/C)GTTA have been identified for some of theseproteins. Related DNA-binding preferences of MYB proteins may be specifiedby combinatorial interactions with other transcription factors and redox control[23]. On the other hand, analysis of representative R2R3-MYBs fromArabidopsis identified unique binding specificities for particular subfamilies,like C/TAACNA/G [25].Several members of this family were shown to be involved in a wide variety of cell

processes, including biotic and abiotic stress responses in plants. In relation toabiotic stress responses, it has been shown that constitutively expressed MYBsconfer drought and salinity tolerance in a diversity of plants. Some recent examplesare summarized in Table 24.1.

24.2 Plant Transcription Factor Families Implicated in Drought and Salinity 645

24.2.2

bHLH Family

The bHLH family is the second largest class of plant transcription factors, whichgroups proteins with a small conserved bipartite domain formed by, the basic andthe HLH regions, which contains approximately 60 amino acids. The basic (b)component contains 13–17 primarily basic amino acids and binds to a consensusE-box (CANNTG) or the more common G-box (CACGTG) in the target promoters.The helix–loop–helix is a region of approximately 50 residues responsible foroligomerization and comprises two amphipathic a-helices, mainly consisting ofhydrophobic amino acids, which are connected by a loop of variable length [23].The bHLH-domain-containing proteins are structurally heterogeneous because

they contain several highly conserved domains, as leucine zippers or PAS domains.In addition, the relative placement of the bHLH domain can vary significantly. ThebHLH domain can be located at the C-terminus of the protein, as in members ofthe MYC plant subfamily [57], named after the structure similarity with theprototypic c-MYC (myelocytomatosis gene) from vertebrates. To date, several bHLHgenes have been implicated in stress responses, including drought and salinity,although only a low percentage of this family has been characterized in plants.Possibly one of the most emblematic bHLH (MYC-type) transcription factorscharacterized is ICE1 (inducer of CBF expression 1), a regulator of freezingtolerance in Arabidopsis [58] through the activation of DREB1/CBF proteins (seeSection 24.4). Members of this family associated with drought or salinity toleranceare mentioned in Table 24.1.

24.2.3

bZIP Family

The bZIP transcription factors possess a distinctive structural domain, the bZIP, ofaround 65 amino acids, that consists of two modules: one that directly contactsDNA and an adjacent homo- and/or heterodimerization domain. The DNA-bindingregion (18 amino acids) comprises a number of basic amino acids, and thedimerization domain, the leucine zipper, is composed of heptad repeats of leucinesand other hydrophobic amino acids. Plant bZIP proteins present a relaxed bindingspecificity for DNA motifs containing an ACGTcore, and preferentially bind the G-box (CACGTG), C-box (GACGTC), and A-box (TACGTA) [59]. A stretch similar tothe G-box, PyACGTGG/TC, constitutes the cis-acting sequence that is bound bytrans-elements regulated by abscisic acid (ABA). According to that, the sequence isnamed the ABA-responsive element (ABRE), which is recognized by ABRE-bindingproteins (AREB) and ABRE-binding factors (ABFs). Although the G-box resemblesthe ABRE motif, the G-box functions in the regulation of plant genes in a variety ofenvironmental conditions like red light, UV light, anaerobiosis, and wounding [60],which do not necessarily overlap with the functions in which the ABRE elementparticipates. Not only are the nucleotides around the ACGT core motif importantfor determining binding, but also additional copies or other elements are critical to


give the appropriate response. In this sense, a single ABRE copy is not sufficientfor triggering a transcriptional response to the ABA phytohormone [61] and site-specific mutagenesis of G-box-flanking I-box sequences in the Arabidopsis ribulose1,5-biphosphate small subunit gene, rbcS-1A, rendered the promoter unresponsiveto light [62].The bZIP family is one of the largest and most diverse families present in

eukaryotes. Taking into account the available data, plant bZIP transcription factorsrange from 75 in Arabidopsis to 131 in soybean; much larger than the27 (Drosophila) to 56 (human) size range found in animals [63]. In spite of this,only a small part of bZIP transcription factors have been functionally studied inplants. The majority of the transcription factors known to recognize ABREsequences are bZIP proteins [61] associated to water-deficit and high-salt stresses,among other processes in the ABA signaling pathway (see Section 24.4). So far, allisolated interactors with divergent types of ABRE sequences belong to the bZIPclass of transcription factors and can bind to them, at least in vitro [35]. InArabidopsis, 13 of the 75 bZIP transcription factors belong to group A, whichcontains ABF genes, among 10 groups [64]. In maize, 19 of the 170 bZIPtranscription factor belong to group A, among 11 groups [63]. Examples of bZIPtranscription factors associated with stress tolerance are cited in Table 24.1.

24.2.4

NAC Family

NAC transcription factors belong to one of the largest plant-specific transcriptionfactor families and are involved in different processes, such as growth, develop-ment, and biotic and abiotic stress responses [65]. The family name refers to the N-terminal domain present in three proteins: NAM (no apical meristem) frompetunia, ATAF1/2, and CUC2 (cup-shaped cotyledon 2) from Arabidopsis [66].NAM and CUC2 were first identified as being involved in embryo development.NAC proteins have a conserved N-terminal region (around 150 amino acids) that

contains the DNA-binding domain, which is divided into five subdomains (A–E).The variable C-terminal domain is composed of different transcriptional activationregions (TARs). NAC proteins have been shown to homo- and heterodimerizethrough the NAC domain, but this interaction is affected by the C-terminal regions[67]. More than 10% of the predicted transcription factors in Arabidopsis and ricewere identified as potential NAC proteins [68]. According to a phylogenetic analysiswith the predicted NAC domain amino acid sequences, NAC domains wereclassified into two large groups, groups I and II. Members in the ATAF subgroup,belonging to the group I, were found to be involved in stress responses. Moreover, itwas predicted that four subgroups of the 14 that form group I (ATAF, OsNAC3,AtNAC3, and NAP) could be involved in these responses due to a conservedsubdomain E (DXWVLXRX2–3KK) in the NAC domain [68]. During the last decade,there have been reports of NAC transcription factors belonging to at least three ofthese four subgroups (ATAF, OsNAC3, and AtNAC3) whose overexpression confersdrought and/or salt tolerance in plants [43] (Table 24.1). On the other hand, five


exclusive motifs of TARs were found in these subgroups, which suggestsinvolvement in different responses [68].Information about the cis-recognition sequence of NAC transcription factors

mainly comes from stress-inducible transcription factors. The consensus NACrecognition sites CGTA, CGTC, CGT(G/A), and CACG were identified and namedNACRS or NACBS [40,69]. The DNA sequences that are recognized differ amongfamily members, in most cases with weak base requirements in each position. Thesequences flanking the core site in the promoter of target genes may define thebinding specificity of different NAC transcription factors. Thus, different cis-elements that contain the cores were identified, such as the imperfect palindromicsequence AN5TCN7ACACGCATGT bound by ANAC019, ANAC055, andANAC072 from the AtNAC3 subgroup or the secondary wall NAC binding element(SNBE), (T/A)N2(C/T)(T/C/G)TN7A(A/C)GN(A/C/T)(A/T), bound by regulators ofsecondary wall biosynthesis (SWN proteins [70]).

