understanding and improving salt tolerance in plan

Understanding and Improving Salt Tolerance in Plants

Viswanathan Chinnusamy,Andre ´Jagendorf,and Jian-Kang Zhu*ABSTRACT

even when ECe is Ͻ3.0dS m Ϫ1(Table 1),which in terms of osmotic potential is less than –0.117MPa (osmotic One-fifth of irrigated agriculture is adversely affected by soil salin-potential ϭϪ0.39ϫECe).At these salinity levels,the ity.Hence,developing salt-tolerant crops is essential for sustaining

predominant cause of crop susceptibility appears to be food production.Progress in breeding for salt-tolerant crops has been hampered by the lack of understanding of the molecular basis of salt ion toxicity rather than osmotic stress.Ion cytotoxicity tolerance and lack of availability of genes that confer salt tolerance.is caused by replacement of K ϩby Na ϩin biochemical Genetic evidence suggests that perception of salt stress leads to a reactions and conformational changes and loss of func-cytosolic calcium-signal that activates the calcium sensor protein tion of proteins as Na ϩand Cl Ϫions penetrate the hydra-SOS3.SOS3binds to and activates a ser/thr protein kinase SOS2.tion shells and interfere with noncovalent interactions The activated SOS2kinase regulates activities of SOS1,a plasma between their amino acids.Metabolic imbalances caused membrane Na ؉/H ؉antiporter,and NHX1,a tonoplast Na ؉/H ؉anti-by ionic toxicity,osmotic stress,and nutritional defi-porter.This results in Na ؉efflux and vacuolar compartmentation.ciency under salinity may also lead to oxidative stress A putative osmosensory histidine kinase (AtHK1)-MAPK cascade (Zhu,2002).Hence,engineering crops that are resistant probably regulates osmotic homeostasis and ROS scavenging.Os-to salinity stress is critical for sustaining food production motic stress and ABA (abscisic acid)-mediated regulation of LEA (late-embryogenesis-abundant)-type proteins also play important and achieving future food security.Understanding the roles in plant salt tolerance.Genetic engineering of ion transporters molecular basis of salt-stress signaling and tolerance and their regulators,and of the CBF (C-repeat-binding factor)regu-mechanisms is essential for breeding and genetic engi-lons,holds promise for future development of salt-tolerant crops.

neering of salt tolerance in crop plants.Here,we discuss the molecular basis of cellular ion homeostasis,osmotic homeostasis,stress damage control and repair under S

alinity is one of the major abiotic stresses that ad-salt stress,and their exploitation for genetic engineering versely affect crop productivity and quality.About of salt-tolerant crop plants.

20%of irrigated agricultural land is adversely affected by salinity (Flowers and Yeo,1995).The problem of soil Sensors of Salt Stress

salinity is further increasing because of the use of poor Plants sense salt stress through both ionic (Na ϩ)and quality water for irrigation and poor drainage.In clay osmotic stress signals.Excess Na ϩcan be sensed either soils,improper management of salinity may lead to soil on the surface of the plasma membrane by a transmem-sodicity whereby sodium binds to negatively charged clay brane protein or within the cell by membrane proteins causing clay swelling and dispersal that makes the soil or Na ϩsensitive enzymes (Zhu,2003).In addition to less fit for crop growth.According to the USDA salinity its role as an antiporter,the plasma membrane Na ϩ/H ϩlaboratory,saline soil can be defined as soil having an antiporter SOS1(S alt O verly S ensitive 1),having 10to electrical conductivity of the saturated paste extract (EC e )12transmembrane domains and a long cytoplasmic tail,of 4dS m Ϫ1(4dS m Ϫ1≈40m M NaCl)or more.Most may act as a Na ϩsensor (Zhu,2003).This dual role would grain crops and vegetables are glycophytes and are highly be analogous to the sugar permease BglF in Escherichia susceptible to soil salinity even when the soil EC e is Ͻ4coli and the yeast ammonium transporter Mep2p.When dS m Ϫ1.Different threshold tolerance EC e and different expressed in Xenopus laevis oocytes Na ϩ–K ϩcotrans-rate of reduction in yield beyond threshold tolerance porters from Eucalyptus camaldulensis Dehnh.show in-EC e indicate variation in mechanisms of salt tolerance creased ion uptake under hypoosmotic conditions while,among crop species (Table 1).

their Arabidopsis homolog do not show this osmosen-Soil type and environmental factors,such as vapor sing capacity (Liu et al.,2001).Entry of Na ϩthrough pressure deficit,radiation,and temperature may further nonspecific ion channels under salinity may cause mem-alter salt tolerance.Adverse effects of salinity on plant brane depolarization that activates Ca 2ϩchannels (Sand-growth may be due to ion cytotoxicity (mainly due to

ers et al.,1999),and thus generates Ca 2ϩoscillations,Na ϩ,Cl Ϫ,and SO Ϫ

4),and osmotic stress (reviewed by and signals salt stress.Cell volume decreases because Zhu,2002).Most crop plants are susceptible to salinity

of turgor loss under salinity-induced hyperosmotic stress may lead to retraction of the plasma membrane from Viswanathan Chinnusamy,Water Technology Centre,Indian Agricul-the cell wall,which is probably sensed by both stretch-tural Research Institute,New Delhi,India;Andre

´Jagendorf,Depart-activated channels and transmembrane protein kinases,ment of Plant Biology,Cornell University,Ithaca,NY14853;Jian-such as two component histidine kinases and wall-asso-Kang Zhu,Institute for Integrative Genome Biology and Department ciated kinases (Urao et al.,1999;Kreps et al.,2002;Seki of Botany and Plant Sciences,University of California,Riverside,Cali-fornia 92521.Received 3Dec.2003.Symposia.*Corresponding author et al.,2002).Salinity up-regulates the biosynthesis of (jian-kang.zhu@ucr.edu).

the plant stress hormone ABA (Jia et al.,2002;Xiong and Zhu,2003),and causes accumulation of reactive Published in Crop Sci.45:437–448(2005).oxygen species (ROS)(Smirnoff,1993;Hernandez et al.,©Crop Science Society of America

677S.Segoe Rd.,Madison,WI 53711USA

2001).ABA and ROS also regulate ionic and osmotic 437

Published online January 31, 2005

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Table 1.Many important crops are susceptible to soil salinity†(Maas,1990).

