Evolution of halophytes multiple origins of salt tolerancein land plants

Timothy J FlowersABE Hanaa K GalalAC and Lindell BromhamD

ABiology and Environmental Science School of Life Sciences John Maynard Smith BuildingUniversity of Sussex Falmer Brighton BN1 9QG UK

BSchool of Plant Biology Faculty of Natural and Agricultural Sciences University of Western Australia35 Stirling Highway Crawley WA 6009 Australia

CFaculty of Science Assiut University Assiut 71516 EgyptDCentre for Macroevolution and Macroecology Research School of Biology Australian National UniversityCanberra ACT 0200 Australia

ECorresponding author Email tjflowerssussexacuk

This paper is part of an ongoing series lsquoThe Evolution of Plant Functionsrsquo

Abstract The evolution of salt tolerance is interesting for several reasons First since salt-tolerant plants (halophytes)employ several differentmechanisms todealwith salt the evolutionof salt tolerance represents a fascinating case study in theevolutionof a complex trait Second thediversityofmechanismsemployedbyhalophytes basedonprocesses common toallplants sheds light on the way that a plantrsquos physiology can become adapted to deal with extreme conditions Third as theamount of salt-affected land increases around the globe understanding the origins of the diversity of halophytes shouldprovide abasis for theuseof novel species inbioremediationandconservation In this reviewwepose thequestion howmanytimes has salt tolerance evolved since the emergence of the land plants some 450ndash470million years agoWe summarise thephysiological mechanisms underlying salt-tolerance and provide an overview of the number and diversity of salt-tolerantterrestrial angiosperms (defined as plants that survive to complete their life cycle in at least 200mM salt) We consider theevolution of halophytes using information from fossils and phylogenies Finally we discuss the potential for halophytes tocontribute to agriculture and land management and ask why when there are naturally occurring halophytes it is provingto be difficult to breed salt-tolerant crops

Additional keywords phylogeny saline agriculture salinity

The freshwater origin of land plants

Water relations

All organisms are faced with balancing their water relations withthose of their environment Marine photosynthetic organismsachieve this balance at the water potential of seawater aboutndash23MPa (Harvey 1966) using a mixture of ions and organiccompounds (Bisson and Kirst 1995 Iwamoto and Shiraiwa2005) Terrestrial plants must also adjust their water potentialto be at least as low as that of the soil in which they are growingFor plants living in saline environments the Na+ concentrationcan range from ~100 to 2380mMNa (Flowers 1985) equivalentto a water potential range from ndash05 to ndash11MPa This means thatplants growing in saline soils must adjust osmotically to waterpotentials that are commonly in the range ndash2 to ndash3 MPa they dothis as do marine plants by accumulating a mixture of ions andorganic solutes (eg Flowers et al 1977) For all terrestrial plantshoweverwhether living ina salineornon-saline environment themajor factor affecting their water relations is the large differencein concentration ofwater vapourbetween their stubstomatal poresand the atmosphere ndash the driving force for themovement of water

through the soilndashplantndashatmosphere continuum (even at 90 RHthe atmospheric water potential is ndash142MPa) The consequentflux of water through the plant carries essential nutrients to theshoots but in a saline environment anydissolved solutes from thesoil that enter the transpiration streamare also carried to the leaveswhere they must be accommodated re-circulated or excreted

The first land plants

Before the evolution of embryophytes (terrestrial plants that arenot algae viz bryophytes pteridophytes and spermatophytes)green plants were composed of the Chlorophyta andStreptophyta which split ~1000million years ago (Becker andMarin 2009) Palaeontological and molecular evidence suggestsembryophytes arose from the Streptophyta rather than theChlorophyta from charophycean green algae (Graham 1993Kenrick andCrane1997RavenandEdwards2001Waters 2003)in theOrdovician period some 450million years ago This date issupported by molecular data (Sanderson 2003) although thetransition to terrestrial habitats happened on several occasions(Lewis and McCourt 2004) While there is some uncertainty

CSIRO PUBLISHING Review

wwwpublishcsiroaujournalsfpb Functional Plant Biology 2010 37 604ndash612

CSIRO 2010 101071FP09269 1445-440810070604

concerning the origins of the embryophytes with support for theCharales Coleochaetales and the Zygnematales (Becker andMarin 2009) being sister to the embryophytes recent evidencefavours the Charales (Becker and Marin 2009)

The first records of land plants are microfossils consisting ofspores and plant parts which appeared in the mid-Ordovician(~470million years ago) they are found through to the EarlySilurian period (Wellman and Gray 2000) The earliest clearlyrecognisable land plants date from ~40million years later in theEarly Late Silurian (Wellman and Gray 2000) in which plantssuch asCooksonia can be seen in the fossil record (Graham1993)The origin of these early embryophytes is crucial to the origin ofsalt tolerance in land plants as it could have evolved from speciesthat lived in fresh or salt water by the time the first transition ofplants to a terrestrial environment occurred the seas were saline(Railsback et al 1990) even if a little less so (26ndash29 g per 1000 gseawater) than todayrsquos oceans (30ndash40 g per 1000 g seawater)

Did terrestrial plants derive from charophycean green algaeliving in fresh or salt water SinceCharacean algae occupied bothfreshwater and marine habitats in the mid-Paleozoic (Kenrickand Crane 1997) early embryophytes could have arisen frommarine or freshwater species Any marine organism making thetransition to the land on the edge of a saline pool would haveto cope not only with water loss from its cells to the atmospherebut alsowith hyper-saline conditionswhichwouldhaveoccurredas water evaporated from the pool If however the first landplants evolved at the edges of freshwater pools (Raven andEdwards 2001) then land plants would have arisen from anorganism (or organisms) that had adapted to living in fresh water

A particularly important adaptation to freshwater life is thedevelopment of systems for acquiring nutrients from the lowconcentrations of minerals present in fresh water rather thanthe high concentrations of the nutrient rich (eutrophic) marineenvironment (Rodriguez-Navarro and Rubio 2006) This abilitywould be equally important for growth on land where plantsneed to acquire nutrients in a nutrient poor (oligotrophic)environment Any such adaptation to oligotrophy might haveleft intact those systems required for osmotic adjustment andwhich are necessary for growth in seawater or in fluctuatingsalinity If embryophytes originated from freshwater algae thenas suggested by Becker andMarin (2009) the streptophytes werelsquophysiologically pre-adapted to terrestrial existence by theirprimary freshwater lifestyle in contrast to their marine sisterlineage ie Chlorophytarsquo (perhaps because their adaptationto an oligotrophic lifestyle resulted in evolution of specificmechanisms for dealing with fluctuating ion concentration inthe environment see also Ion compartmentation below)Analysis of fossils from Rhynie in Aberdeenshire in Scotlandsuggests that by the Early Devonian ~400million years agoplants were drought and probably salt tolerant (Channing andEdwards 2009) The question then is how many times salttolerance has evolved since the emergence of the land plantssome 450ndash470million years ago

Physiology of salt tolerance

Although there are many aspects of the physiology of salttolerance that are yet to be understood it is clear that the traitis complex in that at a minimum it requires the combination of

several different traits the accumulation and compartmentationof ions for osmotic adjustment the synthesis of compatiblesolutes the ability to accumulate essential nutrients(particularly K) in the presence of high concentrations of theions generating salinity (Na) the ability to limit the entry ofthese saline ions into the transpiration stream and the ability tocontinue to regulate transpiration in the presence of highconcentrations of Na+ and Clndash (Flowers and Colmer 2008)

Commonality of traits

All plants not just halophytes have the main characteristicsnecessary for salt tolerance ndash the ability to acquire ions thatare then compartmentalised in vacuoles (fundamental to turgorgeneration in plants and algae) and discrimination in favour ofK over Na Given that the physiological foundations of salttolerance are found in all plants it is not surprising that plantspecies show a continuum of salt tolerance with glycophytessuch as chickpea (Flowers et al 2010) at one extreme (wherejust 25mMNaCl can be toxic) and halophytes (which can toleratesalt concentrations as high as 500ndash1000mM) at the otherFurthermore salt tolerance is often part of a multi-facetedsyndrome that may include for example tolerance of flooding(Colmer and Voesenek 2009) and drought Indeed physiologicalresponses to salinity are often similar to responses to otherenvironmental stresses (Munns and Tester 2008) and may relyon common stress-tolerance pathways (Tuteja 2007)

Ion compartmentation

Most commonly the ions dominating saline environments arethose of Na and Cl as they predominate in seawaters halophytesuse these ions for most of their osmotic adjustment (Yeo 1983)It is clear however that in the process of ion accumulation forosmotic adjustment some ions can lsquoleakrsquo into the transpirationstream (Yeo et al 1987) and that such leaks must be minimisedif the aerial parts are not to be swamped by ions (Munns 2005)Consequently tight regulation of ion transport from roots toshoots is vital for halophytes Sufficient Na and Cl must beaccumulated to achieve osmotic adjustment but excess must beavoided as both ions are toxic at the concentrations requiredfor osmotic adjustment In practice Na+ and Clndash are largelycompartmentalised in vacuoles in halophytes (Flowers et al1977 1986) A range of metabolically inert organiccompounds is also present and utilised to adjust the osmoticpotential of the cytoplasm (Flowers and Colmer 2008) In somespecies excess ions are secreted throughglands that have evolvedon the surface of the aerial partsRecirculation of ions fromshootsback to the roots is unlikely to be an important aspect of salttolerance as the ions would have to be carried in the symplasm ofthe phloem where they would be toxic (Flowers et al 1986Munns and Tester 2008) Furthermore phloem transport deliverssolutes to growing regions where ions would cause metabolicdamage unless re-exported to the environment The latter wouldnot remove ions from the rhizosphere So ions reaching theleaves must be accommodated in vacuoles in the leaf cells aprocess that involves the transport of ions across both plasma andvacuolar membranes

Landplants includingmosses and ferns regulate their internalNa+ concentrations through the control of influx and efflux

Evolution of halophytes Functional Plant Biology 605

Within the eukaryotes Na+ efflux is mediated by Na+H+

antiporters and Na+-pump ATPases of which there are twotypes the Na+K+-ATPases of animal cells and the fungalNa+-ATPases Higher plants do not appear to express Na+-ATPases (Garciadeblas et al 2001) meaning that they mustrely on Na+H+ antiporters to transport Na+ out of theircytoplasm whether it be into the apoplast or the vacuolesBenito and Rodriguez-Navarro (2003) argued that the absenceof the Na+-ATPases in higher plants was evidence for theirevolution in non-saline oligotrophic environments where Na+

concentrations were low and there was no need for the system(s)than mediated extensive Na+ efflux This would presumablyhave occurred during the early development of land organismsin non-saline environments (Horodyski and Knauth 1994) orthrough the loss of such a system(s) following the migrationof marine organisms to freshwater Interestingly the mossPhyscomitrella patens (Bryopsida) expresses two ATPasessimilar to fungal ENA-Na+-ATPases (Benito and Rodriguez-Navarro 2003 Lunde et al 2007) The presence of the Na+-ATPases in amoss suggests these enzymeswere lost during earlyevolution of land plants (Benito and Rodriguez-Navarro 2003)which then rely on the Na+H+ antiporters (also present inP patens Benito and Rodriguez-Navarro 2003) to regulatecytosolic Na+ The question of how plants living in seawaterachieve this function remains The Na+H+ antiporters in plantsare electroneutral (Munns and Tester 2008) which means theywould not facilitate Na+ efflux at the alkaline pH values ofseawater (Benito and Rodriguez-Navarro 2003) Howeverseagrasses do presumably efflux Na+ but how their Na+H+

antiporters function in this respect is unclear (Garciadeblaacuteset al 2007 Touchette 2007) The same question can be posedfor halophytes although it is possible that the rhizosphere pHcould be lower than that of seawater the operation of Na-effluxthrough antiporters has been recently questioned (Britto andKronzucker 2009)

KNa selectivity

The selectivity of halophytes for K over Na varies betweenfamilies of flowering plants (Flowers et al 1986) Netselectivity (net SK Na) calculated as the ratio of Kconcentration in the plant to that in the medium divided by theratio ofNa concentration in the plant to that in themedium rangesbetween average values of 9 and 60 (Flowers and Colmer 2008)with an overall mean of 19 it is only in the Poales that net SK Na

values of the order of 60 are foundWithin the monocots (Fig 1)there are three orders with halophytes but no data are availablefor the net SK Na values of species within the Arecales In theAlismatales the average net SK Na (across just three species) is16 (range 10 to 22) suggesting that high selectivity has evolvedonly in the Poales (for halophytes within this order averageselectivities of are 58 in the Juncaginaceae (two species) and 60in the Poaceae (nine species)) There is too little data to analysethe net SK Na values within the dicots but the average value is11 compared with 60 in the Poales (Flowers and Colmer 2008)

Salt glands

Glandular structures are not uncommon on plants they cansecrete a range of organic compounds (Wagner 1991

Wagner et al 2004) However the ability to secrete saltappears to have evolved less frequently than salt toleranceSalt glands epidermal appendages of one to a few cells thatsecrete salt to the exterior of a plant (Thomson et al 1988) havebeendescribed in just a feworders offloweringplantsndash the Poales(eg in Aeluropus littoralis and Chloris gayana) Myrtales (egthe mangrove Laguncularia racemosa) Caryophyllales (egMesembryanthemum crystallinum and the saltbush Atriplexhalimus) Lamiales (eg the mangroves Avicennia marina andAvicennia germinans) and the Solanales (eg Cressa cretica)Their distribution across the orders of flowering plants suggestsat least three origins although there may have been moreindependent origins within orders (see for example within theCaryophyllales Fig 2b) Whether salt glands evolved from

Fig 1 The distribution of halophytes in the main tree by Stevens (2001)based on species listed by Aronson (1989) that survive at least 200mMNaClThose orders containing halophytes are boxed in red and the white circleindicates some species within the order have salt glands Indicates a nodewith rather weak support according to Stevens (2001)

606 Functional Plant Biology T J Flowers et al

glands that originally performed some other function is unclearbut it is difficult at least in the Poaceae to get glandular hairs onnon-halophytes (such as Zea mays L) to secrete salt (Ramadanand Flowers 2004)

Compatible solutes

The compatible solutes synthesised by halophytes range fromquaternary ammonium compounds through sulfonium analoguesto proline and sugar alcoholsmost arewidely distributed throughthe orders of flowering plants reflecting both phylogeny andfunctional needs (Flowers and Colmer 2008) There is no clearpattern of particular solutes being exclusive to particular ordersother than of sorbitol to the Plantaginaceae so that it appearsthat although the ability to synthesise and compartmentalise acompatible solute is a requirement in halophytes the nature ofthat solute depends on the habitat and the availability of NCompatible solutes comprise a small proportion of the totalsolutes accumulated by halophytes (Yeo 1983)

How many halophytes are there

As a trait salt tolerance has been recognised in plants for morethan 200 years (Flowers et al 1986) and there have been severaldefinitions of what constitutes a lsquohalophytersquo Angiospermspecies show a wide range of levels of salt tolerance frominhibition of growth at low salt concentrations (eg sensitivechickpea genotypes dying in 25mM NaCl Flowers et al 2010)to tolerance of salt concentrations as high or higher thanseawater (eg the halophyte Arthrocnemum macrostachyumsurvives in up to 1000mM NaCl Khan et al 2005) Anydefinition of a lsquohalophytersquo must include a threshold value ofsalt concentration and the number of halophytes clearly dependson the salt concentration used in that definition By setting adividing line between halophytes and glycophytes (salt sensitiveplants) at a salt concentration of ~80mM NaCl (a conductivityin the soil solution of 78 dS mndash1) Aronson (1989) listed ~1550salt-tolerant plants Using the same definition Menzel and Lieth(2003) recorded over 2600 species These comprehensive lists

(a) (b)

Fig 2 The distribution of halophytes in phylogenetic trees by Stevens (2001) based on species listed byAronson (1989) (a) Asterales and (b) CaryophyllalesThose orders containing halophytes are boxed in red and the white circle indicates some species within the order have salt glands For (a) denotes brancheswith 100 posterior probability and curren denotes branches with 70ndash80 jackknife support all other branches have gt80 support (according to Stevens 2001)In (b) denotes nodeswith 50ndash80bootstrap support unmarked branches havegt80support [1] and [3] have 50ndash80bootstrap support inmatK tree only and[2] is not recognised at all in matK tree only (according to Stevens 2001)

Evolution of halophytes Functional Plant Biology 607

define salt tolerant plants using a salt concentration that ismuch lower than that present in seawaters (an averageof ~480mM Na+ and 580mM Clndash) Since there are speciesthat have evolved tolerance to seawater salinities we havechosen to use a salt concentration of lsquo~200mM saltrsquo as ourdefining limit (as did Flowers and Colmer 2008) precisionis not required as salt concentrations in natural habitatsfluctuate with evaporation and precipitation of waterApplying the definition of lsquoplants that survive to completetheir life cycle in at least 200mM saltrsquo to Aronsonrsquos (1989)database generates a list of some 350 species which wecall lsquohalophytesrsquo (whether this group of the most salttolerant of plant species should be given a separate namesuch as lsquoeuhalophytersquo is an issue that could be furtherdebated) Our halophytes are not uniformly distributedacross the taxa of flowering plants listed by Stevens (2001)The orders with the greatest number of halophytes(Table 1) are the Caryophyllales (which includes thosespecies that were formerly in the Chenopodiaceae such asthe saltbushes in the genus Atriplex genera with reducedleaves such Salicornia and Tectomia and succulent membersof the genus Suaeda) and the Alismatales (which includesmarine flowering plants belonging to genera such as Zosteraand Thalassia) Although our list of halophytes is notcomprehensive as the limits of tolerance for many species arenot known our analysis suggests that halophytes are notmore than ~025 of the known species of angiosperms

Salt tolerance in bryophytes and pteridophytes

Halophytes are uncommon among the higher plants and thissituation is also true for bryophytes and pteridophytes There aresome mosses that occur in areas that are inundated by tides(Table 2) for example four species in Northern England(Adam 1976) and five species in Nova Scotia one of whichhas been shown to survive immersion in seawater for 24 h(Garbary et al 2008) Aronson (1989) lists only one genus ofa salt resistant fern This low frequency of salt tolerance inbryophytes and pteridophytes indicates that they also haverarely evolved the ability to tolerate salt concentrationsapproaching those of seawater although some are able to growin lower concentrations (eg Physcomitrella patens in 100mMNaCl see Lunde et al 2007)

Repeated evolution of salt-tolerance in angiosperms

In an earlier analysis based on the salt tolerance criterion usedby Aronson (1989) and a phylogeny constructed by Takhtajan(1969) Flowers et al (1977) concluded that salt tolerance waswidely distributed amongst the families of flowering plants andsuggested a polyphyletic origin something more recentlyestablished for other complex traits such as C4 photosynthesis(Sage 2004) and crassulacean acid metabolism (Crayn et al2004) In Fig 1 we have plotted the distribution of cladescontaining halophytes (as defined above) on an order-levelphylogeny of angiosperms based on the main tree published

Table 1 The distribution of halophytes amongst orders of flowering plantsThe estimates of species frequency are approximate due to the inexact numbers of specieswithin families the change in taxonomy that hasoccurred since the species were listed by Aronson (1989) and as the limits of tolerance for many species are not known Examples of

halophytes and suggestions for model species can be found in Flowers and Colmer (2008)

Order Number offamilies

Number offamilies withhalophytes

Families withhalophytes within

the order

Number ofhalophyticspecies

of all halophyticspecies (345)A

Caryophyllales 34 9 26 74 214Alismatales 14 7 50 61 177Malpighiales 40 4 10 35 101Poales 16 4 25 28 81Lamiales 25 5 20 24 70Myrtales 12 4 33 22 64Fabales 4 1 25 21 61Malvales 10 1 10 18 52Ericales 25 6 24 13 38Arecales 1 1 100 11 32Gentianales 5 4 80 9 26Sapindales 9 3 33 8 23Asterales 11 2 18 8 23Zygophyllales 2 1 50 3 09Apiales 7 1 14 3 09Magnoliales 6 1 17 2 06Solanales 5 1 20 2 06Celastrales 4 1 25 1 03Fagales 8 1 13 1 03Brassicales 18 1 6 1 03

Totals 256 58 ndash 345 100

A345 is the total number of halophytes included in this analysis

608 Functional Plant Biology T J Flowers et al

by Stevens (Stevens 2001) From this order-level tree we can seethat salt tolerance seems to have evolved multiple times

We cannot determine the exact number of origins of salttolerance from this phylogeny for several reasons Firstwithout information on the number of halophytes within eachorder and their relationships to each other we cannot knowwhether each family containing halophytes represents a singleorigin or multiple origins (see below) Second if a clade containsboth halophytes and non-halophytes we cannot be sure whethersalt tolerance was gained independently in multiple lineages orwhether the condition is ancestral to the clade and then lost insome families Third the evidence for the presence of halophytesin some orders is based on incomplete data (eg there are just twospecies reported as able to grow in seawater in the Magnoliales(Annonaceae) and without any physiological data) In mostorders that contain families with halophytes the percentage ofhalophytes is 1or less (Table 1)whichmakes it unlikely that thecondition is ancestral and the non-halophytes lost salt-toleranceTherefore it seems likely that virtually each of the red boxes inFig 1 represents at least one independent origin of salt tolerance

Many of the orders marked in Fig 1 may contain multipleorigins of halophytes For examplewithin theAsterales (Fig 2a)we have recorded eight halophytes in two families (seven in theAsteraceae and one in the Goodeniaceae) but the remainingfamilies do not have any halophytes It is parsimonious to assumethat at least two lineages within the Asterales evolved salttolerance independently Examining the presences of particulartraits associated with salt tolerance may also reveal someinteresting evolutionary patterns For example five of the ninesalt-tolerant lineages within the Caryophyllales use salt glands toexcrete excess salt from the leaves This illustrates how evenrelated lineages may develop different strategies for dealing withenvironmental salt The distribution of salt glands within thephylogeny (Fig 2b) suggests that even within this single clade offlowering plants a key salt tolerance mechanism has evolvedindependently several times

It also seems likely that the distribution of halophyte-containing families is not random over the phylogeny with

some clades containing more putative origins of salt tolerancethan others If true this would seem to offer support for the ideathat some clades are more likely to give rise to halophytes thanothers It may be profitable to look at the physiology of membersof those clades to see if they share certain characteristics thatmakeadapting to environmental salt easier than in other cladesMapping key components of salt tolerance on phylogeniesmay help to reveal whether some lineages are predisposed toevolving particular solutions to salinity whether some traits thatconfer salt tolerance aremore easily acquired than others or evenwhether the component traits of salt tolerance are usually acquiredin a particular order Looking at the patterns of origination withinfamilies will be important for answering the questions of whetherthere can be an ancestral condition that favours the evolutionof salt tolerance

Importance of halophytes to agricultureand land management

Salinity is an expanding problem More than 800million ha ofland is salt-affected which is over 6 of the worldrsquos land area(Flowers and Yeo 1995) Salt-affected land is increasingworldwide through vegetation clearance and irrigation both ofwhich raise the watertable bringing dissolved salts to the surfaceIt is estimated that up to half of irrigation schemes worldwideare affected by salinity (Flowers and Yeo 1995) Althoughirrigated land is a relatively small proportion of the total globalareaof foodproduction it produces a third of the food (Munns andTester 2008) Salt stress has been identified as one of the mostserious environmental factors limiting the productivity of cropplants (Flowers and Yeo 1995 Barkla et al 1999) with a hugeimpact on agricultural productivity The global annual cost ofsalt-affected land is likely to be well over US$12 billion (Qadiret al 2008) Future agricultural production will rely increasinglyon our ability to grow food and fibre plants in salt-affected land(Rozema and Flowers 2008 Qadir et al 2008)

Breeding for salt tolerance

Although it is abundantly clear that at some level salinity reducesthe growth of plants the reason is generally not clear Both Na+

and Clndash can reach toxic concentrations if not compartmentalisedin vacuoles but within the complexity of the whole plant theexact cause of any damage is generally unknown However theconsequences are that salinity reduces plant growth and enhancessenescence ofmature leaves resulting in a reduction in functionalleaf area in crop plants this decreases yield (Munns et al 2006Munns and Tester 2008) There is therefore a great incentive toincrease the salt tolerance of crop plants to allow greater yields insalt-affected soils However breeding of salt-tolerant crops ishampered by the complexity of salt tolerance as seen inhalophytes (see above) So despite a large amount of effortthat has been directed towards increasing the salt tolerance ofcrops ranging fromwide-crossing to transgenics few productivesalt-tolerant varieties have been produced (Flowers and Yeo1995 Flowers 2004 Flowers and Flowers 2005 Witcombeet al 2008) The realisation that halophytes have evolvedseveral times should encourage attempts to generate new salt-tolerant crops and may assist plant breeders in several waysFirst itmay illuminate specific traits associatedwith salt tolerance