24.2.5

AP2/ERF Family

The AP2/ERF family (also named AP2/EREB) is a large family of transcriptionfactors that share a common DNA-binding domain, the AP2/ERF, with no apparentsimilarity outside this domain [71,72]. In Arabidopsis, AP2/ERF, MYB, bHLH, andNAC families are the largest, with more than 100 members each [73]. This domain(AP2, around 70 residues) was first found in the Arabidopsis homeotic geneAPETALA2, which has a key role in the establishment of the floral meristem and thespecification of floral organ identity, and it was at the same time identified in AP2-like genes isolated from petunia, snapdragon, and rice [74]. A year later, a similar andsmaller domain (ERF, around 60 residues) was identified in tobacco (Nicotianatabacum) ethylene-responsive element-binding proteins (EREBPs) [75].Activation and repression domains that mediate transcriptional regulation have

been defined for a few members [73]. There is not enough evidence of dimerizationbetween plant AP2 domains, but it has been shown that they interact with othertranscription factors [76,77]. A common assumption is that this family is specific toplants, but in fact it was possibly mainly expanded in this kingdom. A group ofsequences encoding a putative member of this family was annotated in 2006 in thegreen alga Chlamydomonas reinhardtii [78]. Moreover, in apicomplexans, a lineage-specific family of proteins was discovered with a version of the AP2-integrase DNA-binding domain (AP2-IDBD), which is predicted to function in the regulation oftheir developmental cycle progression [79]. The domain was also identified inbacterial and viral endonucleases [80], and it was concluded, based on the results ofphylogenetical analyses, that AP2-IDBD should be associated with differentendonuclease domains on multiple occasions during evolution. It appears that thisdomain has contributed the primary transcription factors in both plants andapicomplexans through lineage-specific expansions [79].The AP2/ERF proteins have been subdivided into five subfamilies [81], according

to the number of copies and conservation of the AP2/ERF domain: AP2, DREB


(dehydration-responsive element-binding protein), ERF, RAV (related to ABI3/VP1), and others. The AP2 subfamily contains proteins with two AP2/ERFdomains connected by a conserved linker of 25 amino acids. The DREB, ERF, andother subfamilies contain a single AP2/ERF domain, and the RAV subfamilyincludes genes with two different conserved DNA-binding domains – AP2/ERF atthe N-terminus and B3 at the C-terminus.Several DREBs activate the expression of abiotic stress-responsive genes via

specific binding to the dehydration-responsive element (DRE)/C-repeat (CRT) cis-acting element in their promoters. They are members of the groups DREB1/CBFand DREB2, two of the six subgroups in which the DREB subfamily was divided.These proteins have the highest affinity for the DRE core sequence A/GCCGAC[81] and variations of it. DREB1/CBF transcription factors are major regulators ofcold stress responses, contributing to cold tolerance, although overexpression ofsome members in plants produces significant phenotypic variations, includingtolerance to drought and high salinity [72]. Unlike the DREB1 genes, DREB2Aexpression is highly inducible by high salinity and drought stress rather than cold.However, phenotypic changes in insertion mutants of DREB2A under water stressconditions were not detected, probably because of the redundant function of theDREB2A family genes, which has eight copies in Arabidopsis. Overexpression ofconstitutive active DREB2A (a deletion mutant of DREB2A) resulted in significantdrought stress tolerance, but only slight freezing tolerance, in transgenicArabidopsis plants [45,81], which supports the redundancy and involvement indrought/high-salinity response hypotheses.There are also reports associating ERF subfamily members to abiotic stress

responses. These proteins bind with highest affinity to the GCC-box sequence(AGCCGCC), which is the core sequence of the ethylene-responsive element(ERE), in the promoters of genes whose expression they activate or repress. Theyinclude members that are induced by drought and high salinity, and can confertolerance to these stresses by overexpression in transgenic plants [48,49,51].Examples of AP2/ERF transcription factors associated with stress tolerance focusedon this work are detailed in Table 24.1.

24.2.6

WRKY Family

The WRKY transcription factor family is among the 10 largest families oftranscription factors in higher plants and, although not plant specific, it isparticularly expanded in this kingdom [82]. It is composed of proteins that share aDNA-binding domain of around 60 amino acids, which contains the four commonWRKY amino acids and a zinc finger-like motif (named altogether the WRKYdomain). They recognize the cis-binding sequence TTGAC[C/T], known as the W-box, and are influenced by adjacent sequences [82]. They were first classified inArabidopsis on the basis of both the number of WRKY domains and the pattern oftheir zinc finger-like motifs into three main groups: I, II, and III, the second ofwhich was divided in five subgroups, IIa, IIb, IIc, IId, and IIe. Depending on the


subgroup, some members also present putative leucine zippers (LZs), calmodulin-binding sites, serine/threonine-, glutamine-, or proline-rich regions, between otherdomains, which reflects their multifunctional nature [84,85]. Homo- and hetero-dimerization between these proteins occur, probably through the N-terminal LZ, aswas reported using different techniques: yeast two-hybrid, immunoprecipitation,and electrophoretic mobility shift assays (EMSAs) [86,87], in planta using thehomolog system [87], and measuring an effect in the strength and specificity oftheir target genes expression [88]. WRKYproteins can act as activators or repressorsof transcription, or both, and are mostly known as regulators in responses topathogens [82]. However, they are involved in a variety of processes includinggrowth and development, and abiotic stress responses. Recent functional analyseshave provided direct evidence of the significant roles they have in tolerance tostresses caused by drought, salt, osmotic, UV-B, and cold, and their participation asactivators or repressors in ABA signaling [82,89]. Some examples of WRKYproteins whose overexpression induces drought and/or salinity tolerance are listedin Table 24.1.

24.2.7

HD Family

The HD is a DNA-binding domain of 60 residues present in transcription factorsfrom organisms of all the main eukaryotic lineages. Structurally, it is composed ofthree helixes: helix I and II are connected by a loop and their arrangement isantiparallel; helix II and III are connected by a turn, with helix III, the DNArecognition helix, positioned perpendicularly to the other two [90]. In plants they werefirst discovered around 20 years ago when the sequence of the Knotted-1 gene frommaize was determined [91]. HD transcription factors isolated afterwards revealed thata considerable diversity of domains accompany this DNA-binding domain in differentcombinations, which allowed the classification of the HD superfamily in families orclasses [92,93]. Taking advantage of the available whole-genome sequences, aclassification supported by a phylogenetic clustering defined a total of 14 classes: HD-ZIP I, HD-ZIP II, HD-ZIP III, HD-ZIP IV, PLINC, WOX, NDX, DDT, PHD, LD,SAWADEE, PINTOX, BEL, and KNOX [93]. Most of the transcription factors of theseclasses are involved in different developmental processes, such as the maintenance ofthe stem cells in the shoot apical meristem, embryo patterning, anther and ovuledevelopment, vascular pattering, and leaf polarity. However, within the HD-ZIPfamily, whose members present a LZ (also called ZIP) adjacent to the HD domain,subfamilies I and II mediate responses to environmental stimuli [94]. While HD-ZIPII proteins are mainly related to light responses, most HD-ZIP I transcription factorshave been associated to abiotic stress responses.SELEX (systematic evolution of ligands by exponential enrichment) assays [95]

and competitive EMSAs revealed that HD-ZIP I transcription factors bind thepseudopalindromic sequence CAATNATTG with maximum affinity in vitro andwith a slight preference for an A/T base at the central position [96–99]. In vivobinding of this element was also demonstrated in yeast and plant cells [99–101].


Recently, a chromatin immunoprecipitation assay with the HB1 transcriptionfactor of Medicago truncatula confirmed that this sequence, present in thepromoter of a target gene, is bound by the transcription factor to exert itsregulatory function [102].A phylogenetic and sequence analysis of the HD-ZIP I subfamily enabled the

identification of conserved motifs in the C-terminal region of most of the 178proteins studied [103]. A motif with activation capacity was recognized and itsactivity in one transcription factor was verified in yeasts. Conserved sites forsumoylation and phosphorylation were also identified, in accordance with previousexperimental information [104,105].Only a few HD transcription factors from other classes have been associated with

abiotic stress responses. Among them there are transcription factors from thePLINC class (also called ZF-HD), which have two zinc finger-like motifs located inthe N-terminus relative to the HD [106,107], and the WOX class, which have acharacteristic HD and a WUS-box in the C-terminus [93,108].

