Improving Crop Resistance to Abiotic Stress (TUTEJA:PLANT STRESS OMICS O-BK) || Tomato: Genomic Approaches for Salt and Drought Stress Tolerance

43Tomato: Genomic Approaches for Saltand Drought Stress ToleranceBenito Pineda, José Osvaldo García-Abellán, Teresa Antón, Fernando Pérez,Elena Moyano, Begoña García Sogo, Juan francisco Campos, Trinidad Angosto,Belén Morales, Juan Capel, Vicente Moreno, Rafael Lozano, Mari Carmen Bolarín, andAlejandro Atarés

Tomato is considered one of the most economically important vegetable crops in theworld, particularly in temperate areas. Abiotic stresses as those promoted by saltaccumulation and water deficiency entail significant losses of productivity. Despitethe great efforts for increasing tolerance in such species of agronomic interest astomato, the results so far obtained both with conventional breeding methods andwith somebiotechnological approaches have been rather scarce due to the complexityof the response to salt and drought stress. Moreover, only a small number of genesplaying important roles in tolerance mechanisms to drought and/or salinity havebeen identified so far. Thus, novel tomato genes involved in abiotic stress toleranceneed to be isolated and functionally characterized to help increase the level of salt anddrought tolerance by means of gene transformation. This chapter focuses on theapplications of genomic tools to the genetic dissection of those complex traits intomato and related halotolerant wild species. First, the opportunities and limitationsof the genome-wide expression profiling approaches to identify the genes associatedwith the stress response are discussed. Likewise, the advances achieved throughforward and reverse genetics approaches such as insertional and chemical muta-genesis, TILLING, and other gene tagging approaches are reviewed. Hopefully, thecombined use of all these genomics tools will lead to important advances in thegenetic and physiologicalmechanisms of tolerance to drought and salinity in tomato,thus allowing the proper design of future breeding programs.

43.1Introduction

Tomato is considered one of the most economically important vegetable crops in theworld. Abiotic stresses, like those promoted by soil salinity andwater deficiency, havea huge impact on tomato production and mainly affect arid and semiarid zones.Although production losses are very difficult to estimate, it is considered that drought

Improving Crop Resistance to Abiotic Stress, First Edition.Edited by Narendra Tuteja, Sarvajeet Singh Gill, Antonio F. Tiburcio, and Renu Tuteja� 2012 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2012 by Wiley-VCH Verlag GmbH & Co. KGaA.

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is a major abiotic stress that affects agriculture in 45% of the world�s geography,where 38% of the world population resides [1]. Moreover, 20% of the irrigatedagricultural lands are considered saline, yet production losses are higher due to thecontinuously increasing secondary salinization brought about by the low-qualitywater used for irrigation [2]. This risk will increase as the population increasesbecause cities and industry will pay for the best quality water, leaving the worst toagriculture. Development of crop plants tolerant to stress is vital to meet the growingfood demand through sustainable agriculture. Therefore, the greatest challenge forthe coming years will be to increase crop production in abiotic stress-affected lands.

Despite the economic relevance of tomato, themechanisms that govern responsesto these abiotic stresses in this horticultural species are not well characterized, andonly a very small number of genes playing a role in tomato tolerance to salinity anddrought have so far been identified. Similarly, despite the existence of a great wealthof sources of variation in salinity and drought tolerance in accessions of tomato wildspecies, we still do not know the main physiological processes determining theirability to grow and reproduce in stressed lands and still less about the key genescontrolling the high level of tolerance. Results from several laboratories have shownthat it is possible to increase the level of salt and/or drought tolerance through atransgenic approach; however, it is not possible to conclude for the moment thatcultivars with a sufficient level of tolerance from an agronomic point of view havebeen obtained via genetic transformation. In order to overcome the present limita-tions, it would first be necessary to perform the genetic dissection of those complextraits in tomato and related halotolerant wild species, which in turn would enable theidentification of the targets for future breeding programs. In this respect, large-scaleprograms based on the use of genomic approaches should usher in a new era in theknowledge of the genetic and physiological bases of the response andmechanisms oftolerance to salinity and drought, thus allowing the design ofmore effective strategiesfor breeding for abiotic stress tolerance in tomato.

43.2Tolerance Mechanisms to Drought and Salinity in Tomato and Related Wild Species

Tolerance to drought and salt stress is a complex phenomenon at both thewhole-plantlevel and the cellular level, and intense research efforts have focused on under-standing the physiological basis of tolerance in higher plants [3–7]. In physiologicalterms, drought and salinity share osmotic stress, such as a decrease in soil wateravailability under drought or a decrease in water potential of soil solution undersalinity, causing osmotic stress, which leads to decreased water uptake and loss ofturgor [8]. Drought and salt stresses also provoke oxidative stress, which leads to theacceleration of reactive oxygen species (ROS) scavenging systems [9]. The differentialeffect induced by salinity is the toxic effect of the salt induced by the root uptake andshoot transport of saline ions. While the osmotic effect starts immediately after theimposition of salt stress (i.e., before the saline ions are taken up by the roots), the toxiceffect starts later when the saline ions are transported to the shoot and build up to

1086j 43 Tomato: Genomic Approaches for Salt and Drought Stress Tolerance

toxic levels within the leaves. However, the timescale over which ion-specific damageis manifested depends on the salt sensitivity of the genotype and the stress level [10].Although the two phases are generally separated in time for most plants, it is alsopossible for ion toxicity to take effect during the first phase itself and for osmoticeffects to persist in the second phase [11, 12].

43.2.1Physiological Response to Osmotic Stress Induced by Drought and Salinity

Drought and salinity are known to induce stomatal closure, slowingCO2 assimilationand, consequently, reducing the photosynthetic rate, although the causes ofdecreased photosynthetic rate under abiotic stress are still not well established, andthere remains substantial controversy about the main physiological targets respon-sible for photosynthetic impairment [13, 14]. In addition to the effects on CO2

diffusion, ATP synthesis and reductant status, abiotic stresses can also negativelyaffect the Calvin cycle by reducing the content and activity of photosynthetic carbonreduction cycle enzymes, including the key enzyme Rubisco. In tomato, thephotosynthetic rate and the carbohydrate availability do not seem to be the firstlimiting factors for plant growth under saline conditions. Rather, these are thedistribution and the use of photoassimilates in the sink organs [15, 16].

One important mechanism of the maintenance of water uptake and cell turgorunder drought and salinity is osmotic adjustment. One important difference betweendrought and salinity is the main solutes contributing to osmotic adjustment. Underdrought stress, the compatible solutes (or osmolytes), together with Kþ and NO3

�,are the most important ones contributing to the osmotic adjustment, while undersalinity the most important solutes are the saline ions. The use of organic solutes forosmotic adjustment is energetically much more expensive than the use of the salineions proceeding from the substrate [17], which could make tomato more sensitive tothe osmotic component of NaCl than the ionic component [18]. Thus, in the salt-tolerant wild tomato species, the greater salt tolerance has been associated with highNaþ accumulation in leaves and fruits [19, 20], through the use in the main of thecheapest solutes from an energetic point of view for osmotic adjustment. Within thecultivated species, the salt tolerance is not always associated with low Naþ concen-tration in the leaves. Thus, a direct relationship between the fruit yield and theaccumulation of leaf saline ions was found in tomato when plants are grown at low-mid levels [21]. Other evidence on the importance of the osmotic component in thesalt tolerance of tomato was apparent when the salt response of tomato transgeniclines with different expression levels ofHAL1 gene, involved in Naþ regulation, wasstudied. In plants of a homozygous line proceeding from a transgenic plant with avery high expression level ofHAL1 gene, the fruit yield under saline conditions notonly did not increase but was also even lower than that of azygous plants, and this inspite of themuch lower Naþ uptake andNaþ translocation to shootmaintained overtime in the homozygous line [12]. The deeper physiological characterization of theseplants allowed it to be elucidated that the greater ability of Naþ exclusion in thehomozygous line caused another type of osmotic problem, as leaves required an

43.2 Tolerance Mechanisms to Drought and Salinity in Tomato and Related Wild Species j1087

increased synthesis of organic solutes to maintain osmotic balance, thus leading togrowth penalty that negatively reflected on fruit yield. These results demonstrate theimportance of considering the osmotic component of salt stress in tomato.

