Section IIC Other Approaches

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.

j419

18Molecular Breeding for Enhancing Abiotic Stress ToleranceUsing HalophytesAjay Parida, Suja George, and K. Kavita

Halophytes are plants that survive in environments where the salt concentration isaround 200mM NaCl. The tolerance of halophytes to salinity relies on controlleduptake and compartmentalization of Naþ and the synthesis of compatible solutes.Identification and isolation of novel genes by genomic approaches will advance theunderstanding of mechanisms of high salt tolerance. Plant stress tolerance can beimproved by manipulating stress-associated genes and proteins and/or overexpres-sion of stress-associated metabolites. Here, we discuss the role of possible genes instress mitigation and tolerance in halophytes and their overexpression to generatestress-tolerant crop plants.

18.1Introduction

Abiotic stress limits crop productivity and their effect on plants in both natural andagricultural settings is a topic that is receiving increasing attention. Salt and droughtare the twomajor abiotic stresses causing yield losses in crop plants. Seven percent ofthe land�s surface and five percent of cultivated lands are affected by salinity [1], withsalt stress being one of the most serious environmental factors limiting theproductivity of crop plants [2]. Extensive research in plant salt tolerance has beencarried out, with the aim of improving the tolerance of crop plants. Salt tolerance isthe ability of plants to grow and complete their life cycle on a substrate that containshigh concentrations of soluble salt. Plants have been categorized into halophytes andglycophytes depending upon their behavior in saline environments [1]. Plants thatcan survive on high concentrations of salt in the rhizosphere and growwell are calledhalophytes. Depending on their salt-tolerating capacity, halophytes are either obligateor characterized by lowmorphological and taxonomical diversity with relative growthrates increasing up to 50% sea water or facultative and found in less saline habitatsalong the border between saline and nonsaline upland and characterized by broaderphysiological diversity that enables them to cope with saline and nonsalineconditions [3].

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.

j421

Classification of halophytes has been based on the characteristics of naturalsaline habitats [3, 4], the chemical composition of the shoots [5], or the ability tosecrete ions [6]. Saline habitats do differ in many regards (e.g., soil water content)and differences do exist among species in the balance of Naþ and Kþ in shoottissues [7]. The halophytes have been discussed over the past three decadesincluding review on general physiology of halophytes [1, 8], ecophysiology [4, 6,9–11], photosynthesis [12], response to oxidative stress [13], flooding tolerance, andsalinity tolerance [14].

18.1.1Halophytes and their Adaptations to Salinity

Halophytes have the capacity to tolerate extreme salinity because of very specialanatomical and morphological adaptations or avoidance mechanisms [15]. Sodiumsecretion through specialized cells is a strategy used by many halophytic plants [16].Salts may, however, also be released through the cuticle or in guttation fluid. Inaddition, they may be retransported back to the roots and soil via the phloem orbecome concentrated in salt hairs [17]. Halophytes become succulent in response toincreasing salinity, and such influential changes seem to integral to halophyticdevelopment [3]. Dropping off salt-saturated organs also removes large quantities ofsalt from some halophytes [18].

Halophytes utilize various physiological and biochemical mechanisms thatinclude (1) exclusion of Naþ at the soil root boundary and therefore from all tissues;(2) exclusion of Naþ from the xylem and therefore from leaf, thus preventingdisruption of photosynthesis; (3) inclusion of Naþ and synthesis of compatiblesolutes to maintain osmotic adjustment; (4) inclusion of Naþ and its subsequentsequestration in vacuoles [19]; and (5) inclusion of Naþ and its eventual eliminationthrough secretion by leaves [20].

Halophytes store about 90%ofNaþ in the shoot, at least 80% in the leaves, while theroot system has a much lower Naþ concentration compared to the aerial parts of theplant [20]. Ithasbeenfoundthatalthoughhalophytesaccumulate largeamountsofNaþ

in thecells, theconcentrationofNaCl inthecytosol ismaintainedwithinnontoxic limitsbythecompartmentationofNaClinvacuoles [21]. InductionoftheCAMpathway,whichincreases water use efficiency, is also reported in some halophytes [22]. However,individualhalophytesutilizedifferent salt tolerance traits indifferentsituations.Sinceawide range of salt tolerance mechanisms are employed by halophytes, the precisecombination of one or more mechanisms used to tolerate salinity varies between andacross species and makes the study of salinity tolerance more complex.

18.1.2Halophytes as a Source for Gene Mining

The mechanism of salinity tolerance is a very complex phenomenon. Studies haveshown that components of various pathways are involved in imparting the salinitytolerance to the plants. Understanding themolecular basis of the salt stress signaling

422j 18 Molecular Breeding for Enhancing Abiotic Stress Tolerance Using Halophytes

and tolerance mechanisms is essential to breeding and genetic engineering of salt-tolerant plants [23]. Genetically engineering the plants by introducing and/oroverexpressing selected genes seems to be a viable option to hasten the breedingof �improved� plants, while the introgression of genomic portions (QTL) involvedin stress tolerance often brings along undesirable agronomic characteristics fromthe donor parents. Intuitively, genetic engineering would be a faster way to insertbeneficial genes than through conventional or molecular breeding. Also, it wouldbe the only option when genes of interest originate from cross barrier species,distant relatives, or from nonplant sources. Attempts on plant stress tolerance canbe made by manipulating stress-associated genes and proteins and/or overexpres-sion of stress-associatedmetabolites that would confer increased tolerance to salt ordrought.