24.3

Crop Domestication: Examples of the Major Role of Transcription Factors

Domestication involves the accumulation of genetic changes resulting fromselection of the offspring of one or more wild species with phenotypes moretractable for harvest and more edible for humans. It is recognized that domestica-tion is a continuous evolutionary process [109], which produces a diversity of traitalterations with subtle to deep phenotypic changes. Most of the major changeswere produced by monogenic traits and several of them by transcription factors.Transcriptional regulators are now genetic tools for crop improvement, but theywere also major actors at the beginning of crop domestication. This subject istreated in different reviews [109–111]. We will focus on three examples of geneticloci encoding transcription factors responsible for major alterations of plantstructure and/or reproductive physiology, which introduced drastic phenotypicchanges in crops along the path of domestication.

24.3.1

Maize Domestication: Increasing Apical Dominance

Crop domestication involved suppression of axillary buds development, concomi-tantly with increased apical dominance. Domesticated maize is a classic exampleand the teosinte branched 1 (tb1) gene was identified more than 15 years ago as themajor gene responsible for the increased apical dominance [110].tb1 of maize was the first described member of the later characterized TCP (TB1,

cycloidea, PCF-domain protein) family of transcription factors [112]. Thesetranscription factors are involved in the transcriptional regulation of genesassociated with growth and cell proliferation, as cell cycle genes. tb1 was identifiedas a major quantitative trait locus (QTL) controlling the difference in apical


dominance between maize and its progenitor, teosinte. Maize and teosinte wouldboth carry functional alleles of tb1, but the maize allele is expressed at twice thelevel of the teosinte allele in the ear primordia and immature shank of the maize.tb1 also regulates the sex of the inflorescences terminating the lateral branches andis required for the normal formation of ears [113]. For this gene, the effects ofselection were limited to the regulatory region and could not be detected in theprotein-coding region [114].The current model is that tb1 impairs the outgrowth of the axillary meristems

and branch elongation as a consequence of its higher expression in maize than inteosinte, and its repressive action on the cell cycle gene expression. This repressionmay result from competitive binding of TB1 (a repressor) to TCP-specific bindingsites in the promoters of cell cycle genes, thus blocking other TCP genes fromactivating these genes [110,115].Another transcription factor controls a relevant difference in ear development

between teosinte and maize, the teosinte glume architecture 1 (tga1) gene. Firstdescribed as a QTL associated to the maize phenotype [116], it was later identifiedas a member of the SBP (squamosa promoter-binding protein) family [117]. Themutated transcription factor alters the development of the teosinte cupulatefruitcase so that the kernel is borne uncovered on the ear, which makes harvesteasier [118]. From the six sequence differences found between maize and teosinte(mostly single nucleotide polymorphisms in the regulatory region), the site mostprobably responsible for the functional difference was identified as a nucleotidesubstitution that renders a non-conservative amino acid substitution K6N in maize.No differences in the level of tga1messenger accumulation or in the pattern of tga1expression were found; moreover, the protein encoded by the teosinte allele wasmore abundant over a range of developmental stages, suggesting instability ordysfunction of the maize protein.

24.3.2

Rice Domestication: Reducing Grain Shattering

Natural seed dispersal in wild plants, particularly in wild grasses, frequentlyinvolves seed shattering, which has beneficial consequences in plant propagation.Indeed, one of the main steps in crop domestication is the selection of plants withimpaired seed shattering, to make harvest easier for farmers. This trait wasacquired early by wild rice progenies during the long path towards moderncultivated varieties, but the molecular basis of this key event remained to beelucidated until a few years ago, when two transcription factors involved in this traitwere independently identified.Following a classical genetics approach, in 2006 and 2007, two independent

research groups [119,120] identified a locus encoding a MYB3 transcriptionalregulator. First, a QTL named sh4 was identified in a 1.7-kb region of a gene ofunknown function. The comparison of the 1.7-kb sequences between the mappingparents (the cultivated species O. sativa ssp. indica and the wild annual species O.nivara) revealed seven mutations. The remaining accessions of the wild species

24.3 Crop Domestication: Examples of the Major Role of Transcription Factors 655

with confirmed shattering differed invariably from the cultivars by one mutationthat caused an amino acid substitution (K79N) in the predicted DNA-bindingdomain encoded by the gene [119]. In a similar approach, an allelic form of sh4,SHA1, was mapped to a 5.5-kb genomic fragment that contained a single openreading frame. The predicted amino acid sequence of SHA1 in the perennial wildrice O. rufipogon was distinguished in eight domesticated rice cultivars of O. sativassp. indica by the same amino acid substitution found in Sh4 (K79N) caused by asingle nucleotide change. The same mutation was also identified in japonicacultivated varieties [120].Although this substitution undermined the gene function, it appears not to be

exactly the same for each allele. The normal development of the abscission layer iscompromised in O. sativa varieties that bear sh4, as reflected by the discontinuousabscission layer that can be observed in longitudinal sections at an early stage offlower development, and is completely absent near the vascular bundle. This wassupported by a complementation assay in which transgenic plants with reducedgrain attachment had a more continuous and extended abscission layer [119].Conversely, SHA1 is not involved in the development of the abscission layer. Nosignificant histological differences in abscission layer formation were observedbetween wild and domesticated varieties; seed shattering was caused by thecomplete separation of the abscission layer from the pedicel after pollination. It wassuggested that the polymorphic differences between the alleles could be the causeof the divergence in their roles [120].Also in 2006, another locus associated with grain shattering was identified by

back-crossings and mapping, the QTL of seed shattering in chromosome 1 (qSH1),which encodes a BEL1-type homeobox gene [121]. No abscission layer was observedin the non-shattering cultivated variety and the involvement of qSH1 in shatteringwas confirmed through the analysis of a near-isogenic line bearing the wild locus inthe same genotypic context. Moreover, qSH1 expression was detected at theabscission layer in the near-isogenic line, but not in the non-shattering variety. Asingle nucleotide polymorphism in the 50-regulatory region of the qSH1 genecaused loss of expression and seed shattering, owing to the absence of abscissionlayer formation.

24.3.3

Barley Domestication: Yield to the Yield

Barley (Hordeum vulgare) domestication occurred more than 8000 years ago in theNear East [122]. Two main important traits were selected during the process: non-brittle rachis and six-rowed spikes. Spikes with brittle rachis disintegrate atmaturity, shedding the grains and significantly reducing harvest yields. Theselection pressure imposed by farmers harvesting more frequently non-brittlerachis spikes than brittle ones might explain how the trait prevailed [122].Barley spikes present a triplet at each rachis node (i.e., three spikelets: one central

and two lateral). In wild barley, spikes are of the two-rowed type, the lateral spikeletsare reduced and sterile; whereas in modern cultivars the full development of the


lateral spikelets generates six-rowed spikes [122]. The production of 3 times moreseeds per spike has a direct impact in the yield of this crop.The six-rowed spike phenotype is controlled by the recessive allele vrs1. Recently,

it was demonstrated that VRS1 is a subfamily I HD-ZIP transcription factor [123].Following the identification of HvHox2, a paralog of Vrs1, and based on thestructure and expression patterns of both genes, a model for the development ofthe spikelets was proposed [122–124]. In this model, VRS1, which is only expressedin the lateral spikelets, heterodimerizes with HvHOX2 and/or competes with it forthe binding to the same cis-elements, therefore suppressing the activity ofHvHOX2. As a result, the dominant allele VRS1 causes abortion of the lateralspikelet development, resulting in the two-rowed spike phenotype. The recessiveallele vrs1, incapable of generating a protein that competes with HvHOX2, isresponsible for the six-rowed spike phenotype.