With respect to the role of osmolytes in drought and salinity, plants accumulatemany metabolites in the cytoplasm to increase their osmotic tolerance against waterloss from the cells inducedbydrought and salt stress, especially soluble sugars (mainlyfructose and glucose) and organic acids [22]. Accumulation of compatible solutes suchas proline, glycine-betaine, and trehalose has also been proposed as playing a role intolerance to abiotic stress by protecting protein and membrane structure, regulatingredox status, or acting as a scavenger of ROS [23–26]. Trehalose is present in somedesiccation-tolerant higher plants [27] and the quaternary ammonium compoundglycine-betaine is accumulated in numerous halophytes fromseveral families [28–30].Proline is one of themost studied osmolytes in tomato, as its concentration increasessignificantly after stress exposure, although consensus has not been reached on therelationship between stress tolerance and accumulation of proline [31]. Thus, prolineincrease in the leaf was deemed to be a symptomof salt injury rather than a trait of salttolerance, whereas the opposite response was also observed, mainly when the salttolerance mechanisms were studied at short term [32, 33]. Santa-Cruz et al. [34]observed a higher proline accumulation in the salt-tolerant species Solanum pennelliiafter24 hof salt treatment,whichwas associatedwith the compensationof lowerpHinthe cytoplasmof the stressed cells. Taken together, the changes inducedby stress in theosmolyte contentsmay vary dependingon the intensity, duration, andprogression rateof stress; proline changes may show adaptive responses of the plants in order toreestablish osmotic homeostasis at the short mid-term, but they may also show adefense strategy of the plant to tackle the harmful effects induced by stress after a longexposure and may be even part of the damage caused by the stress.

43.2.2Physiological Response to Oxidative Stress Induced by Drought and Salinity

Whenplants are exposed to drought and salinity and the availability of CO2within theleaf is restricted and/or the synthesis of ATP is impaired, the concentration of thefinal electron acceptor NADPþ is generally very low, which leads to an excess ofexcitation energy in the photosystems.High-energy statesmay be dissipated by eithernonphotochemical quenching (e.g., xanthophyll cycle) or alternative processes, suchas photorespiratory metabolism [35]. If not dissipated, electrons accumulate in theelectron transport chain and are transferred to oxygen (Mehler reaction), generatingROS. Because of their high reactive potential, ROS react with, and damage, manycellular components (e.g., proteins, DNA, and lipids), constituting oxidative stress.ROS also inactivate the photochemical reaction center of PSII, causing photoinhibi-tion. It has been proposed that most environmental stresses inactivate PSII byinhibiting the mechanisms of repairing photodamage rather than by directlyattacking it [36]. Plants have defensive mechanisms and utilize several biochemicalstrategies to overcome drought and salt-mediated oxidative stress. Plant enzymaticdefenses include antioxidant enzymes such as the phenol peroxidase, ascorbate


peroxidase, glutathione peroxidase, superoxide dismutase, and catalase that, togetherwith other enzymes of the ascorbate-glutathione cycle, promote the scavenging ofROS [37, 38]. The biochemical defense system also includes carotenoids, ascorbate,glutathione, and tocopherols. Several authors have suggested that the function ofsugars, poliols, glycine-betaine, and proline could be to protect cells against thehydroxyl radical [39]. Acorrelation between antioxidant capacity and salinity tolerancehas been reported in tomato through comparative studies between cultivated andwild species [25, 40, 41].

In contrast to the negative termused for the increased ROS production, implying aharmful process, recent studies have shown that ROS play a key role in plants assignal transduction molecules involved in mediating responses to abiotic stress,suggesting that ROS signaling is an integral part of the adaptation response of plantsto drought and salinity stresses [42, 43]. Thus, H2O2 seems tomodulate the activitiesof many components that contribute to cell signaling, including Ca2þ and Kþ

channels [44]. Although the sources of ROS under stress, mechanisms of ROSdetoxification, and the role of ROS in stress signaling are all active areas of researchand have been extensively studied and reviewed [43, 45], more studies are necessarybefore any definitive conclusion can be reached about the role of the ROS productionunder stress in tomato. Perhaps, ROS levels could be the key factor, producingfavorable action (signaling) at low concentrations and oxidative stress at high ROSconcentrations.

43.2.3Plant Response to Ionic and Nutritional Stress Induced by Salinity

Ionic stress due to the accumulation of toxic saline ions, especially Naþ and Cl�,induces a nutritional stress due to the altered nutrient uptake, especially of Kþ ions.Thus, salt tolerance of the cultivated species has generally been correlated with anefficient Naþ and Cl� exclusion mechanism and with a better maintenance of leafKþ concentration. In most studies on salinity, it has not been possible to determinewhether the toxic effects observed are due to Naþ and Cl� or both. In tomato, it isinteresting to point out that similar relationship between fruit yield and leaf ionicconcentrations for Naþ and Cl� were observed, which suggests that the toxic effectsare, at least in the long term, due to the contribution of both ions [21]. Despite a widebody of literature, the mechanisms that govern tomato response to salt stress are notwell characterized, and a very small number of genes playing a role in the transport ofsaline ions have been identified to date [46, 47]. AnionCl� transporters are not knownin tomato yet, and themost important advances in the transport processes have beenachieved for the Naþ and Kþ transport [48, 49].

According to Plett et al. [50], salinity tolerance in plants is derived from thecontributions of three components: tolerance to the osmotic stress imposed bysalinity, exclusion of Naþ from the shoot, and tissue tolerance of the Naþ accu-mulated either by vacuolar storage or by tolerance to cytoplasmic Naþ . It is likely thatall three components operate simultaneously and interact to a greater or lesser extentto provide a plant with its overall salinity tolerance. The osmotic tolerance mech-


anism for salt tolerance, which has not received as much attention as the Naþ

exclusion mechanism, appears to be equally important in providing salt tolerance totomato plants. It has been reported that the salt-tolerant wild species and sometomato genotypes show growth stimulation on addition of NaCl to a growthmediumwhen NaCl is rapidly accumulated and employed preferentially as an osmoticum,both in the leaves or vegetative organs [10, 21] and in the fruits [20]. Thus, the Naþ

transport to the shoot and its accumulation in leavesmay be amore effective strategythan exclusion for improving tolerance of tomato whenmoderately saline waters areused for irrigation.

However, it is necessary to take into account that mechanisms to tolerate poten-tially toxic levels of Naþ in the leaf tissues may be valid up to a certain salinity level,but not when the limit of tolerance to cytoplasmic Naþ is exceeded. In tolerantgenotypes, theNaþ uptakewas not proportional to external salinity, but was curtailedat high salinities or longer time [21, 51]. Moreover, sometimes the major differencesinNaþ accumulation aremainly observed when the concentrations are expressed ona dry weight basis instead of on a cell water basis, such as was observed bycomparatively studying the response of cultivated and wild tomato species at thecell and whole-plant level [19]. Taken together, the salinity tolerance mechanisms intomato and, especially, in wild tolerant species seem to be mainly associated withtheir increased capacity to uptake water throughout osmotic adjustment, diluting thetoxic ions and maintaining shoot growth.

In spite of the advances in the tomato gene identification involved in the Naþ andKþ homeostasis in past years, more advances are necessary to understand the roleand regulation of some genes involved in the reestablishment of ion homeostasisunder salt stress. In this respect, tomato is a very good model for studying longdistance transport of saline ions because of its physiological and anatomical structure,as tomatoes have ways of partitioning the salt arriving at the shoot, either retaining itin the leaf base or stemand preventingNaþ from reaching the photosynthetic tissuesor directing salt away from younger leaves toward older ones [10]. Recently, Ol�ıaset al. [49] showed that the relevant role of SlSOS1 gene was associated with thepartitioning of Naþ in plant tomato shoot. Another important factor is to maintain alow ratio of Naþ to Kþ reaching the shoot tissues, although tomato appears to have apoor correlation between salinity tolerance and Naþ /Kþ ratio [12, 52]. However, Kþ

ions are one of the essential elements required for growth, as alterations in Kþ candisturb the osmotic balance and the function of stomata and some enzymes, andconsequently more advances are necessary in order to fully understand the transportprocesses of this important nutrient under stress conditions [53].

43.2.4Long-Distance Signaling Pathways and their Relationship with Drought and SalinityResponse

There is evidence for a variety of long-distance signaling pathways, involvinghormones and nutrient ions moving in the xylem sap, which regulate the plantgrowth under abiotic stress [54, 55]. It is well known that drought and salinity induce


stomatal closure and that this process ismediated by abscisic acid (ABA) and possiblyby other signals generated in response to abiotic stress [35]. It has been suggested thatearlier and higher ABA accumulation at short term is related to increased tolerance todrought stress. In support of results for a growth-promoting role of ABA in tomato,Makela et al. [56] showed that shoot growth of an ABA-deficient tomato mutant wasaffected by salt stress during the first phase (osmotic phase) to a significantly greaterextent than its ABA-producing wild type. In a recent study, the higher droughttolerance induced by the overexpression of a tomato dehydrin (TAS14) was associatedwith a rapid increase in ABA in leaves [12], which corroborates the role of ABA in thetolerance. The action of ABA may be involved in the suppression of ethyleneproduction [57–59]. Moreover, ABA-induced accumulation of compatible solutes,as proline, can be crucial for dehydration avoidance [60]. Thus, at the level of theorganism, it seems that amain function of ABA is to coordinate the various aspects ofabiotic stress response [31, 61].