Halophytic plants are a very important genetic resource for the isolation of novelpromoters and/or genes that are involved in the adaptation to salinity that could betransferred to salt-sensitive glycophytes. Identification and isolation of novel genesby genomic approaches will advance the understanding of these mechanisms [24].Hence, different efforts in large-scale ESTsequencing and analysis have beenmadein a number of dicotyledonous halophytes, such as Suaeda salsa [25], Thellungiellahalophila [26], Mesembryanthemum crystallinum [27], Aegiceras corniculatum [28],Avicennia marina [29], Tamarix hispida [30], and Limonium sinense [31], andmonocotyledonous halophytes such as Leymus chinensis [32], Puccinellia tenui-flora [33], Lolium temulentum [34], Aeluropus littoralis [35], and Spartinaalterniflora [36].

This chapter summarizes the involvement of various genes in sensing andresponse to salt stress in halophytes according to their putative functions such as(1) genes for reestablishing ionic homeostasis or preventing damage, (2) genes withan osmotic or unknown protective function, and (3) genes for signal transduction.This paper focuses on the different genes explored from halophytes for the geneticenhancement of crop plants for abiotic stress tolerance using transgenic approach.

18.2Genes for Reestablishing Ionic Homeostasis/Preventing Damage

Excessive intracellular or extracellular Naþ triggers a cytoplasmic Ca2þ signal thatinvolves an SOS pathway. This leads to enhanced expression of transporters for ionssuch as Naþ , Kþ , and Hþ . Naþ /Hþ antiporter located in the plasma membraneexcludes Naþ from the cells [37]. The compartmentalization of Naþ into vacuolesprovides an efficient mechanism for averting the toxic effects of Naþ in the cytosol.The transport of Naþ into vacuoles mediated by vacuolar Naþ /Hþ antiporters isdriven by the electrochemical gradient of protons. The proton-motive force generatedby the vacuolar ATPase (V-ATPase) and vacuolar pyrophosphatase (V-PPase) can drivesecondary transporters, such as the Naþ /Hþ antiporter and the Ca2þ /Hþ anti-porter, as well as organic acids, sugars, and other compound transporters tomaintaincell turgor.

18.2 Genes for Reestablishing Ionic Homeostasis/Preventing Damage j423

18.2.1Vacuolar Naþ /Hþ Antiporter

Salt-tolerant plants such as halophytes efficiently sequester Naþ into vacuoles, tomaintain low cytosolic concentrations of Naþ and thus allow antiporter genes to beisolatedfromhalophytesandtransformedintotransgenicplants.Intransgenicsystems,it was found that vacuolar antiporters from glycophytes and halophytes confer salttolerance to varying limits. Genes encoding vacuole-type Naþ /Hþ antiporters havebeen isolated fromanumber of halophytes such asAtriplex gmelini (AgNHX1) [38],M.crystallinum (McNHX1) [39],A. dimorphostegia (AdNHX1) [40],Chenopodium glaucum(CgNHX1)[41],S.salsa(SsNHX1)[42],Porteresiacoarctata(PcNHX1)[43],andSalicorniabrachiata (SbNHX1) [44]. A recent study identified six putative vacuolar Naþ /Hþ

antiporter genes in P. euphratica (PeNHX1–6), a salt-resistant tree species [45].Overexpression of SsNHX1 [46] and AgNHX1 [47] in rice markedly enhanced the

tolerance to salt stress (300mM NaCl). Increased Naþ /Hþ antiport activity in thetransgenic plants caused larger amounts of Naþ to be excluded into vacuolesin individual cells, thus rendering the transgenic rice plants more tolerant tosalinity [46, 47]. But another study reported that overexpression of NHX1 genesfrom both glycophytic (OsNHX1) and halophytic (AdNHX1, CgNHX1) species led tosimilar degree of salt tolerance in transgenic rice plants [41]. The better salt tolerancein halophytes might result from a different regulation system of NHX1 genes ormechanisms other than vacuolar Naþ pump [48].

18.2.2Plasma Membrane Naþ /Hþ Antiporter

In addition to Naþ influx control and vacuolar compartmentation, Naþ efflux is alsoimportant in maintaining a low Naþ concentration in the cytoplasm. Unlike animalcells, which have Naþ /KþATPases, or fungal and perhaps some algal cells, whichhave NaþATPases for Naþ efflux, plant cells do not appear to contain NaþATPases.In higher plants, themainmechanism forNaþ extrusion is powered by the operationof the plasma membrane HþATPase. HþATPase allows the operation of plasmamembrane Naþ /Hþ antiporter that couples the downhill movement of Hþ into thecell along its electrochemical gradient to the extrusion of Naþ against its electro-chemical gradient [49]. Naþ /Hþ antiporter activity has been reported to occuracross the plasma membrane of A. nummularia [50]. In A. thaliana salt overlysensitive 1 (SOS1) is a plasma membrane Naþ /Hþ antiporter that retrieves andloads Naþ ions from and into the xylem [51]. A comparison of SOS1 transcript inunstressed plants of A. thaliana and T. halophila revealed that the two species hadsimilar levels of SOS1 transcript in their shoots, while T. halophila possessedthreefoldmore SOS1 transcript in its roots thanA. thaliana under control conditions.A. thaliana plants that overexpress SOS1 aremore tolerant to salt because of thisNaþ

retrieval [52]. InT. halophila, the salt-mediated induction of shootThSOS1 expressioncoupled with high basal root ThSOS1 expression is likely to be a crucial factor intightly controlling the extent of shoot Naþ accumulation [53].