Crop

Threshold salinity

Decrease in yield dS m Ϫ1

Slope %per dS m Ϫ1

Bean (Phaseolus vulgaris L.)

1.019.0Eggplant (Solanum melongena L.) 1.1 6.9Onion (Allium cepa L.)

1.216.0Pepper (Capsicum annuum L.) 1.514.0Corn (Zea mays L.)

1.71

2.0Sugarcane (Saccharum officinarum L.) 1.7 5.9Potato (Solanum tuberosum L.)

1.71

2.0Cabbage (Brassica oleracea var.capitata L.) 1..7Tomato (Lycopersicon esculentum Mill.) 2.59.9Rice,paddy (Oryza sativa L.)

3.012.0Peanut (Arachis hypogaea L.) 3.229.0Soybean [Glycine max (L.)Merr.] 5.020.0Wheat (Triticum aestivum L.) 6.07.1Sugar beet (Beta vulgaris L.)7.0 5.9Cotton (Gossypium hirsutum L.)7.7 5.2Barley (Hordeum vulgare L.)

8.0

5.0

†Lack of a direct correlation between the threshold salinity and yield decrease per unit increase in salinity may be attributed to the differences in salt exclusion,uptake,compartmentation and other mechanisms of salt tolerance among these crop species.

homeostasis as well as stress damage control and re-1997).Silica deposition and polymerization of silicate in the endodermis and rhizodermis blocks Na ϩinflux pair processes.

Regulation of K ϩuptake and/or prevention of Na ϩthrough the apoplastic pathway in roots of rice (Yeo et al.,1999).Restriction of sodium influx either into the entry,efflux of Na ϩfrom the cell,and utilization of Na ϩfor osmotic adjustment are strategies commonly used root cells or into the xylem stream is one way of main-taining the optimum cytosolic K ϩ/Na ϩratio of plants by plants to maintain desirable K ϩ/Na ϩratios in the cytosol.Osmotic homeostasis is established either by Na ϩunder salt stress.The hkt1mutation suppresses the salt hypersensitivity and K ϩ-deficient phenotype of the Ara-compartmentation into the vacuole or by biosynthesis and accumulation of compatible solutes.ROS detoxifi-bidopsis S alt O verly S ensitive 3(sos3)mutant (Rus et al.,2001a).Antisense expression of wheat HKT1in cation systems as well as stress proteins belonging to the LEA protein family contribute to prevention of salt-transgenic wheat causes significantly less 22Na uptake and enhances growth under salinity when compared stress damage (Zhu,2002).In addition to these mecha-nisms,Na ϩsecretion is a strategy used by some halo-with control plants (Laurie et al.,2002).These results suggest that either inactivation of the low affinity Na ϩphytic plants.Thus,precise regulation of ion transport systems is critical for salt tolerance.Important insights transporter (HKT)activity or suppression of its expres-sion can considerably improve plant salt tolerance.into ion homeostasis under salt stress have emerged from the molecular genetic analysis of s alt o verly s ensi-In saline conditions,cellular potassium levels can be maintained by activity or expression of potassium-spe-tive (sos )mutants of Arabidopsis (Fig.1;Zhu,2003).

cific transporters.In Mesembryanthemum crystallinum L.,high affinity K ϩtransporter–K ϩuptake genes are Sodium Influx and K ؉/Na ؉Balance

up-regulated under NaCl stress (Su et al.,2002).In yeast,A high K ϩ/Na ϩratio in the cytosol is essential for HAL1and HAL3regulate K ϩuptake and Na ϩefflux.normal cellular functions of plants.Na ϩcompetes with Overexpression of the Arabidopsis HAL3a gene en-K ϩuptake through Na ϩ–K ϩcotransporters,and may hances salt tolerance of transgenic Arabidopsis (Es-also block the K ϩ-specific transporters of root cells un-pinosa-Ruiz et al.,1999).Similarly,transgenic tomato der salinity (Zhu,2003).This results in toxic levels of plants overexpressing yeast HAL1gene show a higher sodium as well as insufficient K ϩconcentration for enzy-K ϩ/Na ϩratio and improved salt tolerance than control matic reactions and osmotic adjustment.Under salinity,plants.Transgenic tomato plants exhibit lower reduction sodium gains entry into root cell cytosol through cation in fruit yield than that of control plants when irrigated channels or transporters (selective and nonselective)or with 35m M NaCl (Rus et al.,2001b).Signaling events into the root xylem stream via an apoplastic pathway that regulate the potassium-specific transporters under depending on the plant species.In Arabidopsis (Uozumi salinity should be understood.

et al.,2000),Eucalyptus (Liu et al.,2001),and wheat,it has been shown that high affinity K ϩtransporters Sodium Efflux

(HKT)act as low affinity Na ϩtransporters (Rubio et al.,1995;Gorham et al.,1997)under salinity.The HKT Sodium efflux from root cells prevents accumulation of toxic levels of Na ϩin the cytosol and transport of transporters of Eucalyptus camaldulensis are more per-meable to Na ϩthan they are to K ϩwhen extracellular Na ϩto the shoot.Molecular genetic analysis in Arabi-dopsis sos mutants have led to the identification of a concentrations of Na ϩand K ϩare equal (Liu et al.,2001).Hence,under salinity HKT homologs may con-plasma membrane Na ϩ/H ϩantiporter,SOS1,which plays a crucial role in sodium extrusion from root epider-tribute to Na ϩinflux.However,in rice,sodium influx into the xylem through the apoplastic pathway appears mal cells under salinity.The SOS1transcript level is up-regulated under salt stress.The sos1mutant plants show

to be more significant (Yadav et al.,1996;Garcia et al.,

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Fig.1.SOS signaling pathway for ion homeostasis under salt stress in Arabidopsis .Salt stress elicited Ca 2؉signals are perceived by SOS3,which activates the protein kinase SOS2.Activated SOS2phosphorylates SOS1,a plasma membrane Na ؉/H ؉antiporter,which then transports Na ؉out of the cytosol.The transcript level of SOS1is regulated by the SOS3-SOS2kinase complex.SOS2also activates the tonoplast Na ؉/H ؉antiporter that sequesters Na ؉into the vacuole.Na ؉entry into the cytosol through the Na ؉transporter HKT1may also be restricted by SOS2.ABI1regulates the gene expression of NHX1,while ABI2interacts with SOS2and negatively regulates ion homeostasis either by inhibiting SOS2kinase activity or the activities of SOS2targets.Double arrow indicates SOS3-independent and SOS2-dependent pathway.