43.2.5Tolerance to Drought and Salinity Varies with the Developmental Stage

An important factor to be taken into consideration at the time of evaluating thetolerance to drought and salinity is that the tolerance at one stage of plant develop-ment is often not correlated with tolerance at other developmental stages [10]. Thus,efforts have been made to identify QTL for salinity tolerance during seed germina-tion, vegetative growth, and later stages in tomato [46]. The overall results support thesuggestion that different genetic and physiological mechanisms contribute to salttolerance during different stages of plant development. This complicates indirectselection and comparison of results coming from different experiments andresearchers. Furthermore, it requires knowledge of the physiological traits contrib-uting to the tolerance at different plant developmental stages.

In comparison to the research conducted during seed germination and thevegetative stage, limited research has been conducted to identify QTL for salttolerance during reproductive development in tomato [62, 63]. It is interesting topoint out that a QTL involved in Naþ accumulation has been recently identified inRIL lines proceeding from the cross between the cultivated and the wild species S.cheesmaniae [52]. For osmotic stress, it would be of agricultural importance to evaluatethe tolerance at vegetative and reproductive developmental stages, which are keywater-demanding periods of growth. Furthermore, it should be taken into accountthat the incidence of stress is unpredictable and plants may be exposed to droughtstress at any time during their life cycle under field conditions.

In order to answer the questions �what is known so far and what remains to beknown,� it is essential to recognize the important work done in the past decade, aswell as the important advances described here. However, more advances arenecessary to understand themechanisms underlying drought and salinity in tomato.Moreover, to obtain the knowledge required to develop genotypes with enhancedtolerance to field conditions, it is very important to combine the descriptive power ofphysiological analysiswith thenew tools of functional genomics that have emerged in


recent years, including the high-throughput methods for transcriptomic, proteomic,and ionomic analysis. Using this integrated analysis would make it possible toelucidate the dynamics of plant metabolism in the context of the plant–environmentsystem as a whole.

43.2.6Tomato Genes Involved in or Related to Salinity and Drought Tolerance

Although studies inmodel species such asArabidopsishave led to important advancesin the drought and salinity tolerance, the main genes involved in the toleranceprocess have to be identified in crops or wild species since the role could varyaccording to the species, as seems to occur with the SlSOS1 gene [64]. Recently,Nagata et al. [65] performeda comparativemolecular biological analysis ofmembranetransport genes in different organisms, ranging from bacteria to animals and plants.They compared the numbers ofmembrane transporter genes inArabidopsis and rice.According to these authors, although many transporter genes are similar in theseplants,Arabidopsis has amore diverse array of genes formultiefflux transport and forresponse to stress signals, while rice has more secondary transporter genes forcarbohydrate and nutrient transport. After stress perception, plants must triggersignal transduction cascades, which in turn activate stress-responsive genes andultimately lead to changes at the physiological and biochemical levels (Figure 43.1).The majority of studies have aimed to decipher the function of genes encodingdownstream components (effectors), such as those coding for antiporters, heatshock proteins, superoxide dismutases (SODs), and LEA proteins, rather thanupstream components (regulators), such as those coding for transcription factorsand kinases.

Since the most important effect induced by salinity for a long term is the toxiceffect, most approaches have been directed to studying cation transporters andtheir regulation, especially the Naþ transporter genes, such as SOS1, HKT, andNHX. Thus, the tomato genes belonging to the SOS pathway, homologues to SOS1,SOS2, and SOS3 genes from Arabidopsis, were isolated [49, 64]. SlSOS1, whichencodes a putative Naþ /Hþ antiporter from tomato, highly homologous toAtSOS1, seems to play a relevant role in maintaining ion homeostasis in tomato,as SlSOS1-silenced plants weremore sensitive to salt stress thanwild type (WT) andshowed higherNaþ accumulation in leaves and roots andKþ deficiency [49]. Theseauthors concluded that besides its main action in extruding Naþ out of the root,SlSOS1 is critical for the partitioning of Naþ in plant tomato shoot, retaining Naþ

in the stems, and preventing Naþ from reaching the photosynthetic tissues.Preliminary experiments with transgenic tomato plants constitutively overexpres-sing SlSOS2 and SlSOS3 suggest a relevant role for these genes in tomato salttolerance [64]. On the other hand, two tomatoHKTgenes, SlHKT1.1 and SlHKT1.2,encoding putative Naþ or Kþ transporters, have also recently been isolated (Belver,unpublished results). Ol�ıas et al. [49] proposed that the transport function of theSOS1 and HKT systems in tomato may be coordinated to achieve Naþ and Kþ

homeostasis.


Important work has been carried out by Pardo�s Group in order to determine therole ofNHXantiporters, whichwere thought tomediate the compartmentalization ofNaþ into vacuoles [66]. Recently, they showed that transgenic tomato plants over-expressingAtNHX1 had larger Kþ vacuolar pools under all growth conditions tested,but no steady enhancement of Naþ accumulation was observed under saltstress [111]. In tomato, the SlNHX1, SlNHX2, and SlNHX3 (LeNHX1, LeNHHX2,

Drought

SalinityOsmotic stress

Oxidativet

Disruption of osmotic and ionic homeostasis; damage of functional and

structural proteins and membranes

Ionic stress stress

Osmosensors, phospholipid-cleaving enzymes, second messengers, MAP kinases (e.g., DY523508), Ca2+

sensors, calcium-dependent protein kinases, protein-phosphatase (e.g., DY523345)

SlSOS3/SlSOS2 protein kinase complex?

Signal-sensing perception and transduction

Chaperone funtions (e.g., Detoxification

Transcription factorAP2/ERF (e.g., TERF1), bZIP, NAC, MYB (e.g., SlAIM1), MYC, Cys2His2 zinc finger, WRKY

Transcription control

S i

Water and ion movement(e.g., SlSOS1, SlHKT1.1.,

SlHKT1.2, SlNHX1, SlNHX2,SlNHX3, LeHAK5)

TAS14, SlNCED1 )

Osmoprotection (e.g., SlFUM1)

Sucrose transporter genes?

Stress-responsivemechanisms Gene activation

Reestablishment of cellular homeostasis, functional and structural protection of

proteins and membranes

Salinity and/or drought tolerance

Figure 43.1 Generic pathway under salinity and drought stresses, where the regulator andresponse genes identified so far in tomato are included along with some genes whose role intolerance is not yet sufficiently known.


and LeNHX3, respectively, in theNCBI database) genes that encodeNHX antiporterswere also identified, and the function of some of them, such as SlNHX2, wasstudied [67].

Molecular approaches have allowed the identification of a tomato Kþ transporter,LeHAK5 [68].However, the low levels of the LeHAK5 expression inKþ -starved plantsgrown with NaCl showed no correlation between root Kþ concentrations andtranscript accumulation. The regulation of the expression of this gene was furtherassociated with a more negative electrical potential difference across the plasmamembrane of root epidermal and cortical cells, and the depolarized root plasmamembrane potential of tomato plants grown with NaCl prevented the induction ofLeHAK5 produced by Kþ deprivation [48]. Therefore, under salt stress, the beneficialeffect of a Kþ -uptake systemwith a high discrimination betweenKþ andNaþ seemsto be missing, although more studies are necessary in order to elucidate the maingenes involved in the Kþ transport mechanisms under drought and salinity [69].

There is evenmore limited knowledge on the role of regulator genes, for example,protein kinases and transcription factors. In a study focused on identifying salt-responsive genes in the root tissues of tomato seedlings, 24 cDNAs corresponding toearly induced transcription factors were isolated [70]. Furthermore, several compo-nents of the phosphorylation/dephosphorylation cascades, such as a protein phos-phatase 2C (DY523345) and a MAPKKK (DY523508), were identified as beingupregulated at the later stages of the salt stress response [70]. More recently, theABA-induced SlAIM1 gene, which encodes a R2R3MYB transcription factor, has alsobeen cloned [71]. SlAIM1 RNAi plants accumulate more Naþ , whereas the over-expression lines accumulate less Naþ relative to wild-type plants, suggesting thatSlAIM1 regulates ion fluxes. Furthermore, a previously uncharacterized connectionwas observed between ABA, Naþ homeostasis, oxidative stress, and pathogenresponse, suggesting SlAIM1 has a function in both biotic and abiotic stressresponses and in the existence of a crosstalk between these stress responses intomato [71].