424j 18 Molecular Breeding for Enhancing Abiotic Stress Tolerance Using Halophytes

18.2.3Vacuolar Pyrophosphatase

The vacuolar Hþ -PPase is a single subunit protein located in the vacuolarmembrane [54]. It pumps Hþ from the cytoplasm into vacuoles with PPi-depen-dentHþ transport. Theoretically, overexpression ofHþ -PPase should enhance theability to form the pHgradient between the cytoplasm and the vacuoles, resulting ina stronger proton-motive force for the Naþ /Hþ antiporter, Ca2þ /Hþ antiporter,and other secondary transporters. Overexpression of Hþ -PPase genes from T.halophila (TsVP) enhanced the salt tolerance of tobacco [55] and cotton [56]. Thetransgenic lines had higher Hþ -PPase hydrolytic activity and the plants accumu-latedmore Naþ under salt stress conditions [55, 56]. A comparison of promoters ofvacuolar Hþ -PPase genes from T. halophila (TsVP1) and Arabidopsis (AVP1)indicated that these two promoters had seven similar motifs at similar positions.But analysis of transgenic plants expressingGUS reporter gene under the control ofthese promoters indicated that TsVP1 promoter was responsible for strong reportergene activity in almost all tissues except the seeds, and the activity was induced inboth shoots and roots, especially in the root tips, when treated with salt stress. Suchinduction was not found in transgenic Arabidopsis with the AVP1 promoter.Deletion analysis revealed the presence of enhancer elements in TsVP1 promoterthat increased gene expression levels [57]. These results point out the importance ofcloning stress tolerance genes under stress-inducible promoters for improvedtolerance.

18.2.4Potassium Transporters

Salt tolerance requires not only the adaptation to sodium toxicity but also theacquisition of potassium whose uptake is affected by high external sodium concen-tration. Therefore, potassium transport systems involving good selectivity ofpotassium over sodium can also be considered an important salt tolerance deter-minant [58]. In some halophytes, salinity increases the Kþ concentration of thetissue. Kþ transport is mediated by Kþ channels and high-affinity Kþ transportersboth in the plasmalemma and in the tonoplast of plant cells [59]. The Kþ channelsand transporters may regulate Naþ transport – either directly because they may beincompletely selective for Kþ and transport Naþ when presented with a high Naþ

concentration or a high Naþ /Kþ ratio or indirectly because they may buffer the cellagainst Naþ uptake by maintaining rigorous Kþ homeostasis.

The families in Kþ transporters include HAK/KUP/KT and HKT transporters.These transporters control Kþ uptake and Kþ/Naþ selectivity. In M. crystallinum,HAK-type proteinsmediates the transport of Kþ , Rbþ , andCsþ but not that of Naþ .McHAKs seem to have a role inmediating root Kþ uptake and that could be involvedin plant long-distance Kþ transport through loading and/or unloading in thevasculature [60]. The capacity of HKT to mediate Naþ uptake in some speciesmakes it a candidate that could have amajor function formaintaining or breaking ion

18.2 Genes for Reestablishing Ionic Homeostasis/Preventing Damage j425

homeostasis under saline conditions. Unlike the HAK transporters, transporters inthe HKT family display different ion selectivity and transport mechanisms.

McHKT1, isolated from M. crystallinum, is a potassium transporter localized inthe plasma membrane of cells of both the leaves and the roots. The expression ofMcHKT1 is upregulated after a sudden increase in external salinity (400mmNaCl) [61], as is the expression of SOS1 and some HAK transporters. The decreasedstorage of Naþ in the root and enhanced transport to the shoot, with theupregulation of McHKT1, suggested to contribute to storage of Naþ in the leavesin ice plant [61]. In Xenopus oocytes, McHKT1 transports Naþ and Kþ equally [61].In S. salsa, SsHKT1 transcript was developmentally controlled and significantlyupregulated by Kþ deprivation and NaCl treatment suggesting its role in ionhomeostasis and salt tolerance [62]. An AKT1-type K(þ ) channel gene fromPuccinellia tenuiflora, a salt-tolerant plant, was found to be localized in the plasmamembrane and preferentially expressed in the roots. The expression of PutAKT1was induced by K(þ )-starvation stress in the roots and was not downregulated bythe presence of excess Na(þ ). Arabidopsis plants overexpressing PutAKT1 showedenhanced salt tolerance compared to wild-type plants. PutAKT1 transgenic plantsalso showed a decrease in Na(þ ) accumulation both in the shoot and in the root. Itis possible that PutAKT1 is involved in mediating K(þ ) uptake (i) both in low- andin high-affinity K(þ ) uptake range and (ii) unlike its homologues in rice, evenunder salt stress condition [63]. Evidence from the range of studies discussedindicates that there may be considerable variation in the transporters involved inthe uptake of Naþ , not only between glycophytes and halophytes but also betweenspecies of halophyte and even at different external salt concentrations. InM. crystallinum coordinate regulation of multiplicity of channels, transporters,symporters, and antiporters results in irreversible transport of NaCl from root toshoot, accumulation in leaves, and sequestration of Naþ into the vacuoles of cells inthe leaves and shoot [61].

18.2.5ROS Scavengers

The accumulation of ROS during salt stress is mainly attributed to the inhibition ofphotosynthesis and a decline in CO2 fixation. Some of the ROS are highly toxic andneed to be detoxified rapidly. In order to control the level of ROS and protectthe cells from oxidative injury, plants have developed a complex antioxidant defensesystem to scavenge the ROS. These antioxidant systems include various enzymesand nonenzymatic metabolites that may also play a significant role in ROS signalingin plants [64]. A number of transgenic improvements in abiotic stress tolerancehave been achieved through detoxification strategy. These include transgenic plantsoverexpressing enzymes involved in oxidative protection, such as glutathioneperoxidase, superoxide dismutase, ascorbate peroxidases, and glutathione reduc-tases. In Bruguiera parviflora, salt treatment preferentially enhanced both thecontent of H2O2 and the activity of ascorbate peroxidase (APX), guaiacol peroxidase(GPX), glutathione reductase (GR), and superoxide dismutase (SOD), while induc-

426j 18 Molecular Breeding for Enhancing Abiotic Stress Tolerance Using Halophytes

ing a decrease in total ascorbate and glutathione (GSHþGSSG) content and incatalase (CAT) activity [65]. Expression of cytosolic Cu/Zn SOD in B. gymnorrhizawas increased after NaCl treatment and also in the presence of mannitol andabscisic acid (ABA) [66]. In A. marina, the mRNA transcripts of Cu/Zn SOD (Sod1),catalase (Cat1) [67], and ascorbate peroxidase (AmAPX1) [68] were upregulatedunder both salinity and oxidative stress. Overexpression of A. marina cytosoliccopper/zinc SOD conferred enhanced tolerance to both salt and drought treatmentsin transgenic rice [69].