hypersensitivity to salt stress (100m M NaCl),and accu-ing EF hands and an N-myristoylation motif (Liu and Zhu,1998;Ishitani et al.,2000).Mutations that disrupt mulate more Na ϩin shoots than wild-type plants.So-dium efflux by SOS1is also vital for salt tolerance of either calcium binding (sos3-1)or myristoylation (G2A)of SOS3cause salt-stress hypersensitivity in Arabidopsis meristem cells such as growing root-tips and shoot apex as these cells do not have large vacuoles for sodium plants (Ishitani et al.,2000).The SOS3gene product transduces a salt stress-elicited calcium signal by activat-compartmentation (Shi et al.,2000&2002).Isolated plasma membrane vesicles from sos1mutants show ing SOS2,a ser/thr protein kinase with an N-terminal kinase catalytic domain that is similar to that of yeast significantly less inherent as well as salt stress-induced Na ϩ/H ϩantiporter activity than vesicles from wild-type SNF1and animal AMP-activated kinase,and a unique C-terminal regulatory domain.The C-terminal regula-plants (Qiu et al.,2002).The expression of SOS1is ubiquitous,but stronger in epidermal cells surrounding tory domain of SOS2consists of an autoinhibitory FISL motif (Liu et al.,2000),deletion of which results in the root-tip,as well as parenchyma cells bordering the xylem.Thus,SOS1functions as a Na ϩ/H ϩantiporter constitutive activation of SOS2(Guo et al.,2001).Under salt stress,SOS3binds to the FISL motif of SOS2and on the plasma membrane and plays a crucial role in sodium efflux from root cells and the long distance Na ϩactivates its substrate phosphorylation (protein kinase)activity (Halfter et al.,2000).Activated SOS2then phos-transport from root to shoot (Shi et al.,2002).Indeed,transgenic Arabidopsis plants overexpressing SOS1phorylates SOS1,and results in activation of antiporter activity of SOS1.The Na ϩ–H ϩexchange activity of iso-have lower Na ϩin the xylem transpirational stream and in shoots compared with wild-type plants.These plants lated plasma membranes vesicles from sos3and sos2mutants is significantly less than that of wild-type plants.also show enhanced salt tolerance,measured in terms of their growth,ability to bolt and flower at increasing Consistent with this finding,these mutants also accumu-late higher levels of Na ϩ,similar to those accumulated concentrations of salt stress (50–200m M NaCl);while,control plants became necrotic and have failed to bolt by the sos1mutant (Quintero et al.,2002).Overexpres-sion of an active form of SOS2could overcome the salt (Shi et al.,2003).

Sodium efflux through SOS1under salinity is regu-hypersensitivity of sos2and sos3mutants and enhanced the salt tolerance of transgenic Arabidopsis (Guo et al.,lated by SOS3–SOS2kinase complex (Fig.1).In Arabi-dopsis ,salt-stress induced calcium signatures are sensed 2004).The SOS1up-regulation under salt stress is also impaired in sos2and sos3mutants.Hence,the SOS3–

by SOS3,a Ca 2ϩsensor protein with three calcium bind-

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SOS2signaling pathway positively regulate salt-stress proteins such as SOS3,SCaBP1(SOS3-like calcium-binding proteins 1),SCaBP3,SCaBP5,and SCaBP6.induced SOS1gene expression and/or transcript stabil-ity as well as SOS1transporter activity (Shi et al.,2003).One of these SCaBPs may signal SOS2to regulate the tonoplast Na ϩ/H ϩ-exchange activity (Fig.1;Qiu et al.,In addition to increasing cytosolic calcium,salt-stress induced ABA accumulation also appears to regulate 2003).

Transgenic Arabidopsis plants overexpressing AtNHX1the SOS pathway through the ABA insensitive 2(ABI2)protein phosphatase 2C.ABI2interacts with the protein have showed significantly higher salt (200m M NaCl)tolerance than wild-type plants (Apse et al.,1999).Since phosphatase interaction (PPI)motif of SOS2.This inter-action is abolished by the abi2-1mutation,which en-tomato is a highly salt-sensitive crop (Table 1),an effort has been made to improve its salt tolerance by overex-hances tolerance of seedlings to salt shock (150m M NaCl)and causes ABA insensitivity.Hence,the wild-pressing AtNHX1.These tomato transgenics grow and produce fruits in the presence of very high salt concen-type ABI2may negatively regulate salt tolerance either by inactivating SOS2,or the SOS2regulated Na ϩ/H ϩtrations (200m M NaCl).Yield and fruit quality of trans-genic tomato plants under salinity are equivalent to antiporters such as SOS1or NHX1(Fig.1;Ohta et al.,2003).

those of control plants grown under nonstress conditions (Zhang and Blumwald,2001).Similar results have been reported for transgenic canola (Brassica napus L.)over-Sodium Compartmentation

expressing AtNHX1(Zhang et al.,2001).

A positive turgor is indispensable for expansion growth of cells and stomatal openings in plants.A de-Compatible Osmolytes

crease in water potential due to soil salinity causes os-motic stress that leads to turgor loss.Plants have evolved Although use of ions for osmotic adjustment may be energetically more favorable than biosynthesis of organic an osmotic adjustment (active solute accumulation)mechanism that maintains water uptake and turgor un-osmolyte under osmotic stresses,many plants accumulate organic osmolytes to tolerate osmotic stresses.These os-der osmotic stress conditions.For osmotic adjustment,plants use inorganic ions such as Na ϩand K ϩand/or molytes include proline,betaine,polyols,sugar alcohols,and soluble sugars.Glycine betaine and trehalose act as synthesize organic compatible solutes such as proline,betaine,polyols,and soluble sugars.Vacuolar seques-osmoprotectants by stabilizing quaternary structures of proteins and highly ordered states of membranes.Manni-tration of Na ϩis an important and cost-effective strategy for osmotic adjustment that also reduces the Na ϩcon-tol serves as a free-radical scavenger.Proline serves as a storage sink for carbon and nitrogen and a free-radical centration in the cytosol.Na ϩsequestration into the vacu-ole depends on expression and activity of Na ϩ/H ϩanti-scavenger.It also stabilizes subcellular structures (mem-branes and proteins),and buffers cellular redox potential porters as well as on V-type H ϩ-ATPase and H ϩ-PPase.These phosphatases generate the necessary proton gra-under stress.Hence,these organic osmolytes are known as osmoprotectants (Bohnert and Jensen,1996;Chen and dient required for activity of Na ϩ/H ϩantiporters.