With respect to genes playing a role in water deficit stress tolerance of tomato, theinvolvement of ASR stress response proteins in physiological adaptation of wildtomato to dry climates is strongly supported by different studies [72]. Some of themost studied proteins that accumulate in response to drought stress in higher plantsare the group 2 LEAproteins or dehydrins. In tomato, the expression of TAS14 gene,which shows sequence similarities to other dehydrins, is upregulated by ABA, salt,and osmotic stress [73]. We have observed that the overexpression of TAS14 intransgenic tomato plants improves drought and salt tolerance and that the toleranceis associated with a rapid ABA increase in the leaves of transgenic plants afterapplying the stress (unpublished results). In thewiltedmutant, flacca (flc), induced byX-ray irradiation, it was shown that the genetic lesion impaired the last step of ABAbiosynthesis. The mutant has played an invaluable role in elucidating many impor-tant features of ABA biosynthesis [74], it being known that ABA is an essentialmediator in triggering the plant response to dehydration, cold, and osmotic stress.Recently, Tung et al. [75] cloned the tomatoSlNCED1 (LeNCD1 in theNCBIDatabase)gene and demonstrated that the overexpression of this gene, which is theWTallele of


the classical ABA-deficient tomatomutant notabilis, enhances thewater use efficiencyin tomato. However, these plants exhibited important developmental alterations andgreatly reduced growth, which shows the adverse consequences of a very high ABAaccumulation for long term. Tung et al. [75] suggested that only more moderateincreases in ABA biosynthesis are likely to be useful in the context of agriculture.Furthermore, valuable work has been carried out by Botella�s group using muta-genesis (EMS) to identify plant genes required for salt tolerance in tomato [76, 77].Thus, Rosado et al. [78] showed that crosstalk occurs between the ABA and theethylene signaling pathways in tomato and that the TSS2 and TOS1 loci appear to beregulators of this crosstalk. From a spontaneous mutant (Aco1) of the wild tolerantspecies S. pennellii, Nunes-Nesi et al. [79] cloned its homologue of tomato, SlFUM1gene, which has an important role in the stomatal function and consequently in theosmotic tolerance.

Furthermore, several sucrose transporter genes (LeSUT1, LeSUT2, and LeSUT4 inthe NCBI database) were isolated from tomato and it has been shown that theirinhibition affects tomato fruit development [80]. Although the role of sucrosetransporters in the abiotic stress response of tomato has not been studied yet, thesegenesmight be involved in the tomato responses to water stress, as plants usemainlysugars to reduce leaf osmotic potential and avoid dehydrationunder these conditions.

Taking into account the scant number of tomato genes identified to date in relationto salinity and, especially, water deficit stress (Figure 43.1), the identification of newgenes playing pivotal roles in the response/tolerancemechanisms to drought and/orsalinity is a priority objective.

43.2.7Increasing Salt Tolerance through a Transgenic Approach: Advances and LimitationsSurvey of Previous Results

In numerous papers published from the early 1990s onward, several authors haveclaimed enhancement of drought and salt tolerance through either overexpression ofendogenous genes or, more frequently, heterologous expression of genes thatsupposedly act on different mechanisms involved in the process [6, 10].

Genes that have proven quite effective in providing stress tolerance using atransgenic approach belong to different categories (Table 43.1). Preliminary researchin this field focused mainly on the overproduction of metabolically compatible(organic) solutes [27, 60, 81] in transgenic plants. In this respect, trehalose biosyn-thesis in transgenic tobacco, Arabidopsis, potato, or rice improved drought or salttolerance [82]. In tomato, the yeast gene for trehalose-6-phosphate synthase (TPS1),driven by the 35S promoter of CaMV, has been used to enhance stress tolerance [83].Under drought, salt, and oxidative stress TPS1 tomato plants improved tolerancewith respect to thewild type.However, the plants displayed abnormal phenotypes dueto trehalose-6-phosphate accumulation. It has been reported that these problems canbe overcome by using a microbial TPS-TPP fusion gene together with a stress-inducible promoter, directing the gene product into chloroplasts [84–87], or using adifferent type of trehalose biosynthetic gene (trehalose phosphorylase) that bypasses


Table 43.1 Relevant examples of genes conferring salt and drought tolerance in tomato.

Gene/Source Function Phenotype observed References

TPS1/Yeast Osmoprotection(trehalose-6 phosphatesynthase)

Enhanced tolerance todrought and salt

[84]

BADH-1/Sorghum Osmoprotection(glycine-betaine)

Maintenance of theosmotic potential undersalt stress

[88]

BADH/A. hortensis Osmoprotection;glycine-betaine

Improved salt tolerance [90, 91]

KatE/E. coli Oxidative stress (catalase) Tolerance increased to thephotooxidative stressinduced by drought

[97]

APX/Pisum sativum Oxidative stress(ascorbate peroxidase)

Tolerance increased to theoxidative injury inducedby salt stress

[93]

HAL2/Yeast Cation-sensitivenucleotidase requiredfor sulfate assimilationand RNA processing

Salt tolerance in calli androoting

[96]

HAL1/Yeast Ion transport Increased Kþ accumula-tion and higher salt tol-erance under salt stress

[104, 105]

AtNHX1/A. thaliana Ion transportCompartmentalization ofNaþ in vacuolesCompartmentalization ofKþ in vacuoles

High level of salt toleranceHigh level of salt tolerance

[109][111]

AVP1/A. thaliana Ion transport (vacuolarHþ -pyrophosphatase)

Enhanced recovery ofplants in droughtconditions

[117]

SlNHX2/tomato Ion transport (tonoplastKþ/Hþ antiporter)

Silencing of SlNHX2increased sensitivity toNaCl

[67]

SlSOS1/tomato Ion transport (plasmamembrane Naþ /Hþ

antiporter)

Silencing of SlSOS1increased sensitivity toNaCl

[49]

CBF1/A. thaliana Transcriptional regula-tion (CRT/DRE-bindingprotein)

Enhanced tolerance tochilling,water deficit, andsalt stress

[149–151]

CaKR1/pepper Transcriptional regula-tion (Ankyrin repeatdomain zinc finger)

Enhanced tolerance tosalt and oxidative stress

[133]

Osmyb4/rice Transcriptional regula-tion (MYB transcriptionfactor)

Enhanced tolerance todrought stress and virusdisease

[134]


the trehalose-6-phosphate [89]. On the other hand, glycine-betaine has been shown toprotect higher plants against salt/osmotic stresses by playing an osmolyte role andprotecting the photosystem II (PSII) complex under salinity [24]. In tomato, it hasbeen reported that transgenic plants with the BADH gene from Atriplex hortensisimproved salt tolerance [90]. Notably, it has been shown that the accumulation ofglycine-betaine in genetically modified plants of tomato is more effective in thechloroplasts than in the cytosol [91], in a similar way to that previously observed inrice [92].

Another strategy to increase the level of salt tolerance has been the transfer of genescodifying different kinds of proteins functionally associated with the protection ofmacromolecules, such as LEA proteins, osmotin, chaperones, mRNA bindingproteins [93–95], or with the protection of metabolism key enzymes [96].

Salinity and drought are well established as inducing oxidative stress. In tomato,the overexpression of the Escherichia coli catalase encoded by the katE gene increasedthe tolerance to the photooxidative stresses imposed by drought stress or chillingstress [97]. Interestingly, it has been reported that the coexpression of more than onegene involved in oxidative stress protection in both the chloroplasts and the cytosolgives rise to plants with increased tolerance to different types of abiotic stress[98–100].

Genetic manipulation with genes encoding membrane proteins involved in theuptake and transport of water and ions, such as water channel proteins and iontransporters, has been an alternative approach [101–103]. Thus, overexpression ofyeast gene HAL1, a regulator of Kþ transport, in tomato resulted in increased Kþ

accumulation and higher salt tolerance under salt stress [104, 105]. As ion transportacross the tonoplast into vacuoles is energized by a proton moving force [106], thestrategy based on the use of antiporters has generated large expectations in recentyears. By overexpressing the vacuolar antiporter AtNHX1, a high level of salttolerance was reported in genetically modified plants of Arabidopsis [107] andcanola [108]. In tomato, Zhang and Blumwald [109] reported similar fruit yields inboth 200mMNaCl-treated transgenic plants overexpressingAtNHX1 and the controlplants grown under normal conditions (5mM NaCl). However, these results havebeen questioned [10, 110] as has the action mechanism of AtNHX1 gene [66]. Thus,Leidi et al. [111] have shown that the overexpression of AtNHX1 in tomato canincrease salt tolerance without enhancing Naþ accumulation into vacuoles; theseauthors suggest that this tolerance derives from the significant role that theAtNHX1antiporter plays in Kþ homeostasis by capturing Kþ in the vacuoles.