18.2.6Genes with an Osmotic/Protective Function

Compatible solutes or osmolytes would be essential for coordinated regulation ofvacuolar and cytoplasmic volumes. Compatible solutes are nontoxic solutes thatcould increase inhigh concentrations in the cytosol andbe compatiblewithmetabolicactivity. They would be important to adapt plants to drought, as they could enhanceosmotic adjustment and allow turgor maintenance of cells that would otherwisedehydrate. In addition, certain solutes have a metabolic protective role. They couldstabilize soluble or membrane proteins and thus maintain growth at high salinity,and the termosmoprotectant has arisen for this function. There are fourmain classesof solutes that could have an osmotic or protective role: N-containing solutes such asproline and glycine betaine; sugars such as sucrose and raffinose; straight-chainpolyhydric alcohols (polyols) such as mannitol and sorbitol; and cyclic polyhydricalcohols (cyclic polyols) [70].

Many crops lack the ability to synthesize the special osmoprotectants that arenaturally accumulated by stress-tolerant organisms. It is believed that osmoregula-tion would be the best strategy for abiotic stress tolerance, especially if osmoregu-latory genes could be triggered in response to drought and salinity. Therefore, awidely adopted strategy has been to engineer crops with such osmolytes for abioticstress tolerance.

18.2.7Amines

Glycine-betaine (GB) highly accumulates as a compatible solute in certain plants andhas been considered to play a role in the protection from salt stress. In plants, glycine-betaine is synthesized from choline in two steps, the first being catalyzed by cholinemonooxygenase (CMO) that requires phosphoethanolamine N-methyltransferase(PEAMT) and S-adenosyl-L-methionine (SAM) leading to synthesis of betaine-alde-hyde, which is further oxidized by betaine-aldehyde dehydrogenase (BADH). CMOhas been cloned and characterized from halophytes such as Beta vulgaris subsp.maritime (BvCMO) [71], A. hortensis (AhCMO) [72], A. prostrata (ApCMO) [73], andA. nummularia (AmCMO) [74]. CMO expression was highly induced upon salttreatment in A. hortensis [75] and A. prostrata [73]. Drought stress also induced theexpression of AhCMO, but with ABA treatment AhCMO was induced only slight-

18.2 Genes for Reestablishing Ionic Homeostasis/Preventing Damage j427

ly [75]. ABA treatment did not induce ApCMO in A. prostrate showing that ApCMOmRNA does not depend upon exogenous ABA [73].

Metabolic engineering of GlyBet in all the plants suffered from one feature: theGlyBet level in transformants is lower than that in the natural accumulator to adjustthe osmotic pressure in vivo [76]. The transport of choline into the chloroplastconstrains GlyBet accumulation in CMOþ tobacco [77]. If supplied with extraneoussubstrate, transgenic plants would then synthesize enough GlyBet and greatlypromote stress tolerance [78, 79]. Overexpression of AhCMO improved droughttolerance in transgenic tobacco and the transgenic plants also performedbetter undersalt stress [75]. Transplastomic tobacco plants overexpressing BvCMOgene exhibitedincreased tolerance to salt and drought stress. Accumulation of GlyBet in transplas-tomic plants enhances the net photosynthetic rate and apparent quantum yield ofphotosynthesis under salt stress condition [80].

BADH gene has been isolated and characterized from halophytes such asA. hortensis (AhBADH) [81], A. marina (AmBADH) [82], and S. liaotungensis(SlBADH) [40]. Overexpression of AhBADH gene into rice [83], wheat [84], andwhite clover [85] and SlBADH into tobacco plants [40] improved salt tolerance.Cotargetingmultiple steps in the same pathway was found to be a successful strategyfor overexpressing glycine-betaines in plants. A study by Yilmaz and B€ulow [86]reported that salt tolerance can be enhanced by genetic engineering of tobacco plantswith the betaine aldehyde–choline dehydrogenase fusion protein.

18.2.8Proline

Among compatible osmolytes, proline accumulates in many plants in response toabiotic stress [87]. Proline accumulation was correlated with improved plantperformance under salt stress. In plants, the proline biosynthetic pathway fromglutamate proceeds through the action of a determining enzyme, D1-pyrroline-5-carboxylate synthetase (P5CS). Proline catabolism is catalyzed by proline dehy-drogenases (PDHs). Stress-induced increase in proline content is caused byconcerted induction of proline biosynthesis genes and by repression of prolinecatabolism genes [87]. Undetectable level of PDH transcripts after NaCl stress in T.halophila resulted in reduced capacity for proline catabolism in T. halophila. Higherproline accumulation in T. halophila suggests that changes in T. halophila PDHexpression could cause significant increases in free proline levels. Increasedaccumulation of proline leads to improved salt stress tolerance and is possibly areason for improved salt stress tolerance of T. halophila in comparison to A.thaliana [53]. Increased levels of proline accumulation have also been observedin salt-stressed calli of S. nudiflora suggesting that proline protects the callus cellsfrommembrane damage caused by free radicals formed during salt stress [88]. Theability of NaCl to improve the performance of Sesuvium portulacastrum undermannitol-induced water stress may be due to its effect on osmotic adjustmentthrough Naþ and proline accumulation, which is coupled with an improvement inphotosynthetic activity [89].