Table 2 Salt tolerant mosses and ferns

Species names Location Reference

MossesPottia heimi

Amblystegium serpensEurhynchium praelongumCratoneuron filiciniumand C stellatumA

Northern England Adam (1976)

Campylium stellatumBryum capillareDidymodon rigidulusMnium hornumand Am serpens

Nova Scotia Garbary et al (2008)

FernsAcrostichum aureum

Ar danaeifolium andAr Speciosum

Tropics Aronson (1989)

ACratoneuron stellatumwas shown to survive immersion in seawater for 24 h(Garbary et al 2008)

Evolution of halophytes Functional Plant Biology 609

that may be selected in established crop plants (eg Huang et al2008) second it may lead to the domestication of naturally salttolerant species as crop plants

Halophytes as crops

Naturally salt-tolerant species are now being promoted inagriculture particularly to provide forage medicinal plantsaromatic plants (Qadir et al 2008) and for forestry (Marcarand Crawford 2004) For example Barrett-Lennard (2002)identified 26 salt-tolerant plant species capable of producingproducts (or services) of value to agriculture in AustraliaExamples of useful halophytes include the potential oil-seedcrops Kosteletzkya virginica (eg Ruan et al 2008) Salvadorapersica (eg Reddy et al 2008) Salicornia bigelovii (Glenn et al1991) and Batis maritima (Marcone 2003) fodder crops such asAtriplex spp (egEl Shaer 2003)Distichlis palmeri (egMasterset al 2007) and biofuels (Y Waisel pers comm Qadir et al2008) Growing salt-tolerant biofuel crops on marginalagricultural land would help to counter concerns that thebiofuel industry reduces the amount of land available forfood production (Qadir et al 2008) At the extreme plants thatcan grow productively at very high salt levels could be irrigatedwith brackish water or seawater (Rozema and Flowers 2008)Although plants that put resources (cf Yeo 1983) intodeveloping salt-tolerance mechanisms (eg the production ofcompatible solutes to maintain osmotic balance is an energeticcost) may do so at the expense of other functions manyhalophytes show optimal growth in saline conditions (Flowersand Colmer 2008) and salt marshes have high productivity(Colmer and Flowers 2008 Flowers and Colmer 2008) Thefact that dicolytedonous halophytes can grow at similar rates toglycophytes (see Flowers and Colmer 2008 for a more detaileddiscussion) suggests that salt tolerance per se will not limitproductivity Here the contrast with drought tolerance is starkwithout water plants do not grow but may survive with saltwater some plants can grow well

Apart from direct use as crops we may increasingly need torely on halophytes for revegetation and remediation of salt-affected land Over the last 200 years industrialisation inEurope and elsewhere has lead to an enormous increase ofproduction use and release of traces of heavy metals into theenvironment A large portion of these toxic materials includingCd Cu Pb and Zn accumulate in sediments including the soilsof tidal marshes Recent studies showed that some seagrassesand salt marsh plants are capable of extracting heavy metalsfrom sediments and accumulating them in belowground oraboveground tissues (Weis and Weis 2004 Cambrolle et al2008 Lewis and Devereux 2009) The processes and potentialapplication of these aquatic halophytes merits much greaterresearch and development Growing salt-tolerant plantsincluding species of Kochia Bassia Cynodon MedicagoPortulaca Sesbania and Brachiaria may also improve othersoil properties such as increasing water conductance orincreasing soil fertility (Qadir et al 2008) Halophytes mayalso lower the watertable thereby allowing growth of salt-sensitive species in salt-affected land (Barrett-Lennard 2002)

Finally conservation and land management in salt-affectedregions will benefit from improved knowledge of the salt

tolerance of local species Halophytes are being targeted forlsquoecosystem engineeringrsquo projects such as stabilising wetlandsto increase the success of revegetation programs (Fogel et al2004) Halophytes are also being used to develop sustainableagricultural practise for example using indigenous halophytesto restore overgrazed pasture and to reduce reliance on irrigation(Peacock et al 2003) Salt-tolerant species are increasinglybeing considered for landscape remediation For example wellplanned revegetation can reduceboth the in situ salt concentrationat mine sites and also reduce the movement of salts from themine sites in runoff (Carroll and Tucker 2000) However caremust be taken in introducing species for bioremediation as theymay become an environmental problem in their own right Forexample Kochia planted for forage and bioremediation inWestern Australia in 1990 soon spread and was declared aweed in 1992 (Qadir et al 2008)

The halophyte paradox

In this review we have shown that salt tolerance has evolvedmany different times in angiosperms and in different ways Wehave also noted that despite increasing salinisation of agriculturalland few salt-tolerant crops have been produced This raises animportant paradox if all plants contain the basic physiologicalmechanisms on which salt tolerance is based and if halophyteshave evolved in many different lineages using a variety ofadaptations thenwhy is it so difficult to breed salt-tolerant crops

The reasons for the slowprogress in changing the salt toleranceof plants were discussed by Flowers andYeo (1995) who arguedthat this was because salinity had not become a problem ofsufficient priority that the necessary resource was beingapplied to its solution Moreover they also concluded thattreating salinity resistance as a single character could accountfor the limited success We have already established that the salttolerance of halophytes is a multi-genic trait and the same is truefor non-halophytes (Flowers 2004) It is not our intention hereto rehearse the arguments over approaches to breeding forcomplex traits However it is clear that breeding for yield hasnot delivered many salt-resistant varieties of the commoncrops (Flowers 2004 Witcombe et al 2008) The probabilityof combining two complex traits ndash yield and salt tolerance ndash tomaximise yield under saline conditions is evidentially very lowAs a consequence Yeo et al (1990) advocated understandingthe physiology of tolerance and pooling a range of traits anapproach that had some success for rice (Dedolph and Hettel1997) A similar approach has been advocated for durum wheatcombining traits for regulating sodiumaccumulation and osmoticadjustment (Munns et al 2002 James et al 2006 2008) Thetraits that necessarily need to be pooled for wheat (and barley)includeNa lsquoexclusionrsquo KNa discrimination the retention of ionsin sheaths tissue tolerance ion partitioning into different leavesosmotic adjustment enhanced vigour water use efficiency andearly flowering (Colmer et al 2005) In addition other traits suchas tolerance to waterndashlogging (see Colmer and Flowers 2008 fora discussion of flooding tolerance in halophytes) are vitallyimportant further complicating any breeding program (Colmeret al 2005) Given the complexity of the task it is not surprisingthat the transfer of one or two genes has generally failed to changethe tolerance of transgenic plants what is surprising is that there

610 Functional Plant Biology T J Flowers et al

are instances where such simple gene transfers appear to havealtered yield (Flowers 2004)

Evolutionary studies of halophytes which reconstruct theorigin and development of salt tolerance in a variety of plantlineages may help us to understand why artificial breedinghas failed to produce robust and productive salt tolerant cropsSuch studies may also help us develop new salt-tolerant lines byrevealing the order of acquisition of components of salt toleranceor indicating favourable genetic backgrounds on which salttolerance may be developed By examining the repeatedevolution of this complex trait we may identify particular traitsor conditions that predispose species to evolve a complex multi-faceted trait such as salt tolerance and give rise to halophytelineages More generally this may shed light on the adaptationof angiosperm lineages to extreme environments In order toachieve these ends more information is required on at aminimum the effects of salt on the growth and ion relations ofa wider range of plant species that may prove to be halophytes

Acknowledgements

HKGwould like to than the Egyptian Government for financial support whileat the University of Sussex and our thanks also go to Jessica Thomas andKatherine Byron for assistance in earlier stages of this project

References

Adam P (1976) Occurrence of bryophytes on British salt marshes Journalof Bryology 9 265ndash274

Aronson JA (1989) lsquoHALOPHa data base of salt tolerant plants of the worldrsquo(Office of Arid Land Studies University of Arizona Tucson AZ)

Barkla BJ Vera Estrella R Pantoja O (1999) Towards the production ofsalt-tolerant crops Advances in Experimental Medicine and Biology464 77ndash89

Barrett-Lennard EG (2002) Restoration of saline land through revegetationAgricultural Water Management 53 213ndash226 doi101016S0378-3774(01)00166-4

Becker BMarinB (2009) Streptophyte algae and the origin of embryophytesAnnals of Botany 103 999ndash1004 doi101093aobmcp044

Benito B Rodriguez-Navarro A (2003) Molecular cloning andcharacterization of a sodium-pump ATPase of the moss Physcomitrellapatens The Plant Journal 36 382ndash389 doi101046j1365-313X200301883x

Bisson MA Kirst GO (1995) Osmotic acclimation and turgor pressureregulation in algae Naturwissenschaften 82 461ndash471 doi101007BF01131597

Britto DT Kronzucker HJ (2009) Ussingrsquos conundrum and the search fortransport mechanisms in plants New Phytologist 183 243ndash246doi101111j1469-8137200902872x

Cambrolle J Redondo-Gomez S Mateos-Naranjo E Figueroa ME (2008)Comparison of the role of two Spartina species in terms ofphytostabilization and bioaccumulation of metals in the estuarinesediment Marine Pollution Bulletin 56 2037ndash2042 doi101016jmarpolbul200808008

Carroll C Tucker A (2000) Effects of pasture cover on soil erosion andwater quality on central Queensland coal mine rehabilitation TropicalGrasslands 34 254ndash262

Channing A Edwards D (2009) Yellowstone hot spring environmentsand the paleo-ecophysiology of Rhynie chert plants towards asynthesis Plant Ecology amp Diversity 2 111ndash143 doi10108017550870903349359

Colmer TD Flowers TJ (2008) Flooding tolerance in halophytes NewPhytologist 179 964ndash974 doi101111j1469-8137200802483x

Colmer TD Voesenek L (2009) Flooding tolerance suites of plant traitsin variable environments Functional Plant Biology 36 665ndash681doi101071FP09144

Colmer TD Munns R Flowers TJ (2005) Improving salt tolerance of wheatand barley future prospects Australian Journal of ExperimentalAgriculture 45 1425ndash1443 doi101071EA04162

Crayn DM Winter K Smith JAC (2004) Multiple origins of crassulaceanacid metabolism and the epiphytic habit in the neotropical familyBromeliaceae Proceedings of the National Academy of Sciences ofthe United States of America 101 3703ndash3708 doi101073pnas0400366101

Dedolph C Hettel G (Eds) (1997) lsquoPartners making a differencersquo(International Rice Research Institute Manila)

El Shaer HM (2003) Potential of halophytes as animal fodder in EgyptIn lsquoTasks for vegetation science 38 cash crop halophytesrsquo (Eds H LiethM Mochtchenko) pp 111ndash119 (Kluwer Dordrecht the Netherlands)

Flowers TJ (1985) Physiology of halophytes Plant and Soil 89 41ndash56doi101007BF02182232

Flowers TJ (2004) Improving crop salt tolerance Journal of ExperimentalBotany 55 307ndash319 doi101093jxberh003

Flowers TJ Colmer TD (2008) Salinity tolerance in halophytes NewPhytologist 179 945ndash963 doi101111j1469-8137200802531x

Flowers TJ Flowers SA (2005) Why does salinity pose such a difficultproblem for plant breeders Agricultural Water Management 78 15ndash24doi101016jagwat200504015

Flowers TJ Yeo AR (1995) Breeding for salinity resistance in crop plants ndashwhere next Australian Journal of Plant Physiology 22 875ndash884doi101071PP9950875

Flowers TJ Troke PF Yeo AR (1977) The mechanism of salt tolerance inhalophytesAnnual Review of Plant Physiology 28 89ndash121 doi101146annurevpp28060177000513

Flowers TJ Hajibagheri MA Clipson NJW (1986) Halophytes TheQuarterly Review of Biology 61 313ndash337 doi101086415032

Flowers TJGaur PMLaxmipathiGowdaCLKrishnamurthy L Samineni SSiddique KHM Turner NC Vadez V Varshney RK Colmer TD (2010)Salt sensitivity in chickpea Plant Cell amp Environment 33 490ndash509doi101111j1365-3040200902051x

Fogel BN Crain CM Bertness MD (2004) Community level engineeringeffects of Triglochin maritima (seaside arrowgrass) in a salt marsh innorthern New England USA Journal of Ecology 92 589ndash597doi101111j0022-0477200400903x

Garbary DJ Miller AG Scrosati R Kim KY Schofield WB (2008)Distribution and salinity tolerance of intertidal mosses from NovaScotian salt marshes The Bryologist 111 282ndash291 doi1016390007-2745(2008)111[282DASTOI]20CO2

Garciadeblas B Benito B Rodriacuteguez-Navarro A (2001) Plant cells expressseveral stress calcium ATPases but apparently no sodium ATPase Plantand Soil 235 181ndash192 doi101023A1011949626191

Garciadeblaacutes B Haro R Benito B (2007) Cloning of two SOS1transporters from the seagrass Cymodocea nodosa SOS1transporters from Cymodocea and Arabidopsis mediate potassiumuptake in bacteria Plant Molecular Biology 63 479ndash490doi101007s11103-006-9102-2

Glenn EP Oleary JW Watson MC Thompson TL Kuehl RO (1991)Salicornia-bigelovii Torr ndash an oilseed halophyte for seawater irrigationScience 251 1065ndash1067 doi101126science25149971065

Graham LE (1993) lsquoOrigin of land plantsrsquo (John Wiley amp Sons New York)Harvey HW (1966) lsquoThe chemistry and fertility of sea watersrsquo (University

Press Cambridge)Horodyski RJ Knauth LP (1994) Life on land in the Precambrian Science

263 494ndash498 doi101126science2635146494HuangSSpielmeyerWLagudahESMunnsR (2008)Comparativemapping

of HKT genes in wheat barley and rice key determinants of Na+transport and salt tolerance Journal of Experimental Botany 59927ndash937 doi101093jxbern033

Evolution of halophytes Functional Plant Biology 611

concerning the origins of the embryophytes with support for theCharales Coleochaetales and the Zygnematales (Becker andMarin 2009) being sister to the embryophytes recent evidencefavours the Charales (Becker and Marin 2009)

The first records of land plants are microfossils consisting ofspores and plant parts which appeared in the mid-Ordovician(~470million years ago) they are found through to the EarlySilurian period (Wellman and Gray 2000) The earliest clearlyrecognisable land plants date from ~40million years later in theEarly Late Silurian (Wellman and Gray 2000) in which plantssuch asCooksonia can be seen in the fossil record (Graham1993)The origin of these early embryophytes is crucial to the origin ofsalt tolerance in land plants as it could have evolved from speciesthat lived in fresh or salt water by the time the first transition ofplants to a terrestrial environment occurred the seas were saline(Railsback et al 1990) even if a little less so (26ndash29 g per 1000 gseawater) than todayrsquos oceans (30ndash40 g per 1000 g seawater)

Did terrestrial plants derive from charophycean green algaeliving in fresh or salt water SinceCharacean algae occupied bothfreshwater and marine habitats in the mid-Paleozoic (Kenrickand Crane 1997) early embryophytes could have arisen frommarine or freshwater species Any marine organism making thetransition to the land on the edge of a saline pool would haveto cope not only with water loss from its cells to the atmospherebut alsowith hyper-saline conditionswhichwouldhaveoccurredas water evaporated from the pool If however the first landplants evolved at the edges of freshwater pools (Raven andEdwards 2001) then land plants would have arisen from anorganism (or organisms) that had adapted to living in fresh water

A particularly important adaptation to freshwater life is thedevelopment of systems for acquiring nutrients from the lowconcentrations of minerals present in fresh water rather thanthe high concentrations of the nutrient rich (eutrophic) marineenvironment (Rodriguez-Navarro and Rubio 2006) This abilitywould be equally important for growth on land where plantsneed to acquire nutrients in a nutrient poor (oligotrophic)environment Any such adaptation to oligotrophy might haveleft intact those systems required for osmotic adjustment andwhich are necessary for growth in seawater or in fluctuatingsalinity If embryophytes originated from freshwater algae thenas suggested by Becker andMarin (2009) the streptophytes werelsquophysiologically pre-adapted to terrestrial existence by theirprimary freshwater lifestyle in contrast to their marine sisterlineage ie Chlorophytarsquo (perhaps because their adaptationto an oligotrophic lifestyle resulted in evolution of specificmechanisms for dealing with fluctuating ion concentration inthe environment see also Ion compartmentation below)Analysis of fossils from Rhynie in Aberdeenshire in Scotlandsuggests that by the Early Devonian ~400million years agoplants were drought and probably salt tolerant (Channing andEdwards 2009) The question then is how many times salttolerance has evolved since the emergence of the land plantssome 450ndash470million years ago

Physiology of salt tolerance

Although there are many aspects of the physiology of salttolerance that are yet to be understood it is clear that the traitis complex in that at a minimum it requires the combination of

several different traits the accumulation and compartmentationof ions for osmotic adjustment the synthesis of compatiblesolutes the ability to accumulate essential nutrients(particularly K) in the presence of high concentrations of theions generating salinity (Na) the ability to limit the entry ofthese saline ions into the transpiration stream and the ability tocontinue to regulate transpiration in the presence of highconcentrations of Na+ and Clndash (Flowers and Colmer 2008)

Commonality of traits

All plants not just halophytes have the main characteristicsnecessary for salt tolerance ndash the ability to acquire ions thatare then compartmentalised in vacuoles (fundamental to turgorgeneration in plants and algae) and discrimination in favour ofK over Na Given that the physiological foundations of salttolerance are found in all plants it is not surprising that plantspecies show a continuum of salt tolerance with glycophytessuch as chickpea (Flowers et al 2010) at one extreme (wherejust 25mMNaCl can be toxic) and halophytes (which can toleratesalt concentrations as high as 500ndash1000mM) at the otherFurthermore salt tolerance is often part of a multi-facetedsyndrome that may include for example tolerance of flooding(Colmer and Voesenek 2009) and drought Indeed physiologicalresponses to salinity are often similar to responses to otherenvironmental stresses (Munns and Tester 2008) and may relyon common stress-tolerance pathways (Tuteja 2007)

Ion compartmentation

Most commonly the ions dominating saline environments arethose of Na and Cl as they predominate in seawaters halophytesuse these ions for most of their osmotic adjustment (Yeo 1983)It is clear however that in the process of ion accumulation forosmotic adjustment some ions can lsquoleakrsquo into the transpirationstream (Yeo et al 1987) and that such leaks must be minimisedif the aerial parts are not to be swamped by ions (Munns 2005)Consequently tight regulation of ion transport from roots toshoots is vital for halophytes Sufficient Na and Cl must beaccumulated to achieve osmotic adjustment but excess must beavoided as both ions are toxic at the concentrations requiredfor osmotic adjustment In practice Na+ and Clndash are largelycompartmentalised in vacuoles in halophytes (Flowers et al1977 1986) A range of metabolically inert organiccompounds is also present and utilised to adjust the osmoticpotential of the cytoplasm (Flowers and Colmer 2008) In somespecies excess ions are secreted throughglands that have evolvedon the surface of the aerial partsRecirculation of ions fromshootsback to the roots is unlikely to be an important aspect of salttolerance as the ions would have to be carried in the symplasm ofthe phloem where they would be toxic (Flowers et al 1986Munns and Tester 2008) Furthermore phloem transport deliverssolutes to growing regions where ions would cause metabolicdamage unless re-exported to the environment The latter wouldnot remove ions from the rhizosphere So ions reaching theleaves must be accommodated in vacuoles in the leaf cells aprocess that involves the transport of ions across both plasma andvacuolar membranes

Landplants includingmosses and ferns regulate their internalNa+ concentrations through the control of influx and efflux

Evolution of halophytes Functional Plant Biology 605

Within the eukaryotes Na+ efflux is mediated by Na+H+

antiporters and Na+-pump ATPases of which there are twotypes the Na+K+-ATPases of animal cells and the fungalNa+-ATPases Higher plants do not appear to express Na+-ATPases (Garciadeblas et al 2001) meaning that they mustrely on Na+H+ antiporters to transport Na+ out of theircytoplasm whether it be into the apoplast or the vacuolesBenito and Rodriguez-Navarro (2003) argued that the absenceof the Na+-ATPases in higher plants was evidence for theirevolution in non-saline oligotrophic environments where Na+

concentrations were low and there was no need for the system(s)than mediated extensive Na+ efflux This would presumablyhave occurred during the early development of land organismsin non-saline environments (Horodyski and Knauth 1994) orthrough the loss of such a system(s) following the migrationof marine organisms to freshwater Interestingly the mossPhyscomitrella patens (Bryopsida) expresses two ATPasessimilar to fungal ENA-Na+-ATPases (Benito and Rodriguez-Navarro 2003 Lunde et al 2007) The presence of the Na+-ATPases in amoss suggests these enzymeswere lost during earlyevolution of land plants (Benito and Rodriguez-Navarro 2003)which then rely on the Na+H+ antiporters (also present inP patens Benito and Rodriguez-Navarro 2003) to regulatecytosolic Na+ The question of how plants living in seawaterachieve this function remains The Na+H+ antiporters in plantsare electroneutral (Munns and Tester 2008) which means theywould not facilitate Na+ efflux at the alkaline pH values ofseawater (Benito and Rodriguez-Navarro 2003) Howeverseagrasses do presumably efflux Na+ but how their Na+H+

antiporters function in this respect is unclear (Garciadeblaacuteset al 2007 Touchette 2007) The same question can be posedfor halophytes although it is possible that the rhizosphere pHcould be lower than that of seawater the operation of Na-effluxthrough antiporters has been recently questioned (Britto andKronzucker 2009)

KNa selectivity

The selectivity of halophytes for K over Na varies betweenfamilies of flowering plants (Flowers et al 1986) Netselectivity (net SK Na) calculated as the ratio of Kconcentration in the plant to that in the medium divided by theratio ofNa concentration in the plant to that in themedium rangesbetween average values of 9 and 60 (Flowers and Colmer 2008)with an overall mean of 19 it is only in the Poales that net SK Na

values of the order of 60 are foundWithin the monocots (Fig 1)there are three orders with halophytes but no data are availablefor the net SK Na values of species within the Arecales In theAlismatales the average net SK Na (across just three species) is16 (range 10 to 22) suggesting that high selectivity has evolvedonly in the Poales (for halophytes within this order averageselectivities of are 58 in the Juncaginaceae (two species) and 60in the Poaceae (nine species)) There is too little data to analysethe net SK Na values within the dicots but the average value is11 compared with 60 in the Poales (Flowers and Colmer 2008)

Salt glands

Glandular structures are not uncommon on plants they cansecrete a range of organic compounds (Wagner 1991

Wagner et al 2004) However the ability to secrete saltappears to have evolved less frequently than salt toleranceSalt glands epidermal appendages of one to a few cells thatsecrete salt to the exterior of a plant (Thomson et al 1988) havebeendescribed in just a feworders offloweringplantsndash the Poales(eg in Aeluropus littoralis and Chloris gayana) Myrtales (egthe mangrove Laguncularia racemosa) Caryophyllales (egMesembryanthemum crystallinum and the saltbush Atriplexhalimus) Lamiales (eg the mangroves Avicennia marina andAvicennia germinans) and the Solanales (eg Cressa cretica)Their distribution across the orders of flowering plants suggestsat least three origins although there may have been moreindependent origins within orders (see for example within theCaryophyllales Fig 2b) Whether salt glands evolved from

Fig 1 The distribution of halophytes in the main tree by Stevens (2001)based on species listed by Aronson (1989) that survive at least 200mMNaClThose orders containing halophytes are boxed in red and the white circleindicates some species within the order have salt glands Indicates a nodewith rather weak support according to Stevens (2001)

606 Functional Plant Biology T J Flowers et al

glands that originally performed some other function is unclearbut it is difficult at least in the Poaceae to get glandular hairs onnon-halophytes (such as Zea mays L) to secrete salt (Ramadanand Flowers 2004)