24.4

Drought and Salinity: From Perception to Gene Expression

There is undeniable evidence that human activity, especially since the industrialrevolution, has caused severe environmental changes at a global scale. In particular,the emission of large quantities of greenhouse gases has generated a relativelyrapid and sustained increase in temperature – a phenomenon known as “globalwarming” [125]. As a result, different regions of the planet are experiencingconsiderable alterations in precipitation patterns, which are causing increasingdrought at low latitudes.In addition to global warming, multiple human activities reduce water availability

(industrial and agriculture demand, water pollution, etc.). From an agriculturalperspective, this implies that crops are increasingly challenged to develop instressful conditions, particularly drought and high salinity, which directly impacton their yield [126].Plants exposed to these stresses suffer an ion and osmotic homeostasis

imbalance, the former mainly in high-salinity stress [127]. The response theydevelop to cope with these adverse conditions follows the general signalingschema: stimuli sensing, intracellular signaling transduction cascades, geneexpression regulation, modification of metabolism, and alteration in activity ofeffector proteins [128].The primary goal of this response is, in the short term, the re-establishment of

cellular homeostasis. An immediate and well-known plant reaction to drought andsalinity is stomatal closure, which reduces water loss by evapotranspiration and ionflux to the shoot in the case of high salinity, although at the expense of limitingcarbon fixation [129].There is also a set of developmental responses which, under prolonged stress,

produce large modifications in the whole-plant architecture [130]. These responsesare stress-avoidance mechanisms that mainly involve a reduction in shoot growthand leaf expansion, and a reduction in shoot branching and lateral root emergence

24.4 Drought and Salinity: From Perception to Gene Expression 657

[128,130,131]. Specifically in roots under salt stress, these developmental responsespresent variations with salt (NaCl) concentration [132]. Additionally, roots arecapable of a hydrotropic response that further shapes the architecture of the rootsystem of plants in a natural environment [133].The signaling pathways triggered by drought and high salinity have been

extensively studied, and there are excellent specific reviews that comprehensivelycover this topic [127,129,134–136]. Hereafter, we will briefly deal with the mainsignaling components and events in these pathways.

24.4.1

Early Signaling Events

As described, the first event in the response to the stress is its perception. In spiteof the significant advances that have been made in the field, particularly inArabidopsis, relatively little is known about the sensors or receptors responsible forsensing drought and high salinity stresses and triggering the responses.Both environmental conditions are sources of osmotic stress, which also explains

the common signaling pathways elicited by them. A candidate osmosensor protein,ATHK1/AHK1, was identified in Arabidopsis mainly because of its capability ofcomplementing different mutant yeast strains with deficiencies in one or bothyeast osmosensor proteins, SLN1 and SHO1 [137]. ATHK1 is a histidine kinasewith high similarity to the yeast SLN1 histidine kinase and its transcripts areaccumulated in response to changes in osmolarity [137]. The study of knockdownmutants and overexpressor plants showed that ATHK1 is a positive regulator ofdrought and salt stress responses, and ABA signaling [138,139]. Recently, Kumaret al. [140] analyzed ahk1 mutant plants in moderately low-water-potential assaysand proved that some responses triggered by osmotic stress (e.g., ABA and prolineaccumulation) were not impaired. These authors concluded that ATHK1 may notbe the main plant osmosensor.Although the perception of the osmotic state through ATHK1 and other

osmosensors would be shared in drought and high-salinity conditions, one ormore specific high-salinity sensors should exist since the response to this stresspresents differences to the drought response [130]. It has been proposed that thesesensors could be located at the plasma membrane, but currently there are no goodcandidates for this important role.The signaling cascades downstream of stress perception have been extensively

studied. One of the earliest events involves an increase in cytosolic Ca2þ

concentration [141]. This is the input signal to the salt stress-specific SOS (saltoverly sensitive) pathway. The calcineurin B-like (CBL) protein CBL4 (also knownas SOS3) senses the change in Ca2þ concentration, dimerizes, and interacts withCBL-interacting protein kinase CIPK24 (originally, SOS2). The complex formed istargeted to the plasma membrane, which results in the phosphorylation andactivation of SOS1, a membrane Naþ/Hþ antiporter [130].Other important components of the signaling cascades are mitogen-activated

protein kinases, lipid messengers, and reactive oxygen species [127].


A key player in drought and high-salinity responses is the phytohormone ABA.Its levels rise in plants subjected to these stresses, and it is well known that ABA-deficient mutants (e.g., aba1, aba2, and aba3) are extremely sensitive to droughtand high-salt treatments [127]. ABA is responsible for the stress-regulatedexpression of a large set of genes; however, gene expression studies showed thatseveral genes remained responsive to stress in ABA-deficient or ABA-insensitivemutants [135]. This evidence led to the conclusion that the response to drought andhigh salinity can be divided into ABA-dependent and ABA-independent pathways.

24.4.2

ABA-Dependent Pathway

The ABA signaling pathway starts with the perception of the hormone by therecently recognized pyrabactin resistance 1/PYR1-like/regulatory components ofABA receptor (PYR/PYL/RCAR) family of protein receptors [142]. Upon ABAbinding, these proteins are capable of forming complexes with protein phospha-tases 2C (PP2C), inhibiting their function as negative regulators of ABA signaling.Many targets of PP2C phosphatases have been identified; among them, kinases ofthe SNF1-related protein kinases 2 (SnRK2) family represent one of the best-characterized links to the regulation of transcription factors by ABA and thus to theregulation of gene expression by this hormone [134,142].In the absence of ABA, SnRK2 kinases form complexes with PP2C phosphatases

and are inactive. The disruption of these complexes by ABA-bound PYR/PYL/RCAR proteins relieves the repression, as has been demonstrated in dicots andmonocots. Active SnRK2 kinases, specifically SnRK2.2, SnRK2.3, and SnRK2.6,phosphorylate and activate AREB/ABF transcription factors [61,134,142–144]. Thecentral role of these three kinases in the ABA response is shown in the Arabidopsistriple-knockout mutant. In this mutant the expression of some of the mostcharacteristic ABA-responsive genes (i.e., RD29A, COR15A, RAB18, RD22, andNCED3) is not induced after ABA treatment [145].Arabidopsis AREB/ABF bZIP transcription factors were independently discov-

ered using yeast one-hybrid assays with promoter fragments containing multipleABREs [61,146]. Arabidopsis plants expressing a constitutively active version ofAREB1 displayed enhanced drought tolerance when subjected to severe treatments,although through a mechanism not involving stomatal closure [35]. AREB1,AREB2, and ABF3 cooperatively regulate the expression of many ABA-responsivegenes during drought and high salinity [147]. The Arabidopsis areb1 areb2 abf3 triplemutant presented, under dehydration and salt treatments, many significantlydownregulated transcription factors, LEA, and PP2C genes, among others. Thismutant plant also showed reduced drought stress tolerance and enhancedinsensitivity to ABA in primary root growth [147].The ABA-insensitive 5 (ABI5) family is another clade of bZIP transcription

factors that acts in the ABA signaling cascade downstream of the SnRK2 kinasesand is composed of five members: ABI5, EEL, DPBF2/AtbZIP67, DPBF4, andAREB3 [134]. In contrast to AREB/ABF transcription factors, which are expressed


in vegetative tissues, ABI5 family transcription factors are mainly expressed in theseeds [134]. However expression directed by ABI5 promoter has been detected inseedlings and mature vegetative and reproductive tissues, especially in response toABA, glucose, cold, and NaCl treatments [148].ABI5 is under a complex combination of post-translational regulatory mechan-

isms. It is phosphorylated by SnRK2 kinases [134,149], and bimolecular fluores-cence complementation assays show that it interacts in vivo with the CPK11 kinaseand the PP2C phosphatases AHG1 and AHG3 [150]. ABI5 protein stability iscontrolled by multiple proteins: the KEG RING E3 ligase [151,152], the ABI fivebinding proteins (AFPs) [153,154], and DWA1 and DWA2 DWD proteincomponents of the CUL4-based E3 ligases [155]. Additionally, sumoylationmediated by the SUMO E3 ligase SIZ1 is another modification that negativelyregulates ABI5 function [156].ABI5 is also capable of interacting with ABI3, a transcription factor with a B3