New AtNHX genes have been cloned and characterized [112–115] and significantefforts have been carried out to identify the orthologous genes in different species,including tomato, and to perform the functional analysis, usually by overexpressionand silencing in genetically modified plants. Recently, Rodriguez-Rosales et al. [67]studied the function of the tomato Kþ/Hþ antiporter LeNHX2 using 35S-drivengene overexpression of a histagged LeNHX2 protein in Arabidopsis thaliana andLeNHX2 gene silencing in tomato. Transgenic Arabidopsis plants overexpressing thehistagged tomato antiporter LeNHX2 exhibited inhibited growth in the absence ofKþ in the growth medium, but were more tolerant to high concentrations of Naþ


than untransformed controls. When grown in the presence of NaCl, transgenicplants contained lower concentrations of intracellular Naþ , but more Kþ , comparedto untransformed controls. On the other hand, silencing of LeNHX2 in tomato plantscaused both significant inhibition of plant growth and fruit and seed production andan increased sensitivity to NaCl.

It has also been reported that the overexpression of a vacuolar Hþ -pyropho-sphatase (AVP1) from A. thaliana in transgenic plants of the same species increasesthe level of salt tolerance [116]. In tomato, the overexpression of AVP1 resulted ingreater pyrophosphate-driven cation transport into root vacuolar fractions, increasedroot biomass, and enhanced recovery of plants from an episode of soil water deficitstress. The more robust root systems allowed transgenic tomato plants to take upgreater amounts of water during the imposed water deficit stress, resulting in amorefavorable plant water status and less injury [117].

Likewise, a higher level of salt tolerance has been described through the over-expression of genes that codify plasma membrane Naþ /Hþ antiports cloned fromdifferent sources (e.g., AtSOS1 from A. thaliana, [118]; SOD2 from Schizosacchar-omyces pombe, [98, 119]; nhaA from E. coli [120]; andOsSOS1 fromOryza sativa [121].Using posttranscriptional gene silencing, Ol�ıas et al. [49] evaluated the role played bySlSOS1, the functional homologue ofAtSOS1, in salt tolerance of tomato. Transgenictomato plants with reduced expression of SlSOS1 exhibited reduced growth ratecompared to WT plants under saline conditions.

Other targets in this field have been regulatory genes, such as transcription factorsand those codifying signal transduction components or receptor-related pro-teins [122, 123]. Cloning of genes codifying transcription factors is a promisingfield, as they lie upstream with respect to many other genes. Recent research has ledto the identification of several transcription factor families (e.g., AP2/ERF, bZIP,NAC,MYB,MYC, Cys2His2 zinc finger, andWRKY) that are important in regulatingstress plant responses, including not only different kinds of abiotic stresses but alsopathogen-induced defense responses, various physiological processes, hormonalsignaling pathways, and several developmental processes [123–130]. For example,it has been documented that ERF proteins integrate signals from different planthormone pathways and play roles in stress responses [131, 132]. Huang et al. [133]reported a novel member of ERF proteins from tomato designated tomato ethylene-responsive factor 1 (TERF1). Overexpression of TERF1 in tobacco activated theexpression of GCC box-containing pathogen-related genes and also gave rise to thetypical ethylene triple response. Further investigation indicated that transgenicTERF1 tobacco exhibited salt tolerance. In another work, Seong et al. [134] reportedenhanced resistance to Phytophthora infestans and salt and oxidative stress tolerancein tomato plants overexpressing CaKR1 gene, which encodes an ankyrin repeatdomain zinc finger and is involved in transcriptional regulation in response topathogens and abiotic stresses. Likewise, tomato plants overexpressing the riceOsmyb4 gene, coding for a MYB transcription factor, acquired a higher tolerance todrought stress and viral disease [135].

In the search for different approaches, it has been suggested that genes codifyingcalcium sensors [136] or even DNA helicases and RNA helicases [137, 138] could be


involved in the salt tolerance process. The role of siRNAs under stress conditions isalso under study [139, 140]. Finally, the knowledge of processes related to DNA/RNAmetabolism andG-protein signaling pathways could be useful in elucidating the lessknown stress signaling networks and thereby be helpful in engineering salinitytolerance in crop plants [23].

Overall, the results obtained in thisfield show that the expression of different kindsof genes in transgenic plants can increase salinity and/or drought tolerance, at least tosome extent (Table 43.1). Unfortunately, it is not possible to conclude for themomentthat true tolerant cultivars (i.e., with a sufficient tolerance level from an agronomicpoint of view) have been obtained via transformation.

43.2.7.1 Aspects Related to the Evaluation of Transgenic PlantsWhen performing the evaluation of genetically modified plants or the functionalanalysis of a tolerance-related gene, it would be advisable to take into considerationsome questions, such as the procedure for evaluating tolerance to salinity or drought,the plant material used for the evaluation, and the complexity of those traits.

Regarding the procedure for evaluating the tolerance to salinity, if the publishedresults are scrutinized, some of the methods of evaluation of transgenic materialsappear to be of doubtful value [110]. Responses to salinity are frequently studied withsmall samples, in the very short term, by using shock treatments and,moreover, datacollected for very specific growth periods, in spite of the fact that salt sensitivity oftomato depends on the growth stage [10, 51]. The usefulness of in vitro tests,frequently used for the evaluation of salt tolerance, could also be questioned becausetranspiring conditions have a major influence on Naþ transport and tolerance [141].However, a clear relationship between tolerance to salinity in vitro (callus) and in vivo(plants grown in greenhouse) has been observed for cultivated and wild tomatospecies [105, 142]. In vitro tests can provide complementary information on the effectof some transgenes (e.g., genes involved in ionic homeostasis) and can be useful forthe preselection of transgenic lines (if an in vitro and in vivo correlation has beenpreviously shown), but they should not be used as the only criterion to determine thedegree of salt tolerance.

The plant developmental stage can also be a critical issue for the evaluation ofwaterdeficit stress tolerance as seedlings, young (e.g., 2–4 leaves), and older plants willshow different levels of relative tolerance. Drought stress can be imposed in vitro byraising the osmotic pressure (e.g., mannitol and sorbitol) or using, in vivo or in vitro,chemical agents (e.g., polyethylene glycol) limiting root water availability. In bothcases, it is essential to avoid unnatural treatments leading to artifactual results.Drought tolerance is more frequently evaluated by reducing the level of water ormineral solution, in which case it is necessary to decide the level of water reduction,length treatment, and the nutrient supply during the stress period. Alternatively, thedrought stress can be imposed as watering/dehydration cycles, in which case it isnecessary to select the number of cycles and length of each cycle. Treatmentsperformed in walking chambers may produce different results in the greenhouse.In any case, the relative humidity in the environment is of crucial importance as itaffects stomatal closure and the water status of the plant. Moreover, as stated above, it


should be taken into account that in nature the incidence of stress is unpredictableand plants may be exposed to drought at any time during their life cycle. As a result,the evaluation for drought stress tolerance can be evenmore difficult than for salinity.

Another important aspect in the evaluation of saline or water deficit stresstolerance is the plant material to be used. The use of TG1 plants (primary transfor-mants) is questionable because epigenetic effects (which are very important in somecases) may lead to erroneous conclusions. The evaluation in TG2 avoids the aboveproblem, but it is necessary to take into account that this is a segregant progeny. In theauthors� opinion, the best materials are the homozygous and azygous lines obtainedin TG3. Thus, each homozygous line should be compared with two controls: theWTand the corresponding azygous line without the transgene. Positional effects cangenerate great differences in the expression of a given transgene in independenttransgenic lines, indicating the necessity of selecting those with the best expressionfor the trait [143]. Dose effects of the transgene can be estimated by comparing thebehavior of homozygous lines with that of hemizygous lines (i.e., those derived fromthe sexual crossing between the homozygous and the azygous lines). The relativetolerance of these lines can be estimated in the short andmid-term, although, finally,the long-term response (estimating yield with quantitative data) must also bereported.

Apart fromall these considerations, in evaluating the tolerance of transgenic crops,it is important to perform long-term experiments, focus on growth and yield, andprovide quantitative data [7, 110].

43.2.7.2 Overexpression versus Spatial and TemporaryModulation of Gene ExpressionThe choice of promoters can significantly affect the result of a transgenic manip-ulation [7]. Overexpression has so far been the most widely used strategy forincreasing salinity or drought tolerance in transgenic plants. The underlying ideais that by overexpressing a certain gene or by expressing it in a constitutive way itwould always have a positive effect on the phenotype. But increasing evidencesupports the idea that sometimes strong and constitutive promoters (e.g., CaMV-35S) involve a high energetic cost and yield penalty in transgenic plants [12, 105, 143,144] and, in other cases, the beneficial effects of the transgene are masked bypleiotropic effects derived from the use of strong promoters [145–147]. Evidencefrom research in this field supports the advantages of using inducible promoters [85,130, 148, 149].