Compatible solutes

The compatible solutes synthesised by halophytes range fromquaternary ammonium compounds through sulfonium analoguesto proline and sugar alcoholsmost arewidely distributed throughthe orders of flowering plants reflecting both phylogeny andfunctional needs (Flowers and Colmer 2008) There is no clearpattern of particular solutes being exclusive to particular ordersother than of sorbitol to the Plantaginaceae so that it appearsthat although the ability to synthesise and compartmentalise acompatible solute is a requirement in halophytes the nature ofthat solute depends on the habitat and the availability of NCompatible solutes comprise a small proportion of the totalsolutes accumulated by halophytes (Yeo 1983)

How many halophytes are there

As a trait salt tolerance has been recognised in plants for morethan 200 years (Flowers et al 1986) and there have been severaldefinitions of what constitutes a lsquohalophytersquo Angiospermspecies show a wide range of levels of salt tolerance frominhibition of growth at low salt concentrations (eg sensitivechickpea genotypes dying in 25mM NaCl Flowers et al 2010)to tolerance of salt concentrations as high or higher thanseawater (eg the halophyte Arthrocnemum macrostachyumsurvives in up to 1000mM NaCl Khan et al 2005) Anydefinition of a lsquohalophytersquo must include a threshold value ofsalt concentration and the number of halophytes clearly dependson the salt concentration used in that definition By setting adividing line between halophytes and glycophytes (salt sensitiveplants) at a salt concentration of ~80mM NaCl (a conductivityin the soil solution of 78 dS mndash1) Aronson (1989) listed ~1550salt-tolerant plants Using the same definition Menzel and Lieth(2003) recorded over 2600 species These comprehensive lists

(a) (b)

Fig 2 The distribution of halophytes in phylogenetic trees by Stevens (2001) based on species listed byAronson (1989) (a) Asterales and (b) CaryophyllalesThose orders containing halophytes are boxed in red and the white circle indicates some species within the order have salt glands For (a) denotes brancheswith 100 posterior probability and curren denotes branches with 70ndash80 jackknife support all other branches have gt80 support (according to Stevens 2001)In (b) denotes nodeswith 50ndash80bootstrap support unmarked branches havegt80support [1] and [3] have 50ndash80bootstrap support inmatK tree only and[2] is not recognised at all in matK tree only (according to Stevens 2001)

Evolution of halophytes Functional Plant Biology 607

define salt tolerant plants using a salt concentration that ismuch lower than that present in seawaters (an averageof ~480mM Na+ and 580mM Clndash) Since there are speciesthat have evolved tolerance to seawater salinities we havechosen to use a salt concentration of lsquo~200mM saltrsquo as ourdefining limit (as did Flowers and Colmer 2008) precisionis not required as salt concentrations in natural habitatsfluctuate with evaporation and precipitation of waterApplying the definition of lsquoplants that survive to completetheir life cycle in at least 200mM saltrsquo to Aronsonrsquos (1989)database generates a list of some 350 species which wecall lsquohalophytesrsquo (whether this group of the most salttolerant of plant species should be given a separate namesuch as lsquoeuhalophytersquo is an issue that could be furtherdebated) Our halophytes are not uniformly distributedacross the taxa of flowering plants listed by Stevens (2001)The orders with the greatest number of halophytes(Table 1) are the Caryophyllales (which includes thosespecies that were formerly in the Chenopodiaceae such asthe saltbushes in the genus Atriplex genera with reducedleaves such Salicornia and Tectomia and succulent membersof the genus Suaeda) and the Alismatales (which includesmarine flowering plants belonging to genera such as Zosteraand Thalassia) Although our list of halophytes is notcomprehensive as the limits of tolerance for many species arenot known our analysis suggests that halophytes are notmore than ~025 of the known species of angiosperms

Salt tolerance in bryophytes and pteridophytes

Halophytes are uncommon among the higher plants and thissituation is also true for bryophytes and pteridophytes There aresome mosses that occur in areas that are inundated by tides(Table 2) for example four species in Northern England(Adam 1976) and five species in Nova Scotia one of whichhas been shown to survive immersion in seawater for 24 h(Garbary et al 2008) Aronson (1989) lists only one genus ofa salt resistant fern This low frequency of salt tolerance inbryophytes and pteridophytes indicates that they also haverarely evolved the ability to tolerate salt concentrationsapproaching those of seawater although some are able to growin lower concentrations (eg Physcomitrella patens in 100mMNaCl see Lunde et al 2007)

Repeated evolution of salt-tolerance in angiosperms

In an earlier analysis based on the salt tolerance criterion usedby Aronson (1989) and a phylogeny constructed by Takhtajan(1969) Flowers et al (1977) concluded that salt tolerance waswidely distributed amongst the families of flowering plants andsuggested a polyphyletic origin something more recentlyestablished for other complex traits such as C4 photosynthesis(Sage 2004) and crassulacean acid metabolism (Crayn et al2004) In Fig 1 we have plotted the distribution of cladescontaining halophytes (as defined above) on an order-levelphylogeny of angiosperms based on the main tree published

Table 1 The distribution of halophytes amongst orders of flowering plantsThe estimates of species frequency are approximate due to the inexact numbers of specieswithin families the change in taxonomy that hasoccurred since the species were listed by Aronson (1989) and as the limits of tolerance for many species are not known Examples of

halophytes and suggestions for model species can be found in Flowers and Colmer (2008)

Order Number offamilies

Number offamilies withhalophytes

Families withhalophytes within

the order

Number ofhalophyticspecies

of all halophyticspecies (345)A

Caryophyllales 34 9 26 74 214Alismatales 14 7 50 61 177Malpighiales 40 4 10 35 101Poales 16 4 25 28 81Lamiales 25 5 20 24 70Myrtales 12 4 33 22 64Fabales 4 1 25 21 61Malvales 10 1 10 18 52Ericales 25 6 24 13 38Arecales 1 1 100 11 32Gentianales 5 4 80 9 26Sapindales 9 3 33 8 23Asterales 11 2 18 8 23Zygophyllales 2 1 50 3 09Apiales 7 1 14 3 09Magnoliales 6 1 17 2 06Solanales 5 1 20 2 06Celastrales 4 1 25 1 03Fagales 8 1 13 1 03Brassicales 18 1 6 1 03

Totals 256 58 ndash 345 100

A345 is the total number of halophytes included in this analysis

608 Functional Plant Biology T J Flowers et al

by Stevens (Stevens 2001) From this order-level tree we can seethat salt tolerance seems to have evolved multiple times

We cannot determine the exact number of origins of salttolerance from this phylogeny for several reasons Firstwithout information on the number of halophytes within eachorder and their relationships to each other we cannot knowwhether each family containing halophytes represents a singleorigin or multiple origins (see below) Second if a clade containsboth halophytes and non-halophytes we cannot be sure whethersalt tolerance was gained independently in multiple lineages orwhether the condition is ancestral to the clade and then lost insome families Third the evidence for the presence of halophytesin some orders is based on incomplete data (eg there are just twospecies reported as able to grow in seawater in the Magnoliales(Annonaceae) and without any physiological data) In mostorders that contain families with halophytes the percentage ofhalophytes is 1or less (Table 1)whichmakes it unlikely that thecondition is ancestral and the non-halophytes lost salt-toleranceTherefore it seems likely that virtually each of the red boxes inFig 1 represents at least one independent origin of salt tolerance

Many of the orders marked in Fig 1 may contain multipleorigins of halophytes For examplewithin theAsterales (Fig 2a)we have recorded eight halophytes in two families (seven in theAsteraceae and one in the Goodeniaceae) but the remainingfamilies do not have any halophytes It is parsimonious to assumethat at least two lineages within the Asterales evolved salttolerance independently Examining the presences of particulartraits associated with salt tolerance may also reveal someinteresting evolutionary patterns For example five of the ninesalt-tolerant lineages within the Caryophyllales use salt glands toexcrete excess salt from the leaves This illustrates how evenrelated lineages may develop different strategies for dealing withenvironmental salt The distribution of salt glands within thephylogeny (Fig 2b) suggests that even within this single clade offlowering plants a key salt tolerance mechanism has evolvedindependently several times

It also seems likely that the distribution of halophyte-containing families is not random over the phylogeny with

some clades containing more putative origins of salt tolerancethan others If true this would seem to offer support for the ideathat some clades are more likely to give rise to halophytes thanothers It may be profitable to look at the physiology of membersof those clades to see if they share certain characteristics thatmakeadapting to environmental salt easier than in other cladesMapping key components of salt tolerance on phylogeniesmay help to reveal whether some lineages are predisposed toevolving particular solutions to salinity whether some traits thatconfer salt tolerance aremore easily acquired than others or evenwhether the component traits of salt tolerance are usually acquiredin a particular order Looking at the patterns of origination withinfamilies will be important for answering the questions of whetherthere can be an ancestral condition that favours the evolutionof salt tolerance

Importance of halophytes to agricultureand land management

Salinity is an expanding problem More than 800million ha ofland is salt-affected which is over 6 of the worldrsquos land area(Flowers and Yeo 1995) Salt-affected land is increasingworldwide through vegetation clearance and irrigation both ofwhich raise the watertable bringing dissolved salts to the surfaceIt is estimated that up to half of irrigation schemes worldwideare affected by salinity (Flowers and Yeo 1995) Althoughirrigated land is a relatively small proportion of the total globalareaof foodproduction it produces a third of the food (Munns andTester 2008) Salt stress has been identified as one of the mostserious environmental factors limiting the productivity of cropplants (Flowers and Yeo 1995 Barkla et al 1999) with a hugeimpact on agricultural productivity The global annual cost ofsalt-affected land is likely to be well over US$12 billion (Qadiret al 2008) Future agricultural production will rely increasinglyon our ability to grow food and fibre plants in salt-affected land(Rozema and Flowers 2008 Qadir et al 2008)

Breeding for salt tolerance

Although it is abundantly clear that at some level salinity reducesthe growth of plants the reason is generally not clear Both Na+

and Clndash can reach toxic concentrations if not compartmentalisedin vacuoles but within the complexity of the whole plant theexact cause of any damage is generally unknown However theconsequences are that salinity reduces plant growth and enhancessenescence ofmature leaves resulting in a reduction in functionalleaf area in crop plants this decreases yield (Munns et al 2006Munns and Tester 2008) There is therefore a great incentive toincrease the salt tolerance of crop plants to allow greater yields insalt-affected soils However breeding of salt-tolerant crops ishampered by the complexity of salt tolerance as seen inhalophytes (see above) So despite a large amount of effortthat has been directed towards increasing the salt tolerance ofcrops ranging fromwide-crossing to transgenics few productivesalt-tolerant varieties have been produced (Flowers and Yeo1995 Flowers 2004 Flowers and Flowers 2005 Witcombeet al 2008) The realisation that halophytes have evolvedseveral times should encourage attempts to generate new salt-tolerant crops and may assist plant breeders in several waysFirst itmay illuminate specific traits associatedwith salt tolerance

Table 2 Salt tolerant mosses and ferns

Species names Location Reference

MossesPottia heimi

Amblystegium serpensEurhynchium praelongumCratoneuron filiciniumand C stellatumA

Northern England Adam (1976)

Campylium stellatumBryum capillareDidymodon rigidulusMnium hornumand Am serpens

Nova Scotia Garbary et al (2008)

FernsAcrostichum aureum

Ar danaeifolium andAr Speciosum

Tropics Aronson (1989)

ACratoneuron stellatumwas shown to survive immersion in seawater for 24 h(Garbary et al 2008)

Evolution of halophytes Functional Plant Biology 609

that may be selected in established crop plants (eg Huang et al2008) second it may lead to the domestication of naturally salttolerant species as crop plants

Halophytes as crops

Naturally salt-tolerant species are now being promoted inagriculture particularly to provide forage medicinal plantsaromatic plants (Qadir et al 2008) and for forestry (Marcarand Crawford 2004) For example Barrett-Lennard (2002)identified 26 salt-tolerant plant species capable of producingproducts (or services) of value to agriculture in AustraliaExamples of useful halophytes include the potential oil-seedcrops Kosteletzkya virginica (eg Ruan et al 2008) Salvadorapersica (eg Reddy et al 2008) Salicornia bigelovii (Glenn et al1991) and Batis maritima (Marcone 2003) fodder crops such asAtriplex spp (egEl Shaer 2003)Distichlis palmeri (egMasterset al 2007) and biofuels (Y Waisel pers comm Qadir et al2008) Growing salt-tolerant biofuel crops on marginalagricultural land would help to counter concerns that thebiofuel industry reduces the amount of land available forfood production (Qadir et al 2008) At the extreme plants thatcan grow productively at very high salt levels could be irrigatedwith brackish water or seawater (Rozema and Flowers 2008)Although plants that put resources (cf Yeo 1983) intodeveloping salt-tolerance mechanisms (eg the production ofcompatible solutes to maintain osmotic balance is an energeticcost) may do so at the expense of other functions manyhalophytes show optimal growth in saline conditions (Flowersand Colmer 2008) and salt marshes have high productivity(Colmer and Flowers 2008 Flowers and Colmer 2008) Thefact that dicolytedonous halophytes can grow at similar rates toglycophytes (see Flowers and Colmer 2008 for a more detaileddiscussion) suggests that salt tolerance per se will not limitproductivity Here the contrast with drought tolerance is starkwithout water plants do not grow but may survive with saltwater some plants can grow well

Apart from direct use as crops we may increasingly need torely on halophytes for revegetation and remediation of salt-affected land Over the last 200 years industrialisation inEurope and elsewhere has lead to an enormous increase ofproduction use and release of traces of heavy metals into theenvironment A large portion of these toxic materials includingCd Cu Pb and Zn accumulate in sediments including the soilsof tidal marshes Recent studies showed that some seagrassesand salt marsh plants are capable of extracting heavy metalsfrom sediments and accumulating them in belowground oraboveground tissues (Weis and Weis 2004 Cambrolle et al2008 Lewis and Devereux 2009) The processes and potentialapplication of these aquatic halophytes merits much greaterresearch and development Growing salt-tolerant plantsincluding species of Kochia Bassia Cynodon MedicagoPortulaca Sesbania and Brachiaria may also improve othersoil properties such as increasing water conductance orincreasing soil fertility (Qadir et al 2008) Halophytes mayalso lower the watertable thereby allowing growth of salt-sensitive species in salt-affected land (Barrett-Lennard 2002)

Finally conservation and land management in salt-affectedregions will benefit from improved knowledge of the salt

tolerance of local species Halophytes are being targeted forlsquoecosystem engineeringrsquo projects such as stabilising wetlandsto increase the success of revegetation programs (Fogel et al2004) Halophytes are also being used to develop sustainableagricultural practise for example using indigenous halophytesto restore overgrazed pasture and to reduce reliance on irrigation(Peacock et al 2003) Salt-tolerant species are increasinglybeing considered for landscape remediation For example wellplanned revegetation can reduceboth the in situ salt concentrationat mine sites and also reduce the movement of salts from themine sites in runoff (Carroll and Tucker 2000) However caremust be taken in introducing species for bioremediation as theymay become an environmental problem in their own right Forexample Kochia planted for forage and bioremediation inWestern Australia in 1990 soon spread and was declared aweed in 1992 (Qadir et al 2008)

The halophyte paradox

In this review we have shown that salt tolerance has evolvedmany different times in angiosperms and in different ways Wehave also noted that despite increasing salinisation of agriculturalland few salt-tolerant crops have been produced This raises animportant paradox if all plants contain the basic physiologicalmechanisms on which salt tolerance is based and if halophyteshave evolved in many different lineages using a variety ofadaptations thenwhy is it so difficult to breed salt-tolerant crops

The reasons for the slowprogress in changing the salt toleranceof plants were discussed by Flowers andYeo (1995) who arguedthat this was because salinity had not become a problem ofsufficient priority that the necessary resource was beingapplied to its solution Moreover they also concluded thattreating salinity resistance as a single character could accountfor the limited success We have already established that the salttolerance of halophytes is a multi-genic trait and the same is truefor non-halophytes (Flowers 2004) It is not our intention hereto rehearse the arguments over approaches to breeding forcomplex traits However it is clear that breeding for yield hasnot delivered many salt-resistant varieties of the commoncrops (Flowers 2004 Witcombe et al 2008) The probabilityof combining two complex traits ndash yield and salt tolerance ndash tomaximise yield under saline conditions is evidentially very lowAs a consequence Yeo et al (1990) advocated understandingthe physiology of tolerance and pooling a range of traits anapproach that had some success for rice (Dedolph and Hettel1997) A similar approach has been advocated for durum wheatcombining traits for regulating sodiumaccumulation and osmoticadjustment (Munns et al 2002 James et al 2006 2008) Thetraits that necessarily need to be pooled for wheat (and barley)includeNa lsquoexclusionrsquo KNa discrimination the retention of ionsin sheaths tissue tolerance ion partitioning into different leavesosmotic adjustment enhanced vigour water use efficiency andearly flowering (Colmer et al 2005) In addition other traits suchas tolerance to waterndashlogging (see Colmer and Flowers 2008 fora discussion of flooding tolerance in halophytes) are vitallyimportant further complicating any breeding program (Colmeret al 2005) Given the complexity of the task it is not surprisingthat the transfer of one or two genes has generally failed to changethe tolerance of transgenic plants what is surprising is that there

610 Functional Plant Biology T J Flowers et al

are instances where such simple gene transfers appear to havealtered yield (Flowers 2004)

Evolutionary studies of halophytes which reconstruct theorigin and development of salt tolerance in a variety of plantlineages may help us to understand why artificial breedinghas failed to produce robust and productive salt tolerant cropsSuch studies may also help us develop new salt-tolerant lines byrevealing the order of acquisition of components of salt toleranceor indicating favourable genetic backgrounds on which salttolerance may be developed By examining the repeatedevolution of this complex trait we may identify particular traitsor conditions that predispose species to evolve a complex multi-faceted trait such as salt tolerance and give rise to halophytelineages More generally this may shed light on the adaptationof angiosperm lineages to extreme environments In order toachieve these ends more information is required on at aminimum the effects of salt on the growth and ion relations ofa wider range of plant species that may prove to be halophytes

Acknowledgements

HKGwould like to than the Egyptian Government for financial support whileat the University of Sussex and our thanks also go to Jessica Thomas andKatherine Byron for assistance in earlier stages of this project

References

Adam P (1976) Occurrence of bryophytes on British salt marshes Journalof Bryology 9 265ndash274

Aronson JA (1989) lsquoHALOPHa data base of salt tolerant plants of the worldrsquo(Office of Arid Land Studies University of Arizona Tucson AZ)

Barkla BJ Vera Estrella R Pantoja O (1999) Towards the production ofsalt-tolerant crops Advances in Experimental Medicine and Biology464 77ndash89

Barrett-Lennard EG (2002) Restoration of saline land through revegetationAgricultural Water Management 53 213ndash226 doi101016S0378-3774(01)00166-4

Becker BMarinB (2009) Streptophyte algae and the origin of embryophytesAnnals of Botany 103 999ndash1004 doi101093aobmcp044

Benito B Rodriguez-Navarro A (2003) Molecular cloning andcharacterization of a sodium-pump ATPase of the moss Physcomitrellapatens The Plant Journal 36 382ndash389 doi101046j1365-313X200301883x

Bisson MA Kirst GO (1995) Osmotic acclimation and turgor pressureregulation in algae Naturwissenschaften 82 461ndash471 doi101007BF01131597

Britto DT Kronzucker HJ (2009) Ussingrsquos conundrum and the search fortransport mechanisms in plants New Phytologist 183 243ndash246doi101111j1469-8137200902872x

Cambrolle J Redondo-Gomez S Mateos-Naranjo E Figueroa ME (2008)Comparison of the role of two Spartina species in terms ofphytostabilization and bioaccumulation of metals in the estuarinesediment Marine Pollution Bulletin 56 2037ndash2042 doi101016jmarpolbul200808008

Carroll C Tucker A (2000) Effects of pasture cover on soil erosion andwater quality on central Queensland coal mine rehabilitation TropicalGrasslands 34 254ndash262

Channing A Edwards D (2009) Yellowstone hot spring environmentsand the paleo-ecophysiology of Rhynie chert plants towards asynthesis Plant Ecology amp Diversity 2 111ndash143 doi10108017550870903349359

Colmer TD Flowers TJ (2008) Flooding tolerance in halophytes NewPhytologist 179 964ndash974 doi101111j1469-8137200802483x

Colmer TD Voesenek L (2009) Flooding tolerance suites of plant traitsin variable environments Functional Plant Biology 36 665ndash681doi101071FP09144

Colmer TD Munns R Flowers TJ (2005) Improving salt tolerance of wheatand barley future prospects Australian Journal of ExperimentalAgriculture 45 1425ndash1443 doi101071EA04162

Crayn DM Winter K Smith JAC (2004) Multiple origins of crassulaceanacid metabolism and the epiphytic habit in the neotropical familyBromeliaceae Proceedings of the National Academy of Sciences ofthe United States of America 101 3703ndash3708 doi101073pnas0400366101

Dedolph C Hettel G (Eds) (1997) lsquoPartners making a differencersquo(International Rice Research Institute Manila)

El Shaer HM (2003) Potential of halophytes as animal fodder in EgyptIn lsquoTasks for vegetation science 38 cash crop halophytesrsquo (Eds H LiethM Mochtchenko) pp 111ndash119 (Kluwer Dordrecht the Netherlands)

Flowers TJ (1985) Physiology of halophytes Plant and Soil 89 41ndash56doi101007BF02182232

Flowers TJ (2004) Improving crop salt tolerance Journal of ExperimentalBotany 55 307ndash319 doi101093jxberh003

Flowers TJ Colmer TD (2008) Salinity tolerance in halophytes NewPhytologist 179 945ndash963 doi101111j1469-8137200802531x

Flowers TJ Flowers SA (2005) Why does salinity pose such a difficultproblem for plant breeders Agricultural Water Management 78 15ndash24doi101016jagwat200504015

Flowers TJ Yeo AR (1995) Breeding for salinity resistance in crop plants ndashwhere next Australian Journal of Plant Physiology 22 875ndash884doi101071PP9950875

Flowers TJ Troke PF Yeo AR (1977) The mechanism of salt tolerance inhalophytesAnnual Review of Plant Physiology 28 89ndash121 doi101146annurevpp28060177000513

Flowers TJ Hajibagheri MA Clipson NJW (1986) Halophytes TheQuarterly Review of Biology 61 313ndash337 doi101086415032

Flowers TJGaur PMLaxmipathiGowdaCLKrishnamurthy L Samineni SSiddique KHM Turner NC Vadez V Varshney RK Colmer TD (2010)Salt sensitivity in chickpea Plant Cell amp Environment 33 490ndash509doi101111j1365-3040200902051x

Fogel BN Crain CM Bertness MD (2004) Community level engineeringeffects of Triglochin maritima (seaside arrowgrass) in a salt marsh innorthern New England USA Journal of Ecology 92 589ndash597doi101111j0022-0477200400903x

Garbary DJ Miller AG Scrosati R Kim KY Schofield WB (2008)Distribution and salinity tolerance of intertidal mosses from NovaScotian salt marshes The Bryologist 111 282ndash291 doi1016390007-2745(2008)111[282DASTOI]20CO2

Garciadeblas B Benito B Rodriacuteguez-Navarro A (2001) Plant cells expressseveral stress calcium ATPases but apparently no sodium ATPase Plantand Soil 235 181ndash192 doi101023A1011949626191

Garciadeblaacutes B Haro R Benito B (2007) Cloning of two SOS1transporters from the seagrass Cymodocea nodosa SOS1transporters from Cymodocea and Arabidopsis mediate potassiumuptake in bacteria Plant Molecular Biology 63 479ndash490doi101007s11103-006-9102-2

Glenn EP Oleary JW Watson MC Thompson TL Kuehl RO (1991)Salicornia-bigelovii Torr ndash an oilseed halophyte for seawater irrigationScience 251 1065ndash1067 doi101126science25149971065

Graham LE (1993) lsquoOrigin of land plantsrsquo (John Wiley amp Sons New York)Harvey HW (1966) lsquoThe chemistry and fertility of sea watersrsquo (University

Press Cambridge)Horodyski RJ Knauth LP (1994) Life on land in the Precambrian Science

263 494ndash498 doi101126science2635146494HuangSSpielmeyerWLagudahESMunnsR (2008)Comparativemapping

of HKT genes in wheat barley and rice key determinants of Na+transport and salt tolerance Journal of Experimental Botany 59927ndash937 doi101093jxbern033