Overexpression of AVP1,a vacuolar H ϩ-pyrophos-Murata,2000).Genes involved in osmoprotectant biosyn-thesis are up-regulated under salt stress,and concentra-phatase in Arabidopsis enhanced sequestration of Na ϩinto the vacuole and maintained higher relative water tions of accumulated osmoprotectants correlate with os-motic stress tolerance (Zhu,2002).Analysis of the content in leaves.These plants also show higher salt-and drought-stress tolerance than that of wild type Arabidopsis t365mutant supports the involvement of os-moprotectants in salt tolerance.The t365mutant is im-(Gaxiola et al.,2001).The tonoplast Na ϩ/H ϩantiporter NHX1gene is induced by both salinity and ABA in paired in the S -adenosyl-L-methionine phosphoethano-lamine N -methyltransferase (PEAMT )gene.The PEAMT Arabidopsis (Shi and Zhu,2002)and rice (Fukuda et al.,1999).The AtNHX1promoter contains putative ABA enzyme catalyzes conversion of phosphoethanolamine to phosphocholine,which is a precursor of glycinebetaine responsive elements (ABRE)between –736and –728from the initiation codon.AtNHX1expression under biosynthesis (Mou et al.,2002).

Salt tolerance of transgenic tobacco engineered to salt stress is partially dependent on ABA biosynthesis and ABA signaling through ABI1.Salt-stress induced over-accumulate mannitol was first demonstrated by Tarczynski et al.(1993).Genetically engineered over-up-regulation of AtNHX1expression is lower in ABA deficient mutants (aba2-1and aba3-1)and in the ABA production of compatible osmolytes in transgenic plants such as Arabidopsis ,rice,wheat,and Brassica has also insensitive mutant,abi1-1(Shi and Zhu,2002).Compar-ing tonoplast Na ϩ/H ϩ-exchange activity (mainly due to been shown to enhance stress tolerance as measured by germination,seedling growth,survival,recovery,pho-AtNHX1)between wild type and mutants (sos1,sos2,and sos3)shows that SOS2also regulates the tonoplast tosystem II yield,and seed production under very high salt and osmotic stresses.The observed salt tolerance exchange.The impaired tonoplast Na ϩ/H ϩ-exchange activity in vitro from isolated sos2tonoplasts could be was attributed to the osmoprotectant effect of compati-ble osmolytes rather than their contribution to osmotic restored to levels in wild type by adding activated SOS2protein.Since the tonoplast Na ϩ/H ϩ-exchange activity adjustment (Table 2).It is interesting to note that gly-cine betaine-(Kishitani et al.,2000)and trehalose-(Garg is not affected in the sos3mutant,the tonoplast Na ϩ/H ϩ-exchange activity is not regulated by SOS3.SOS2et al.,2002)overproducing transgenic rice plants accu-mulated fewer Na ϩions,and maintained K ϩuptake,

has been found to interact with plant calcium sensor

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Table 2.Salt-stress tolerance of transgenic plants over-producing compatible osmolytes.

Gene and source

Transgenic plants

Stress tolerant traits

Reference Mannitol

E.coli mt1D (mannitol-1-phosphate tobacco fresh weight,plant height and flowering under

Tarczynski et al.,1993dehydrogenase)salinity stress

E.coli mt1D Arabidopsis germination at 400m M NaCl Thomas et al.,1995E.coli mt1D tobacco salt-stress tolerance;mannitol contributed only to

Karakas et al.,199730-40%of the osmotic adjustment

E.coli mt1D

wheat (Triticum aestivum L.)only 8%biomass reduction when compared to

Abebe et al.,2003

56%reduction in control plants in 150m M NaCl stress

D-Ononitol

IMT1(myo-inositol O -methyl trans-tobacco drought and salinity stress

Sheveleva et al.,1997

ferase)of common ice plant Sorbitol

Stpd1(sorbitol-6-phosphate dehy-Japanese persimmon tolerance in Fv/Fm ratio under NaCl stress Gao et al.,2001

drogenase)of apple,driven by CaMV 35S promoter

Glycine betaine

Arthrobacter globiformis CodA Arabidopsis germination at 300m M NaCl;seedling growth Hayashi et al.,1997(choline oxidase)

at 200m M NaCl;retention of PSII activity at 400m M NaCl

A.globiformis CodA targeted to the rice

faster recovery after 150m M NaCl stress Sakamoto et al.,1998;chloroplasts or cytosol Mohanty et al.,2002A.globiformis CodA

Brassica juncea (L.)Czernj.germination in 100–150m M NaCl;seedling Prasad et al.,2000growth in 200m M NaCl

E.coli choline dehydrogenase (betA )tobacco

biomass production of greenhouse grown plants Holmstrom et al.,2000

and betaine aldehyde dehydroge-under salt stress;faster recovery from photo nase (betB )genes

inhibition under high light,salt stress and cold stresses

Atriplex hortensis betaine aldehyde wheat (Triticum aestivum L.)seedling growth in 0.7%(ϭ120m M )NaCl Guo et al.,2000dehydrogenase (BADH )gene under maize ubiquitin promoter Barley peroxisomal BADH gene

rice

stability in chlorophyll fluorescence under Kishitani et al.,2000

100m M NaCl stress;accumulates less Na ؉and Cl Ϫions but maintained K ؉uptake Proline