Thus, Kasuga et al. [147] overexpressed the cDNA encoding DREB1a under thecontrol of a 35S promoter in transgenic Arabidopsis plants. As a result, transgenicplants exhibited improved tolerance to drought, salinity, and freezing stresses.However, constitutive expression of DREB1a resulted in severe growth retardationunder normal growing conditions. In contrast, expression ofDREB1a gene under thecontrol of a stress-inducible promoter rd29A led to minimal effects on plant growthunder normal growing conditions and provided even greater tolerance to abioticstress treatments. Similar results have been observed in tomato. Hsieh et al.[150, 151] reported that the use of a 35S promoter to drive the expression ofArabidopsis CBF1 in tomato improved tolerance to cold, drought, and salt loading,


at the expense of growth and yield under normal growth conditions. Lee et al. [152]expressed theArabidopsis CBF1 driven by three copies of anABA-responsive complex(ABRC1) from the barley HAV22. Transgenic tomato plants exhibited enhancedtolerance to chilling, water deficit, and salt stresses in comparison to untransformedplants; but under normal growing conditions the ABRC1-CBF1 tomato plants alsomaintained normal growth and yield similar to the untransformed plants. Likewise,the constitutive expression of genes encoding compatible solutes often causesabnormalities in plants grown under normal conditions, for example, constitutiveoverproduction of molecules such a trehalose [145], polyamines [146], or manni-tol [153]. The use of stress-inducible specific promotersmay protect transgenic plantsfrom such growth abnormalities [154].

When the scientific literature is critically reviewed, it is difficult to estimate theproportion of genes whose overexpression in tomato transgenic plants leads toundesirable pleiotropic effects and/or yield penalty, as in most cases data on salinityor drought tolerance of transgenic plants are not accompanied by a thoroughphenotypical characterization, and, even less, fruit yield with and without stressconditions. Our results on the functional analysis of several genes putatively relatedto salinity and drought have revealed that the overexpression of some of them islinked to these kinds of collateral and undesirable effects while others are not. Forexample, as stated above, the expression of HAL1 gene driven by 35s promoterenhanced the level of salt tolerance, but this positive effect was counteracted by yieldpenalty under control conditions [104]. However, the tomato transgenic plantsoverexpressing the dehydrinTAS14 gene did not exhibitmorphological or significantgrowth differences compared to wild-type plants when the former were grown underunstressed conditions, which indicates that in this case the yield was not penalizedunder normal conditions [12].

In any case, the use of inducible or specific promoters will be essential whentackling the cotransference and coexpression of several genes to avoid homology-based gene silencing [10, 144]. It is to be expected that the identification of new cis-regulator elements, which allow a proper expression in time and space, will be amajor target in the near future [10, 101, 154, 155].

43.2.7.3 Complexity of the Trait and Sources of Genetic VariationSalt and drought tolerance are complex traits [6, 7, 23, 156]. If one takes into accountthe diversity of mechanisms involved, the question that immediately arises iswhether the introduction of a single gene can produce a sufficient level of toleranceor whether it is necessary to introduce several genes involved in different processes(e.g., osmotic adjustment, osmoprotection, ionic homeostasis, oxygen free radicalscavenging, stress response, restoration of enzymatic activity, photorespiration, etc.).Of course, a particular gene (e.g., one that codes a transcription factor) can havea cascade effect, thus modifying the expression of many genes. Alternatively,the expression of a gene involved in the compartmentalization of ions in thevacuoles may alleviate toxic effects. Even so, it seems unlikely that a single genecould affect all the processes influenced by salinity. What is most likely is that thetransfer and expression, in a coordinated way, of a series of genes, each of which


would affect one of the principal mechanisms of the process, would produce tolerantplants. The problem is that there is still no clear idea of which genes have to betransferred.

In this respect, rather than looking for salinity or drought tolerance-related genesin sensitive species, such asArabidopsis, it would be better to focus on tolerant plants.As far as salinity is concerned, Flowers and Colmer [29] have recently reviewed themechanisms of salt tolerance in halophytes, plants that are able to survive andreproduce in environments where the salt concentration is around 200mMNaCl ormore. The authors have proposed that research should be concentrated on a numberof �model� (halotolerant) species that are representative of the various mechanismsthat might be involved in tolerance. Nevertheless, as these halophytes are evolution-arily far from the main crop species, from a breeding point of view it would perhapsbe better to take advantage of the existence of halotolerant accessions of wild speciesrelated to a given crop. In this respect, in the genus Solanum there are accessions ofwild species (e.g., S. pennellii, S. cheesmaniae, and S. pimpipinellifolium) with a highlevel of tolerance to salinity and/or drought [10]. Unfortunately, despite the wealth ofsources of variation, it is still not knownwhich are the key genes determining thehightolerance level in those plants.

43.2.8Genomic Tools for the Genetic Dissection of those Complex Traits

43.2.8.1 Transcriptomics, Proteomics, and other �Omic� ApproachesSome �omic� approaches should provide very useful information with respect to thegenes actually involved in salinity and drought tolerance. Gene, metabolite, andprotein discovery is being revolutionized through the combination of genomesequencing, microarray analysis, and other �omic� approaches [157]. Thus, tran-scriptomic analysis provides the expression profiles of hundreds or thousands ofgenes. At present, this kind of approach is being used to identify those genes that areup- or downregulated in response to saline or other types of abiotic stresses [10]. Inthis sense, several transcriptomic studies in model species such as Arabidopsis andrice have revealed new stress-related pathways in addition to the previously well-described stress-related genes [158]. Valuable information on the involvement oftranscription factors in root apex response to salt stress has also been obtained in themodel species Medicago truncatula [159]. To this purpose, the authors used twocomplementary transcriptomic approaches. Forty-six salt-regulated TF genes wereidentified using massive quantitative real-time PCR TF profiling, whereas Mt16Kþ

microarray analysis revealed 824 genes (including 84 TF) showing significantchanges in their expression in salt-treated root apexes.

In tomato, Wei et al. [160] observed changes in the accumulation of a number ofdifferent RNA from salt-treated and nontreated roots and identified 20 cDNAs thatare responsive to salt treatment. The results indicated that the majority of the salt-induced changes in the rootmRNAprofile occurred in anABA-independentmanner.Using microarray analysis focused on early-response genes after salt stress in thecotyledons and shoot tip of tomato seedling (cv. Money Maker), Zhou et al. [161]


found 1757 genes regulated by salt, of which 563 were downregulated and 1194 wereupregulated. Using a similar approach, Ouyang et al. [70] identified 201 nonredun-dant genes that were differentially expressed upon 30min of severe salt stress in twocultivated tomato genotypes with different levels of salt tolerance. Interestingly, alarge number of early-response genes regulated by salt stress encoded unknownproteins, indicating that there is still a great deal to discover about the mechanism ofthe salt tolerance in tomato. At present, we are studying the differences in theregulation of salt tolerance between cultivated tomato and its relatedwild salt-tolerantspecies S. pennellii using microarray analysis. It is interesting to point out that ahigher number of genes with salinity (100mM NaCl applied for 24 h and 7 days)changed their expression level in cultivated tomato, compared to the wild tolerantspecies, with a predominance of genes upregulated over genes downregulated.However, in wild species an induction of the expression of putative key genesoccurred in response to saline stress, including several families of transcriptionfactors, drought response genes, such as aquaporins and dehydrins, and genesinvolved in bioenergetics and membrane ion transport, including some ATPasesubunits [162].

Transcriptomic analysis can be useful both for identifying new stress-relatedpathways and genes regulated by stress encoding unknown proteins (and putativelynew functions) and for inferring the main mechanisms responsible for differentstress tolerance between cultivated and wild species. Nevertheless, these methodsusually lead to an overestimation of the number of genes supposedly involved,which makes the identification of relevant genes among an enormous number ofother genes with purely secondary or irrelevant functions more difficult. Despitethis, it is foreseeable that transcriptomics will become a valuable tool in the nearfuture. However, in order to fulfill the expectations created in this field, it would besensible to take into account the state of development at which the stress treatmentis applied as well as the intensity and exposure time to stress. On the other hand,rather than apply these approaches to model (salt-sensitive) species, it would bebetter to apply them in both crop species and halotolerant accessions of related wildspecies and thus, by comparison, try to identify the genes responsible for toler-ance [10, 163].

The rapid expansion of new molecular �omics� tools has opened up newperspectives in the identification of the major determinants involved in salt anddrought tolerance. Thus, the proteomic analysis of plant under normal and stressedconditions (salt or drought stress) can play an important role in qualitatively andquantitatively studying the changes in protein expression patterns [164, 165]. Therehave been observed water deficit stress-induced changes in polypeptide accumu-lation in the leaves and roots of different species including tomato [166]. Likewise,salt stress resulted in the altered synthesis and accumulation of a number ofprominent polypeptides in tomato roots [167]. Recently, Chen et al. [169] carried outa proteomic analysis to investigate the molecular differences between two tomatophenotypes differing in their salt tolerance to salinity. They identified 23 salt stressresponse proteins, classified into 6 functional categories, and almost all of theseproteins increased their abundance in the salt-tolerant phenotype. These authors


also evaluated the effect of exogenously applied glycine-betaine and found that thiscompatible solute could alleviate the inhibition of tomato growth induced by saltstress by changing the expression abundance of six proteins in the salt-tolerantphenotype and two proteins in the salt-sensitive phenotype compared to salt-stressed seedlings.