DNA-binding domain that is also involved in ABA signaling [157]. ABI3 has acentral role in seed development by determining ABA sensitivity, and participatingin the establishment of desiccation tolerance and dormancy during zygoticembryogenesis. Mutant complementation studies showed that ABI5 acts down-stream of ABI3 [158]. As ABI5, this transcription factor can also be targeted fordegradation by the 26S proteasome, in this case by the AIP2 E3 ligase [159]. Arecent study of the homologs of ABI3 in Physcomitrella patents demonstrated thatthey are required for the vegetative tissue of the moss to survive desiccation [160].The authors proposed that the gene-regulatory pathways which originally evolvedfor cellular protection have been used to provide desiccation tolerance in differenttissues: in seeds in the case of angiosperms and in vegetative tissues in that ofmosses. This raises the question of whether ABI3 could be engineered to conferdesiccation tolerance in vegetative tissues of higher plants, particularly in crops.The mutant plants of ABI4, an AP2/ERF transcription factor, were recognized in

the same ABA-resistant germination screening as abi5 plants [161]. ABI4 is post-transcriptionally regulated and the protein is mainly accumulated in roots [162]where it has been shown to mediate ABA and cytokinin inhibition of lateral rootformation [163]. These results indicate that beyond its best-characterized role (i.e.,the regulation of seed development), ABI4 participates in post-germinationprocesses.A recent study of ABI4 and ABI5 ABA-regulated direct target genes enabled the

identification of 95 and 59 regulated genes, respectively [164]. There was only 11%overlap between these groups, suggesting relatively little functional redundancy atthis direct level of regulation. ABRE and ABRE-like motifs were enriched in thepromoters of both gene sets, but the ABI4 group was not particularly enriched insequences bound by this transcription factor according to previous works, like theS-box and the CE1-like motif [164].As mentioned in Section 24.2, the DRE/CRT-binding proteins (DREB/CBF) are

AP2/ERF transcription factors encoded by a clade of genes involved in abioticstress signaling pathways. The Arabidopsis DREB1D/CBF4 was induced bydrought [165]; conversely, DREB1A/CBF3, DREB1B/CBF1, and DREB1C/CBF2


have been associated with cold stress [166]. However, when DREB1D/CBF4 wasoverexpressed in Arabidopsis, it generated plants with drought and freezingtolerance. DREB1D/CBF4 was induced by ABA and this induction was dramaticallyreduced in an aba1 mutant [165]. The transcription factors DREB1E/DDF2 andDREB1F/DDF1 were induced by high salinity, while only DDF2 responded todrought [167]. It has been demonstrated that DREB1A, DREB2A, and DREB2C arecapable of physically interacting with ABF2 in vitro and in yeast, and with ABF4 inyeast. The interaction between members of the DREB/CBF family and the AREB/ABF family may constitute an important cross-talk mechanism between thepathways controlled by these proteins [168].The transcription factor members and families previously mentioned constitute

the first and best-characterized transcriptional regulators in the ABA response. Assuch, they epitomize the complexity of the signaling networks in which there aremultiple levels of regulation, multiple points of cross-talk, and functionalredundancy. However, numerous works showed that several additional transcrip-tion factors, many belonging to other families, also participate in ABA-mediateddrought and salt responses.A transcription factor family strongly associated with abiotic stress and ABA is

the subfamily I of HD-ZIP proteins [92,94]. Together with the AREB/ABF group ofbZIP genes and the NAC genes, they represent the clades of transcription factorswith more concentration of members functionally related to ABA [134]. Theexpression of several of the 17 Arabidopsis HD-ZIP I genes is induced by ABA andhigh salinity [101,169]. AtHB7 and AtHB12 have also been implicated in thedrought and osmotic stress responses, and their induction is dependent on ABI1,ABI2, and ABA3 [170,171]. Recently, it was demonstrated that both transcriptionfactors directly regulate the expression of many key components of the ABAsignaling pathway: the PP2C phosphatases ABI1, ABI2, HAB1, HAB2, and AHG3,the ABA receptors PYL5 and PYL8, and the kinases SnRK2.3 and SnRK2.8 [172]The Arabidopsis AtHB6 HD-ZIP gene is also induced by drought [169], and isinvolved in stomatal closure, germination, and is capable of interacting with thePP2C phosphatase ABII in vitro and in a yeast two-hybrid system [104]. AtHB5expression also responds to ABA and its expression under the 35S promotergenerates ABA hypersensibility [98]. Another Arabidopsis gene, AtHB20, is relatedto ABA through the regulation of germination in response to this hormone [173].HD-ZIP I transcription factors from other species have also been associated with

ABA responses. HB1 from M. truncatula represses lateral root emergence underhigh salinity stress [102]. HaHB4 from sunflower (Helianthus annuus) is inducedby drought, salinity, and ABA, and generates drought tolerance when expressedunder the 35S promoter in Arabidopsis [55,174]. It participates in the cross-talk withthe hormones ethylene, jasmonic acid, and salicylic acid, hence altering senescenceand biotic defense mechanisms [175,176]. Another relevant sunflower HD-ZIP Igene involved in the response to abiotic stress is HaHB1 and its Arabidopsisortholog AtHB13. HaHB1 expression under the 35S promoter enhances toleranceto freezing, high salinity, and drought through a mechanism that involves cellmembrane stabilization [56,177]. The NaHD20 HD-ZIP I transcription factor from


Nicotiana attenuata induces the accumulation of ABA under water stress, andcoordinates the responses to dehydration and its integration with changes in flowertransitions [178].Many transcription factors from other families have been found to participate in

the ABA signaling pathways in association with abiotic stress. There is evidence fortranscription factors of the bHLH, C2H2-ZF, HD-ZF, MYB, NAC, WRKY, andnuclear factor Y (NF-Y) families [134]. Various members are discussed in Section24.4 in relation to their potential use in crop improvement.

24.4.3

ABA-Independent Pathway

ABA-deficient and ABA-insensitive mutant plants still respond to drought andhigh salinity, exhibiting altered gene expression through an ABA-independentpathway. This signaling cascade partially overlaps with the cold stress pathway,and is mainly caused by the osmotic component of drought and high salinitystresses [135].Members of the AP2/ERF family of transcription factors are involved in the

ABA-independent responses to osmotic stress. DREB2A was isolated for itscapacity of binding the DRE motif in a fragment of the RD29A promoter.Expression studies showed that together with its homolog, DREB2B, they aresignificantly induced by dehydration and high salinity, while ABA has no effect[166,179]. The stability of the DREB2A protein is controlled by the DRIP1 andDRIP2 RING E3 ligases [180]. This regulation is potentially related to a PEST-like sequence because when these residues are deleted the DREB2A protein isconverted to a constitutive active form [45]. Overall, there is much lessknowledge about the components of this pathway.The expression of the gene early responsive to dehydration stress 1 (ERD1) is

upregulated by drought, but not by ABA or cold. Based on this, additionaltranscription factors participating in the ABA-independent pathway were isolatedusing its promoter in yeast one-hybrid screenings. Three NAC transcription factorswere recovered using a 63-bp fragment: ANAC019, ANAC055, and ANAC072.These three genes were induced by drought, high salinity, and ABA. Transgenicoverexpressor lines generated with these genes lacked induction of ERD1expression; nonetheless, they had improved drought tolerance when subjected to asevere stress [40]. Using a different fragment of the promoter of ERD1, a screeningallowed the isolation of the zinc finger homeodomain 1 (ZFHD1) transcriptionfactor and homologs. ZFHD1 expression was induced by ABA, drought, and highsalinity. Transactivation assays in which ZFHD1 and any of the three previouslyidentified NACs were coexpressed, together with yeast two-hybrid assays, provedthat these transcription factors act cooperatively and are capable of interacting. 35S:ZFHD1 plants showed no induction of ERD1, but presented enhanced droughttolerance. However, the overexpression of ZFHD1 together with any of the NACsnot only produced plants with improved drought tolerance, but also generated theinduction of ERD1 [106].