Evolution of halophytes Functional Plant Biology 611

Within the eukaryotes Na+ efflux is mediated by Na+H+

antiporters and Na+-pump ATPases of which there are twotypes the Na+K+-ATPases of animal cells and the fungalNa+-ATPases Higher plants do not appear to express Na+-ATPases (Garciadeblas et al 2001) meaning that they mustrely on Na+H+ antiporters to transport Na+ out of theircytoplasm whether it be into the apoplast or the vacuolesBenito and Rodriguez-Navarro (2003) argued that the absenceof the Na+-ATPases in higher plants was evidence for theirevolution in non-saline oligotrophic environments where Na+

concentrations were low and there was no need for the system(s)than mediated extensive Na+ efflux This would presumablyhave occurred during the early development of land organismsin non-saline environments (Horodyski and Knauth 1994) orthrough the loss of such a system(s) following the migrationof marine organisms to freshwater Interestingly the mossPhyscomitrella patens (Bryopsida) expresses two ATPasessimilar to fungal ENA-Na+-ATPases (Benito and Rodriguez-Navarro 2003 Lunde et al 2007) The presence of the Na+-ATPases in amoss suggests these enzymeswere lost during earlyevolution of land plants (Benito and Rodriguez-Navarro 2003)which then rely on the Na+H+ antiporters (also present inP patens Benito and Rodriguez-Navarro 2003) to regulatecytosolic Na+ The question of how plants living in seawaterachieve this function remains The Na+H+ antiporters in plantsare electroneutral (Munns and Tester 2008) which means theywould not facilitate Na+ efflux at the alkaline pH values ofseawater (Benito and Rodriguez-Navarro 2003) Howeverseagrasses do presumably efflux Na+ but how their Na+H+

antiporters function in this respect is unclear (Garciadeblaacuteset al 2007 Touchette 2007) The same question can be posedfor halophytes although it is possible that the rhizosphere pHcould be lower than that of seawater the operation of Na-effluxthrough antiporters has been recently questioned (Britto andKronzucker 2009)

KNa selectivity

The selectivity of halophytes for K over Na varies betweenfamilies of flowering plants (Flowers et al 1986) Netselectivity (net SK Na) calculated as the ratio of Kconcentration in the plant to that in the medium divided by theratio ofNa concentration in the plant to that in themedium rangesbetween average values of 9 and 60 (Flowers and Colmer 2008)with an overall mean of 19 it is only in the Poales that net SK Na

values of the order of 60 are foundWithin the monocots (Fig 1)there are three orders with halophytes but no data are availablefor the net SK Na values of species within the Arecales In theAlismatales the average net SK Na (across just three species) is16 (range 10 to 22) suggesting that high selectivity has evolvedonly in the Poales (for halophytes within this order averageselectivities of are 58 in the Juncaginaceae (two species) and 60in the Poaceae (nine species)) There is too little data to analysethe net SK Na values within the dicots but the average value is11 compared with 60 in the Poales (Flowers and Colmer 2008)

Salt glands

Glandular structures are not uncommon on plants they cansecrete a range of organic compounds (Wagner 1991

Wagner et al 2004) However the ability to secrete saltappears to have evolved less frequently than salt toleranceSalt glands epidermal appendages of one to a few cells thatsecrete salt to the exterior of a plant (Thomson et al 1988) havebeendescribed in just a feworders offloweringplantsndash the Poales(eg in Aeluropus littoralis and Chloris gayana) Myrtales (egthe mangrove Laguncularia racemosa) Caryophyllales (egMesembryanthemum crystallinum and the saltbush Atriplexhalimus) Lamiales (eg the mangroves Avicennia marina andAvicennia germinans) and the Solanales (eg Cressa cretica)Their distribution across the orders of flowering plants suggestsat least three origins although there may have been moreindependent origins within orders (see for example within theCaryophyllales Fig 2b) Whether salt glands evolved from

Fig 1 The distribution of halophytes in the main tree by Stevens (2001)based on species listed by Aronson (1989) that survive at least 200mMNaClThose orders containing halophytes are boxed in red and the white circleindicates some species within the order have salt glands Indicates a nodewith rather weak support according to Stevens (2001)

606 Functional Plant Biology T J Flowers et al

glands that originally performed some other function is unclearbut it is difficult at least in the Poaceae to get glandular hairs onnon-halophytes (such as Zea mays L) to secrete salt (Ramadanand Flowers 2004)

Compatible solutes

The compatible solutes synthesised by halophytes range fromquaternary ammonium compounds through sulfonium analoguesto proline and sugar alcoholsmost arewidely distributed throughthe orders of flowering plants reflecting both phylogeny andfunctional needs (Flowers and Colmer 2008) There is no clearpattern of particular solutes being exclusive to particular ordersother than of sorbitol to the Plantaginaceae so that it appearsthat although the ability to synthesise and compartmentalise acompatible solute is a requirement in halophytes the nature ofthat solute depends on the habitat and the availability of NCompatible solutes comprise a small proportion of the totalsolutes accumulated by halophytes (Yeo 1983)

How many halophytes are there

As a trait salt tolerance has been recognised in plants for morethan 200 years (Flowers et al 1986) and there have been severaldefinitions of what constitutes a lsquohalophytersquo Angiospermspecies show a wide range of levels of salt tolerance frominhibition of growth at low salt concentrations (eg sensitivechickpea genotypes dying in 25mM NaCl Flowers et al 2010)to tolerance of salt concentrations as high or higher thanseawater (eg the halophyte Arthrocnemum macrostachyumsurvives in up to 1000mM NaCl Khan et al 2005) Anydefinition of a lsquohalophytersquo must include a threshold value ofsalt concentration and the number of halophytes clearly dependson the salt concentration used in that definition By setting adividing line between halophytes and glycophytes (salt sensitiveplants) at a salt concentration of ~80mM NaCl (a conductivityin the soil solution of 78 dS mndash1) Aronson (1989) listed ~1550salt-tolerant plants Using the same definition Menzel and Lieth(2003) recorded over 2600 species These comprehensive lists

(a) (b)

Fig 2 The distribution of halophytes in phylogenetic trees by Stevens (2001) based on species listed byAronson (1989) (a) Asterales and (b) CaryophyllalesThose orders containing halophytes are boxed in red and the white circle indicates some species within the order have salt glands For (a) denotes brancheswith 100 posterior probability and curren denotes branches with 70ndash80 jackknife support all other branches have gt80 support (according to Stevens 2001)In (b) denotes nodeswith 50ndash80bootstrap support unmarked branches havegt80support [1] and [3] have 50ndash80bootstrap support inmatK tree only and[2] is not recognised at all in matK tree only (according to Stevens 2001)

Evolution of halophytes Functional Plant Biology 607

define salt tolerant plants using a salt concentration that ismuch lower than that present in seawaters (an averageof ~480mM Na+ and 580mM Clndash) Since there are speciesthat have evolved tolerance to seawater salinities we havechosen to use a salt concentration of lsquo~200mM saltrsquo as ourdefining limit (as did Flowers and Colmer 2008) precisionis not required as salt concentrations in natural habitatsfluctuate with evaporation and precipitation of waterApplying the definition of lsquoplants that survive to completetheir life cycle in at least 200mM saltrsquo to Aronsonrsquos (1989)database generates a list of some 350 species which wecall lsquohalophytesrsquo (whether this group of the most salttolerant of plant species should be given a separate namesuch as lsquoeuhalophytersquo is an issue that could be furtherdebated) Our halophytes are not uniformly distributedacross the taxa of flowering plants listed by Stevens (2001)The orders with the greatest number of halophytes(Table 1) are the Caryophyllales (which includes thosespecies that were formerly in the Chenopodiaceae such asthe saltbushes in the genus Atriplex genera with reducedleaves such Salicornia and Tectomia and succulent membersof the genus Suaeda) and the Alismatales (which includesmarine flowering plants belonging to genera such as Zosteraand Thalassia) Although our list of halophytes is notcomprehensive as the limits of tolerance for many species arenot known our analysis suggests that halophytes are notmore than ~025 of the known species of angiosperms

Salt tolerance in bryophytes and pteridophytes

Halophytes are uncommon among the higher plants and thissituation is also true for bryophytes and pteridophytes There aresome mosses that occur in areas that are inundated by tides(Table 2) for example four species in Northern England(Adam 1976) and five species in Nova Scotia one of whichhas been shown to survive immersion in seawater for 24 h(Garbary et al 2008) Aronson (1989) lists only one genus ofa salt resistant fern This low frequency of salt tolerance inbryophytes and pteridophytes indicates that they also haverarely evolved the ability to tolerate salt concentrationsapproaching those of seawater although some are able to growin lower concentrations (eg Physcomitrella patens in 100mMNaCl see Lunde et al 2007)

Repeated evolution of salt-tolerance in angiosperms

In an earlier analysis based on the salt tolerance criterion usedby Aronson (1989) and a phylogeny constructed by Takhtajan(1969) Flowers et al (1977) concluded that salt tolerance waswidely distributed amongst the families of flowering plants andsuggested a polyphyletic origin something more recentlyestablished for other complex traits such as C4 photosynthesis(Sage 2004) and crassulacean acid metabolism (Crayn et al2004) In Fig 1 we have plotted the distribution of cladescontaining halophytes (as defined above) on an order-levelphylogeny of angiosperms based on the main tree published

Table 1 The distribution of halophytes amongst orders of flowering plantsThe estimates of species frequency are approximate due to the inexact numbers of specieswithin families the change in taxonomy that hasoccurred since the species were listed by Aronson (1989) and as the limits of tolerance for many species are not known Examples of

halophytes and suggestions for model species can be found in Flowers and Colmer (2008)

Order Number offamilies

Number offamilies withhalophytes

Families withhalophytes within

the order

Number ofhalophyticspecies

of all halophyticspecies (345)A

Caryophyllales 34 9 26 74 214Alismatales 14 7 50 61 177Malpighiales 40 4 10 35 101Poales 16 4 25 28 81Lamiales 25 5 20 24 70Myrtales 12 4 33 22 64Fabales 4 1 25 21 61Malvales 10 1 10 18 52Ericales 25 6 24 13 38Arecales 1 1 100 11 32Gentianales 5 4 80 9 26Sapindales 9 3 33 8 23Asterales 11 2 18 8 23Zygophyllales 2 1 50 3 09Apiales 7 1 14 3 09Magnoliales 6 1 17 2 06Solanales 5 1 20 2 06Celastrales 4 1 25 1 03Fagales 8 1 13 1 03Brassicales 18 1 6 1 03

Totals 256 58 ndash 345 100

A345 is the total number of halophytes included in this analysis

608 Functional Plant Biology T J Flowers et al

by Stevens (Stevens 2001) From this order-level tree we can seethat salt tolerance seems to have evolved multiple times

We cannot determine the exact number of origins of salttolerance from this phylogeny for several reasons Firstwithout information on the number of halophytes within eachorder and their relationships to each other we cannot knowwhether each family containing halophytes represents a singleorigin or multiple origins (see below) Second if a clade containsboth halophytes and non-halophytes we cannot be sure whethersalt tolerance was gained independently in multiple lineages orwhether the condition is ancestral to the clade and then lost insome families Third the evidence for the presence of halophytesin some orders is based on incomplete data (eg there are just twospecies reported as able to grow in seawater in the Magnoliales(Annonaceae) and without any physiological data) In mostorders that contain families with halophytes the percentage ofhalophytes is 1or less (Table 1)whichmakes it unlikely that thecondition is ancestral and the non-halophytes lost salt-toleranceTherefore it seems likely that virtually each of the red boxes inFig 1 represents at least one independent origin of salt tolerance

Many of the orders marked in Fig 1 may contain multipleorigins of halophytes For examplewithin theAsterales (Fig 2a)we have recorded eight halophytes in two families (seven in theAsteraceae and one in the Goodeniaceae) but the remainingfamilies do not have any halophytes It is parsimonious to assumethat at least two lineages within the Asterales evolved salttolerance independently Examining the presences of particulartraits associated with salt tolerance may also reveal someinteresting evolutionary patterns For example five of the ninesalt-tolerant lineages within the Caryophyllales use salt glands toexcrete excess salt from the leaves This illustrates how evenrelated lineages may develop different strategies for dealing withenvironmental salt The distribution of salt glands within thephylogeny (Fig 2b) suggests that even within this single clade offlowering plants a key salt tolerance mechanism has evolvedindependently several times

It also seems likely that the distribution of halophyte-containing families is not random over the phylogeny with

some clades containing more putative origins of salt tolerancethan others If true this would seem to offer support for the ideathat some clades are more likely to give rise to halophytes thanothers It may be profitable to look at the physiology of membersof those clades to see if they share certain characteristics thatmakeadapting to environmental salt easier than in other cladesMapping key components of salt tolerance on phylogeniesmay help to reveal whether some lineages are predisposed toevolving particular solutions to salinity whether some traits thatconfer salt tolerance aremore easily acquired than others or evenwhether the component traits of salt tolerance are usually acquiredin a particular order Looking at the patterns of origination withinfamilies will be important for answering the questions of whetherthere can be an ancestral condition that favours the evolutionof salt tolerance

Importance of halophytes to agricultureand land management

Salinity is an expanding problem More than 800million ha ofland is salt-affected which is over 6 of the worldrsquos land area(Flowers and Yeo 1995) Salt-affected land is increasingworldwide through vegetation clearance and irrigation both ofwhich raise the watertable bringing dissolved salts to the surfaceIt is estimated that up to half of irrigation schemes worldwideare affected by salinity (Flowers and Yeo 1995) Althoughirrigated land is a relatively small proportion of the total globalareaof foodproduction it produces a third of the food (Munns andTester 2008) Salt stress has been identified as one of the mostserious environmental factors limiting the productivity of cropplants (Flowers and Yeo 1995 Barkla et al 1999) with a hugeimpact on agricultural productivity The global annual cost ofsalt-affected land is likely to be well over US$12 billion (Qadiret al 2008) Future agricultural production will rely increasinglyon our ability to grow food and fibre plants in salt-affected land(Rozema and Flowers 2008 Qadir et al 2008)

Breeding for salt tolerance

Although it is abundantly clear that at some level salinity reducesthe growth of plants the reason is generally not clear Both Na+

and Clndash can reach toxic concentrations if not compartmentalisedin vacuoles but within the complexity of the whole plant theexact cause of any damage is generally unknown However theconsequences are that salinity reduces plant growth and enhancessenescence ofmature leaves resulting in a reduction in functionalleaf area in crop plants this decreases yield (Munns et al 2006Munns and Tester 2008) There is therefore a great incentive toincrease the salt tolerance of crop plants to allow greater yields insalt-affected soils However breeding of salt-tolerant crops ishampered by the complexity of salt tolerance as seen inhalophytes (see above) So despite a large amount of effortthat has been directed towards increasing the salt tolerance ofcrops ranging fromwide-crossing to transgenics few productivesalt-tolerant varieties have been produced (Flowers and Yeo1995 Flowers 2004 Flowers and Flowers 2005 Witcombeet al 2008) The realisation that halophytes have evolvedseveral times should encourage attempts to generate new salt-tolerant crops and may assist plant breeders in several waysFirst itmay illuminate specific traits associatedwith salt tolerance

Table 2 Salt tolerant mosses and ferns

Species names Location Reference

MossesPottia heimi

Amblystegium serpensEurhynchium praelongumCratoneuron filiciniumand C stellatumA

Northern England Adam (1976)

Campylium stellatumBryum capillareDidymodon rigidulusMnium hornumand Am serpens

Nova Scotia Garbary et al (2008)

FernsAcrostichum aureum

Ar danaeifolium andAr Speciosum

Tropics Aronson (1989)

ACratoneuron stellatumwas shown to survive immersion in seawater for 24 h(Garbary et al 2008)

Evolution of halophytes Functional Plant Biology 609

that may be selected in established crop plants (eg Huang et al2008) second it may lead to the domestication of naturally salttolerant species as crop plants

Halophytes as crops

Naturally salt-tolerant species are now being promoted inagriculture particularly to provide forage medicinal plantsaromatic plants (Qadir et al 2008) and for forestry (Marcarand Crawford 2004) For example Barrett-Lennard (2002)identified 26 salt-tolerant plant species capable of producingproducts (or services) of value to agriculture in AustraliaExamples of useful halophytes include the potential oil-seedcrops Kosteletzkya virginica (eg Ruan et al 2008) Salvadorapersica (eg Reddy et al 2008) Salicornia bigelovii (Glenn et al1991) and Batis maritima (Marcone 2003) fodder crops such asAtriplex spp (egEl Shaer 2003)Distichlis palmeri (egMasterset al 2007) and biofuels (Y Waisel pers comm Qadir et al2008) Growing salt-tolerant biofuel crops on marginalagricultural land would help to counter concerns that thebiofuel industry reduces the amount of land available forfood production (Qadir et al 2008) At the extreme plants thatcan grow productively at very high salt levels could be irrigatedwith brackish water or seawater (Rozema and Flowers 2008)Although plants that put resources (cf Yeo 1983) intodeveloping salt-tolerance mechanisms (eg the production ofcompatible solutes to maintain osmotic balance is an energeticcost) may do so at the expense of other functions manyhalophytes show optimal growth in saline conditions (Flowersand Colmer 2008) and salt marshes have high productivity(Colmer and Flowers 2008 Flowers and Colmer 2008) Thefact that dicolytedonous halophytes can grow at similar rates toglycophytes (see Flowers and Colmer 2008 for a more detaileddiscussion) suggests that salt tolerance per se will not limitproductivity Here the contrast with drought tolerance is starkwithout water plants do not grow but may survive with saltwater some plants can grow well

Apart from direct use as crops we may increasingly need torely on halophytes for revegetation and remediation of salt-affected land Over the last 200 years industrialisation inEurope and elsewhere has lead to an enormous increase ofproduction use and release of traces of heavy metals into theenvironment A large portion of these toxic materials includingCd Cu Pb and Zn accumulate in sediments including the soilsof tidal marshes Recent studies showed that some seagrassesand salt marsh plants are capable of extracting heavy metalsfrom sediments and accumulating them in belowground oraboveground tissues (Weis and Weis 2004 Cambrolle et al2008 Lewis and Devereux 2009) The processes and potentialapplication of these aquatic halophytes merits much greaterresearch and development Growing salt-tolerant plantsincluding species of Kochia Bassia Cynodon MedicagoPortulaca Sesbania and Brachiaria may also improve othersoil properties such as increasing water conductance orincreasing soil fertility (Qadir et al 2008) Halophytes mayalso lower the watertable thereby allowing growth of salt-sensitive species in salt-affected land (Barrett-Lennard 2002)

Finally conservation and land management in salt-affectedregions will benefit from improved knowledge of the salt

tolerance of local species Halophytes are being targeted forlsquoecosystem engineeringrsquo projects such as stabilising wetlandsto increase the success of revegetation programs (Fogel et al2004) Halophytes are also being used to develop sustainableagricultural practise for example using indigenous halophytesto restore overgrazed pasture and to reduce reliance on irrigation(Peacock et al 2003) Salt-tolerant species are increasinglybeing considered for landscape remediation For example wellplanned revegetation can reduceboth the in situ salt concentrationat mine sites and also reduce the movement of salts from themine sites in runoff (Carroll and Tucker 2000) However caremust be taken in introducing species for bioremediation as theymay become an environmental problem in their own right Forexample Kochia planted for forage and bioremediation inWestern Australia in 1990 soon spread and was declared aweed in 1992 (Qadir et al 2008)

The halophyte paradox

In this review we have shown that salt tolerance has evolvedmany different times in angiosperms and in different ways Wehave also noted that despite increasing salinisation of agriculturalland few salt-tolerant crops have been produced This raises animportant paradox if all plants contain the basic physiologicalmechanisms on which salt tolerance is based and if halophyteshave evolved in many different lineages using a variety ofadaptations thenwhy is it so difficult to breed salt-tolerant crops

The reasons for the slowprogress in changing the salt toleranceof plants were discussed by Flowers andYeo (1995) who arguedthat this was because salinity had not become a problem ofsufficient priority that the necessary resource was beingapplied to its solution Moreover they also concluded thattreating salinity resistance as a single character could accountfor the limited success We have already established that the salttolerance of halophytes is a multi-genic trait and the same is truefor non-halophytes (Flowers 2004) It is not our intention hereto rehearse the arguments over approaches to breeding forcomplex traits However it is clear that breeding for yield hasnot delivered many salt-resistant varieties of the commoncrops (Flowers 2004 Witcombe et al 2008) The probabilityof combining two complex traits ndash yield and salt tolerance ndash tomaximise yield under saline conditions is evidentially very lowAs a consequence Yeo et al (1990) advocated understandingthe physiology of tolerance and pooling a range of traits anapproach that had some success for rice (Dedolph and Hettel1997) A similar approach has been advocated for durum wheatcombining traits for regulating sodiumaccumulation and osmoticadjustment (Munns et al 2002 James et al 2006 2008) Thetraits that necessarily need to be pooled for wheat (and barley)includeNa lsquoexclusionrsquo KNa discrimination the retention of ionsin sheaths tissue tolerance ion partitioning into different leavesosmotic adjustment enhanced vigour water use efficiency andearly flowering (Colmer et al 2005) In addition other traits suchas tolerance to waterndashlogging (see Colmer and Flowers 2008 fora discussion of flooding tolerance in halophytes) are vitallyimportant further complicating any breeding program (Colmeret al 2005) Given the complexity of the task it is not surprisingthat the transfer of one or two genes has generally failed to changethe tolerance of transgenic plants what is surprising is that there

610 Functional Plant Biology T J Flowers et al

are instances where such simple gene transfers appear to havealtered yield (Flowers 2004)

Evolutionary studies of halophytes which reconstruct theorigin and development of salt tolerance in a variety of plantlineages may help us to understand why artificial breedinghas failed to produce robust and productive salt tolerant cropsSuch studies may also help us develop new salt-tolerant lines byrevealing the order of acquisition of components of salt toleranceor indicating favourable genetic backgrounds on which salttolerance may be developed By examining the repeatedevolution of this complex trait we may identify particular traitsor conditions that predispose species to evolve a complex multi-faceted trait such as salt tolerance and give rise to halophytelineages More generally this may shed light on the adaptationof angiosperm lineages to extreme environments In order toachieve these ends more information is required on at aminimum the effects of salt on the growth and ion relations ofa wider range of plant species that may prove to be halophytes

Acknowledgements

HKGwould like to than the Egyptian Government for financial support whileat the University of Sussex and our thanks also go to Jessica Thomas andKatherine Byron for assistance in earlier stages of this project

References

Adam P (1976) Occurrence of bryophytes on British salt marshes Journalof Bryology 9 265ndash274

Aronson JA (1989) lsquoHALOPHa data base of salt tolerant plants of the worldrsquo(Office of Arid Land Studies University of Arizona Tucson AZ)

Barkla BJ Vera Estrella R Pantoja O (1999) Towards the production ofsalt-tolerant crops Advances in Experimental Medicine and Biology464 77ndash89

Barrett-Lennard EG (2002) Restoration of saline land through revegetationAgricultural Water Management 53 213ndash226 doi101016S0378-3774(01)00166-4

Becker BMarinB (2009) Streptophyte algae and the origin of embryophytesAnnals of Botany 103 999ndash1004 doi101093aobmcp044

Benito B Rodriguez-Navarro A (2003) Molecular cloning andcharacterization of a sodium-pump ATPase of the moss Physcomitrellapatens The Plant Journal 36 382ndash389 doi101046j1365-313X200301883x

Bisson MA Kirst GO (1995) Osmotic acclimation and turgor pressureregulation in algae Naturwissenschaften 82 461ndash471 doi101007BF01131597

Britto DT Kronzucker HJ (2009) Ussingrsquos conundrum and the search fortransport mechanisms in plants New Phytologist 183 243ndash246doi101111j1469-8137200902872x

Cambrolle J Redondo-Gomez S Mateos-Naranjo E Figueroa ME (2008)Comparison of the role of two Spartina species in terms ofphytostabilization and bioaccumulation of metals in the estuarinesediment Marine Pollution Bulletin 56 2037ndash2042 doi101016jmarpolbul200808008

Carroll C Tucker A (2000) Effects of pasture cover on soil erosion andwater quality on central Queensland coal mine rehabilitation TropicalGrasslands 34 254ndash262

Channing A Edwards D (2009) Yellowstone hot spring environmentsand the paleo-ecophysiology of Rhynie chert plants towards asynthesis Plant Ecology amp Diversity 2 111ndash143 doi10108017550870903349359