Microarray analysis and proteomics of plant stress tolerance report on theregulation of many genes simultaneously through changes in transcript levelsand protein levels, respectively. However, it has been reported that microarraystudies provide no information about changes in the corresponding proteinexpression patterns [165]. Only poor or moderate correlation between changes inthe levels of specificmRNAs and their corresponding proteins has been reported instudies involving yeast (Saccharomyces cerevisiae) [169] or Arabidopsis [170, 171]. Acombination of microarray and proteomic analysis can indicate whether generegulation is controlled at the level of transcription, translation, posttranslationalmodification, or protein accumulation. Although proteomics in higher plants isstill in its infancy compared to prokaryotes, yeast, and humans [165], it isforeseeable that it may serve to shed light on some of the mechanisms of salt anddrought stress tolerance.

Furthermore, by comparing the behavior of salt-sensitive and salt-tolerant geno-types (e.g., cultivated species versus accessions of the related tolerant wild species),ionomic approaches [172] can provide new insights both into the key mechanismsresponsible for ion homeostasis and into the underlying cause of the different abilityto use saline ions in the osmotic adjustment of halotolerant genotypes. Moreover,ionomics can be a valuable tool for thoroughly characterizing newmutants altered inthe level of salt tolerance.

Large-scale programs based on the use of these omic approaches should usher innew era in the knowledge of the genetic and physiological basis of the response andmechanisms of tolerance to salinity and drought, thus allowing the design of moreeffective strategies for breeding cultivated species for abiotic stress tolerance.However, in order to fulfill these expectations it is necessary to focus these genomictools to identify the main determinants of tolerance, avoiding background noise thatcan mask what actually is essential. Transcriptomic analysis has so far provided ageneral picture of genes that are down- or upregulated under abiotic stress situationsas well as the number of genes belonging to different functional categories whoseexpression is significantly changed. Though this is a valuable information, it does notprovide the basis to design a breeding program (e.g., via transgenesis), as it is verydifficult to tackle the functional analysis of the hundreds (or thousands) of geneswhose expression changes under abiotic stress. Thus, in the near future the mostimportant challenge in transcriptomic analysis is to shed light onwhich are the key orpivotal genes responsible for salinity and drought tolerance. In the same way, themost important objective in proteomic and ionomic studies should be to clarify themain physiological processes determining the tolerance to those kinds of abioticstress. In other words, research in this field must not be focused on increasing thecomplexity of traits that themselves are very complex but, on the contrary, onidentifying the targets for future breeding programs.


43.2.8.2 Posttranscriptional Gene Silencing (PTGS) Large-Scale ProgramsMajor advances have been achieved in the study of mechanisms of PTGS and high-throughput systemsare available to infer gene function [154, 173, 174]. For example, tohelp identify the functions of genes in rice, Miki and Shimamoto [175] developed agateway vector, pANDA, for RNA interference of rice genes. Analysis of rice genesusing this vector showed that suppression ofmRNAexpressionwas observed inmorethan 90% of transgenic plants examined. Hilson et al. [176] generated a collection ofgene-specific tags (GSTs) representing at least 21 500 genes that can be used to createRNAi vectors for functional genomics studies. Preliminary analysis showed effectivesilencing for three genes coding for vacuolar-type Hþ -ATPase subunit B3, a compo-nent of cellulose synthase andpentatricopeptide repeats [176]. According toXiong andZhu [177], RNAi is very efficient as an alternative to knockoutmutants in componentsof stress signalingpathways. To thebest of ourknowledge, a similar strategyhasnot yetbeen applied to the tomato. However, we foresee that if systematically applied on alarge-scale program for tomato and tolerant accessions of related wild species, thisapproachwould be particularly valuable for the identification of genes related to stressresponse (cultivated species) or genes involved in different mechanisms responsiblefor salinity and drought tolerance (related wild species).

Moreover, after the identification through microarray analysis of the hundreds ofgenes that are differentially expressed under stress conditions, the use of a PTGS-based strategy could be particularly valuable in discriminating the key genes fromthose that have a merely secondary role in the tolerance process.

43.2.8.3 Spontaneous and Induced MutantsIn order to overcome the difficulties in performing the genetic dissection of thesecomplex traits, the use of mutants as genomic tools needs to be one of the mainresearch areas in the coming years. One of the key factors explaining our presentknowledge in several areas of plant development lies in the detection and charac-terization of mutants altered in developmental traits, for example, those affected intomato fruit development and maturation [178–181].

Mutagenesis in Arabidopsis has been employed to identify genes involved in salttolerance [182] and thanks to the identification of salt overly sensitive (sos) mutants andthe cloning and characterization of the SOS genes, an important and novel signalingpathway, called the SOS pathway that mediates ion homeostasis and salt tolerance,has been discovered [183–187]. Salt-tolerant mutants in Arabidopsis have likewisebeen obtained [188, 189].

By comparison, the number of mutants affected in the level of salt tolerance inspecies other thanArabidopsis that are already available to the scientific community israther few. Occasional spontaneous mutants or, alternatively, those generated bychemical (e.g., EMS) or physical (e.g., X- and d-rays or fast neutrons) methods couldprovide the basis for advancing in the knowledge of physiological processes related tosalt tolerance. For example, by analyzing tomato salt-hypersensitive (tss) mutants,Borsani et al. [76] were able to identify two loci, theTSS1 andTSS2. Of these, theTSS1locus is essential for potassium nutrition and salt tolerance, while TSS2 plays animportant role in the interactions between salt tolerance and abscisic acid signaling.


However, in the absence of obvious candidate genes, isolation of the gene altered inthe mutant through a positional cloning strategy supposes huge effort. Fortunately,we can today overcome these problems by using alternative approaches.

43.2.8.4 Targeting Induced Local Lesions in Genomes ApproachesThere is no generally applicable method as yet to generate a plant bearing a mutantallele of a concrete gene. Despite several technical advances, homologous recombi-nation in plants is a technology that is still far from being considered efficient andapplicable to all putative predicted genes of a given genome [190]. The completion ofthe genome sequence project of several cultivated plant species such as rice,cucumber, melon, and more recently tomato (http://solgenomics.net/) opens upa new era where high-throughput reverse genetic approaches are needed to meet thedemand of functional genomics as well as new breeding programs.

Targeting induced local lesions in genomes (TILLING), initially developed forArabidopsis, has been used successfully for high-throughput screening of librariesmutagenized with EMS or other chemical mutagens in several plant and animalspecies [191]. The TILLING method is based on the detection of single-nucleotidepolymorphisms (SNPs) induced by the mutagen, which presumably affects thegenes of interest. Induced mutations were initially identified by denaturing high-performance liquid chromatography (dHPLC), which detected conformationalchanges induced in heteroduplex originated by an SNP [192]. Sensitivity of theselection method was optimized later using mismatch-specific endonucleasesCEL1 or ENDO1 [193, 194], which cleave at mismatches within heteroduplexesformed betweenmutant and wild-type DNA strands. Recently, two novel, sensitive,and very high-throughput mutation screening techniques used in human geneticdiagnostic – conformation-sensitive capillary electrophoresis (CSCE) and high-resolution melt curve analysis (HRM) – have been successfully described to detectnovel mutations in EMS mutagenized libraries of tomato [195]. The resultsobtained by Christian Bachem�s group demonstrated that both newmethodologicalapproaches are fast and reliable and permit identification by TILLING of severalnew alleles in genes responsible for fruit quality such as phytoene synthase, whichis involved in the synthesis of the characteristic red color of fruits, and sucrosesynthase 2, a gene that participates in sucrose metabolism in young fruits. Thisgroup also identified 19 mutations in the coding sequence of the tomato prolinedehydrogenase (ProDH) gene, with the aim of inactivating this proline degradationenzyme. Unfortunately, the effect of all these mutations on the sequence of thecorresponding enzymes has not been reported; even if some of the mutations maybe silent, due to synonymous substitution, others should be senseless or nonsensemutations. It is expected that inactivation of ProDHwill increase proline content inall plant cells and the effect, if any, of proline accumulation in tomato resistance toabiotic stress will be determined.