The fact that many of the transcription factors involved in the ABA-independentresponse are induced by this hormone highlights the complexity of this poorlyunderstood pathway.

24.5

Transcription Factor Gene Discovery in Stress Responses

The selection of candidate genes for crop improvement generally starts in a modelorganism with the characterization of genes involved in the trait of interest. Twomain approaches have dominated this process of discovery and characterization:forward and reverse genetics.Approaches based on forward genetics originated before the development of

molecular biology, but have been greatly and continuously improved with theintroduction of new techniques and genomic information. The duration of theprocess of gene identi fication once the mutant has been isolated has beendramatically reduced by, for example, the use of next-generation sequencing inapproaches like the SHOREmap pipeline [181 –183].Although many of the most important master regulator transcription factors have

been discovered by means of forward genetics, this approach is inherently non-speci fic. Nonetheless, some strategies can partially overcome this limitation, suchas the use of plants bearing a reporter gene under the control of a stress-regulatedpromoter for the generation of the mutant population [184].Reverse genetics approaches (i.e., “from gene to function ”) took a quantitative

leap with the sequencing of complete genomes, which started with Arabidopsis andrice [185,186], and has currently reached a total of at least 41 genomes from speciesof the plant kingdom (Phytozome v9.0) [187] (http://www.phytozome.net). The firststep of the process is gene selection, which is essential to maximize the success ofthe approach, in particular when there is a specific goal such as crop improvement.The wealth of “omic” information available (genomic, transcriptomic, metabolo-mics, etc.), predominantly in model species, has made bioinformatic analysesalmost a must in guiding the selection, from a simple BLAST search [188] andphylogenetic analysis to more sophisticated techniques like integrative approachesor genome-wide association studies [189]. These, together with the indispensablebibliographic information, will conduct the selection of the candidate gene, in ourcase a transcription factor with the potential of conferring tolerance to droughtand/or salt stresses.The following step is the assessment of the potential of the selected gene to be used

as a biotechnological tool. This generally involves the characterization of gene functionand study of the phenotypic alterations caused by its introduction in the homologousor a heterologous plant species. When a forward genetics approach is carried out andthe gene identified, the study is very similar, with an a priori greater probability ofsuccess as a consequence of the screening for a specific mutant phenotype.There is a vast repertoire of techniques and methods to study transcription

factors; however, some general approaches should be highlighted because they

24.5 Transcription Factor Gene Discovery in Stress Responses 663

http://www.phytozome.net

have been extensively used and with great success (Figure 24.2). The phenotypicand molecular characterization of mutant and/or overexpressing plants (generallyusing the 35S promoter) proved to be extremely useful tools in understanding theregulatory network in which the transcription factor participates and in evaluatingthe effects at the whole-plant level of its absence or its ectopic and constitutiveexpression.A detailed analysis of the expression pattern and its regulation is also

fundamental in elucidating the function of the transcription factor. This isgenerally investigated in wild-type plants using qPCR or in transgenic plant inwhich the promoter of the transcription factor gene controls a reporter gene suchas b-glucuronidase (GUS), green fluorescent protein (GFP), or luciferase (LUC). Ithas been proposed that the own promoter of the gene could become a goodsubstitute for the 35S promoter because its induction is generally associated withthe stress and may prevent undesirable phenotypic effects of the constitutiveexpression [111,190].Biochemical studies of the recombinant protein obtained in a convenient

expression system, generally Escherichia coli, have also significantly helped. Thein vitro determination of the DNA sequence recognized by the transcription factor

Figure 24.2 Gene discovery and characterization approaches used in the identification of

potentially useful transcription factors for crop improvement.


(e.g., using SELEX [95]) allowed the identification of cis-elements in the promotersof putative target genes.Knowing the function of the transcription factor is extremely important, but as

the ultimate goal is obtaining stress tolerance in crops, the critical step is thecorrect evaluation of this response in transgenic model organisms and later incrops. Skirycz et al. [191] have recently shown that many genes reported to confertolerance to severe drought do not provide improved growth performance whentransgenic Arabidopsis lines are subjected to mild drought stress. Field stressconditions tend to be mild and growth performance is intimately related to yield,therefore these results reveal a major issue in the way tolerance has been assessedin most of the studies.There are two fundamental impediments in performing mild stress assays: being

able of accurately controlling stress severity and using a “large” sample size. Thislast point derives from the fact that plant responses under mild stress are expectedto be relatively small; hence, sample size must be increased to achieve a significantstatistical power that allows the recognition of differential behaviors. The logicalsolution to both issues is automation, which was applied by Skirycz et al. [191] inwhat they called the “weighing imaging and watering machine.” Previous workshad already recognized the power of high-throughput phenotyping platforms forquantitative traits [192,193]; probably the high cost of this type of facility preventstheir wider adoption. A detailed and comprehensive review on phenotyping traitsrelevant to drought stress has been recently published [194].Finally, it is important to perform at least primary assays to test whether the

regulatory pathways controlled by the transcription factor in the model organismare conserved in crops. If the model organism used was rice, then the results aredirectly applicable to it as a crop. However, when the effects of the gene areevaluated in Arabidopsis or any species different from the crop that will be target ofthe modification, this type of assays should be carried out. A fast and valuableapproach to assess if the transcription factor preserves its function in the crop is toconduct an Agrobacterium-mediated transient transformation followed by a geneexpression analysis [175,195–197].

24.6

The Long and Winding Road to Crop Improvement

The first generation of genetically modified crops [198] was based on one traitacquisition through one transgene involved in a metabolic gain of function. At thesame time, the long-standing “Green Revolution” gained a new ally. Based on landfertilization and the dissemination of dwarf wheat and rice cultivars, whichdeveloped shorter stems and higher overall yield faster than the traditional varieties[199], the “Green Revolution” had the limitation of the genetic barriers to expandthe traits to other species. The identification of the gibberellic acid insensitive (GAI)gene in Arabidopsis, a modulator of the gibberellin pathway and ortholog of thewheat gene [200,201], overcame this barrier. According to this early finding, the use

24.6 The Long and Winding Road to Crop Improvement 665

of transgenic technologies would allow the insertion of potentially any planttranscription factor gene in the genome of another plant – a procedurecomparatively faster than breeding and selection. Likewise, there have been severalexamples of transcription factors reported as potential biotechnological tools tomake crops tolerant to stresses [166,190,202].In 2003, the concept of the “Blue Revolution ” was installed during the Third