Colmer TD Flowers TJ (2008) Flooding tolerance in halophytes NewPhytologist 179 964ndash974 doi101111j1469-8137200802483x

Colmer TD Voesenek L (2009) Flooding tolerance suites of plant traitsin variable environments Functional Plant Biology 36 665ndash681doi101071FP09144

Colmer TD Munns R Flowers TJ (2005) Improving salt tolerance of wheatand barley future prospects Australian Journal of ExperimentalAgriculture 45 1425ndash1443 doi101071EA04162

Crayn DM Winter K Smith JAC (2004) Multiple origins of crassulaceanacid metabolism and the epiphytic habit in the neotropical familyBromeliaceae Proceedings of the National Academy of Sciences ofthe United States of America 101 3703ndash3708 doi101073pnas0400366101

Dedolph C Hettel G (Eds) (1997) lsquoPartners making a differencersquo(International Rice Research Institute Manila)

El Shaer HM (2003) Potential of halophytes as animal fodder in EgyptIn lsquoTasks for vegetation science 38 cash crop halophytesrsquo (Eds H LiethM Mochtchenko) pp 111ndash119 (Kluwer Dordrecht the Netherlands)

Flowers TJ (1985) Physiology of halophytes Plant and Soil 89 41ndash56doi101007BF02182232

Flowers TJ (2004) Improving crop salt tolerance Journal of ExperimentalBotany 55 307ndash319 doi101093jxberh003

Flowers TJ Colmer TD (2008) Salinity tolerance in halophytes NewPhytologist 179 945ndash963 doi101111j1469-8137200802531x

Flowers TJ Flowers SA (2005) Why does salinity pose such a difficultproblem for plant breeders Agricultural Water Management 78 15ndash24doi101016jagwat200504015

Flowers TJ Yeo AR (1995) Breeding for salinity resistance in crop plants ndashwhere next Australian Journal of Plant Physiology 22 875ndash884doi101071PP9950875

Flowers TJ Troke PF Yeo AR (1977) The mechanism of salt tolerance inhalophytesAnnual Review of Plant Physiology 28 89ndash121 doi101146annurevpp28060177000513

Flowers TJ Hajibagheri MA Clipson NJW (1986) Halophytes TheQuarterly Review of Biology 61 313ndash337 doi101086415032

Flowers TJGaur PMLaxmipathiGowdaCLKrishnamurthy L Samineni SSiddique KHM Turner NC Vadez V Varshney RK Colmer TD (2010)Salt sensitivity in chickpea Plant Cell amp Environment 33 490ndash509doi101111j1365-3040200902051x

Fogel BN Crain CM Bertness MD (2004) Community level engineeringeffects of Triglochin maritima (seaside arrowgrass) in a salt marsh innorthern New England USA Journal of Ecology 92 589ndash597doi101111j0022-0477200400903x

Garbary DJ Miller AG Scrosati R Kim KY Schofield WB (2008)Distribution and salinity tolerance of intertidal mosses from NovaScotian salt marshes The Bryologist 111 282ndash291 doi1016390007-2745(2008)111[282DASTOI]20CO2

Garciadeblas B Benito B Rodriacuteguez-Navarro A (2001) Plant cells expressseveral stress calcium ATPases but apparently no sodium ATPase Plantand Soil 235 181ndash192 doi101023A1011949626191

Garciadeblaacutes B Haro R Benito B (2007) Cloning of two SOS1transporters from the seagrass Cymodocea nodosa SOS1transporters from Cymodocea and Arabidopsis mediate potassiumuptake in bacteria Plant Molecular Biology 63 479ndash490doi101007s11103-006-9102-2

Glenn EP Oleary JW Watson MC Thompson TL Kuehl RO (1991)Salicornia-bigelovii Torr ndash an oilseed halophyte for seawater irrigationScience 251 1065ndash1067 doi101126science25149971065

Graham LE (1993) lsquoOrigin of land plantsrsquo (John Wiley amp Sons New York)Harvey HW (1966) lsquoThe chemistry and fertility of sea watersrsquo (University

Press Cambridge)Horodyski RJ Knauth LP (1994) Life on land in the Precambrian Science

263 494ndash498 doi101126science2635146494HuangSSpielmeyerWLagudahESMunnsR (2008)Comparativemapping

of HKT genes in wheat barley and rice key determinants of Na+transport and salt tolerance Journal of Experimental Botany 59927ndash937 doi101093jxbern033

Evolution of halophytes Functional Plant Biology 611

glands that originally performed some other function is unclearbut it is difficult at least in the Poaceae to get glandular hairs onnon-halophytes (such as Zea mays L) to secrete salt (Ramadanand Flowers 2004)

Compatible solutes

The compatible solutes synthesised by halophytes range fromquaternary ammonium compounds through sulfonium analoguesto proline and sugar alcoholsmost arewidely distributed throughthe orders of flowering plants reflecting both phylogeny andfunctional needs (Flowers and Colmer 2008) There is no clearpattern of particular solutes being exclusive to particular ordersother than of sorbitol to the Plantaginaceae so that it appearsthat although the ability to synthesise and compartmentalise acompatible solute is a requirement in halophytes the nature ofthat solute depends on the habitat and the availability of NCompatible solutes comprise a small proportion of the totalsolutes accumulated by halophytes (Yeo 1983)

How many halophytes are there

As a trait salt tolerance has been recognised in plants for morethan 200 years (Flowers et al 1986) and there have been severaldefinitions of what constitutes a lsquohalophytersquo Angiospermspecies show a wide range of levels of salt tolerance frominhibition of growth at low salt concentrations (eg sensitivechickpea genotypes dying in 25mM NaCl Flowers et al 2010)to tolerance of salt concentrations as high or higher thanseawater (eg the halophyte Arthrocnemum macrostachyumsurvives in up to 1000mM NaCl Khan et al 2005) Anydefinition of a lsquohalophytersquo must include a threshold value ofsalt concentration and the number of halophytes clearly dependson the salt concentration used in that definition By setting adividing line between halophytes and glycophytes (salt sensitiveplants) at a salt concentration of ~80mM NaCl (a conductivityin the soil solution of 78 dS mndash1) Aronson (1989) listed ~1550salt-tolerant plants Using the same definition Menzel and Lieth(2003) recorded over 2600 species These comprehensive lists

(a) (b)

Fig 2 The distribution of halophytes in phylogenetic trees by Stevens (2001) based on species listed byAronson (1989) (a) Asterales and (b) CaryophyllalesThose orders containing halophytes are boxed in red and the white circle indicates some species within the order have salt glands For (a) denotes brancheswith 100 posterior probability and curren denotes branches with 70ndash80 jackknife support all other branches have gt80 support (according to Stevens 2001)In (b) denotes nodeswith 50ndash80bootstrap support unmarked branches havegt80support [1] and [3] have 50ndash80bootstrap support inmatK tree only and[2] is not recognised at all in matK tree only (according to Stevens 2001)

Evolution of halophytes Functional Plant Biology 607

define salt tolerant plants using a salt concentration that ismuch lower than that present in seawaters (an averageof ~480mM Na+ and 580mM Clndash) Since there are speciesthat have evolved tolerance to seawater salinities we havechosen to use a salt concentration of lsquo~200mM saltrsquo as ourdefining limit (as did Flowers and Colmer 2008) precisionis not required as salt concentrations in natural habitatsfluctuate with evaporation and precipitation of waterApplying the definition of lsquoplants that survive to completetheir life cycle in at least 200mM saltrsquo to Aronsonrsquos (1989)database generates a list of some 350 species which wecall lsquohalophytesrsquo (whether this group of the most salttolerant of plant species should be given a separate namesuch as lsquoeuhalophytersquo is an issue that could be furtherdebated) Our halophytes are not uniformly distributedacross the taxa of flowering plants listed by Stevens (2001)The orders with the greatest number of halophytes(Table 1) are the Caryophyllales (which includes thosespecies that were formerly in the Chenopodiaceae such asthe saltbushes in the genus Atriplex genera with reducedleaves such Salicornia and Tectomia and succulent membersof the genus Suaeda) and the Alismatales (which includesmarine flowering plants belonging to genera such as Zosteraand Thalassia) Although our list of halophytes is notcomprehensive as the limits of tolerance for many species arenot known our analysis suggests that halophytes are notmore than ~025 of the known species of angiosperms

Salt tolerance in bryophytes and pteridophytes

Halophytes are uncommon among the higher plants and thissituation is also true for bryophytes and pteridophytes There aresome mosses that occur in areas that are inundated by tides(Table 2) for example four species in Northern England(Adam 1976) and five species in Nova Scotia one of whichhas been shown to survive immersion in seawater for 24 h(Garbary et al 2008) Aronson (1989) lists only one genus ofa salt resistant fern This low frequency of salt tolerance inbryophytes and pteridophytes indicates that they also haverarely evolved the ability to tolerate salt concentrationsapproaching those of seawater although some are able to growin lower concentrations (eg Physcomitrella patens in 100mMNaCl see Lunde et al 2007)

Repeated evolution of salt-tolerance in angiosperms

In an earlier analysis based on the salt tolerance criterion usedby Aronson (1989) and a phylogeny constructed by Takhtajan(1969) Flowers et al (1977) concluded that salt tolerance waswidely distributed amongst the families of flowering plants andsuggested a polyphyletic origin something more recentlyestablished for other complex traits such as C4 photosynthesis(Sage 2004) and crassulacean acid metabolism (Crayn et al2004) In Fig 1 we have plotted the distribution of cladescontaining halophytes (as defined above) on an order-levelphylogeny of angiosperms based on the main tree published

Table 1 The distribution of halophytes amongst orders of flowering plantsThe estimates of species frequency are approximate due to the inexact numbers of specieswithin families the change in taxonomy that hasoccurred since the species were listed by Aronson (1989) and as the limits of tolerance for many species are not known Examples of

halophytes and suggestions for model species can be found in Flowers and Colmer (2008)

Order Number offamilies

Number offamilies withhalophytes

Families withhalophytes within

the order

Number ofhalophyticspecies

of all halophyticspecies (345)A

Caryophyllales 34 9 26 74 214Alismatales 14 7 50 61 177Malpighiales 40 4 10 35 101Poales 16 4 25 28 81Lamiales 25 5 20 24 70Myrtales 12 4 33 22 64Fabales 4 1 25 21 61Malvales 10 1 10 18 52Ericales 25 6 24 13 38Arecales 1 1 100 11 32Gentianales 5 4 80 9 26Sapindales 9 3 33 8 23Asterales 11 2 18 8 23Zygophyllales 2 1 50 3 09Apiales 7 1 14 3 09Magnoliales 6 1 17 2 06Solanales 5 1 20 2 06Celastrales 4 1 25 1 03Fagales 8 1 13 1 03Brassicales 18 1 6 1 03

Totals 256 58 ndash 345 100

A345 is the total number of halophytes included in this analysis

608 Functional Plant Biology T J Flowers et al

by Stevens (Stevens 2001) From this order-level tree we can seethat salt tolerance seems to have evolved multiple times

We cannot determine the exact number of origins of salttolerance from this phylogeny for several reasons Firstwithout information on the number of halophytes within eachorder and their relationships to each other we cannot knowwhether each family containing halophytes represents a singleorigin or multiple origins (see below) Second if a clade containsboth halophytes and non-halophytes we cannot be sure whethersalt tolerance was gained independently in multiple lineages orwhether the condition is ancestral to the clade and then lost insome families Third the evidence for the presence of halophytesin some orders is based on incomplete data (eg there are just twospecies reported as able to grow in seawater in the Magnoliales(Annonaceae) and without any physiological data) In mostorders that contain families with halophytes the percentage ofhalophytes is 1or less (Table 1)whichmakes it unlikely that thecondition is ancestral and the non-halophytes lost salt-toleranceTherefore it seems likely that virtually each of the red boxes inFig 1 represents at least one independent origin of salt tolerance

Many of the orders marked in Fig 1 may contain multipleorigins of halophytes For examplewithin theAsterales (Fig 2a)we have recorded eight halophytes in two families (seven in theAsteraceae and one in the Goodeniaceae) but the remainingfamilies do not have any halophytes It is parsimonious to assumethat at least two lineages within the Asterales evolved salttolerance independently Examining the presences of particulartraits associated with salt tolerance may also reveal someinteresting evolutionary patterns For example five of the ninesalt-tolerant lineages within the Caryophyllales use salt glands toexcrete excess salt from the leaves This illustrates how evenrelated lineages may develop different strategies for dealing withenvironmental salt The distribution of salt glands within thephylogeny (Fig 2b) suggests that even within this single clade offlowering plants a key salt tolerance mechanism has evolvedindependently several times

It also seems likely that the distribution of halophyte-containing families is not random over the phylogeny with

some clades containing more putative origins of salt tolerancethan others If true this would seem to offer support for the ideathat some clades are more likely to give rise to halophytes thanothers It may be profitable to look at the physiology of membersof those clades to see if they share certain characteristics thatmakeadapting to environmental salt easier than in other cladesMapping key components of salt tolerance on phylogeniesmay help to reveal whether some lineages are predisposed toevolving particular solutions to salinity whether some traits thatconfer salt tolerance aremore easily acquired than others or evenwhether the component traits of salt tolerance are usually acquiredin a particular order Looking at the patterns of origination withinfamilies will be important for answering the questions of whetherthere can be an ancestral condition that favours the evolutionof salt tolerance

Importance of halophytes to agricultureand land management

Salinity is an expanding problem More than 800million ha ofland is salt-affected which is over 6 of the worldrsquos land area(Flowers and Yeo 1995) Salt-affected land is increasingworldwide through vegetation clearance and irrigation both ofwhich raise the watertable bringing dissolved salts to the surfaceIt is estimated that up to half of irrigation schemes worldwideare affected by salinity (Flowers and Yeo 1995) Althoughirrigated land is a relatively small proportion of the total globalareaof foodproduction it produces a third of the food (Munns andTester 2008) Salt stress has been identified as one of the mostserious environmental factors limiting the productivity of cropplants (Flowers and Yeo 1995 Barkla et al 1999) with a hugeimpact on agricultural productivity The global annual cost ofsalt-affected land is likely to be well over US$12 billion (Qadiret al 2008) Future agricultural production will rely increasinglyon our ability to grow food and fibre plants in salt-affected land(Rozema and Flowers 2008 Qadir et al 2008)

Breeding for salt tolerance

Although it is abundantly clear that at some level salinity reducesthe growth of plants the reason is generally not clear Both Na+

and Clndash can reach toxic concentrations if not compartmentalisedin vacuoles but within the complexity of the whole plant theexact cause of any damage is generally unknown However theconsequences are that salinity reduces plant growth and enhancessenescence ofmature leaves resulting in a reduction in functionalleaf area in crop plants this decreases yield (Munns et al 2006Munns and Tester 2008) There is therefore a great incentive toincrease the salt tolerance of crop plants to allow greater yields insalt-affected soils However breeding of salt-tolerant crops ishampered by the complexity of salt tolerance as seen inhalophytes (see above) So despite a large amount of effortthat has been directed towards increasing the salt tolerance ofcrops ranging fromwide-crossing to transgenics few productivesalt-tolerant varieties have been produced (Flowers and Yeo1995 Flowers 2004 Flowers and Flowers 2005 Witcombeet al 2008) The realisation that halophytes have evolvedseveral times should encourage attempts to generate new salt-tolerant crops and may assist plant breeders in several waysFirst itmay illuminate specific traits associatedwith salt tolerance

Table 2 Salt tolerant mosses and ferns

Species names Location Reference

MossesPottia heimi

Amblystegium serpensEurhynchium praelongumCratoneuron filiciniumand C stellatumA

Northern England Adam (1976)

Campylium stellatumBryum capillareDidymodon rigidulusMnium hornumand Am serpens

Nova Scotia Garbary et al (2008)

FernsAcrostichum aureum

Ar danaeifolium andAr Speciosum

Tropics Aronson (1989)

ACratoneuron stellatumwas shown to survive immersion in seawater for 24 h(Garbary et al 2008)

Evolution of halophytes Functional Plant Biology 609

that may be selected in established crop plants (eg Huang et al2008) second it may lead to the domestication of naturally salttolerant species as crop plants

Halophytes as crops

Naturally salt-tolerant species are now being promoted inagriculture particularly to provide forage medicinal plantsaromatic plants (Qadir et al 2008) and for forestry (Marcarand Crawford 2004) For example Barrett-Lennard (2002)identified 26 salt-tolerant plant species capable of producingproducts (or services) of value to agriculture in AustraliaExamples of useful halophytes include the potential oil-seedcrops Kosteletzkya virginica (eg Ruan et al 2008) Salvadorapersica (eg Reddy et al 2008) Salicornia bigelovii (Glenn et al1991) and Batis maritima (Marcone 2003) fodder crops such asAtriplex spp (egEl Shaer 2003)Distichlis palmeri (egMasterset al 2007) and biofuels (Y Waisel pers comm Qadir et al2008) Growing salt-tolerant biofuel crops on marginalagricultural land would help to counter concerns that thebiofuel industry reduces the amount of land available forfood production (Qadir et al 2008) At the extreme plants thatcan grow productively at very high salt levels could be irrigatedwith brackish water or seawater (Rozema and Flowers 2008)Although plants that put resources (cf Yeo 1983) intodeveloping salt-tolerance mechanisms (eg the production ofcompatible solutes to maintain osmotic balance is an energeticcost) may do so at the expense of other functions manyhalophytes show optimal growth in saline conditions (Flowersand Colmer 2008) and salt marshes have high productivity(Colmer and Flowers 2008 Flowers and Colmer 2008) Thefact that dicolytedonous halophytes can grow at similar rates toglycophytes (see Flowers and Colmer 2008 for a more detaileddiscussion) suggests that salt tolerance per se will not limitproductivity Here the contrast with drought tolerance is starkwithout water plants do not grow but may survive with saltwater some plants can grow well

Apart from direct use as crops we may increasingly need torely on halophytes for revegetation and remediation of salt-affected land Over the last 200 years industrialisation inEurope and elsewhere has lead to an enormous increase ofproduction use and release of traces of heavy metals into theenvironment A large portion of these toxic materials includingCd Cu Pb and Zn accumulate in sediments including the soilsof tidal marshes Recent studies showed that some seagrassesand salt marsh plants are capable of extracting heavy metalsfrom sediments and accumulating them in belowground oraboveground tissues (Weis and Weis 2004 Cambrolle et al2008 Lewis and Devereux 2009) The processes and potentialapplication of these aquatic halophytes merits much greaterresearch and development Growing salt-tolerant plantsincluding species of Kochia Bassia Cynodon MedicagoPortulaca Sesbania and Brachiaria may also improve othersoil properties such as increasing water conductance orincreasing soil fertility (Qadir et al 2008) Halophytes mayalso lower the watertable thereby allowing growth of salt-sensitive species in salt-affected land (Barrett-Lennard 2002)

Finally conservation and land management in salt-affectedregions will benefit from improved knowledge of the salt

tolerance of local species Halophytes are being targeted forlsquoecosystem engineeringrsquo projects such as stabilising wetlandsto increase the success of revegetation programs (Fogel et al2004) Halophytes are also being used to develop sustainableagricultural practise for example using indigenous halophytesto restore overgrazed pasture and to reduce reliance on irrigation(Peacock et al 2003) Salt-tolerant species are increasinglybeing considered for landscape remediation For example wellplanned revegetation can reduceboth the in situ salt concentrationat mine sites and also reduce the movement of salts from themine sites in runoff (Carroll and Tucker 2000) However caremust be taken in introducing species for bioremediation as theymay become an environmental problem in their own right Forexample Kochia planted for forage and bioremediation inWestern Australia in 1990 soon spread and was declared aweed in 1992 (Qadir et al 2008)

The halophyte paradox

In this review we have shown that salt tolerance has evolvedmany different times in angiosperms and in different ways Wehave also noted that despite increasing salinisation of agriculturalland few salt-tolerant crops have been produced This raises animportant paradox if all plants contain the basic physiologicalmechanisms on which salt tolerance is based and if halophyteshave evolved in many different lineages using a variety ofadaptations thenwhy is it so difficult to breed salt-tolerant crops

The reasons for the slowprogress in changing the salt toleranceof plants were discussed by Flowers andYeo (1995) who arguedthat this was because salinity had not become a problem ofsufficient priority that the necessary resource was beingapplied to its solution Moreover they also concluded thattreating salinity resistance as a single character could accountfor the limited success We have already established that the salttolerance of halophytes is a multi-genic trait and the same is truefor non-halophytes (Flowers 2004) It is not our intention hereto rehearse the arguments over approaches to breeding forcomplex traits However it is clear that breeding for yield hasnot delivered many salt-resistant varieties of the commoncrops (Flowers 2004 Witcombe et al 2008) The probabilityof combining two complex traits ndash yield and salt tolerance ndash tomaximise yield under saline conditions is evidentially very lowAs a consequence Yeo et al (1990) advocated understandingthe physiology of tolerance and pooling a range of traits anapproach that had some success for rice (Dedolph and Hettel1997) A similar approach has been advocated for durum wheatcombining traits for regulating sodiumaccumulation and osmoticadjustment (Munns et al 2002 James et al 2006 2008) Thetraits that necessarily need to be pooled for wheat (and barley)includeNa lsquoexclusionrsquo KNa discrimination the retention of ionsin sheaths tissue tolerance ion partitioning into different leavesosmotic adjustment enhanced vigour water use efficiency andearly flowering (Colmer et al 2005) In addition other traits suchas tolerance to waterndashlogging (see Colmer and Flowers 2008 fora discussion of flooding tolerance in halophytes) are vitallyimportant further complicating any breeding program (Colmeret al 2005) Given the complexity of the task it is not surprisingthat the transfer of one or two genes has generally failed to changethe tolerance of transgenic plants what is surprising is that there

610 Functional Plant Biology T J Flowers et al

are instances where such simple gene transfers appear to havealtered yield (Flowers 2004)

Evolutionary studies of halophytes which reconstruct theorigin and development of salt tolerance in a variety of plantlineages may help us to understand why artificial breedinghas failed to produce robust and productive salt tolerant cropsSuch studies may also help us develop new salt-tolerant lines byrevealing the order of acquisition of components of salt toleranceor indicating favourable genetic backgrounds on which salttolerance may be developed By examining the repeatedevolution of this complex trait we may identify particular traitsor conditions that predispose species to evolve a complex multi-faceted trait such as salt tolerance and give rise to halophytelineages More generally this may shed light on the adaptationof angiosperm lineages to extreme environments In order toachieve these ends more information is required on at aminimum the effects of salt on the growth and ion relations ofa wider range of plant species that may prove to be halophytes

Acknowledgements

HKGwould like to than the Egyptian Government for financial support whileat the University of Sussex and our thanks also go to Jessica Thomas andKatherine Byron for assistance in earlier stages of this project

References

Adam P (1976) Occurrence of bryophytes on British salt marshes Journalof Bryology 9 265ndash274

Aronson JA (1989) lsquoHALOPHa data base of salt tolerant plants of the worldrsquo(Office of Arid Land Studies University of Arizona Tucson AZ)

Barkla BJ Vera Estrella R Pantoja O (1999) Towards the production ofsalt-tolerant crops Advances in Experimental Medicine and Biology464 77ndash89

Barrett-Lennard EG (2002) Restoration of saline land through revegetationAgricultural Water Management 53 213ndash226 doi101016S0378-3774(01)00166-4

Becker BMarinB (2009) Streptophyte algae and the origin of embryophytesAnnals of Botany 103 999ndash1004 doi101093aobmcp044

Benito B Rodriguez-Navarro A (2003) Molecular cloning andcharacterization of a sodium-pump ATPase of the moss Physcomitrellapatens The Plant Journal 36 382ndash389 doi101046j1365-313X200301883x

Bisson MA Kirst GO (1995) Osmotic acclimation and turgor pressureregulation in algae Naturwissenschaften 82 461ndash471 doi101007BF01131597

Britto DT Kronzucker HJ (2009) Ussingrsquos conundrum and the search fortransport mechanisms in plants New Phytologist 183 243ndash246doi101111j1469-8137200902872x

Cambrolle J Redondo-Gomez S Mateos-Naranjo E Figueroa ME (2008)Comparison of the role of two Spartina species in terms ofphytostabilization and bioaccumulation of metals in the estuarinesediment Marine Pollution Bulletin 56 2037ndash2042 doi101016jmarpolbul200808008

Carroll C Tucker A (2000) Effects of pasture cover on soil erosion andwater quality on central Queensland coal mine rehabilitation TropicalGrasslands 34 254ndash262