Vigna aconitifolia L.P5CS (⌬1]-tobacco root growth;flower development

Kishor et al.,1995pyrroline-5-carboxylate synthe-tase)gene

Vigna aconitifolia L.P5CS gene rice faster recovery after a short period of salt stress Zhu et al,1998under barley HVA22promoter Mutated gene of Vigna aconitifolia L.tobacco

improved seedlings tolerance and low free radical Hong et al,2000

P5CS which encode P5CS enzyme levels at 200m M NaCl

that lacks end product (proline)inhibition

Antisense proline dehydrogenase Arabidopsis tolerant to high salinity (600m M NaCl);constitu-Nanjo et al.,1999

gene

tive freezing tolerance (Ϫ7؇C)

Trehalose

E.coli otsA (Trehalose-6-phosphate rice root and shoot growth at 4wk of 100m M NaCl Garg et al.,2002

synthase)and otsB (Trehalose-stress;survival under prolonged salt stress;6-phosphate phosphatase)bi-func-maintenance of high K ؉/Na ؉ratio;Low Na ؉tional fusion gene (TPSP )under accumulation in the shoot;maintained high the control of ABA responsive PSII activity and soluble sugar levels

promoter or Rubisco small subunit (rbcS )promoter

E.coli TPSP under maize ubiquitin rice better seedling growth and PSII yield under salt,Jang et al.,2003

promoter

drought and cold stresses

Thus,these plants retained optimal K ϩ/Na ϩratios nec-component hybrid histidine kinase,ATHK1,from Ara-bidopsis is implicated in osmosensing under salt stress essary for cellular functions.Whether ion homeostasis in these transgenics was either due to direct regulation based on induced expression and ability to complement the yeast double mutant lacking both osmosensors of ion transporters or to maintenance of cellular integ-rity by protecting membranes and proteins from oxida-(sln1⌬sho1⌬).By analogy to SLN1of yeast,the Arabi-dopsis ATHK1is also probably active at low osmolarity.tive damage was not known and needs to be determined.Although enhanced synthesis and accumulation of Active ATHK1may inactivate a response regulator by phosphorylation.Inactivation of ATHK1under high compatible solutes under osmotic stresses are well docu-mented,little is known about the signaling cascades that osmolarity may result in the accumulation of nonphos-phorylated active form of the response regulator,which regulate the compatible solute biosynthesis in higher plants.A signaling cascade similar to that of the yeast then stimulates osmolyte biosynthesis in plants by acti-vating a MAPK pathway(s)(Urao et al.,1999).Tran-Mitogen Activated Protein Kinase-High Osmotic Glyc-erol 1(MAPK-HOG1)pathway may regulate osmolyte scriptome analyses also show induction of receptor-like kinase genes in Arabidopsis under salt stress (Kreps

biosynthesis (Zhu,2002).A putative osmosensory two-

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et al.,2002;Seki et al.,2002).However,genetic and by both ABA-dependent and independent signaling molecular evidences to support the role of these proteins pathways (Fig.2).Promoters of LEA-like genes contain in osmotic stress sensing and compatible osmolyte bio-dehydration responsive elements/C-Repeat (DRE/CRT),synthesis are lacking.

ABA-responsive elements (ABREs),and/or MYB/MYC ABA may also regulate osmolyte biosynthesis in recognition elements.The DRE/CRT elements regulate plants under salt stress.Osmotic stress-induced ABA gene expression in response to dehydration (salt,drought,accumulation has been shown to regulate the P5CS gene and cold stresses);while,ABRE and MYB/MYC ele-involved in proline biosynthesis (Xiong et al.,2001a).ments control gene expression in response to ABA un-Proline induces the expression of salt-stress responsive der abiotic stresses (Thomashow,1999;Shinozaki and genes,which have proline responsive elements (PRE ,Yamaguchi-Shinozaki,2000).

ACTCAT)in their promoters (Satoh et al.,2002;Oono Genetic analysis of ABA-deficient Arabidopsis mu-et al.,2003).Better understanding of the salt-stress sig-tants,los5and los6,has revealed that ABA is necessary naling pathway that regulates compatible osmolyte bio-for the salt-stress induced expression of some Arabi-synthesis will help to devise better breeding and genetic dopsis LEA genes (Xiong et al.,2001a;Xiong et al.,2002).engineering strategies.

Ca 2ϩand/or H 2O 2act as second messengers of ABA induced stomatal closure and gene expression under LEA-Type Proteins

abiotic stresses (Leung and Giraudat,1998;Schroeder et al.,2001).Transient expression analysis has revealed Osmotic stresses induce late-embryogenesis-abun-that IP 3and cADPR-gated calcium channels are in-dant (LEA)proteins in vegetative tissues,which impart volved in ABA induced Ca 2ϩconcentration changes,dehydration tolerance to vegetative tissues of plants.and these Ca 2ϩtransients regulate expression of LEA-These LEA-type proteins are encoded by RD (respon-type genes,such as RD29A and KIN2(Wu et al.,1997).sive to dehydration),ERD (early responsive to dehydra-Genetic evidence from the fry1(fiery 1)mutant,defec-tion),KIN (cold inducible),COR (cold regulated),and tive in inositol polyphosphate 1-phosphatase,has dem-RAB (responsive to ABA)genes in different plant spe-onstrated that IP 3metabolism is critical for ABA and cies (Shinozaki and Yamaguchi-Shinozaki,2000;Zhu,abiotic stress signaling (Xiong et al.,2001b).Salt-stress/2002).Accumulation levels of these proteins correlate ABA induced Ca 2ϩsignals are at least partially transduced with stress tolerance in various plant species suggesting through calcium-dependent protein kinases (CDPKs).protective roles under osmotic stress.Transgenic rice Transient expression analyses in maize protoplasts have plants engineered to overexpress a barley LEA gene,shown that an increase in cytosolic Ca 2ϩconcentration HVA1,under control of the rice actin 1promoter exhibit activates CDPKs,which in turn induce the stress respon-better stress tolerance under 200m M NaCl and drought sive HVA1promoter.Moreover,expression of CDPKs stress than wild-type plants (Xu et al.,1996).Expression of LEA-type genes under osmotic stresses is regulated

is under the negative control of ABI1protein

phospha-

Fig.2.LEA-type gene transcription under abiotic stresses in Arabidopsis .ABA-independent DREB2and ABA-dependent CBF4transcription factors transactivate DRE/CRT cis-elements in the promoters of LEA type genes.ABA-dependent pathways regulate LEA type genes through MYC/MYB and bZIP type transcription factors.ABA-dependent signaling is mediated through IP3and Ca 2؉.FRY1negatively regulates IP3levels.ABA induced Ca 2؉signaling is negatively regulated by ABI1/2protein phosphatase 2C.Low temperature stress activates ICE1a myc-like bHLH transcription factor,which binds to myc type cis-elements of CBF3promoter and induces CBF3expression.CBFs bind to the CRT/DRE cis-elements on the promoter of LEA-type genes and induce expression of these genes.