428j 18 Molecular Breeding for Enhancing Abiotic Stress Tolerance Using Halophytes

18.2.9Polyols

Accumulation of polyols, either straight-chain metabolites such as mannitol andsorbitol or cyclic polyols such as myo-inositol or its methylated derivatives such aspinitol is correlated with tolerance to drought and/or salinity [90]. Many naturallyoccurring salt or drought-tolerant plants accumulate such compounds during stress.These metabolites are considered compatible solutes that act by providing osmoticadjustment and by lowering the osmotic potential, thus increasing the waterretention capacity of the plant [91]. Myo-inositol, the precursor of pinitol, is synthe-sized through L-myo-inositol 1-phosphate synthase (MIPS) coded by the INO1 gene.The enzymatic product of MIPS is specifically dephosphorylated by a Mgþ þ -dependent L-myo-inositol 1-phosphate phosphatase to form free inositol. Inositol ismethylated to pinitol by the inositol methyl transferase coded by an IMT1 gene in anS-adenosylmethionine (SAM)-dependent reaction [90]. IMT1 gene in halophytes hasbeen cloned and characterized from M. crystallinum [90] and P. coarctata [92]. Thetranscript and protein content of PcIMT1 was substantially upregulated in salinityand ABA in P. coarctata. The halophytic ice plant accumulates predominantlyD pinitol under salinity and low-temperature stresses.

Several halophytic and nonhalophytic species are reported to contain pinitol as themajor soluble carbohydrate in their leaves. The halophytic wild rice P. coarctataharbors a unique salt-tolerant MIPS coded by PcINO1 that is able to generate myo-inositol even at high salt concentration, and the inositol pool in the plant is wellmaintained during salinity [93, 94]. Overexpression of PcINO1 has been reported toconfer salt-tolerant phenotype with unabated photosynthetic functions to trans-formed tobacco plants. In M. crystallinum, McINO1 is induced upon salt stress butin A. thaliana AtINO1 is not induced by salt stress [95]. A coordinated functioning ofboth the INO1 gene and the IMT1 gene is expected to be operative during salt stressin M. crystallinum [95] and P. coarctata [92] for synthesis of pinitol.

18.3Genes for Signal Transduction

Candidate genes controlling growth are probably involved in signaling pathways andrespond to hormones, transcription factors, protein kinases, protein phosphatases,and other signaling molecules. Transcription factors are proteins that respond toenvironmental stimuli through a signaling cascade and bind to specific regulatorysites upstream of constituent genes in a regulatory network by direct physicalinteraction or in combination with other proteins. Consequently, an alteration of theexpression of transcription factor genes results in dramatic differences in theexpression of multiple genes in a plant [96]. NAC proteins form a large family ofplant-specificDNAbinding transcription factors that are gaining importance in recenttimes with respect to understanding plant development and adaptation. InA.marina,AmNAC1 transcript expression was upregulated by NaCl and ABA treatment [97].

18.3 Genes for Signal Transduction j429

The zinc finger proteins (ZPT2) have been previously used as candidates to showhow a subset of transcription factorsmight be involved in thewater stress response atthe level of transcriptional regulation [98]. In T. halophila, ThZF1, encoding a plant-specific transcription factor, is induced at the transcription level by both drought andsalt [99]. Overexpression of AhDREB1 of A. hortensis improved the salt tolerance intransgenic tobacco through functioning as a regulatory molecule in response to saltstress [100]. Recently, a novel zinc finger gene designated AlSAP was isolated fromthe halophyte grass A. littoralis. Sequence homology analysis showed that the AlSAPprotein is characterized by the presence of two conserved zinc finger domains A20and AN1. AlSAPwas found to be induced not only by various abiotic stresses such assalt, osmotic pressure, heat, and cold but also by abscisic acid and salicylic acid (SA).Tobacco plants expressing the AlSAP gene exhibited an enhanced tolerance to high-salinity stress. Moreover, the transgenic plants were able to complete their life cycleand to produce viable seeds under high salt conditions, while the wild-type plantsdied at the vegetative stage [101]. Such studies characterizing novel genes add on tothe available information on salt tolerance mechanisms in halophytes.

18.4Conclusions

Halophytes are a diverse group of plants with varying degrees of salt tolerance, yetthey appear to share in common the ability to sequester NaCl in cell vacuoles as themajor plant osmoticum. Efforts to produce salt-tolerant crops were aimed mainly atincreasing the salt exclusion capacity of glycophytes. However, these efforts have notproduced breakthroughs in salt tolerance [102]. Progress in producinghighly tolerantcrops may require a change in strategy, to attempt to introduce halophyte genesdirectly into glycophytes [103]. This chapter focuses on the combination of the genesthat impart salt tolerance in halophytes and hence their use in the geneticengineering of salt-tolerant crops. Thus, engineering for accumulation of salt invacuolated cells, together with the active extrusion of Naþ from nonvacuolated cells(i.e., young and meristematic tissue) will allow the maintenance of a high cytosolicKþ/Naþ ratio. This in combination with the enhanced production of compatiblesolutes will generate transgenic crop plants that can tolerate and grow in high soil saltconcentrations.

References

1 Flowers, T.J., Garcia, A., Koyama,M., andYeo, A.R. (1997) Acta Physio. Plant., 19,427–433.

2 Ashraf, M. (1999) Crit. Rev. Plant Sci., 13,17–42.

3 Waisel, Y. (1972) The Biology ofHalophytes, Academic Press, London,UK.

4 Le Hou�erou, H.N. (1993) Salt-tolerantplants for the arid regions of themediterranean isoclimatic zone, inTowards the Rational Use of HighSalinity Tolerant Plants (eds H. Lieth andA. Masoon), Kluwer AcademicPublishers, Dordrecht, The Netherlands,pp. 403–422.