Channing A Edwards D (2009) Yellowstone hot spring environmentsand the paleo-ecophysiology of Rhynie chert plants towards asynthesis Plant Ecology amp Diversity 2 111ndash143 doi10108017550870903349359

Colmer TD Flowers TJ (2008) Flooding tolerance in halophytes NewPhytologist 179 964ndash974 doi101111j1469-8137200802483x

Colmer TD Voesenek L (2009) Flooding tolerance suites of plant traitsin variable environments Functional Plant Biology 36 665ndash681doi101071FP09144

Colmer TD Munns R Flowers TJ (2005) Improving salt tolerance of wheatand barley future prospects Australian Journal of ExperimentalAgriculture 45 1425ndash1443 doi101071EA04162

Crayn DM Winter K Smith JAC (2004) Multiple origins of crassulaceanacid metabolism and the epiphytic habit in the neotropical familyBromeliaceae Proceedings of the National Academy of Sciences ofthe United States of America 101 3703ndash3708 doi101073pnas0400366101

Dedolph C Hettel G (Eds) (1997) lsquoPartners making a differencersquo(International Rice Research Institute Manila)

El Shaer HM (2003) Potential of halophytes as animal fodder in EgyptIn lsquoTasks for vegetation science 38 cash crop halophytesrsquo (Eds H LiethM Mochtchenko) pp 111ndash119 (Kluwer Dordrecht the Netherlands)

Flowers TJ (1985) Physiology of halophytes Plant and Soil 89 41ndash56doi101007BF02182232

Flowers TJ (2004) Improving crop salt tolerance Journal of ExperimentalBotany 55 307ndash319 doi101093jxberh003

Flowers TJ Colmer TD (2008) Salinity tolerance in halophytes NewPhytologist 179 945ndash963 doi101111j1469-8137200802531x

Flowers TJ Flowers SA (2005) Why does salinity pose such a difficultproblem for plant breeders Agricultural Water Management 78 15ndash24doi101016jagwat200504015

Flowers TJ Yeo AR (1995) Breeding for salinity resistance in crop plants ndashwhere next Australian Journal of Plant Physiology 22 875ndash884doi101071PP9950875

Flowers TJ Troke PF Yeo AR (1977) The mechanism of salt tolerance inhalophytesAnnual Review of Plant Physiology 28 89ndash121 doi101146annurevpp28060177000513

Flowers TJ Hajibagheri MA Clipson NJW (1986) Halophytes TheQuarterly Review of Biology 61 313ndash337 doi101086415032

Flowers TJGaur PMLaxmipathiGowdaCLKrishnamurthy L Samineni SSiddique KHM Turner NC Vadez V Varshney RK Colmer TD (2010)Salt sensitivity in chickpea Plant Cell amp Environment 33 490ndash509doi101111j1365-3040200902051x

Fogel BN Crain CM Bertness MD (2004) Community level engineeringeffects of Triglochin maritima (seaside arrowgrass) in a salt marsh innorthern New England USA Journal of Ecology 92 589ndash597doi101111j0022-0477200400903x

Garbary DJ Miller AG Scrosati R Kim KY Schofield WB (2008)Distribution and salinity tolerance of intertidal mosses from NovaScotian salt marshes The Bryologist 111 282ndash291 doi1016390007-2745(2008)111[282DASTOI]20CO2

Garciadeblas B Benito B Rodriacuteguez-Navarro A (2001) Plant cells expressseveral stress calcium ATPases but apparently no sodium ATPase Plantand Soil 235 181ndash192 doi101023A1011949626191

Garciadeblaacutes B Haro R Benito B (2007) Cloning of two SOS1transporters from the seagrass Cymodocea nodosa SOS1transporters from Cymodocea and Arabidopsis mediate potassiumuptake in bacteria Plant Molecular Biology 63 479ndash490doi101007s11103-006-9102-2

Glenn EP Oleary JW Watson MC Thompson TL Kuehl RO (1991)Salicornia-bigelovii Torr ndash an oilseed halophyte for seawater irrigationScience 251 1065ndash1067 doi101126science25149971065

Graham LE (1993) lsquoOrigin of land plantsrsquo (John Wiley amp Sons New York)Harvey HW (1966) lsquoThe chemistry and fertility of sea watersrsquo (University

Press Cambridge)Horodyski RJ Knauth LP (1994) Life on land in the Precambrian Science

263 494ndash498 doi101126science2635146494HuangSSpielmeyerWLagudahESMunnsR (2008)Comparativemapping

of HKT genes in wheat barley and rice key determinants of Na+transport and salt tolerance Journal of Experimental Botany 59927ndash937 doi101093jxbern033

Evolution of halophytes Functional Plant Biology 611

define salt tolerant plants using a salt concentration that ismuch lower than that present in seawaters (an averageof ~480mM Na+ and 580mM Clndash) Since there are speciesthat have evolved tolerance to seawater salinities we havechosen to use a salt concentration of lsquo~200mM saltrsquo as ourdefining limit (as did Flowers and Colmer 2008) precisionis not required as salt concentrations in natural habitatsfluctuate with evaporation and precipitation of waterApplying the definition of lsquoplants that survive to completetheir life cycle in at least 200mM saltrsquo to Aronsonrsquos (1989)database generates a list of some 350 species which wecall lsquohalophytesrsquo (whether this group of the most salttolerant of plant species should be given a separate namesuch as lsquoeuhalophytersquo is an issue that could be furtherdebated) Our halophytes are not uniformly distributedacross the taxa of flowering plants listed by Stevens (2001)The orders with the greatest number of halophytes(Table 1) are the Caryophyllales (which includes thosespecies that were formerly in the Chenopodiaceae such asthe saltbushes in the genus Atriplex genera with reducedleaves such Salicornia and Tectomia and succulent membersof the genus Suaeda) and the Alismatales (which includesmarine flowering plants belonging to genera such as Zosteraand Thalassia) Although our list of halophytes is notcomprehensive as the limits of tolerance for many species arenot known our analysis suggests that halophytes are notmore than ~025 of the known species of angiosperms

Salt tolerance in bryophytes and pteridophytes

Halophytes are uncommon among the higher plants and thissituation is also true for bryophytes and pteridophytes There aresome mosses that occur in areas that are inundated by tides(Table 2) for example four species in Northern England(Adam 1976) and five species in Nova Scotia one of whichhas been shown to survive immersion in seawater for 24 h(Garbary et al 2008) Aronson (1989) lists only one genus ofa salt resistant fern This low frequency of salt tolerance inbryophytes and pteridophytes indicates that they also haverarely evolved the ability to tolerate salt concentrationsapproaching those of seawater although some are able to growin lower concentrations (eg Physcomitrella patens in 100mMNaCl see Lunde et al 2007)

Repeated evolution of salt-tolerance in angiosperms

In an earlier analysis based on the salt tolerance criterion usedby Aronson (1989) and a phylogeny constructed by Takhtajan(1969) Flowers et al (1977) concluded that salt tolerance waswidely distributed amongst the families of flowering plants andsuggested a polyphyletic origin something more recentlyestablished for other complex traits such as C4 photosynthesis(Sage 2004) and crassulacean acid metabolism (Crayn et al2004) In Fig 1 we have plotted the distribution of cladescontaining halophytes (as defined above) on an order-levelphylogeny of angiosperms based on the main tree published

Table 1 The distribution of halophytes amongst orders of flowering plantsThe estimates of species frequency are approximate due to the inexact numbers of specieswithin families the change in taxonomy that hasoccurred since the species were listed by Aronson (1989) and as the limits of tolerance for many species are not known Examples of

halophytes and suggestions for model species can be found in Flowers and Colmer (2008)

Order Number offamilies

Number offamilies withhalophytes

Families withhalophytes within

the order

Number ofhalophyticspecies

of all halophyticspecies (345)A

Caryophyllales 34 9 26 74 214Alismatales 14 7 50 61 177Malpighiales 40 4 10 35 101Poales 16 4 25 28 81Lamiales 25 5 20 24 70Myrtales 12 4 33 22 64Fabales 4 1 25 21 61Malvales 10 1 10 18 52Ericales 25 6 24 13 38Arecales 1 1 100 11 32Gentianales 5 4 80 9 26Sapindales 9 3 33 8 23Asterales 11 2 18 8 23Zygophyllales 2 1 50 3 09Apiales 7 1 14 3 09Magnoliales 6 1 17 2 06Solanales 5 1 20 2 06Celastrales 4 1 25 1 03Fagales 8 1 13 1 03Brassicales 18 1 6 1 03

Totals 256 58 ndash 345 100

A345 is the total number of halophytes included in this analysis

608 Functional Plant Biology T J Flowers et al

by Stevens (Stevens 2001) From this order-level tree we can seethat salt tolerance seems to have evolved multiple times

We cannot determine the exact number of origins of salttolerance from this phylogeny for several reasons Firstwithout information on the number of halophytes within eachorder and their relationships to each other we cannot knowwhether each family containing halophytes represents a singleorigin or multiple origins (see below) Second if a clade containsboth halophytes and non-halophytes we cannot be sure whethersalt tolerance was gained independently in multiple lineages orwhether the condition is ancestral to the clade and then lost insome families Third the evidence for the presence of halophytesin some orders is based on incomplete data (eg there are just twospecies reported as able to grow in seawater in the Magnoliales(Annonaceae) and without any physiological data) In mostorders that contain families with halophytes the percentage ofhalophytes is 1or less (Table 1)whichmakes it unlikely that thecondition is ancestral and the non-halophytes lost salt-toleranceTherefore it seems likely that virtually each of the red boxes inFig 1 represents at least one independent origin of salt tolerance

Many of the orders marked in Fig 1 may contain multipleorigins of halophytes For examplewithin theAsterales (Fig 2a)we have recorded eight halophytes in two families (seven in theAsteraceae and one in the Goodeniaceae) but the remainingfamilies do not have any halophytes It is parsimonious to assumethat at least two lineages within the Asterales evolved salttolerance independently Examining the presences of particulartraits associated with salt tolerance may also reveal someinteresting evolutionary patterns For example five of the ninesalt-tolerant lineages within the Caryophyllales use salt glands toexcrete excess salt from the leaves This illustrates how evenrelated lineages may develop different strategies for dealing withenvironmental salt The distribution of salt glands within thephylogeny (Fig 2b) suggests that even within this single clade offlowering plants a key salt tolerance mechanism has evolvedindependently several times

It also seems likely that the distribution of halophyte-containing families is not random over the phylogeny with

some clades containing more putative origins of salt tolerancethan others If true this would seem to offer support for the ideathat some clades are more likely to give rise to halophytes thanothers It may be profitable to look at the physiology of membersof those clades to see if they share certain characteristics thatmakeadapting to environmental salt easier than in other cladesMapping key components of salt tolerance on phylogeniesmay help to reveal whether some lineages are predisposed toevolving particular solutions to salinity whether some traits thatconfer salt tolerance aremore easily acquired than others or evenwhether the component traits of salt tolerance are usually acquiredin a particular order Looking at the patterns of origination withinfamilies will be important for answering the questions of whetherthere can be an ancestral condition that favours the evolutionof salt tolerance

Importance of halophytes to agricultureand land management

Salinity is an expanding problem More than 800million ha ofland is salt-affected which is over 6 of the worldrsquos land area(Flowers and Yeo 1995) Salt-affected land is increasingworldwide through vegetation clearance and irrigation both ofwhich raise the watertable bringing dissolved salts to the surfaceIt is estimated that up to half of irrigation schemes worldwideare affected by salinity (Flowers and Yeo 1995) Althoughirrigated land is a relatively small proportion of the total globalareaof foodproduction it produces a third of the food (Munns andTester 2008) Salt stress has been identified as one of the mostserious environmental factors limiting the productivity of cropplants (Flowers and Yeo 1995 Barkla et al 1999) with a hugeimpact on agricultural productivity The global annual cost ofsalt-affected land is likely to be well over US$12 billion (Qadiret al 2008) Future agricultural production will rely increasinglyon our ability to grow food and fibre plants in salt-affected land(Rozema and Flowers 2008 Qadir et al 2008)

Breeding for salt tolerance

Although it is abundantly clear that at some level salinity reducesthe growth of plants the reason is generally not clear Both Na+

and Clndash can reach toxic concentrations if not compartmentalisedin vacuoles but within the complexity of the whole plant theexact cause of any damage is generally unknown However theconsequences are that salinity reduces plant growth and enhancessenescence ofmature leaves resulting in a reduction in functionalleaf area in crop plants this decreases yield (Munns et al 2006Munns and Tester 2008) There is therefore a great incentive toincrease the salt tolerance of crop plants to allow greater yields insalt-affected soils However breeding of salt-tolerant crops ishampered by the complexity of salt tolerance as seen inhalophytes (see above) So despite a large amount of effortthat has been directed towards increasing the salt tolerance ofcrops ranging fromwide-crossing to transgenics few productivesalt-tolerant varieties have been produced (Flowers and Yeo1995 Flowers 2004 Flowers and Flowers 2005 Witcombeet al 2008) The realisation that halophytes have evolvedseveral times should encourage attempts to generate new salt-tolerant crops and may assist plant breeders in several waysFirst itmay illuminate specific traits associatedwith salt tolerance

Table 2 Salt tolerant mosses and ferns

Species names Location Reference

MossesPottia heimi

Amblystegium serpensEurhynchium praelongumCratoneuron filiciniumand C stellatumA

Northern England Adam (1976)

Campylium stellatumBryum capillareDidymodon rigidulusMnium hornumand Am serpens

Nova Scotia Garbary et al (2008)

FernsAcrostichum aureum

Ar danaeifolium andAr Speciosum

Tropics Aronson (1989)

ACratoneuron stellatumwas shown to survive immersion in seawater for 24 h(Garbary et al 2008)

Evolution of halophytes Functional Plant Biology 609

that may be selected in established crop plants (eg Huang et al2008) second it may lead to the domestication of naturally salttolerant species as crop plants

Halophytes as crops

Naturally salt-tolerant species are now being promoted inagriculture particularly to provide forage medicinal plantsaromatic plants (Qadir et al 2008) and for forestry (Marcarand Crawford 2004) For example Barrett-Lennard (2002)identified 26 salt-tolerant plant species capable of producingproducts (or services) of value to agriculture in AustraliaExamples of useful halophytes include the potential oil-seedcrops Kosteletzkya virginica (eg Ruan et al 2008) Salvadorapersica (eg Reddy et al 2008) Salicornia bigelovii (Glenn et al1991) and Batis maritima (Marcone 2003) fodder crops such asAtriplex spp (egEl Shaer 2003)Distichlis palmeri (egMasterset al 2007) and biofuels (Y Waisel pers comm Qadir et al2008) Growing salt-tolerant biofuel crops on marginalagricultural land would help to counter concerns that thebiofuel industry reduces the amount of land available forfood production (Qadir et al 2008) At the extreme plants thatcan grow productively at very high salt levels could be irrigatedwith brackish water or seawater (Rozema and Flowers 2008)Although plants that put resources (cf Yeo 1983) intodeveloping salt-tolerance mechanisms (eg the production ofcompatible solutes to maintain osmotic balance is an energeticcost) may do so at the expense of other functions manyhalophytes show optimal growth in saline conditions (Flowersand Colmer 2008) and salt marshes have high productivity(Colmer and Flowers 2008 Flowers and Colmer 2008) Thefact that dicolytedonous halophytes can grow at similar rates toglycophytes (see Flowers and Colmer 2008 for a more detaileddiscussion) suggests that salt tolerance per se will not limitproductivity Here the contrast with drought tolerance is starkwithout water plants do not grow but may survive with saltwater some plants can grow well

Apart from direct use as crops we may increasingly need torely on halophytes for revegetation and remediation of salt-affected land Over the last 200 years industrialisation inEurope and elsewhere has lead to an enormous increase ofproduction use and release of traces of heavy metals into theenvironment A large portion of these toxic materials includingCd Cu Pb and Zn accumulate in sediments including the soilsof tidal marshes Recent studies showed that some seagrassesand salt marsh plants are capable of extracting heavy metalsfrom sediments and accumulating them in belowground oraboveground tissues (Weis and Weis 2004 Cambrolle et al2008 Lewis and Devereux 2009) The processes and potentialapplication of these aquatic halophytes merits much greaterresearch and development Growing salt-tolerant plantsincluding species of Kochia Bassia Cynodon MedicagoPortulaca Sesbania and Brachiaria may also improve othersoil properties such as increasing water conductance orincreasing soil fertility (Qadir et al 2008) Halophytes mayalso lower the watertable thereby allowing growth of salt-sensitive species in salt-affected land (Barrett-Lennard 2002)

Finally conservation and land management in salt-affectedregions will benefit from improved knowledge of the salt

tolerance of local species Halophytes are being targeted forlsquoecosystem engineeringrsquo projects such as stabilising wetlandsto increase the success of revegetation programs (Fogel et al2004) Halophytes are also being used to develop sustainableagricultural practise for example using indigenous halophytesto restore overgrazed pasture and to reduce reliance on irrigation(Peacock et al 2003) Salt-tolerant species are increasinglybeing considered for landscape remediation For example wellplanned revegetation can reduceboth the in situ salt concentrationat mine sites and also reduce the movement of salts from themine sites in runoff (Carroll and Tucker 2000) However caremust be taken in introducing species for bioremediation as theymay become an environmental problem in their own right Forexample Kochia planted for forage and bioremediation inWestern Australia in 1990 soon spread and was declared aweed in 1992 (Qadir et al 2008)

The halophyte paradox

In this review we have shown that salt tolerance has evolvedmany different times in angiosperms and in different ways Wehave also noted that despite increasing salinisation of agriculturalland few salt-tolerant crops have been produced This raises animportant paradox if all plants contain the basic physiologicalmechanisms on which salt tolerance is based and if halophyteshave evolved in many different lineages using a variety ofadaptations thenwhy is it so difficult to breed salt-tolerant crops

The reasons for the slowprogress in changing the salt toleranceof plants were discussed by Flowers andYeo (1995) who arguedthat this was because salinity had not become a problem ofsufficient priority that the necessary resource was beingapplied to its solution Moreover they also concluded thattreating salinity resistance as a single character could accountfor the limited success We have already established that the salttolerance of halophytes is a multi-genic trait and the same is truefor non-halophytes (Flowers 2004) It is not our intention hereto rehearse the arguments over approaches to breeding forcomplex traits However it is clear that breeding for yield hasnot delivered many salt-resistant varieties of the commoncrops (Flowers 2004 Witcombe et al 2008) The probabilityof combining two complex traits ndash yield and salt tolerance ndash tomaximise yield under saline conditions is evidentially very lowAs a consequence Yeo et al (1990) advocated understandingthe physiology of tolerance and pooling a range of traits anapproach that had some success for rice (Dedolph and Hettel1997) A similar approach has been advocated for durum wheatcombining traits for regulating sodiumaccumulation and osmoticadjustment (Munns et al 2002 James et al 2006 2008) Thetraits that necessarily need to be pooled for wheat (and barley)includeNa lsquoexclusionrsquo KNa discrimination the retention of ionsin sheaths tissue tolerance ion partitioning into different leavesosmotic adjustment enhanced vigour water use efficiency andearly flowering (Colmer et al 2005) In addition other traits suchas tolerance to waterndashlogging (see Colmer and Flowers 2008 fora discussion of flooding tolerance in halophytes) are vitallyimportant further complicating any breeding program (Colmeret al 2005) Given the complexity of the task it is not surprisingthat the transfer of one or two genes has generally failed to changethe tolerance of transgenic plants what is surprising is that there

610 Functional Plant Biology T J Flowers et al

are instances where such simple gene transfers appear to havealtered yield (Flowers 2004)

Evolutionary studies of halophytes which reconstruct theorigin and development of salt tolerance in a variety of plantlineages may help us to understand why artificial breedinghas failed to produce robust and productive salt tolerant cropsSuch studies may also help us develop new salt-tolerant lines byrevealing the order of acquisition of components of salt toleranceor indicating favourable genetic backgrounds on which salttolerance may be developed By examining the repeatedevolution of this complex trait we may identify particular traitsor conditions that predispose species to evolve a complex multi-faceted trait such as salt tolerance and give rise to halophytelineages More generally this may shed light on the adaptationof angiosperm lineages to extreme environments In order toachieve these ends more information is required on at aminimum the effects of salt on the growth and ion relations ofa wider range of plant species that may prove to be halophytes

Acknowledgements

HKGwould like to than the Egyptian Government for financial support whileat the University of Sussex and our thanks also go to Jessica Thomas andKatherine Byron for assistance in earlier stages of this project

References

Adam P (1976) Occurrence of bryophytes on British salt marshes Journalof Bryology 9 265ndash274

Aronson JA (1989) lsquoHALOPHa data base of salt tolerant plants of the worldrsquo(Office of Arid Land Studies University of Arizona Tucson AZ)

Barkla BJ Vera Estrella R Pantoja O (1999) Towards the production ofsalt-tolerant crops Advances in Experimental Medicine and Biology464 77ndash89

Barrett-Lennard EG (2002) Restoration of saline land through revegetationAgricultural Water Management 53 213ndash226 doi101016S0378-3774(01)00166-4

Becker BMarinB (2009) Streptophyte algae and the origin of embryophytesAnnals of Botany 103 999ndash1004 doi101093aobmcp044

Benito B Rodriguez-Navarro A (2003) Molecular cloning andcharacterization of a sodium-pump ATPase of the moss Physcomitrellapatens The Plant Journal 36 382ndash389 doi101046j1365-313X200301883x

Bisson MA Kirst GO (1995) Osmotic acclimation and turgor pressureregulation in algae Naturwissenschaften 82 461ndash471 doi101007BF01131597

Britto DT Kronzucker HJ (2009) Ussingrsquos conundrum and the search fortransport mechanisms in plants New Phytologist 183 243ndash246doi101111j1469-8137200902872x

Cambrolle J Redondo-Gomez S Mateos-Naranjo E Figueroa ME (2008)Comparison of the role of two Spartina species in terms ofphytostabilization and bioaccumulation of metals in the estuarinesediment Marine Pollution Bulletin 56 2037ndash2042 doi101016jmarpolbul200808008

Carroll C Tucker A (2000) Effects of pasture cover on soil erosion andwater quality on central Queensland coal mine rehabilitation TropicalGrasslands 34 254ndash262

Channing A Edwards D (2009) Yellowstone hot spring environmentsand the paleo-ecophysiology of Rhynie chert plants towards asynthesis Plant Ecology amp Diversity 2 111ndash143 doi10108017550870903349359

Colmer TD Flowers TJ (2008) Flooding tolerance in halophytes NewPhytologist 179 964ndash974 doi101111j1469-8137200802483x

Colmer TD Voesenek L (2009) Flooding tolerance suites of plant traitsin variable environments Functional Plant Biology 36 665ndash681doi101071FP09144

Colmer TD Munns R Flowers TJ (2005) Improving salt tolerance of wheatand barley future prospects Australian Journal of ExperimentalAgriculture 45 1425ndash1443 doi101071EA04162

Crayn DM Winter K Smith JAC (2004) Multiple origins of crassulaceanacid metabolism and the epiphytic habit in the neotropical familyBromeliaceae Proceedings of the National Academy of Sciences ofthe United States of America 101 3703ndash3708 doi101073pnas0400366101

Dedolph C Hettel G (Eds) (1997) lsquoPartners making a differencersquo(International Rice Research Institute Manila)

El Shaer HM (2003) Potential of halophytes as animal fodder in EgyptIn lsquoTasks for vegetation science 38 cash crop halophytesrsquo (Eds H LiethM Mochtchenko) pp 111ndash119 (Kluwer Dordrecht the Netherlands)

Flowers TJ (1985) Physiology of halophytes Plant and Soil 89 41ndash56doi101007BF02182232

Flowers TJ (2004) Improving crop salt tolerance Journal of ExperimentalBotany 55 307ndash319 doi101093jxberh003

Flowers TJ Colmer TD (2008) Salinity tolerance in halophytes NewPhytologist 179 945ndash963 doi101111j1469-8137200802531x

Flowers TJ Flowers SA (2005) Why does salinity pose such a difficultproblem for plant breeders Agricultural Water Management 78 15ndash24doi101016jagwat200504015

Flowers TJ Yeo AR (1995) Breeding for salinity resistance in crop plants ndashwhere next Australian Journal of Plant Physiology 22 875ndash884doi101071PP9950875