World Water Forum in Tokyo as the necessity to better manage increasingly scarcewater resources, mainly in agriculture that would garner “more crop per drop”[203] (http://www.un.org/apps/news/story.asp?NewsID ¼6542). As described inprevious sections, abiotic stress responses in plants are governed by complexpathways with extensive cross-talk, in which transcription factors execute keyfunctions as modulators of the expression of multiple genes. Consequently,transcription factors became good candidates to be included in the development ofa second generation of genetically modified crops, in which these polygenicpathways and their associated traits could be modified.Crop improvement involves managing a battery of desirable traits, such as

increased yield, easier harvest and processing, better palatability, diminishedgrowing seasons, increased quantity and better quality of nutritional character-istics, and increased biotic and abiotic stress tolerance. Examples of recentgenetically modified and non-genetically modified varieties that increased theirtolerance to drought or high salt concentration were mentioned in Table 24.1.Successful experiments should result in increased stress tolerance while sustainingor improving the other desirable traits, either under benign or stressful environ-mental conditions – a scenario that is not generally achieved.The process of generating a genetically modified crop involves a series of

established stages. From gene discovery to the production of a genetically modifiedcrop with good performance in the field, researchers conduct multiphenotypeevaluations in the laboratory, usually in a model plant (the “proof of concept”);thereafter, a similar evaluation is performed in the destination crop at a greenhouseand then in controlled trials at multiple geographies. There are chances of failure inthese stages, because of a multiplicity of variables.The potential of a transcription factor for the predictive manipulation of plant

metabolism is intimately linked to understanding how it fits in the gene-regulatorynetwork [204]. In this sense, the efforts put on high-throughput sequencing andbioinformatics, together with empirical data, allowed the development of systemsbiology projects; one of their goals is assembling all the genes of a genome intotranscriptional networks, taking into account cell type and environmental variables[111]. Then, it is possible that a third generation of transcription factors will comefrom an upgrade of the available technologies based on the knowledge abouttranscriptional networks [111]. In the meantime, some potential pitfalls can beanalyzed.The hierarchical positioning of the transcription factor in the transcriptional

network and the possibility that the transcription factor function is conservedappear to be two fundamental elements determining the predictability of usingsuch proteins for plant metabolic engineering [204]. The model proposes that the


http://www.un.org/apps/news/story.asp?NewsID=6542

higher the hierarchical position occupied by a transcription factor (e.g., a masterhub transcription factor that controls several pathways has a high hierarchy), themore genes will be affected when its expression is altered and the more conservedthe specific regulatory motifs in which it participates are likely to be. Conversely,transcription factors that participate in less-conserved pathways tend to occupy thelowest positions in the hierarchy because they are under more relaxed selectiveconstrains. A transcription factor in that place has a higher probability of regulatingonly a small subset of genes. There are several examples of transcription factorsassociated to the flavonoid biosynthesis pathway that would be in agreement withthis idea [204].In a simplified model, there is an inverse proportionality between the conserva-

tion of the pathway controlled by a transcription factor and the phylogeneticdistance between donor and receptor species. The more distant two species are, themore probable a pathway will not be conserved, particularly a plant family-specificpathway. As a consequence, the less probable it is that a transcription factor willwork in the receptor species as it does in the donor species. As a result,phylogenetic relationships can be considered to have an indirect impact on thepredictability of using a transcription factor for crop engineering (Figure 24.3).Under mild to moderate stress conditions, quick changes in gene expression in

parallel with physiological and biochemical alterations occur. When compared withdrought, salt stress affects the expression of more genes and more intensely, possiblyreflecting the combined effects of dehydration and osmotic stress in salt-stressedplants [206]. Among the physiological and biochemical changes are partial stomatalclosure, restriction on the mesophyll transport of CO2, and changes in photosyn-thetic metabolism, like an increase in soluble sugars, while starch content decreases[206,207]. Species and cultivars differ with respect to the types of solutes whoseaccumulation contributes to the osmotic adjustment (i.e., the lowering of the osmoticpotential due to the net accumulation of solutes or osmoprotectors). Among themthere are various amino acids (e.g., proline), sugars (e.g., sucrose and fructans),polyols (e.g., mannitol and pinitol), quaternary amines (e.g., glycine betaine), ions(e.g., potassium), and organic acids (e.g., malate and citrate) [208].Under severe drought conditions, photoinhibition eventually occurs with

complete stomatal closure, soluble sugars may decrease [207], there is an osmoticadjustment [208] and, almost always, an inhibition of mitochondrial respiration inactively growing roots and whole plants, although results could be more variable inleaves [209].Reported transgenic plants overexpressing a transcription factor gene with

increased drought and high salt tolerance present different characteristicsassociated to the battery of changes mentioned above, which sometimes causedetriment in yield. The phenotype depends on several characteristics: the kind ofpromoter used to express the gene (constitutive versus inducible), the kind of stressapplied (mild to moderate versus severe), and the species in which the transgene isexpressed, which is related to the mechanisms of gene expression regulation thatcould be absent in the receptor species (e.g., post-transcriptional and post-translational modifications). There are probably other important variables that have


Figure 24.3 In a simplified model, there is an

inversely proportional relationship between the

phylogenetic distance that separates two

species and the possibility that the regulatory

networks that a transcription factor can control

are conserved. Thus, the predictability that a

transcriptionfactortestedinaspecies(generally

amodelorganism)will functionasdesiredwhen

introduced in a particular crop decreases with

thephylogeneticdistancebetweenbothspecies.

Phylogenetic distances between species were

approximatedby themillionsof years (Myr) that

have passed since the split of the lineages

containing the species. These distances were

obtained from the TimeTree database [205].


been less studied, like the locus in which the transgene is inserted or the numberof transgene copies. One undesirable consequence that was reported in severalcases is growth retardation under normal conditions [190,210,211]. Although notcompletely recorded, it is probably produced by stomatal closure – a commonmechanism of drought tolerance, which decreases photosynthesis and, conse-quently, reduces yield.Some examples of processes to improve abiotic stress tolerance, with potentially

good prospects, have been granted patents in the United States and or Europe. Huet al. [41,42,212] reported that two NAC transcription factor genes, SNAC1 andSNAC2, enhance drought resistance and salt tolerance in rice. The tests withSNAC1 transgenic lines were made in the field under severe water-deficit conditionsat the vegetative and the reproductive stages, while showing no phenotypic changesor yield penalty. The transgenic plants lost water more slowly by closing stomata,without displaying a significant difference in the rate of photosynthesis. Moreover,spikelet fertility was the same in wild-type and transgenic rice under well-irrigatedconditions, but the transgenic lines also showed 22–34% higher seed setting thanwild-type plants under severe stress and 17–24% under moderate stress. How couldtransgenic and wild-type plants sustain a similar photosynthetic rate if transgenicplants close stomata? One explanation is that rice leaves function normally withmore opened stomata than may be optimal [41]. Other advantageous characteristicsrecorded by researchers in transgenic plants were delayed leaf rolling and reducedrate of water loss with increased stomatal closure, fast recovery after rehydrationprobably through osmotic adjustment, and cell membrane stability. Besides thedetailed phenotypes observed, the molecular mechanism of stress tolerance beganto be characterized. It is known that SNAC1 is expressed preferentially in guardcells and regulates the expression of other transcription factors (from the MYBfamily) involved in abiotic stress tolerance [41,42].OsDREB1A and OsDREB1B are members of the AP2/ERF family of transcription