43.2.8.5 Insertional Mutagenesis and Gene TrapsInsertional mutagenesis with T-DNA or transposable elements is a basic tool for theidentification of genes and the analysis of their functions. With respect to insertional


mutagenesis with T-DNA, we can approach the tagging of genes by using a simpleconstruction with a marker gene. In this way, the integration of T-DNA within thestructural sequence or the controlling elements of a given gene will lead to itsdisruption and the consequent loss-of-function or, depending on the characteristicsof the T-DNA-insert, gain-of-function, or change in its level of expression [196].Detecting the mutant phenotype in TG1 (in the case of dominant, semidominant, oradditive effects) or TG2 (recessive), as the gene is tagged by the T-DNA, means itscloning can be easier.

By comparison with classical insertional mutagenesis, the trapping systems [197]can be particularly useful for the identification of genes related to salt tolerance. Theadvantage of using enhancer, promoter, or gene traps resides in its self-dual nature.Like any other T-DNA, these traps act as insertion mutagens, and when T-DNA isintegrated into an endogenous gene in the appropriate orientation, the reporter genelies under the control of the regulatory elements of the tagged gene. Thus, byanalyzing the reporter gene expression, one can get a precise picture on the space-temporal expression pattern of the endogenous gene tagged by the trap. In thisrespect, trapping strategies bring great advantages to insertional mutagenesis,allowing identification of functionally redundant genes, those expressed at multipledevelopment states (generating confusion during phenotyping), genes whose dis-ruption causes early lethality, and genes whose disruption causes such a softphenotype that it may not be detected (in this case, the reporter expression providesa clue to identify the phenotype during evaluation). In addition, gene identification isindependent of the expression level of the gene, thus avoiding the risk of rejecting lowexpressed genes even though they havemajor effects on the phenotype. Finally, this isthe best way to identify genes that are activated or repressed in response to either anexternal stimulus or biotic and abiotic stress situations [196].

Using an enhancer trap (kindly provided by Dr. Thomas Jack, Department ofBiological Sciences, Dartmouth College, USA) and a promoter trap (developed in thelaboratory of Drs Rafael Lozano and Trinidad Angosto), a collection of 3500 T-DNAlines of tomato has been generated (unpublished results). Following a preliminaryscrutiny of a sample of this collection of T-DNA lines, we have detected several salt-hypersensitive tomato mutants [198]. At first, we focused on the search of mutantsaltered in Naþ absorption and transport since the plant needs to recover the ionichomeostasis in order to keep growing in a salinemedium for a long term. One of themutantsmost sensitive to ionic stress was detected by in vitro screening (Figure 43.2).Wehave also studied the effect of salinity inmutants altered onphotosynthesis. Someof these mutants could help to clarify the putative role of chloroplasts in Naþ

scavenging from the cytoplasm [199]. Most of the hypersensitive tomatomutants arerecessive, but there are some with dominant heredity. Among them, one mutantexhibits a hypersensitive reaction under salinity (i.e., lower growth, markedly wiltedleaves, and few, seedless fruits). The tagged gene codes a MYB-type transcriptionfactor. The involvement ofMYB transcription factors in the response to these kinds ofstresses has been previously described inmodel species [123, 159, 200, 201], althoughup to now there is only one MYB-TF identified in tomato with this possiblefunction [71]. Moreover, we have cloned the tagged gene in another mutant, which


shows homology with a MAP-KKK protein of Arabidopsis that negatively regulatestolerance to salinity [202].

Apart from the insertional mutagenesis program in tomato, a cultivated speciesusually considered as moderately tolerant to salinity, our main objective is to identifysome of the genes responsible for the high level of drought and salt tolerance inaccessions of the relatedwild species S. pennellii. In order to achieve this objective, wedeveloped an efficient transformation method (Pineda, unpublished results), whichhas allowed us to generate 2800 T-DNA lines of the wild species with enhancer orpromoter traps. We have scrutinized T-DNA lines with reporter gene expression instomata since the control of the stomata aperture contributes to drought toler-ance [203]. We have detected a mutant of S. pennellii that is altered in rootdevelopment and that could be interesting since tolerance to water stress is some-times related to rootmass. Moreover, we have already detected four hypersensitive S.pennellii mutants to water deficit stress. Interestingly, in one of these mutants theexpression of the reporter gene increases after stress in stomata and transportingvessels (Figure 43.3). The scrutiny of this collection is under way to identify newmutants altered in the level of saline and water deficit stress (mainly hypersensitive).Taking into account that the collection of T-DNA lines is going to expand progres-sively, the identification of newdrought and salt tolerance-related genes is expected inthe near future.

Figure 43.2 (a) A recessive mutant of tomatowas identified in vitro (on MS mediumsupplemented with 100mM of NaCl) ashypersensitive to salt. (b) The salt sensitivity ofthe tomato mutant was corroborated in vivo:Under control conditions, the phenotype and

plant growth of the wild-type and tomatomutant were similar, whereas the negative effectinduced by salinity was clearly greater in themutant than in the wild type after 20 days oftreatment with 100mM of NaCl.


The identification of insertion mutants altered in the level of salt or droughttolerance may be particularly useful in the identification of key or pivotal genesinvolved in different tolerance mechanisms. Likewise, in-depth physiological stud-ies, as well as the use of transcriptomic and proteomic approaches on these mutants,may provide valuable information on the key physiological processes altered in thesemutants. We foresee that the combined use of all these genomics tools will allow thegenetic and physiological dissection of those complex traits, thus allowing the properdesign of future breeding programs.

43.3Conclusions and Perspectives

In the genus Solanum, there are accessions of wild species with a high level oftolerance to salinity and drought. Unfortunately, despite the wealth of sources ofvariation, there is need to advance our understanding of themechanisms underlyingdrought and salinity response in tomato and to elucidate how plants coordinate theirresponses to overcome drought and salinity. One of the main challenges for the nearfuture is to shed light on which are the differential physiological mechanismsbetween cultivated and tolerant wild species under stress conditions.

Research in recent years has mainly used genetic transformation as a tool todevelop stress-tolerant tomato plants. Although it has been shown that the expressionof different kinds of genes in tomato transgenic plants can increase salinity and/ordrought tolerance, it is not possible to conclude for the moment that true tolerantcultivars from an agronomic point of view have been obtained via genetic transfor-

Figure 43.3 A dominant mutant of the wildspeciesS. pennelliiwithpositiveGUSexpressionin stomata is sensitive to drought stress. Theplants were subjected to several dehydration–

rehydration cycles (30 and 60 days oftreatment); moreover, the reporter gene (GUS)expression is enhanced in stomata afterexposure to drought stress.

43.3 Conclusions and Perspectives j1109

mation.Moreover, taking into account the scant number of tomato genes identified todate in relation to salinity and, especially, water deficit stress, the identification of newgenes playing a significant role in the response mechanisms to drought and/orsalinity is a priority objective. To fulfill the expectations created in this field, it wouldbe necessary to advance not only in the identification of new genes playing pivotalroles in tolerance but also in the identification of regulatory elementsmodulating theexpression level of the transgenes spatially and temporally.

Transcriptomic analysis can be useful to identity new stress-related pathways andgenes regulated by stress encoding unknown proteins (and putatively new functions)and to infer the main mechanisms responsible for stress tolerance. After theidentification through microarray analysis of the hundreds of genes that aredifferentially expressed under stress conditions, the use of a PTGS-based strategycould be particularly valuable in order to discriminate the key genes for the toleranceprocesses. It is interesting to point out that a combination of microarray andproteomic analysis can indicate whether gene regulation is controlled at the levelof transcription, translation, posttranslationalmodification, or protein accumulation.Furthermore, by comparing the behavior of salt-sensitive and salt-tolerant genotypes,ionomic approaches can provide new insight into the key mechanisms responsiblefor ion homeostasis and the underlying causes of the different abilities to use salineions in the osmotic adjustment of halotolerant genotypes.

The identification of mutants, especially insertion mutants, altered in the level ofsalt or drought tolerance can be particularly useful in the identification of key orpivotal genes involved in different tolerance mechanisms. Likewise, in-depth phys-iological studies and the use of transcriptomic and proteomic approaches for thesemutantsmay provide valuable information on the key physiological processes alteredin these mutants. We foresee that the combined use of all these genomics tools willallow the genetic and physiological dissection of those complex traits, thus allowingthe proper design of future breeding programs. In conclusion, the research oppor-tunities in the coming years are very important, as not only are the -omic tools nowavailable but also will the tomato genome be soon sequenced.

Acknowledgments

We thank Stephen Hasler for his help in editing the English version of themanuscript. This work was supported by research projects AGL2006-15290-C01-C02-C03 (Ministry of Education and Science) and 04553/GERM/06.

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Improving Crop Resistance to Abiotic Stress (TUTEJA:PLANT STRESS OMICS O-BK) || Tomato: Genomic Approaches for Salt and Drought Stress Tolerance