Flowers TJ Troke PF Yeo AR (1977) The mechanism of salt tolerance inhalophytesAnnual Review of Plant Physiology 28 89ndash121 doi101146annurevpp28060177000513

Flowers TJ Hajibagheri MA Clipson NJW (1986) Halophytes TheQuarterly Review of Biology 61 313ndash337 doi101086415032

Flowers TJGaur PMLaxmipathiGowdaCLKrishnamurthy L Samineni SSiddique KHM Turner NC Vadez V Varshney RK Colmer TD (2010)Salt sensitivity in chickpea Plant Cell amp Environment 33 490ndash509doi101111j1365-3040200902051x

Fogel BN Crain CM Bertness MD (2004) Community level engineeringeffects of Triglochin maritima (seaside arrowgrass) in a salt marsh innorthern New England USA Journal of Ecology 92 589ndash597doi101111j0022-0477200400903x

Garbary DJ Miller AG Scrosati R Kim KY Schofield WB (2008)Distribution and salinity tolerance of intertidal mosses from NovaScotian salt marshes The Bryologist 111 282ndash291 doi1016390007-2745(2008)111[282DASTOI]20CO2

Garciadeblas B Benito B Rodriacuteguez-Navarro A (2001) Plant cells expressseveral stress calcium ATPases but apparently no sodium ATPase Plantand Soil 235 181ndash192 doi101023A1011949626191

Garciadeblaacutes B Haro R Benito B (2007) Cloning of two SOS1transporters from the seagrass Cymodocea nodosa SOS1transporters from Cymodocea and Arabidopsis mediate potassiumuptake in bacteria Plant Molecular Biology 63 479ndash490doi101007s11103-006-9102-2

Glenn EP Oleary JW Watson MC Thompson TL Kuehl RO (1991)Salicornia-bigelovii Torr ndash an oilseed halophyte for seawater irrigationScience 251 1065ndash1067 doi101126science25149971065

Graham LE (1993) lsquoOrigin of land plantsrsquo (John Wiley amp Sons New York)Harvey HW (1966) lsquoThe chemistry and fertility of sea watersrsquo (University

Press Cambridge)Horodyski RJ Knauth LP (1994) Life on land in the Precambrian Science

263 494ndash498 doi101126science2635146494HuangSSpielmeyerWLagudahESMunnsR (2008)Comparativemapping

of HKT genes in wheat barley and rice key determinants of Na+transport and salt tolerance Journal of Experimental Botany 59927ndash937 doi101093jxbern033

Evolution of halophytes Functional Plant Biology 611

by Stevens (Stevens 2001) From this order-level tree we can seethat salt tolerance seems to have evolved multiple times

We cannot determine the exact number of origins of salttolerance from this phylogeny for several reasons Firstwithout information on the number of halophytes within eachorder and their relationships to each other we cannot knowwhether each family containing halophytes represents a singleorigin or multiple origins (see below) Second if a clade containsboth halophytes and non-halophytes we cannot be sure whethersalt tolerance was gained independently in multiple lineages orwhether the condition is ancestral to the clade and then lost insome families Third the evidence for the presence of halophytesin some orders is based on incomplete data (eg there are just twospecies reported as able to grow in seawater in the Magnoliales(Annonaceae) and without any physiological data) In mostorders that contain families with halophytes the percentage ofhalophytes is 1or less (Table 1)whichmakes it unlikely that thecondition is ancestral and the non-halophytes lost salt-toleranceTherefore it seems likely that virtually each of the red boxes inFig 1 represents at least one independent origin of salt tolerance

Many of the orders marked in Fig 1 may contain multipleorigins of halophytes For examplewithin theAsterales (Fig 2a)we have recorded eight halophytes in two families (seven in theAsteraceae and one in the Goodeniaceae) but the remainingfamilies do not have any halophytes It is parsimonious to assumethat at least two lineages within the Asterales evolved salttolerance independently Examining the presences of particulartraits associated with salt tolerance may also reveal someinteresting evolutionary patterns For example five of the ninesalt-tolerant lineages within the Caryophyllales use salt glands toexcrete excess salt from the leaves This illustrates how evenrelated lineages may develop different strategies for dealing withenvironmental salt The distribution of salt glands within thephylogeny (Fig 2b) suggests that even within this single clade offlowering plants a key salt tolerance mechanism has evolvedindependently several times

It also seems likely that the distribution of halophyte-containing families is not random over the phylogeny with

some clades containing more putative origins of salt tolerancethan others If true this would seem to offer support for the ideathat some clades are more likely to give rise to halophytes thanothers It may be profitable to look at the physiology of membersof those clades to see if they share certain characteristics thatmakeadapting to environmental salt easier than in other cladesMapping key components of salt tolerance on phylogeniesmay help to reveal whether some lineages are predisposed toevolving particular solutions to salinity whether some traits thatconfer salt tolerance aremore easily acquired than others or evenwhether the component traits of salt tolerance are usually acquiredin a particular order Looking at the patterns of origination withinfamilies will be important for answering the questions of whetherthere can be an ancestral condition that favours the evolutionof salt tolerance

Importance of halophytes to agricultureand land management

Salinity is an expanding problem More than 800million ha ofland is salt-affected which is over 6 of the worldrsquos land area(Flowers and Yeo 1995) Salt-affected land is increasingworldwide through vegetation clearance and irrigation both ofwhich raise the watertable bringing dissolved salts to the surfaceIt is estimated that up to half of irrigation schemes worldwideare affected by salinity (Flowers and Yeo 1995) Althoughirrigated land is a relatively small proportion of the total globalareaof foodproduction it produces a third of the food (Munns andTester 2008) Salt stress has been identified as one of the mostserious environmental factors limiting the productivity of cropplants (Flowers and Yeo 1995 Barkla et al 1999) with a hugeimpact on agricultural productivity The global annual cost ofsalt-affected land is likely to be well over US$12 billion (Qadiret al 2008) Future agricultural production will rely increasinglyon our ability to grow food and fibre plants in salt-affected land(Rozema and Flowers 2008 Qadir et al 2008)

Breeding for salt tolerance

Although it is abundantly clear that at some level salinity reducesthe growth of plants the reason is generally not clear Both Na+

and Clndash can reach toxic concentrations if not compartmentalisedin vacuoles but within the complexity of the whole plant theexact cause of any damage is generally unknown However theconsequences are that salinity reduces plant growth and enhancessenescence ofmature leaves resulting in a reduction in functionalleaf area in crop plants this decreases yield (Munns et al 2006Munns and Tester 2008) There is therefore a great incentive toincrease the salt tolerance of crop plants to allow greater yields insalt-affected soils However breeding of salt-tolerant crops ishampered by the complexity of salt tolerance as seen inhalophytes (see above) So despite a large amount of effortthat has been directed towards increasing the salt tolerance ofcrops ranging fromwide-crossing to transgenics few productivesalt-tolerant varieties have been produced (Flowers and Yeo1995 Flowers 2004 Flowers and Flowers 2005 Witcombeet al 2008) The realisation that halophytes have evolvedseveral times should encourage attempts to generate new salt-tolerant crops and may assist plant breeders in several waysFirst itmay illuminate specific traits associatedwith salt tolerance

Table 2 Salt tolerant mosses and ferns

Species names Location Reference

MossesPottia heimi

Amblystegium serpensEurhynchium praelongumCratoneuron filiciniumand C stellatumA

Northern England Adam (1976)

Campylium stellatumBryum capillareDidymodon rigidulusMnium hornumand Am serpens

Nova Scotia Garbary et al (2008)

FernsAcrostichum aureum

Ar danaeifolium andAr Speciosum

Tropics Aronson (1989)

ACratoneuron stellatumwas shown to survive immersion in seawater for 24 h(Garbary et al 2008)

Evolution of halophytes Functional Plant Biology 609

that may be selected in established crop plants (eg Huang et al2008) second it may lead to the domestication of naturally salttolerant species as crop plants

Halophytes as crops

Naturally salt-tolerant species are now being promoted inagriculture particularly to provide forage medicinal plantsaromatic plants (Qadir et al 2008) and for forestry (Marcarand Crawford 2004) For example Barrett-Lennard (2002)identified 26 salt-tolerant plant species capable of producingproducts (or services) of value to agriculture in AustraliaExamples of useful halophytes include the potential oil-seedcrops Kosteletzkya virginica (eg Ruan et al 2008) Salvadorapersica (eg Reddy et al 2008) Salicornia bigelovii (Glenn et al1991) and Batis maritima (Marcone 2003) fodder crops such asAtriplex spp (egEl Shaer 2003)Distichlis palmeri (egMasterset al 2007) and biofuels (Y Waisel pers comm Qadir et al2008) Growing salt-tolerant biofuel crops on marginalagricultural land would help to counter concerns that thebiofuel industry reduces the amount of land available forfood production (Qadir et al 2008) At the extreme plants thatcan grow productively at very high salt levels could be irrigatedwith brackish water or seawater (Rozema and Flowers 2008)Although plants that put resources (cf Yeo 1983) intodeveloping salt-tolerance mechanisms (eg the production ofcompatible solutes to maintain osmotic balance is an energeticcost) may do so at the expense of other functions manyhalophytes show optimal growth in saline conditions (Flowersand Colmer 2008) and salt marshes have high productivity(Colmer and Flowers 2008 Flowers and Colmer 2008) Thefact that dicolytedonous halophytes can grow at similar rates toglycophytes (see Flowers and Colmer 2008 for a more detaileddiscussion) suggests that salt tolerance per se will not limitproductivity Here the contrast with drought tolerance is starkwithout water plants do not grow but may survive with saltwater some plants can grow well

Apart from direct use as crops we may increasingly need torely on halophytes for revegetation and remediation of salt-affected land Over the last 200 years industrialisation inEurope and elsewhere has lead to an enormous increase ofproduction use and release of traces of heavy metals into theenvironment A large portion of these toxic materials includingCd Cu Pb and Zn accumulate in sediments including the soilsof tidal marshes Recent studies showed that some seagrassesand salt marsh plants are capable of extracting heavy metalsfrom sediments and accumulating them in belowground oraboveground tissues (Weis and Weis 2004 Cambrolle et al2008 Lewis and Devereux 2009) The processes and potentialapplication of these aquatic halophytes merits much greaterresearch and development Growing salt-tolerant plantsincluding species of Kochia Bassia Cynodon MedicagoPortulaca Sesbania and Brachiaria may also improve othersoil properties such as increasing water conductance orincreasing soil fertility (Qadir et al 2008) Halophytes mayalso lower the watertable thereby allowing growth of salt-sensitive species in salt-affected land (Barrett-Lennard 2002)

Finally conservation and land management in salt-affectedregions will benefit from improved knowledge of the salt

tolerance of local species Halophytes are being targeted forlsquoecosystem engineeringrsquo projects such as stabilising wetlandsto increase the success of revegetation programs (Fogel et al2004) Halophytes are also being used to develop sustainableagricultural practise for example using indigenous halophytesto restore overgrazed pasture and to reduce reliance on irrigation(Peacock et al 2003) Salt-tolerant species are increasinglybeing considered for landscape remediation For example wellplanned revegetation can reduceboth the in situ salt concentrationat mine sites and also reduce the movement of salts from themine sites in runoff (Carroll and Tucker 2000) However caremust be taken in introducing species for bioremediation as theymay become an environmental problem in their own right Forexample Kochia planted for forage and bioremediation inWestern Australia in 1990 soon spread and was declared aweed in 1992 (Qadir et al 2008)

The halophyte paradox

In this review we have shown that salt tolerance has evolvedmany different times in angiosperms and in different ways Wehave also noted that despite increasing salinisation of agriculturalland few salt-tolerant crops have been produced This raises animportant paradox if all plants contain the basic physiologicalmechanisms on which salt tolerance is based and if halophyteshave evolved in many different lineages using a variety ofadaptations thenwhy is it so difficult to breed salt-tolerant crops

The reasons for the slowprogress in changing the salt toleranceof plants were discussed by Flowers andYeo (1995) who arguedthat this was because salinity had not become a problem ofsufficient priority that the necessary resource was beingapplied to its solution Moreover they also concluded thattreating salinity resistance as a single character could accountfor the limited success We have already established that the salttolerance of halophytes is a multi-genic trait and the same is truefor non-halophytes (Flowers 2004) It is not our intention hereto rehearse the arguments over approaches to breeding forcomplex traits However it is clear that breeding for yield hasnot delivered many salt-resistant varieties of the commoncrops (Flowers 2004 Witcombe et al 2008) The probabilityof combining two complex traits ndash yield and salt tolerance ndash tomaximise yield under saline conditions is evidentially very lowAs a consequence Yeo et al (1990) advocated understandingthe physiology of tolerance and pooling a range of traits anapproach that had some success for rice (Dedolph and Hettel1997) A similar approach has been advocated for durum wheatcombining traits for regulating sodiumaccumulation and osmoticadjustment (Munns et al 2002 James et al 2006 2008) Thetraits that necessarily need to be pooled for wheat (and barley)includeNa lsquoexclusionrsquo KNa discrimination the retention of ionsin sheaths tissue tolerance ion partitioning into different leavesosmotic adjustment enhanced vigour water use efficiency andearly flowering (Colmer et al 2005) In addition other traits suchas tolerance to waterndashlogging (see Colmer and Flowers 2008 fora discussion of flooding tolerance in halophytes) are vitallyimportant further complicating any breeding program (Colmeret al 2005) Given the complexity of the task it is not surprisingthat the transfer of one or two genes has generally failed to changethe tolerance of transgenic plants what is surprising is that there

610 Functional Plant Biology T J Flowers et al

are instances where such simple gene transfers appear to havealtered yield (Flowers 2004)

Evolutionary studies of halophytes which reconstruct theorigin and development of salt tolerance in a variety of plantlineages may help us to understand why artificial breedinghas failed to produce robust and productive salt tolerant cropsSuch studies may also help us develop new salt-tolerant lines byrevealing the order of acquisition of components of salt toleranceor indicating favourable genetic backgrounds on which salttolerance may be developed By examining the repeatedevolution of this complex trait we may identify particular traitsor conditions that predispose species to evolve a complex multi-faceted trait such as salt tolerance and give rise to halophytelineages More generally this may shed light on the adaptationof angiosperm lineages to extreme environments In order toachieve these ends more information is required on at aminimum the effects of salt on the growth and ion relations ofa wider range of plant species that may prove to be halophytes

Acknowledgements

HKGwould like to than the Egyptian Government for financial support whileat the University of Sussex and our thanks also go to Jessica Thomas andKatherine Byron for assistance in earlier stages of this project

References

Adam P (1976) Occurrence of bryophytes on British salt marshes Journalof Bryology 9 265ndash274

Aronson JA (1989) lsquoHALOPHa data base of salt tolerant plants of the worldrsquo(Office of Arid Land Studies University of Arizona Tucson AZ)

Barkla BJ Vera Estrella R Pantoja O (1999) Towards the production ofsalt-tolerant crops Advances in Experimental Medicine and Biology464 77ndash89

Barrett-Lennard EG (2002) Restoration of saline land through revegetationAgricultural Water Management 53 213ndash226 doi101016S0378-3774(01)00166-4

Becker BMarinB (2009) Streptophyte algae and the origin of embryophytesAnnals of Botany 103 999ndash1004 doi101093aobmcp044

Benito B Rodriguez-Navarro A (2003) Molecular cloning andcharacterization of a sodium-pump ATPase of the moss Physcomitrellapatens The Plant Journal 36 382ndash389 doi101046j1365-313X200301883x

Bisson MA Kirst GO (1995) Osmotic acclimation and turgor pressureregulation in algae Naturwissenschaften 82 461ndash471 doi101007BF01131597

Britto DT Kronzucker HJ (2009) Ussingrsquos conundrum and the search fortransport mechanisms in plants New Phytologist 183 243ndash246doi101111j1469-8137200902872x

Cambrolle J Redondo-Gomez S Mateos-Naranjo E Figueroa ME (2008)Comparison of the role of two Spartina species in terms ofphytostabilization and bioaccumulation of metals in the estuarinesediment Marine Pollution Bulletin 56 2037ndash2042 doi101016jmarpolbul200808008

Carroll C Tucker A (2000) Effects of pasture cover on soil erosion andwater quality on central Queensland coal mine rehabilitation TropicalGrasslands 34 254ndash262

Channing A Edwards D (2009) Yellowstone hot spring environmentsand the paleo-ecophysiology of Rhynie chert plants towards asynthesis Plant Ecology amp Diversity 2 111ndash143 doi10108017550870903349359

Colmer TD Flowers TJ (2008) Flooding tolerance in halophytes NewPhytologist 179 964ndash974 doi101111j1469-8137200802483x

Colmer TD Voesenek L (2009) Flooding tolerance suites of plant traitsin variable environments Functional Plant Biology 36 665ndash681doi101071FP09144

Colmer TD Munns R Flowers TJ (2005) Improving salt tolerance of wheatand barley future prospects Australian Journal of ExperimentalAgriculture 45 1425ndash1443 doi101071EA04162

Crayn DM Winter K Smith JAC (2004) Multiple origins of crassulaceanacid metabolism and the epiphytic habit in the neotropical familyBromeliaceae Proceedings of the National Academy of Sciences ofthe United States of America 101 3703ndash3708 doi101073pnas0400366101

Dedolph C Hettel G (Eds) (1997) lsquoPartners making a differencersquo(International Rice Research Institute Manila)

El Shaer HM (2003) Potential of halophytes as animal fodder in EgyptIn lsquoTasks for vegetation science 38 cash crop halophytesrsquo (Eds H LiethM Mochtchenko) pp 111ndash119 (Kluwer Dordrecht the Netherlands)

Flowers TJ (1985) Physiology of halophytes Plant and Soil 89 41ndash56doi101007BF02182232

Flowers TJ (2004) Improving crop salt tolerance Journal of ExperimentalBotany 55 307ndash319 doi101093jxberh003

Flowers TJ Colmer TD (2008) Salinity tolerance in halophytes NewPhytologist 179 945ndash963 doi101111j1469-8137200802531x

Flowers TJ Flowers SA (2005) Why does salinity pose such a difficultproblem for plant breeders Agricultural Water Management 78 15ndash24doi101016jagwat200504015

Flowers TJ Yeo AR (1995) Breeding for salinity resistance in crop plants ndashwhere next Australian Journal of Plant Physiology 22 875ndash884doi101071PP9950875

Flowers TJ Troke PF Yeo AR (1977) The mechanism of salt tolerance inhalophytesAnnual Review of Plant Physiology 28 89ndash121 doi101146annurevpp28060177000513

Flowers TJ Hajibagheri MA Clipson NJW (1986) Halophytes TheQuarterly Review of Biology 61 313ndash337 doi101086415032

Flowers TJGaur PMLaxmipathiGowdaCLKrishnamurthy L Samineni SSiddique KHM Turner NC Vadez V Varshney RK Colmer TD (2010)Salt sensitivity in chickpea Plant Cell amp Environment 33 490ndash509doi101111j1365-3040200902051x

Fogel BN Crain CM Bertness MD (2004) Community level engineeringeffects of Triglochin maritima (seaside arrowgrass) in a salt marsh innorthern New England USA Journal of Ecology 92 589ndash597doi101111j0022-0477200400903x

Garbary DJ Miller AG Scrosati R Kim KY Schofield WB (2008)Distribution and salinity tolerance of intertidal mosses from NovaScotian salt marshes The Bryologist 111 282ndash291 doi1016390007-2745(2008)111[282DASTOI]20CO2

Garciadeblas B Benito B Rodriacuteguez-Navarro A (2001) Plant cells expressseveral stress calcium ATPases but apparently no sodium ATPase Plantand Soil 235 181ndash192 doi101023A1011949626191

Garciadeblaacutes B Haro R Benito B (2007) Cloning of two SOS1transporters from the seagrass Cymodocea nodosa SOS1transporters from Cymodocea and Arabidopsis mediate potassiumuptake in bacteria Plant Molecular Biology 63 479ndash490doi101007s11103-006-9102-2

Glenn EP Oleary JW Watson MC Thompson TL Kuehl RO (1991)Salicornia-bigelovii Torr ndash an oilseed halophyte for seawater irrigationScience 251 1065ndash1067 doi101126science25149971065

Graham LE (1993) lsquoOrigin of land plantsrsquo (John Wiley amp Sons New York)Harvey HW (1966) lsquoThe chemistry and fertility of sea watersrsquo (University

Press Cambridge)Horodyski RJ Knauth LP (1994) Life on land in the Precambrian Science

263 494ndash498 doi101126science2635146494HuangSSpielmeyerWLagudahESMunnsR (2008)Comparativemapping

of HKT genes in wheat barley and rice key determinants of Na+transport and salt tolerance Journal of Experimental Botany 59927ndash937 doi101093jxbern033

Evolution of halophytes Functional Plant Biology 611

that may be selected in established crop plants (eg Huang et al2008) second it may lead to the domestication of naturally salttolerant species as crop plants

Halophytes as crops

Naturally salt-tolerant species are now being promoted inagriculture particularly to provide forage medicinal plantsaromatic plants (Qadir et al 2008) and for forestry (Marcarand Crawford 2004) For example Barrett-Lennard (2002)identified 26 salt-tolerant plant species capable of producingproducts (or services) of value to agriculture in AustraliaExamples of useful halophytes include the potential oil-seedcrops Kosteletzkya virginica (eg Ruan et al 2008) Salvadorapersica (eg Reddy et al 2008) Salicornia bigelovii (Glenn et al1991) and Batis maritima (Marcone 2003) fodder crops such asAtriplex spp (egEl Shaer 2003)Distichlis palmeri (egMasterset al 2007) and biofuels (Y Waisel pers comm Qadir et al2008) Growing salt-tolerant biofuel crops on marginalagricultural land would help to counter concerns that thebiofuel industry reduces the amount of land available forfood production (Qadir et al 2008) At the extreme plants thatcan grow productively at very high salt levels could be irrigatedwith brackish water or seawater (Rozema and Flowers 2008)Although plants that put resources (cf Yeo 1983) intodeveloping salt-tolerance mechanisms (eg the production ofcompatible solutes to maintain osmotic balance is an energeticcost) may do so at the expense of other functions manyhalophytes show optimal growth in saline conditions (Flowersand Colmer 2008) and salt marshes have high productivity(Colmer and Flowers 2008 Flowers and Colmer 2008) Thefact that dicolytedonous halophytes can grow at similar rates toglycophytes (see Flowers and Colmer 2008 for a more detaileddiscussion) suggests that salt tolerance per se will not limitproductivity Here the contrast with drought tolerance is starkwithout water plants do not grow but may survive with saltwater some plants can grow well

Apart from direct use as crops we may increasingly need torely on halophytes for revegetation and remediation of salt-affected land Over the last 200 years industrialisation inEurope and elsewhere has lead to an enormous increase ofproduction use and release of traces of heavy metals into theenvironment A large portion of these toxic materials includingCd Cu Pb and Zn accumulate in sediments including the soilsof tidal marshes Recent studies showed that some seagrassesand salt marsh plants are capable of extracting heavy metalsfrom sediments and accumulating them in belowground oraboveground tissues (Weis and Weis 2004 Cambrolle et al2008 Lewis and Devereux 2009) The processes and potentialapplication of these aquatic halophytes merits much greaterresearch and development Growing salt-tolerant plantsincluding species of Kochia Bassia Cynodon MedicagoPortulaca Sesbania and Brachiaria may also improve othersoil properties such as increasing water conductance orincreasing soil fertility (Qadir et al 2008) Halophytes mayalso lower the watertable thereby allowing growth of salt-sensitive species in salt-affected land (Barrett-Lennard 2002)

Finally conservation and land management in salt-affectedregions will benefit from improved knowledge of the salt

tolerance of local species Halophytes are being targeted forlsquoecosystem engineeringrsquo projects such as stabilising wetlandsto increase the success of revegetation programs (Fogel et al2004) Halophytes are also being used to develop sustainableagricultural practise for example using indigenous halophytesto restore overgrazed pasture and to reduce reliance on irrigation(Peacock et al 2003) Salt-tolerant species are increasinglybeing considered for landscape remediation For example wellplanned revegetation can reduceboth the in situ salt concentrationat mine sites and also reduce the movement of salts from themine sites in runoff (Carroll and Tucker 2000) However caremust be taken in introducing species for bioremediation as theymay become an environmental problem in their own right Forexample Kochia planted for forage and bioremediation inWestern Australia in 1990 soon spread and was declared aweed in 1992 (Qadir et al 2008)