430j 18 Molecular Breeding for Enhancing Abiotic Stress Tolerance Using Halophytes

5 Albert, R., Pfundner, G., Hertenberger,G., Kastenbauer, T., and Watzka, M.(2000) The physiotype approach tounderstanding halophytes andxerophytes, in Ergebnisse Weltweiter€Okologischer Forschung (eds S.W. Breckle,B. Schweizer, and U. Arndt), VerlagG€unter Heimbach, Stuttgart, Germany,pp. 69–87.

6 Breckle, S.W. (2002) Salinity, halophytesand salt affected natural ecosystems, inSalinity: Environment–Plants–Molecules(eds A. L€auchli and U. L€uttge), KluwerAcademic Publishers, Dordrecht, TheNetherlands, pp. 53–77.

7 Wang, S.M., Zheng, W.J., Ren, J.Z., andZhang, C.L. (2002) J. Arid Environ., 52,457–472.

8 Flowers, T.J. (1985) Plant Soil, 89, 41–56.9 Ball, M.C. (1988) Trees, 2, 129–142.

10 Rozema, J. (1991) Aquat. Bot., 39, 17–33.11 Ungar, I.A. (1991) Ecophysiology of

Vascular Halophytes, CRC Press, BocaRaton, FL.

12 Lovelock, C.E. and Ball, M. (2002)Influence of salinity on photosynthesisof halophytes, in Salinity: Environment–Plant–Molecules (eds A. L€auchli andU. L€uttge), Kluwer Academic Publishers,Dordrecht, The Netherlands,pp. 315–339.

13 Jithesh, M.N., Prashanth, S.R.,Sivaprakash, K.R., and Parida, A.K.(2006) J. Genet., 85, 237–254.

14 Colmer, T.D. and Flowers, T.J. (2008)NewPhytol., 179, 964–974.

15 Flowers, T.J., Hajibagheri, M.A., andClipson, N.J.W. (1986) Quart. Rev. Biol.,61, 313–337.

16 Fitzgerald, M.A., Orlovich, D.A., andAllaway, W.G. (1992) New Phytol., 120,1–7.

17 Stenlid, G. (1956) in Encyclopedia of PlantPhysiology (ed. E. Ruhland), vol. 4,Springer, Berlin, pp. 615–637.

18 Chapman, M. (1968) Vegetation undersaline conditions, in Saline Irrigation forAgriculture and Forestry (ed. H. Buyko),Dr. W. Junk Publishers, The Hague,pp. 210–216.

19 Blumwald, E., Aharon, G.S., and Apse,M.P. (2000) Biochem. Biophys. Acta, 1465,140–151.

20 Flowers, T.J., Troke, P.F., and Yeo, A.R.(1977) Annu. Rev. Plant Physiol., 28,89–121.

21 Binzel, M.L., Hess, F.D., Bressan, R.A.,andHasegawa, P.M. (1988)Plant Physiol.,86, 507–514.

22 Bohnert, H.J., Nelson, D.E., and Jensen,R.G. (1995) Plant Cell, 7, 1099–1111.

23 Chinnusamy, V., Jagendorf, A., and Zhu,J.K. (2005) Crop Sci., 45, 437–448.

24 Bohnert, H.J., Ayoubi, P., Borchert, C.,Bressan, R.A., Burnap, R.L., Cushman,C., Cushman, M.A., Deyholos, M.,Fischer, R., Galbraith, D.W., Hasegawa,P.M., Jenks, M., Kawasaki, S., Koiwa, H.,Kore-eda, S., Lee, B.H., Michalowski,C.B., Misawa, E., Nomura, M., andOzturk, N. (2001) Plant Physiol. Biochem.,39, 295–311.

25 Zhang, L., Ma, X.L., Zhang, Q., Ma, C.L.,Wang, P.P., Sun, Y.F., Zhao, Y.X., andZhang, H. (2001) Gene, 267, 193–200.

26 Wang, Z.L., Li, P.H., Fredricksen, M.,Gong, Z.Z., Kim, C.S., Zhang, C.,Bohnert, H.J., Zhu, J.K., Bressan, R.A.,Hasegawa, P.M., Zhao, Y.X., and Zhang,H. (2004) Plant Sci., 166, 609–616.

27 Kore-eda, S., Cushman, M.A., Akselrod,I., Bufford, D., Fredrickson,M., Clark, E.,and Cushman, J.C. (2004) Gene, 27,83–92.

28 Fu, X., Huang, Y., Deng, S., Zhou, R.,Yang, G., Ni, X., Li, W., and Shi, S. (2005)Plant Sci., 169, 147–154.

29 Mehta, P.A., Sivaprakash, K., Parani, M.,Venkataraman, G., and Parida, A.K.(2005) Theor. Appl. Genet., 110, 416–424.

30 Gao, C.,Wang, Y., Liu, G., Yang, C., Jiang,J., and Li, H. (2008) Plant Mol. Biol., 66,245–258.

31 Chen, S.H., Guo, S.L., Wang, Z.L., Zhao,J.Q., Zhao, Y.X., and Zhang, H. (2007)DNA Seq., 18, 61–67.

32 Jin, H., Plaha, P., Park, J.Y., Hong, C.P.,Lee, I.S., Yang, Z.H., Jiang, G.B., Kwak,S.S., Liu, S.K., Lee, J.S., Kim, Y.A.,and Lim, Y.P. (2006) Plant Sci., 170,1081–1086.