R e p r o d u c e d f r o m C r o p S c i e n c e . P u b l i s h e d b y C r o p S c i e n c e S o c i e t y o f A m e r i c a . A l l c o p y r i g h t s r e s e r v e d .

tase 2C (Sheen,1996).Overexpressing OsCDPK7in vation of DRE/CRT cis elements leading to expression of LEA -type genes (Jaglo-Ottosen et al.,1998;Liu et al.,transgenic rice enhances induction of a LEA-type gene (RAB16A )and salt/drought tolerance;while,transgenic 1998;Kasuga et al.,1999;Jaglo et al.,2001;Haake et al.,2002).The CBF pathway for expression of LEA-type suppression of OsCDPK7causes hypersensitivity to salt/drought stress (Saijo et al.,2000).Signaling compo-genes is conserved across plant species such as Arabi-dopsis ,wheat,Brassica napus (Jaglo et al.,2001),tomato nents regulating CDPK-activated gene expression are yet to be defined.

(Hsieh et al.,2002a,2002b),barley,and rice (Dubouzet et al.,2003).Similar to Arabidopsis DREB2,rice Os-ABA-dependent expression of LEA-type genes un-der osmotic stress is regulated by basic Leucine-Zipper DREB2A is also induced by dehydration and salt stress (Dubouzet et al.,2003).Recently,we have identified and MYB/MYC type transcription factors that recog-nize ABRE (Uno et al.,2000)and MYB/MYC recogni-ICE1(Inducer of CBF Expression 1),a MYC-type basic helix-loop-helix transcription factor,as an upstream regu-tion sequences (Abe et al.,2003),respectively (Fig.2).Arabidopsis bZIP transcription factors,ABREB1(ABA-lator of these DREB/CBF transcription factors under cold stress (Chinnusamy et al.,2003).Upstream transcrip-responsive element binding protein 1ϭABF2)and ABREB2(ϭABF4)genes,are up-regulated by drought,tion factors that regulate the expression of DREB2/CBF4transcription factors under osmotic stresses (salt or dehy-NaCl,and ABA.The induction of the RD29B pro-moter –GUS by ABREB1and ABREB2in Arabidopsis dration)have yet to be identified.

Constitutive overexpression of CBFs strongly acti-leaf protoplasts under osmotic stress is repressed in aba2and abi1mutants but is enhanced in an era1mutant.vated expression of several LEA-type genes,enhancing freezing and osmotic stress tolerance of transgenic Ara-ABA is necessary for the activation of ABREB1and ABREB2(Uno et al.,2000).Constitutive overexpres-bidopsis (Jaglo-Ottosen et al.,1998;Liu et al.,1998;Kasuga et al.,1999)and Brassica napus (Jaglo et al.,sion of ABF3and ABREB2(ϭABF4)in Arabidopsis enhance expression levels of target LEA-type genes 2001),and chilling and drought tolerance of tomato (Hsieh et al.,2002a,2002b).However,constitutive over-(RAB18and RD29B ).These transgenic plants are hy-persensitive to ABA,sugar,and salt stress during germi-expression of CBF s resulted in severe growth retarda-tion and reduction in seed production,even under a nation but are drought tolerant at the seedling stage (Kang et al.,2002).

normal environment (Liu et al.,1998).Transgenic Ara-bidopsis overexpressing CBF3under the transcriptional Salt stress-inducible basic-helix-loop-helix type tran-scription factors as well as AtMYC2(ϭRD22BP1)and control of the stress responsive RD29A promoter showed no growth retardation when compared to con-AtMYB2regulate ABA-responsive gene expression in Arabidopsis .Constitutive overexpression of AtMYC2trol plants.These transgenic Arabidopsis plants showed constitutive low-levels of expression of LEA genes and and AtMYB2results in constitutive expression of RD22and AtADH ,and expression levels are further increased enhanced expression under cold,dehydration,and salt stresses.Both the RD29A::CBF3and CaMV35S::CBF3following ABA treatment.These transgenic plants are hypersensitive to ABA during germination.In contrast,transgenic plants showed enhanced tolerance to freez-ing,drought,and salt stresses,but tolerance levels of atmyc2mutation decreases RD22and AtADH expres-sion.Transgenic Arabidopsis plants overexpressing At-RD29A::CBF3transgenics were significantly higher than those of CaMV35S::CBF3transgenic plants.Re-MYC2and AtMYB2show higher osmotic stress toler-ance as measured by electrolyte leakage from cells (Abe covery and survival of seedlings after soaking in 600m M NaCl solution for 2h was 79and 18%for RD29A::CBF3et al.,2003),although their salt-stress tolerance is not known.ABA signaling via ABFs and MYC/MYB and transgenic and control plants,respectively (Kasuga et al.,1999).Transgenic wheat plants expressing RD29A::their targets must be investigated to better understand sensitivity during germination and tolerance during the CBF3also showed enhanced osmotic stress tolerance (Pellegrineschi et al.,2002).In addition to enhanced vegetative growth in transgenic plants.