The halophyte paradox

In this review we have shown that salt tolerance has evolvedmany different times in angiosperms and in different ways Wehave also noted that despite increasing salinisation of agriculturalland few salt-tolerant crops have been produced This raises animportant paradox if all plants contain the basic physiologicalmechanisms on which salt tolerance is based and if halophyteshave evolved in many different lineages using a variety ofadaptations thenwhy is it so difficult to breed salt-tolerant crops

The reasons for the slowprogress in changing the salt toleranceof plants were discussed by Flowers andYeo (1995) who arguedthat this was because salinity had not become a problem ofsufficient priority that the necessary resource was beingapplied to its solution Moreover they also concluded thattreating salinity resistance as a single character could accountfor the limited success We have already established that the salttolerance of halophytes is a multi-genic trait and the same is truefor non-halophytes (Flowers 2004) It is not our intention hereto rehearse the arguments over approaches to breeding forcomplex traits However it is clear that breeding for yield hasnot delivered many salt-resistant varieties of the commoncrops (Flowers 2004 Witcombe et al 2008) The probabilityof combining two complex traits ndash yield and salt tolerance ndash tomaximise yield under saline conditions is evidentially very lowAs a consequence Yeo et al (1990) advocated understandingthe physiology of tolerance and pooling a range of traits anapproach that had some success for rice (Dedolph and Hettel1997) A similar approach has been advocated for durum wheatcombining traits for regulating sodiumaccumulation and osmoticadjustment (Munns et al 2002 James et al 2006 2008) Thetraits that necessarily need to be pooled for wheat (and barley)includeNa lsquoexclusionrsquo KNa discrimination the retention of ionsin sheaths tissue tolerance ion partitioning into different leavesosmotic adjustment enhanced vigour water use efficiency andearly flowering (Colmer et al 2005) In addition other traits suchas tolerance to waterndashlogging (see Colmer and Flowers 2008 fora discussion of flooding tolerance in halophytes) are vitallyimportant further complicating any breeding program (Colmeret al 2005) Given the complexity of the task it is not surprisingthat the transfer of one or two genes has generally failed to changethe tolerance of transgenic plants what is surprising is that there

610 Functional Plant Biology T J Flowers et al

are instances where such simple gene transfers appear to havealtered yield (Flowers 2004)

Evolutionary studies of halophytes which reconstruct theorigin and development of salt tolerance in a variety of plantlineages may help us to understand why artificial breedinghas failed to produce robust and productive salt tolerant cropsSuch studies may also help us develop new salt-tolerant lines byrevealing the order of acquisition of components of salt toleranceor indicating favourable genetic backgrounds on which salttolerance may be developed By examining the repeatedevolution of this complex trait we may identify particular traitsor conditions that predispose species to evolve a complex multi-faceted trait such as salt tolerance and give rise to halophytelineages More generally this may shed light on the adaptationof angiosperm lineages to extreme environments In order toachieve these ends more information is required on at aminimum the effects of salt on the growth and ion relations ofa wider range of plant species that may prove to be halophytes

Acknowledgements

HKGwould like to than the Egyptian Government for financial support whileat the University of Sussex and our thanks also go to Jessica Thomas andKatherine Byron for assistance in earlier stages of this project

References

Adam P (1976) Occurrence of bryophytes on British salt marshes Journalof Bryology 9 265ndash274

Aronson JA (1989) lsquoHALOPHa data base of salt tolerant plants of the worldrsquo(Office of Arid Land Studies University of Arizona Tucson AZ)

Barkla BJ Vera Estrella R Pantoja O (1999) Towards the production ofsalt-tolerant crops Advances in Experimental Medicine and Biology464 77ndash89

Barrett-Lennard EG (2002) Restoration of saline land through revegetationAgricultural Water Management 53 213ndash226 doi101016S0378-3774(01)00166-4

Becker BMarinB (2009) Streptophyte algae and the origin of embryophytesAnnals of Botany 103 999ndash1004 doi101093aobmcp044

Benito B Rodriguez-Navarro A (2003) Molecular cloning andcharacterization of a sodium-pump ATPase of the moss Physcomitrellapatens The Plant Journal 36 382ndash389 doi101046j1365-313X200301883x

Bisson MA Kirst GO (1995) Osmotic acclimation and turgor pressureregulation in algae Naturwissenschaften 82 461ndash471 doi101007BF01131597

Britto DT Kronzucker HJ (2009) Ussingrsquos conundrum and the search fortransport mechanisms in plants New Phytologist 183 243ndash246doi101111j1469-8137200902872x

Cambrolle J Redondo-Gomez S Mateos-Naranjo E Figueroa ME (2008)Comparison of the role of two Spartina species in terms ofphytostabilization and bioaccumulation of metals in the estuarinesediment Marine Pollution Bulletin 56 2037ndash2042 doi101016jmarpolbul200808008

Carroll C Tucker A (2000) Effects of pasture cover on soil erosion andwater quality on central Queensland coal mine rehabilitation TropicalGrasslands 34 254ndash262

Channing A Edwards D (2009) Yellowstone hot spring environmentsand the paleo-ecophysiology of Rhynie chert plants towards asynthesis Plant Ecology amp Diversity 2 111ndash143 doi10108017550870903349359

Colmer TD Flowers TJ (2008) Flooding tolerance in halophytes NewPhytologist 179 964ndash974 doi101111j1469-8137200802483x

Colmer TD Voesenek L (2009) Flooding tolerance suites of plant traitsin variable environments Functional Plant Biology 36 665ndash681doi101071FP09144

Colmer TD Munns R Flowers TJ (2005) Improving salt tolerance of wheatand barley future prospects Australian Journal of ExperimentalAgriculture 45 1425ndash1443 doi101071EA04162

Crayn DM Winter K Smith JAC (2004) Multiple origins of crassulaceanacid metabolism and the epiphytic habit in the neotropical familyBromeliaceae Proceedings of the National Academy of Sciences ofthe United States of America 101 3703ndash3708 doi101073pnas0400366101

Dedolph C Hettel G (Eds) (1997) lsquoPartners making a differencersquo(International Rice Research Institute Manila)

El Shaer HM (2003) Potential of halophytes as animal fodder in EgyptIn lsquoTasks for vegetation science 38 cash crop halophytesrsquo (Eds H LiethM Mochtchenko) pp 111ndash119 (Kluwer Dordrecht the Netherlands)

Flowers TJ (1985) Physiology of halophytes Plant and Soil 89 41ndash56doi101007BF02182232

Flowers TJ (2004) Improving crop salt tolerance Journal of ExperimentalBotany 55 307ndash319 doi101093jxberh003

Flowers TJ Colmer TD (2008) Salinity tolerance in halophytes NewPhytologist 179 945ndash963 doi101111j1469-8137200802531x

Flowers TJ Flowers SA (2005) Why does salinity pose such a difficultproblem for plant breeders Agricultural Water Management 78 15ndash24doi101016jagwat200504015

Flowers TJ Yeo AR (1995) Breeding for salinity resistance in crop plants ndashwhere next Australian Journal of Plant Physiology 22 875ndash884doi101071PP9950875

Flowers TJ Troke PF Yeo AR (1977) The mechanism of salt tolerance inhalophytesAnnual Review of Plant Physiology 28 89ndash121 doi101146annurevpp28060177000513

Flowers TJ Hajibagheri MA Clipson NJW (1986) Halophytes TheQuarterly Review of Biology 61 313ndash337 doi101086415032

Flowers TJGaur PMLaxmipathiGowdaCLKrishnamurthy L Samineni SSiddique KHM Turner NC Vadez V Varshney RK Colmer TD (2010)Salt sensitivity in chickpea Plant Cell amp Environment 33 490ndash509doi101111j1365-3040200902051x

Fogel BN Crain CM Bertness MD (2004) Community level engineeringeffects of Triglochin maritima (seaside arrowgrass) in a salt marsh innorthern New England USA Journal of Ecology 92 589ndash597doi101111j0022-0477200400903x

Garbary DJ Miller AG Scrosati R Kim KY Schofield WB (2008)Distribution and salinity tolerance of intertidal mosses from NovaScotian salt marshes The Bryologist 111 282ndash291 doi1016390007-2745(2008)111[282DASTOI]20CO2

Garciadeblas B Benito B Rodriacuteguez-Navarro A (2001) Plant cells expressseveral stress calcium ATPases but apparently no sodium ATPase Plantand Soil 235 181ndash192 doi101023A1011949626191

Garciadeblaacutes B Haro R Benito B (2007) Cloning of two SOS1transporters from the seagrass Cymodocea nodosa SOS1transporters from Cymodocea and Arabidopsis mediate potassiumuptake in bacteria Plant Molecular Biology 63 479ndash490doi101007s11103-006-9102-2

Glenn EP Oleary JW Watson MC Thompson TL Kuehl RO (1991)Salicornia-bigelovii Torr ndash an oilseed halophyte for seawater irrigationScience 251 1065ndash1067 doi101126science25149971065

Graham LE (1993) lsquoOrigin of land plantsrsquo (John Wiley amp Sons New York)Harvey HW (1966) lsquoThe chemistry and fertility of sea watersrsquo (University

Press Cambridge)Horodyski RJ Knauth LP (1994) Life on land in the Precambrian Science

263 494ndash498 doi101126science2635146494HuangSSpielmeyerWLagudahESMunnsR (2008)Comparativemapping

of HKT genes in wheat barley and rice key determinants of Na+transport and salt tolerance Journal of Experimental Botany 59927ndash937 doi101093jxbern033

Evolution of halophytes Functional Plant Biology 611

are instances where such simple gene transfers appear to havealtered yield (Flowers 2004)

Evolutionary studies of halophytes which reconstruct theorigin and development of salt tolerance in a variety of plantlineages may help us to understand why artificial breedinghas failed to produce robust and productive salt tolerant cropsSuch studies may also help us develop new salt-tolerant lines byrevealing the order of acquisition of components of salt toleranceor indicating favourable genetic backgrounds on which salttolerance may be developed By examining the repeatedevolution of this complex trait we may identify particular traitsor conditions that predispose species to evolve a complex multi-faceted trait such as salt tolerance and give rise to halophytelineages More generally this may shed light on the adaptationof angiosperm lineages to extreme environments In order toachieve these ends more information is required on at aminimum the effects of salt on the growth and ion relations ofa wider range of plant species that may prove to be halophytes

Acknowledgements

HKGwould like to than the Egyptian Government for financial support whileat the University of Sussex and our thanks also go to Jessica Thomas andKatherine Byron for assistance in earlier stages of this project

References

Adam P (1976) Occurrence of bryophytes on British salt marshes Journalof Bryology 9 265ndash274

Aronson JA (1989) lsquoHALOPHa data base of salt tolerant plants of the worldrsquo(Office of Arid Land Studies University of Arizona Tucson AZ)

Barkla BJ Vera Estrella R Pantoja O (1999) Towards the production ofsalt-tolerant crops Advances in Experimental Medicine and Biology464 77ndash89

Barrett-Lennard EG (2002) Restoration of saline land through revegetationAgricultural Water Management 53 213ndash226 doi101016S0378-3774(01)00166-4

Becker BMarinB (2009) Streptophyte algae and the origin of embryophytesAnnals of Botany 103 999ndash1004 doi101093aobmcp044

Benito B Rodriguez-Navarro A (2003) Molecular cloning andcharacterization of a sodium-pump ATPase of the moss Physcomitrellapatens The Plant Journal 36 382ndash389 doi101046j1365-313X200301883x

Bisson MA Kirst GO (1995) Osmotic acclimation and turgor pressureregulation in algae Naturwissenschaften 82 461ndash471 doi101007BF01131597

Britto DT Kronzucker HJ (2009) Ussingrsquos conundrum and the search fortransport mechanisms in plants New Phytologist 183 243ndash246doi101111j1469-8137200902872x

Cambrolle J Redondo-Gomez S Mateos-Naranjo E Figueroa ME (2008)Comparison of the role of two Spartina species in terms ofphytostabilization and bioaccumulation of metals in the estuarinesediment Marine Pollution Bulletin 56 2037ndash2042 doi101016jmarpolbul200808008

Carroll C Tucker A (2000) Effects of pasture cover on soil erosion andwater quality on central Queensland coal mine rehabilitation TropicalGrasslands 34 254ndash262

Channing A Edwards D (2009) Yellowstone hot spring environmentsand the paleo-ecophysiology of Rhynie chert plants towards asynthesis Plant Ecology amp Diversity 2 111ndash143 doi10108017550870903349359

Colmer TD Flowers TJ (2008) Flooding tolerance in halophytes NewPhytologist 179 964ndash974 doi101111j1469-8137200802483x

Colmer TD Voesenek L (2009) Flooding tolerance suites of plant traitsin variable environments Functional Plant Biology 36 665ndash681doi101071FP09144

Colmer TD Munns R Flowers TJ (2005) Improving salt tolerance of wheatand barley future prospects Australian Journal of ExperimentalAgriculture 45 1425ndash1443 doi101071EA04162

Crayn DM Winter K Smith JAC (2004) Multiple origins of crassulaceanacid metabolism and the epiphytic habit in the neotropical familyBromeliaceae Proceedings of the National Academy of Sciences ofthe United States of America 101 3703ndash3708 doi101073pnas0400366101

Dedolph C Hettel G (Eds) (1997) lsquoPartners making a differencersquo(International Rice Research Institute Manila)

El Shaer HM (2003) Potential of halophytes as animal fodder in EgyptIn lsquoTasks for vegetation science 38 cash crop halophytesrsquo (Eds H LiethM Mochtchenko) pp 111ndash119 (Kluwer Dordrecht the Netherlands)

Flowers TJ (1985) Physiology of halophytes Plant and Soil 89 41ndash56doi101007BF02182232

Flowers TJ (2004) Improving crop salt tolerance Journal of ExperimentalBotany 55 307ndash319 doi101093jxberh003

Flowers TJ Colmer TD (2008) Salinity tolerance in halophytes NewPhytologist 179 945ndash963 doi101111j1469-8137200802531x

Flowers TJ Flowers SA (2005) Why does salinity pose such a difficultproblem for plant breeders Agricultural Water Management 78 15ndash24doi101016jagwat200504015

Flowers TJ Yeo AR (1995) Breeding for salinity resistance in crop plants ndashwhere next Australian Journal of Plant Physiology 22 875ndash884doi101071PP9950875

Flowers TJ Troke PF Yeo AR (1977) The mechanism of salt tolerance inhalophytesAnnual Review of Plant Physiology 28 89ndash121 doi101146annurevpp28060177000513

Flowers TJ Hajibagheri MA Clipson NJW (1986) Halophytes TheQuarterly Review of Biology 61 313ndash337 doi101086415032

Flowers TJGaur PMLaxmipathiGowdaCLKrishnamurthy L Samineni SSiddique KHM Turner NC Vadez V Varshney RK Colmer TD (2010)Salt sensitivity in chickpea Plant Cell amp Environment 33 490ndash509doi101111j1365-3040200902051x

Fogel BN Crain CM Bertness MD (2004) Community level engineeringeffects of Triglochin maritima (seaside arrowgrass) in a salt marsh innorthern New England USA Journal of Ecology 92 589ndash597doi101111j0022-0477200400903x

Garbary DJ Miller AG Scrosati R Kim KY Schofield WB (2008)Distribution and salinity tolerance of intertidal mosses from NovaScotian salt marshes The Bryologist 111 282ndash291 doi1016390007-2745(2008)111[282DASTOI]20CO2

Garciadeblas B Benito B Rodriacuteguez-Navarro A (2001) Plant cells expressseveral stress calcium ATPases but apparently no sodium ATPase Plantand Soil 235 181ndash192 doi101023A1011949626191

Garciadeblaacutes B Haro R Benito B (2007) Cloning of two SOS1transporters from the seagrass Cymodocea nodosa SOS1transporters from Cymodocea and Arabidopsis mediate potassiumuptake in bacteria Plant Molecular Biology 63 479ndash490doi101007s11103-006-9102-2

Glenn EP Oleary JW Watson MC Thompson TL Kuehl RO (1991)Salicornia-bigelovii Torr ndash an oilseed halophyte for seawater irrigationScience 251 1065ndash1067 doi101126science25149971065

Graham LE (1993) lsquoOrigin of land plantsrsquo (John Wiley amp Sons New York)Harvey HW (1966) lsquoThe chemistry and fertility of sea watersrsquo (University

Press Cambridge)Horodyski RJ Knauth LP (1994) Life on land in the Precambrian Science

263 494ndash498 doi101126science2635146494HuangSSpielmeyerWLagudahESMunnsR (2008)Comparativemapping

of HKT genes in wheat barley and rice key determinants of Na+transport and salt tolerance Journal of Experimental Botany 59927ndash937 doi101093jxbern033

Evolution of halophytes Functional Plant Biology 611

Iwamoto K Shiraiwa Y (2005) Salt-regulated mannitol metabolism in algaeMarine Biotechnology 7 407ndash415 doi101007s10126-005-0029-4

James RA Davenport RJ Munns R (2006) Physiological characterizationof two genes for Na+ exclusion in durum wheat Nax1 and Nax2 PlantPhysiology 142 1537ndash1547 doi101104pp106086538

James RA von Caemmerer S Condon AGT Zwart AB Munns R (2008)Genetic variation in tolerance to the osmotic stress component of salinitystress in durum wheat Functional Plant Biology 35 111ndash123doi101071FP07234

Kenrick P Crane PR (1997) The origin and early evolution of plants on landNature 389 33ndash39 doi10103837918

Khan MA Ungar IA Showalter AM (2005) Salt stimulation and tolerancein an intertidal stem-succulent halophyte Journal of Plant Nutrition 281365ndash1374 doi101081PLN-200067462

Lewis MA Devereux R (2009) Nonnutrient anthropogenic chemicals inseagrass ecosystems fate and effects Environmental Toxicology andChemistry 28 644ndash661 doi10189708-2011

Lewis LA McCourt RM (2004) Green algae and the origin of land plantsAmerican Journal of Botany 91 1535ndash1556 doi103732ajb91101535

Lunde C Drew DP Jacobs AK Tester M (2007) Exclusion of Na+ viasodium ATPase (PpENA1) ensures normal growth of Physcomitrellapatens under moderate salt stress Plant Physiology 144 1786ndash1796doi101104pp106094946

Marcar NE Crawford DF (2004) lsquoTrees for saline landscapesrsquo (CSIROPublishing Melbourne)

Marcone MF (2003) Batis maritima (SaltwortBeachwort) a nutritioushalophytic seed bearings perennial shrub for cultivation and recoveryof otherwise unproductive agricultural land affected by salinity FoodResearch International 36 123ndash130 doi101016S0963-9969(02)00117-5

Masters DG Benes SE Norman HC (2007) Biosaline agriculture for forageand livestock production Agriculture Ecosystems amp Environment 119234ndash248 doi101016jagee200608003

Menzel U Lieth H (2003) HALOPHYTE Database V 20 update In lsquoCashcrop halophytesrsquo (Eds H Lieth M Mochtchenko) (CD-ROM) (KluwerDordrecht the Netherlands)

Munns R (2005) Genes and salt tolerance bringing them together NewPhytologist 167 645ndash663 doi101111j1469-8137200501487x

Munns R Tester M (2008) Mechanisms of salinity tolerance Annual Reviewof Plant Biology 59 651ndash681 doi101146annurevarplant59032607092911

Munns R Husain S Rivelli AR James RA Condon AG Lindsay MPLagudah ES Schachtman DP Hare RA (2002) Avenues for increasingsalt tolerance of crops and the role of physiologically based selectiontraits Plant and Soil 247 93ndash105 doi101023A1021119414799

Munns R James RA Lauchli A (2006) Approaches to increasing the salttolerance of wheat and other cereals Journal of Experimental Botany57 1025ndash1043 doi101093jxberj100

Peacock JM FergusonMEAlhadramiGAMcCann IRAlHajojA SalehAKarnik R (2003) Conservation through utilization a case study of theindigenous forage grasses of the Arabian Peninsula Journal of AridEnvironments 54 15ndash28 doi101006jare20010895

Qadir M Tubeileh A Akhtar J Larbi A Minhas PS Khan MA (2008)Productivity enhancement of salt-affected environments through cropdiversification Land Degradation amp Development 19 429ndash453doi101002ldr853

Railsback LB Ackerly SC Anderson TF Cisne JL (1990) Paleontologicaland isotope evidence for warm saline deep waters in ordovician oceansNature 343 156ndash159 doi101038343156a0

Ramadan T Flowers JJ (2004) Effects of salinity and benzyl adenine ondevelopment and function of microhairs of Zea mays L Planta 219639ndash648 doi101007s00425-004-1269-7

Raven JAEdwardsD (2001)Roots evolutionary origins andbiogeochemicalsignificance Journal of Experimental Botany 52 381ndash401

ReddyMP ShahMT Patolia JS (2008) Salvadora persica a potential speciesfor industrial oil production in semiarid saline and alkali soils IndustrialCrops and Products 28 273ndash278 doi101016jindcrop200803001

Rodriguez-Navarro A Rubio F (2006) High-affinity potassium and sodiumtransport systems in plants Journal of Experimental Botany 571149ndash1160 doi101093jxberj068

Rozema J Flowers T (2008) Crops for a salinized world Science 3221478ndash1480 doi101126science1168572

Ruan CJ Li H Guo YQ Qin P Gallagher JL Seliskar DM Lutts S Mahy G(2008) Kosteletzkya virginica an agroecoengineering halophytic speciesfor alternative agricultural production in Chinarsquos east coast ecologicaladaptation and benefits seed yield oil content fatty acid and biodieselproperties Ecological Engineering 32 320ndash328 doi101016jecoleng200712010

Sage RF (2004) The evolution of C4 photosynthesis New Phytologist 161341ndash370 doi101111j1469-8137200400974x

Sanderson MJ (2003) Molecular data from 27 proteins do not support aPrecambrian origin of land plants American Journal of Botany 90954ndash956 doi103732ajb906954

Stevens PF (2001) Angiosperm phylogeny V 9 June 2008 (MissouriBotanical Garden) Available at httpwwwmobotorgMOBOTresearchAPweb

Takhtajan A (1969) lsquoFlowering plants origin and dispersalrsquo (Oliver amp BoydEdinburgh)

Thomson W Faraday C Oross J (1988) Salt glands In lsquoSolute transport inplant cells and tissuesrsquo (Eds D Baker J Hall) pp 498ndash537 (LongmanHarlow UK)

Touchette BW (2007) Seagrass-salinity interactions physiologicalmechanisms used by submersed marine angiosperms for a life at seaJournal of Experimental Marine Biology and Ecology 350 194ndash215doi101016jjembe200705037

Tuteja N (2007) Mechanisms of high salinity tolerance in plants InlsquoOsmosensing and osmosignalingrsquo pp 419ndash438 (Elsevier AcademicPress San Diego CA)

WagnerGJ (1991) Secreting glandular trichomes ndashmore than just hairsPlantPhysiology 96 675ndash679 doi101104pp963675

Wagner GJ Wang E Shepherd RW (2004) New approaches for studyingand exploiting an old protuberance the plant trichome Annals of Botany93 3ndash11 doi101093aobmch011

Waters ER (2003) Molecular adaptation and the origin of land plantsMolecular Phylogenetics and Evolution 29 456ndash463 doi101016jympev200307018

Weis JSWeis P (2004)Metal uptake transport and release bywetland plantsimplications for phytoremediation and restoration EnvironmentInternational 30 685ndash700 doi101016jenvint200311002

Wellman CH Gray J (2000) The microfossil record of early land plantsPhilosophical Transactions of the Royal Society of London Series BBiological Sciences 355 717ndash732 doi101098rstb20000612

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Yeo AR (1983) Salinity resistance physiologies and prices PhysiologiaPlantarum 58 214ndash222 doi101111j1399-30541983tb04172x

Yeo AR Yeo ME Flowers TJ (1987) The contribution of an apoplasticpathway to sodium uptake by rice roots in saline conditions Journal ofExperimental Botany 38 1141ndash1153 doi101093jxb3871141

Yeo AR Yeo ME Flowers SA Flowers TJ (1990) Screening of rice (Oryzasativa L) genotypes for physiological characters contributing to salinityresistance and their relationship to overall performance Theoretical andApplied Genetics 79 377ndash384 doi101007BF01186082

Manuscript received 6 November 2009 accepted 3 March 2010

612 Functional Plant Biology T J Flowers et al

httpwwwpublishcsiroaujournalsfpb