33 Wang, Y., Chu, Y., Liu, G., Wang, M.H.,Jiang, J., Hou, Y., Qu, G., and Yang, C.(2007) J. Plant Physiol., 164, 78–89.

34 Baldwin, J.C. and Dombrowski, J.E.(2006) Plant Sci., 171, 459–469.

References j431

35 Zouari, N., Ben Saad, R., Legavre, T.,Azaza, J., Sabau, X., Jaoua, M.,Masmoudi, K., and Hassairi, A. (2007)Gene, 404, 61–69.

36 Baisakh, N., Subudhi, P.K., andVaradwaj, P. (2008) Funct. Integr.Genomics, 8, 287–300.

37 Zhu, J.K. (2003)Curr. Opin. Plant Biol., 6,441–445.

38 Hamada, A., Shono,M., Xia, T., Ohta,M.,Hayashi, Y., Tanaka, A., andHayakawa, T.(2001) Plant Mol. Biol., 46, 35–42.

39 Chauhan, S., Forsthoefel, N., Ran, Y.,Quigley, F., Nelson, D.E., and Bohnert,H.J. (2000) Plant J., 24, 511–522.

40 Li, J.Y., Zhang, F.C., Ma, J., Cai, L., Bao,Y.G., and Wang, B. (2003) Plant Physiol.Commun., 6, 585–588.

41 Li, J.Y., He, X.W., Xu, L., Zhou, J., Wu, P.,Shou, H.X., and Zhang, F.C. (2008)J. Zhejiang Univ. Sci. B., 9, 132–140.

42 Ma, X.L., Zhang, Q., Shi, H.Z., Zhu, J.K.,Xhao, Y.X., Ma, C.L., and Zhang, H.(2004) Biol. Plant., 48, 219–225.

43 Praseetha, K. (2007) Isolation,characterization and overexpression of atonoplast Naþ /Hþ antiporter from thewild rice Porteresia coarctata in transgenicsystem. PhD thesis, University ofMadras, Chennai, India.

44 Jha, A., Joshi, M., Yadav, N.S., Agarwal,P.K., and Jha, B. (2010)Mol. Biol. Rep.doi:10.1007/s11033-010-0318-5

45 Ye, C.Y., Zhang, H.C., Chen, J.H., andXia, X.L., and Yin, W.L. (2009) Physiol.Plant., 137, 166–174.

46 Zhao, F., Wang, Z., Zhang, Q., Zhao, Y.,and Zhang, H. (2006) J. Plant. Res., 119,95–104.

47 Ohta, M., Hayashi, Y., Nakashima, A.,Hamada, A., Tanaka, A., Nakamura, T.,and Hayakawa, T. (2002) FEBS Lett., 532,279–282.

48 Xiong, L. and Zhu, J.K. (2002) Salttolerance, The Arabidopsis Book.

49 Sussman, M.R. (1994) Annu. Rev. PlantPhysiol. Plant Mol. Biol., 45, 211–234.

50 Hassidim, M., Braun, Y., Lerner, Y.H.R.,and Reinhold, L. (1990) Plant Physiol., 94,1795–1801.

51 Shi, H., Ishitani, M., Kim, C., and Zhu,J.K. (2000) Proc. Natl. Acad. Sci. USA, 97,6896–6901.

52 Shi, H., Lee, B.H.,Wu, S.J., and Zhu, J.K.(2003) Nat. Biotech., 21, 81–85.

53 Kant, S., Kant, P., Raveh, E., and Barak, S.(2006) Plant Cell Environ., 29, 1220–1234.

54 Maeshima, M. (2000) Biochim. Biophys.Acta., 1465, 37–51.

55 Gao, F., Gao, Q., Duan, X.G., Yue, G.D.,Yang, A.F., and Zhang, J.R. (2006) J. Exp.Bot., 57, 3259–3270.

56 Lv, S., Yang, A., Zhang, K., Wang, L.,and Zhang, J. (2008) Mol. Breed., 20,233–248.

57 Sun, Q., Gao, F., Zhao, L., Li, K., andZhang, J. (2010) BMC Plant Biol., 10, 90.

58 Rodriguez-Navarro, A. (2000) Biochem.Biophysiol. Acta, 1469, 1–30.

59 Niu, X., Bressan, R.A., Hasegawa, P.M.,and Pardo, J.M. (1995)Plant Physiol., 109,735–742.

60 Su, H., Golldack, D., Zhao, C., andBohnert, H.J. (2002) Plant Physiol., 129,1482–1493.

61 Su, H., Balderas, E., Vera-Estrella, R.,Golldack, D., Quigley, F., Zhao, C.S.,Pantoja, O., and Bohnert, J.H. (2003)Plant Mol. Biol., 52, 967–980.

62 Shao, Q., Zhao, C., Han, N., and Wang,B.S. (2008) DNA Seq., 19, 106–114.

63 Ardie, S.W., Liu, S., and Takano, T. (2010)Plant Cell Rep., 29, 865–874.

64 Vranov�a, E., Inz�e,D., andVanBreusegem,F. (2002) J. Exp. Bot., 53, 1227–1236.

65 Parida, A.K., Das, A.B., and Mohanty, P.(2004) J. Plant Physiol., 161, 531–542.

66 Takemura, T., Hanagata, N., Dubinsky,Z., and Karube, I. (2002) Trees, 16, 94–99.

67 Jithesh, M.N., Prashanth, S.R.,Sivaprakash, K.R., and Parida, A.K.(2006) Plant Cell Rep., 25, 865–876.

68 Kavitha, K., Venkataraman, G., andParida, A.K. (2008) Plant Physiol.Biochem., 46, 794–804.

69 Prashanth, S.R., Sadhasivam, V., andParida, A.K. (2008) Transgenic Res., 17,281–291.

70 Chen, T.H. and Murata, N. (2002) Curr.Opin. Plant Biol., 5, 250–257.

71 Russell, B.L., Rathinasabapathi, B., andHanson, A.D. (1998) Plant Physiol., 116,859–865.

72 Shen, Y.G., Du, B.X., Zhang, J.S., andChen, S.Y. (2001) Sheng Wu Gong ChengXue Bao, 17, 1–6.

432j 18 Molecular Breeding for Enhancing Abiotic Stress Tolerance Using Halophytes