ABA-independent regulation of LEA-type genes is expression of LEA-type genes,multiple abiotic stress tolerance of CBF -overexpressing transgenic plants might mediated by transcription factors that activate DRE/CRT cis -elements of LEA-type protein encoding genes.also be in part due to accumulation of compatible osmo-lytes (Gilmour et al.,2000)and enhanced oxidative stress These transcription factors are called either C-repeat Binding Proteins (CBF s)or Dehydration Responsive tolerance (Hsieh et al.,2002a,2002b).It was not clear how osmolyte biosynthesis and antioxidant defense pathways Element Binding Proteins (DREB s).Arabidopsis DREB s are classified into two classes,DREB1(DREB1A ϭwere activated in CBF -overexpressing plants.Genome-wide expression analysis showed that CBF overexpression CBF3,DREB1B ϭCBF1,and DREB1C ϭCBF2),and DREB2(DREB2A and DREB2B ).Expression of CBF1,also induced transcription factors such as AP2domain proteins (RAP2.1and RAP2.6),a putative zinc finger pro-CBF2,and CBF3is induced by low temperature stress;while,expression of DREB2A and DREB2B is induced tein and R2R3-MYB73(Fowler and Thomashow,2002),that may regulate osmolyte biosynthesis and antioxidant by dehydration and salt stresses (Liu et al.,1998;Thom-ashow,1999;Shinozaki and Yamaguchi-Shinozaki,2000;defense genes.Hence,genetic engineering of CBFs and potentially other transcription factors under stress spe-Fig.2).Recently,a DREB1homolog of Arabidopsis CBF4has been cloned.CBF4shows ABA-dependent cific promoters in crops appears to be a viable approach for engineering tolerance to multiple stresses,including expression under drought stress (Haake et al.,2002).Ov-erexpression of CBF (CBF1-4)genes has resulted in acti-salt stress.

R e p r o d u c e d f r o m C r o p S c i e n c e . P u b l i s h e d b y C r o p S c i e n c e S o c i e t y o f A m e r i c a . A l l c o p y r i g h t s r e s e r v e d .

Tobacco-stress-induced-gene 1(Tsi1)encoding a DNA-MEKK1;Ichimura et al.,1998),mitogen activated protein kinase kinase (AtMKK2;Teige et al.,2004),and MAPKs binding protein with an EREBP/AP2DNA binding mo-tif is rapidly induced by salt stress but not by drought (ATMPK3,ATMPK4and ATMPK6;Mizoguchi et al.,1996;Ichimura et al.,2000).Activated AtMEKK1has or ABA.Overexpression of TSI1in tobacco enhanced retention of chlorophyll content when leaves were been shown to activate AtMPK4and AtMPK6in vitro and in vivo (Huang et al.,2000;Teige et al.,2004).The floated on 400m M NaCl solution for 48or 72h (Park et al.,2001),although the targets of TSI1are not known.

MAPK phosphatase 1(mkp1)mutant of Arabidopsis is more resistant to salinity stress.A yeast two-hybrid screen showed that MKP1could interact with AtMPK4Oxidative Stress Management

(Ulm et al.,2002).Thus,the salt-stress regulated MAPK Abiotic stresses including salt-stress induce accumula-cascade consisting of AtMEKK1,AtMEK1/AtMKK2,tion of ROS that are detrimental to cells at high concen-and AtMPK4is negatively regulated by MKP1.

trations because they cause oxidative damage to mem-Salt-stress induced ROS signaling in Arabidopsis may brane lipids,proteins,and nucleic acids (Smirnoff,1993;also be transduced by ANP1(a MAPKKK),AtMPK3,Gomez et al.,1999;Hernandez et al.,2001).Plants em-and AtMPK6along with its positive regulator Nucleo-ploy antioxidants (e.g.,ascorbate,glutathione,␣-tocoph-side Diphosphate Kinase 2(AtNDPK2)(Kovtun et al.,erol,and carotenoids)and detoxifying enzymes,such as 2000;Moon et al.,2003).Transgenic tobacco plants ov-superoxide dismutase,catalase,and enzymes of ascor-erexpressing a constitutively active tobacco NPK1(or-bate-glutathione cycle to combat oxidative stress.The tholog of ANP1)show enhanced tolerance to salinity activity and expression levels of the genes encoding and other abiotic stresses (Kovtun et al.,2000).AtNDPK2detoxifying enzymes are probably enhanced by ROS interacts with and activates both ATMPK3and ATMPK6.under abiotic stresses.Transgenic plants overexpressing Transgenic Arabidopsis overexpressing AtNDPK2ac-ROS scavenging enzymes,such as superoxide dismutase cumulate lower levels of ROS and show enhanced toler-(reviewed by Alscher et al.,2002),ascorbate peroxidase ance to salinity and other abiotic stresses (Moon et al.,(Wang et al.,1999),and glutathione S -transferase/gluta-2003).In rice,gene expression as well as kinase activity thione peroxidase (Roxas et al.,1997,2000)showed of rice MAPK (OsMAPK5)is regulated by ABA,by increased tolerance to osmotic,temperature,and oxida-biotic,and abiotic stresses including salt,drought,tive stresses.Overexpression of the tobacco NtGST/wounding,and cold (Xiong and Yang,2003).Thus,di-GPX gene (encoding a bifunctional enzyme with both verse abiotic stress signals converge at MAPK cascades glutathione S -transferase and glutathione peroxidase that appear to regulate antioxidant defense under abi-activity)in transgenic tobacco plants has improved salt-otic stresses in plants.

and chilling-stress tolerance because of enhanced ROS Transgenic overexpression of NPK1in tobacco (Kov-scavenging and prevention of membrane damage (Roxas tun et al.,2000),NDPK2in Arabidopsis (Moon et al.,et al.,1997,2000).

2003),and OsMAPK5in rice (Xiong and Yang,2003),The Arabidopsis pst1(photoautotrophic salt tolerance increased tolerance to several abiotic stresses,including 1)mutant is more tolerant to salt stress than the wild salt stress,probably by enhancing ROS detoxification.type.The observed salt tolerance was attributed to Constitutively active NPK1activated a MAPK cascade higher activities of superoxide dismutase and ascorbate that activates promoters of stress-responsive genes,such peroxidase than those in wild-type Arabidopsis (Tsu-as Glutathione-S -transferase (GST6)and HSP18.2but gane et al.,1999).Thus,ROS detoxification is an impor-not RD29A (Kovtun et al.,2000).Promoters of genes tant part of salt-stress tolerance.

encoding ROS detoxifying enzymes contain antioxidant Salt stress (Gomez et al.,1999;Hernandez et al.,2001)responsive elements (ARE),ABA responsive elements and ABA (Guan et al.,2000;Pei et al.,2000)induce (ABRE),heat shock elements (HSE),and redox-regu-enhanced production of H 2O 2,which may also act as a lated transcription factors:nuclear factor kappa-B (NFkB)second messenger at sublethal concentrations to regu-and the activator protein-1(AP-1)recognition cis-ele-lateantioxidantdefensegenesunderabioticstresses.ABA-ments (Vranova

´et al.,2002).However,transacting fac-dependent ROS production is catalyzed by NADPH tors and their regulators need to be identified.

oxidase as revealed with analysis of the atrbohD/F dou-ble mutant of Arabidopsis ,which is impaired in ABA-Genetic Engineering of Salt-Tolerant Crops

induced ROS production (Kwak et al.,2003).ABA-elic-ited H 2O 2production is negatively regulated by the ABI2Transgenic approaches by manipulation of ion homeo-stasis,osmoprotectant accumulation,LEA-type proteins,protein in guard cells (Murata et al.,2001).