factors that, as with Arabidopsis DREB1, confer not just cold, but drought andhigh-salt tolerance under severe stress conditions, when they are highly expressedin Arabidopsis and rice transgenic plants [190,211,213,214]. The phenotypicalconsequences of transgene expression were variable depending on the assay.Constitutive expression of DREB1A increased drought tolerance under severe stressconditions, but caused growth retardation under normal growth conditions intransgenic Arabidopsis. In agreement with other experiences, the growth retarda-tion was overcome using an abiotic stress-inducible promoter, like the rd29Apromoter [190]. Similarly, constitutive expression of DREB1A/CBF3 increaseddrought tolerance in transgenic rice but, in this case, without producing growth oryield penalty [213]. Conversely, constitutive expression of DREB1B/CBF1 in riceproduced a dwarf phenotype without increasing stress tolerance [215]. Constitutiveexpression of OsDREB1A and OsDREB1B in Arabidopsis and rice also increaseddrought tolerance, which shows the high conservation of the DREB transcriptionalpathways [211]. Nonetheless, growth retardation was observed in both species.Although the use of a constitutive promoter could at first explain the dramatic

morphological consequences observed in many other cases, the previously


described success of Oh [213] indicated that another reason could be significant. Itoet al. [211] postulated that the differences may be due to the cultivars used in thethree cases: they used Kita-ake and Nipponbare, while Lee et al. [215] used Dongjin,and Oh [213] used Nakdong. The hypothesis was sustained by the notabledifference in the number of target genes identified in the different transgeniccultivars through the analysis of microarray experiments (nine out of 12 genesinduced in Nakdong were not overexpressed in Kita-ake and Nipponbare) [211]. Onthe other hand, the stress tolerance could be in part explained by the highaccumulation of osmoprotectants and various soluble sugars in the transgenic riceas in the transgenic Arabidopsis plants, which was measured even under non-stressful conditions. In all the cases, stress tolerance was recorded as the differencebetween transgenic and wild-type survival rates under severe drought or salt stressconditions, without informing about the consequences in yield.Differently from DREB1 genes, the stress tolerance associated with the

expression of DREB2 has other requirements. Liu et al. [166] did not find enhanceddrought tolerance or growth retardation in Arabidopsis plants overexpressingDREB2A. Several years later, Sakuma et al. [45] showed that overexpression of anactive form of DREB2A (without a negative regulatory domain) induced down-stream drought genes and enhanced drought tolerance in Arabidopsis. In the sameway, the activity and stability of GmDREB2A;2, an orthologous gene from soybean,were post-translationally regulated in both Arabidopsis and soybean cells [216]. Inaddition, GmDREB2A;2 could induce the expression of DREB2A target genes andimprove drought tolerance in transgenic Arabidopsis. However, there werevariations in the growth phenotypes of the transgenic Arabidopsis, the inducedgenes, and their induction ratios between GmDREB2A;2 and DREB2A. Therefore,the basic function and regulatory machinery of DREB2 are conserved inArabidopsis and soybean, although some degree of divergence has also occurred[216]. In this sense in other plant species other mechanisms of DREB regulationcan be necessary to induce target genes and obtain the desirable phenotype. Anexample of this is the post-transcriptional control by alternative splicing understress conditions that regulates OsDREB2B expression in rice, similarly to itsorthologs in Poaceae. Only under these conditions the functional form of thistranscript accumulates. Transgenic Arabidopsis plants ectopically expressing theactive form of OsDREB2B under the control of the rd29A promoter displayedincreased drought tolerance and survival with no detriment in plant growth [217].No data about yield penalties were reported.The following examples correspond to two members of the HD-ZIP type family,

type I from sunflower. The first of them, HaHB4, conferred drought tolerancewhen it was ectopically expressed in Arabidopsis, but its constitutive expressionproduced some undesirable morphological and phenological consequences, like adelay in anthesis and flower bud development [55,218]. Consequently, the effects ofits expression were evaluated under the control of its own stress-induciblepromoter. These transgenic plants did not display developmental defects, butreduced their water-deficit tolerance about 25% in relation to the percentages ofsurvivor plants with constitutive expression of the transcription factor [175]. Adding


an expression-enhancer intron to the construct, transgenic plants with normalmorphological phenotypes under well-irrigated and drought conditions wereobtained. They also presented a stress tolerance phenotype similar to that of plantsthat expressed HaHB4 under the control of the 35S promoter [196]. This positiveresult seems to be the consequence of a good combination between enhanced andlocalized expression, which had not been achieved using the previous constructs.Transgenic expression ofHaHB4 in soybean, wheat, or corn also conferred droughttolerance in these crops. Recent results of research trials conducted in the fieldindicate that yield increased between 10% and 100%, depending on crop qualityand local conditions.The mechanism of tolerance acquisition appears to involve a complex regulation

of ABA-independent and ABA-dependent pathways. DREB genes and their knowntargets do not change their transcription levels due to the presence of HaHB4,suggesting that it functions through an unrelated pathway [175,176], which wouldbe conserved in a variety of species. A delay in senescence processes mediated bythe ethylene signaling pathway may also contribute to drought tolerance in thetransgenic plants, making them healthier at all developmental stages due to theirreduced ability to enter into the senescence program [175]. On the other hand,according to transcriptomic analysis of Arabidopsis transgenic plants, a large groupof photosynthesis-related genes was downregulated by HAHB4 and the pigmentcontent of transgenic plants was lower than that of non-transformed plants [218].Does this happen in transgenic crops? If it is the case, why they do not suffer yieldpenalty? The downregulated transcription of the main photosynthetic genes involvedin light harvesting would reduce the formation of reactive oxygen species, whichpartially explains the observed drought tolerance of the transgenic plants [219].A second HD-ZIP gene, HaHB1, conferred drought and salinity tolerance to

Arabidopsis transgenic plants cultivated under severe and mild stressful conditionswhen it was expressed constitutively or under its own inducible promoter [56].Similar results were observed when its previously established ortholog inArabidopsis [103], AtHB13, was overexpressed. Limited water availability rarelycauses plant death in temperate climates, but it rather restricts yield [191]; then, ameasure of the yield is a better indicator of growth performance under mild stressconditions. Under normal watering, the wild-type and HaHB1-overexpressingplants produced a similar seed weight. However, after a continuous mild stress,the decrease in seed production was significantly higher in the wild-type (�43%average) than in the overexpressing plants (�30% average) [56]. The observedimproved yield could be associated with higher chlorophyll content in thetransgenic plants leaves. Several pathogenesis-related proteins were induced intransgenic lines under stress treatments, which could be associated with a cellmembrane stabilization mechanism of stress tolerance, similar to the previouslyreported mechanism for the tolerance of freezing temperatures [177]. There is anpatent application as a result of this research [220].A fifth and last example is that of a member of the NF-Y transcription factor

family, AtNF-YB1. This transcription factor conferred drought tolerance whenoverexpressed in Arabidopsis, the homologous system [221,222]. None of the


CBF- and ABA-response pathway markers showed significant and consistentdifferences in expression between constitutively expressing AtNF-YB1 plants andcontrol plants, suggesting that a novel mechanism would be responsible for thestress tolerance. ZmNF-YB2, an AtNF-YB1 ortholog, was shown to have anequivalent activity in the homologous and heterologous systems, which reveals theexistence of common stress response pathways in maize and Arabidopsis. Underconditions of good water supply, transgenic plants did not differ so much from wild-type plants, but under severe drought stress conditions, transgenic plants showed ahigher chlorophyll index, higher photosynthesis rate, cooler leaf temperature, andhigher stomatal conductance. In field trials, Nelson et al. [221] reported an increasein yield of up to 50%, in transgenic maize plants overexpressing ZmNF-YB2. Fieldstudies carried out subsequently showed that the transcription factors encoded bythese genes increased yields by 10–15% under different stress conditions [223].Currently there is no hypothesis to explain the mechanism responsible for thedrought tolerance observed, but researchers argue that this novel function of the NF-Y transcription factors has presumably evolved through the diversification amongmembers of the gene family encoding the B subunit.

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Climate Change and Plant Abiotic Stress Tolerance || Coping with Drought and Salinity Stresses: Role of Transcription Factors in Crop Improvement