73 Wang, L.W. and Showalter, A.M. (2004)Physiol. Plant., 120, 405–412.

74 Tabuchi, T., Kawaguchi, Y., Azuma, T.,Nanmori, T., and Yasuda, T. (2005) PlantCell Physiol., 46, 505–513.

75 Shen, Y.G., Du, B.X., Zhang, W.K.,Zhang, J.S., and Chen, S.Y. (2002) Theor.Appl. Genet., 105, 815–821.

76 Sakamoto, A. andMurata, N. (2002)PlantCell Environ., 25, 163–171.

77 McNeil, S.D., Rhodes, D., Russell, B.L.,Nuccio, M.L., Yair, S.H., and Hanson,A.D. (2000) Plant Physiol., 124, 153–162.

78 Nuccio, M.L., Russell, B.L., Nolte, K.D.,Rathinasabapathi, B., Gage, D.A., andHanson,A.D. (1998)Plant J.,16, 487–496.

79 Huang, J., Hirji, R., Adam, L.,Rozwadowski, K.L., Hammerlindl, J.K.,Keller, W.A., and Selvaraj, G. (2000) PlantPhysiol., 122, 747–756.

80 Zhang, J.,Tan,W.,Yang,X.H.,andZhang,H.X.(2008)PlantCellRep.,27,1113–1124.

81 Xiao, G., Zhang, G.Y., Liu, F.H.,Wang, J.,Chen, S.Y., Li, C., and Geng, H.Z. (1995)Chin. Sci. Bull., 40, 741–745.

82 Hibino, T., Meng, Y.L., Kawamitsu, Y.,Uehara, N., Matsuda, N., Tanaka, Y.,Ishikawa, H., Baba, S., Takabe, T., Wada,K., Ishii, T., and Takabe, T. (2001) PlantMol. Biol., 45, 353–363.

83 Guo,Y.,Zhang,L.,Xiao,G.,andChen,S.Y.(1997) Sci. China (SeriesC), 27, 151–155.

84 Guo, B.H., Zhang, Y.M., Li, H.J., Du,L.Q., Li, Y.X., Zhang, J.S., Chen, S.Y., andZhu, Z.Q. (2000) Acta Bot. Sin., 42,279–283.

85 Chen, C.F., Li, Y.W., Chen, Y., Bai, J.R.,Li, H., Zhu, Y.F., Chen, S.Y., and Jia, X.(2004) Yi Chuan Xue Bao,31, 97–101.

86 Yilmaz, J.L. and B€ulow, L. (2002)Biotechnol. Prog., 18, 1176–1182.

87 Delauney, A.J. and Verma, D.P.S. (1993)Plant J., 4, 215–223.

88 Cherian, S. and Reddy, M.P. (2003) Biol.Plant., 46, 193–198.

89 Slama,I.,Ghnaya,T.,Messedi,D.,Hessini,K., Labidi, N., Savoure, A., and Abdelly, C.(2007) J. Plant Res., 120, 291–299.

90 Vernon, D.M. and Bohnert, H.J. (1992)Plant Physiol., 99, 1695–1698.

91 LeRudulier, D. and Bouillard, L. (1983)Appl. Environ. Microbiol., 46, 152–159.

92 Sengupta, S., Patra, B., Ray, S., andMajumder, A.L. (2008) Plant CellEnviron., 31, 1442–1459.

93 Majee, M., Maitra, S., Dastidar, K.G.,Pattnaik, S., Chatterjee, A., Hait, N.C.,Das, K.P., and Majumder, A.L. (2004) J.Biol. Chem., 279, 28539–28552.

94 Ghosh-Dastidar, K., Maitra, S., Goswami,L., Roy, D., Das, K.P., andMajumder, A.L.(2006) Plant Physiol., 140, 1279–1296.

95 Ishitani, M., Majumder, A.L.,Bornhouser, A., Michalowski, C.B.,Jensen, R.G., and Bohnert, H.J. (1996)Plant J., 9, 537–548.

96 Liu, L., White, M.J., and MacRae, T.H.(1999) Eur. J. Biochem., 262, 247–257.

97 Ganesan, G., Sankararamasubramanian,H.M., Jithesh, M.N., Sivaprakash, K.R.,and Parida, A.K. (2008) Plant Physiol.Biochem., 46, 928–934.

98 Sakamoto, H., Araki, T., Meshi, T., andIwabuchi, M. (2000) Gene, 248, 23–32.

99 Xu, S.M.,Wang, X.C., andChen, J. (2007)Plant Cell Rep., 26, 497–506.

100 Shen, Y.G., Yan, D.Q., Zhang, W.K., Du,B.X., Zhang, J.S., Liu, Q., and Chen, S.Y.(2003) Acta Bot. Sin., 45, 82–87.

101 Ben Saad, R., Zouari, N., Ben Ramdhan,W., Azaza, J., Meynard, D., Guiderdoni,E., and Hassairi, A. (2010) Plant Mol.Biol., 72, 171–190.

102 Flowers, T.J. and Yeo, A.R. (1995) Aust. J.Plant Physiol., 22, 875–884.

103 Bohnert, H.J. and Jensen, R.G. (1996)Aust. J. Plant Physiol., 23, 661–666.

References j433