Potential sensors of ROS may include redox sensitive and ROS scavenging capacity have demonstrated the capabilities of engineering salt-tolerant crops.Although receptors-like kinases and two component histidine ki-nases that likely activate a mitogen-activated protein ki-abiotic stress tolerance is known to be governed by multiple genes,significant increases in salt tolerance can nase (MAPK)module.Salt stress triggers activation and enhanced gene expression of a MAPK signaling cascade,be achieved by single gene manipulations as revealed by SOS1-(Shi et al.,2003)and NHX1-(Apse et al.,1999;some components of which are common for both salt and ROS (for review,Chinnusamy and Zhu,2003).Salt Zhang and Blumwald,2001;Zhang et al.,2001)overex-pressing transgenic plants.These transgenics are capa-stress rapidly (within 5–10min)activates Arabidopsis mitogen activated protein kinase kinase kinase (AT-

ble of growing and producing flowers at salt concentra-

R e p r o d u c e d f r o m C r o p S c i e n c e . P u b l i s h e d b y C r o p S c i e n c e S o c i e t y o f A m e r i c a . A l l c o p y r i g h t s r e s e r v e d .

tions of up to 200m M NaCl (20dS/m),which is lethal control of a regulatory gene.Transgenic manipulation of a single regulatory gene may be sufficient to regulate a to wild-type plants.Most crop plants are susceptible to this concentration of salinity (Table 1).In addition,gene cluster.Genetic and transgenic analyses have clearly demonstrated that manipulation of upstream transcrip-these transgenics do not exhibit any obvious growth abnormalities or changes in the quality of the consum-tion factor or signaling genes can lead to the activation of multiple target tolerance effector genes,and thus sig-able product,similar to results with NHX1overexpress-ing transgenic tomato and Brassica plants.Hence ge-nificantly improves abiotic stress tolerance.

netic engineering for ion homeostasis by tissue specific overexpression of SOS1,NHX1,and their positive regu-Conclusions and Prospects

lator,the active form of SOS2,will help in significant Only a few facets of myriad salt stress-tolerant traits improvement in salt tolerance.

found in nature have been unraveled today by applica-Transgenic analysis of osmolyte over-production has tion of molecular tools such as gene disruption and shown that osmoprotectants can protect plants against transgenic approaches.The SOS pathway regulates ion short term and high intensity salt stress (Table 2),but homeostasis under salt stress.An unknown salt-stress stress tolerance must be evaluated for the entire life sensor induces cytosolic calcium signals,which are trans-period of plants.Polyol over-accumulating transgenic duced by the SOS3–SOS2kinase complex.Activated plants show growth abnormalities,including sterility SOS2kinase regulates sodium efflux and sequestration (Karakas et al.,1997;Sheveleva et al.,1998).Further,of sodium into the vacuole by activating Na ϩ/H ϩanti-compartmentation of these osmoprotectants may also porters of plasma membrane and tonoplast,respec-be required for enhanced tolerance.For example,rice tively.Osmotic homeostasis and stress damage control transgenic plants overexpressing choline oxidase tar-appear to be regulated by salt stress-induced ABA,ROS,geted to chloroplasts show better tolerance to photo-a putative osmosensory histidine kinase (AtHK1),and inhibition under salt-and low-temperature stresses than MAPK cascades.However,these signaling pathways are plants overexpressing choline oxidase targeted to the not yet understood in terms of their components and cytosol (Sakamoto et al.,1998).Hence,the level of targets.It appears possible to engineer salt-tolerant expression of transgene,substrate requirement,meta-crops by manipulating Na ϩ/H ϩantiporters (plasma bolic flux,and cellular compartmentation of osmopro-membrane and tonoplast)and the CBF transcriptome tectants should be considered for engineering osmopro-in the near future.Exploitation of other signaling com-tectant accumulation.Overexpression of antioxidant ponents,osmolyte over-production,and antioxidant de-systems has been shown to protect transgenic plants fense requires further consideration.In the future,pyra-from abiotic stresses;however,in some cases,transgenic miding regulatory genes controlling various aspects of plants did not show enhanced stress tolerance.Pyramid-salt tolerance (i.e.,ionic and osmotic homeostasis,and ing of chloroplastic and mitochondrial Mn-superoxide damage control)in a single transgenic plant is expected dismutases in alfalfa (Medicago sativa L.)resulted in to yield very high levels of tolerance to salt and other lower biomass production as compared with the trans-related stresses.Most of the transgenic plants discussed genic plants expressing either one of the Mn-superoxide here are model plants,and stress tolerance has been dismutases (Samis et al.,2002).Engineering for antioxi-evaluated under controlled growing conditions for short dant systems may alter the pool size of ROS,involved durations.As the rate of transpiration is one of the in developmental and stress signaling,and hence their major determinants of the concentration of salt accumu-possible effects warrant careful examination.

lation in shoots,salt tolerance must be evaluated in the Overexpression of signaling components and tran-field conditions.The effects of stresses in relation to scription factors lead to expression of their target tran-plant ontogeny should be assessed at realistic stress lev-scriptome,which consists of multiple genes contributing els and under various combinations that naturally occur to stress adaptation.Overexpression of CBF transcrip-in the field.

tion factors from constitutive or stress-inducible pro-moters has been shown to confer enhanced tolerance in ACKNOWLEDGMENTS

the seedling stage to multiple abiotic stresses.However,Research in our laboratory is supported by grants from the constitutive overexpression has led to growth abnormal-U.S.National Institutes of Health,the U.S.National Science ities.Hence,overexpression of CBF transcription fac-Foundation,and the U.S.Department of Agriculture.

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