On the salty side of life: molecular, physiological and anatomical adaptation and acclimation of trees to extreme habitats

Review

On the salty side of life: molecular, physiological andanatomical adaptation and acclimation of trees toextreme habitats

Andrea Polle1 & Shaoliang Chen2

1Forstbotanik und Baumphysiologie, Büsgen-Institut, Georg-August Universität Göttingen, Göttingen 37077, Germany and2College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China

ABSTRACT

Saline and sodic soils that cannot be used for agricultureoccur worldwide. Cultivating stress-tolerant trees to obtainbiomass from salinized areas has been suggested. Varioustree species of economic importance for fruit, fibre andtimber production exhibit high salinity tolerance. Little isknown about the mechanisms enabling tree crops to copewith high salinity for extended periods. Here, the molecular,physiological and anatomical adjustments underlying salttolerance in glycophytic and halophytic model tree species,such as Populus euphratica in terrestrial habitats, and man-grove species along coastlines are reviewed. Key mechanismsthat have been identified as mediating salt tolerance are dis-cussed at scales from the genetic to the morphological level,including leaf succulence and structural adjustments of woodanatomy. The genetic and transcriptomic bases for physio-logical salt acclimation are salt sensing and signalling net-works that activate target genes; the target genes keepreactive oxygen species under control, maintain the ionbalance and restore water status. Evolutionary adaptationincludes gene duplication in these pathways. Strategies forand limitations to tree improvement, particularly transgenicapproaches for increasing salt tolerance by transformingtrees with single and multiple candidate genes, are discussed.

Key-words: adaptive trait; antioxidative systems; leaf succu-lence; mangrove; Populus; salt signalling network; sodium;transcriptome; transformation; wood.

Abbreviations: ABA, abscisic acid; APX, ascorbateperoxidase; [Ca2+]cyt, cytosolic Ca2+ concentration; CaM,calmodulin; CAT, catalase; CBL, calcineurin B-like; CIPK,CBL-interacting protein kinases; DMTU, 1,3-dimethyl-2-thiourea; DPI, diphenylene iodonium; eATP, extracellularATP; GR, glutathione reductase; H-G, hexokinase-glucose;H2O2, hydrogen peroxide; HSF, heat-shock transcriptionfactor; iATP, intracellular ATP; KORCs, outward-rectifyingK+ channels; L-NAME, Nɷ -nitro-L-arginine methyl esterhydrochloride; NO, nitric oxide; NSCCs, non-selective cation

channels; PM, plasma membrane; POD, peroxidase;PPADS, pyridoxalphosphate-6-azophenyl-2′,4′-disulphonicacid; PTIO, 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide; ROS, reactive oxygen species; SOD, superoxidedismutase; SOS, salt overly sensitive; XTH, xyloglucanendotransglucosylase/hydrolases.

INTRODUCTION: SETTING THE STAGE

The demand for food and non-food products is expected toincrease enormously as the world’s population increasesfrom a current estimate of 7 billion to approximately 9 billionpeople by the middle of this century (Godfray et al. 2010; UN2013). Furthermore, dwindling fossil fuel resources are inten-sifying the pressure to replace oil-based technologies withalternative production systems for energy and raw materials,heightening the demand for feedstocks for the emerging bio-economy (Mathur & Vyas 2013). Consequently, competinginterests are already struggling over whether land is to beused for feed and food production or for biomass as a rawmaterial for industrial purposes and energy. It is obvious thatmultiple measures have to be undertaken to meet this chal-lenge. A massive increase in farmland is unlikely becauseonly a very limited percentage of the world’s land area,approximately 14%, is suitable for intense cropping systems.Most land worldwide can only be used extensively or isunsuitable for agriculture because of unfavourable climaticand soil conditions (Eswaran et al. 1999; Václavík et al. 2013).As in the past, when productivity was increased by 40% butagricultural areas only expanded by 9% (Godfray et al.2010), it will be necessary to use the available land moreefficiently and to develop means to share land fairly andsustainably for food and non-food products; other importantgoals such as carbon sequestration and diversity conservationcannot be neglected.

In this review, we will focus on trees as a valuable sourcefor non-food products. Wood and wood products are part ofthe global economy, with forestry industries contributingapproximately US$ 468 billion annually of the global grossvalue added (FAO 2011). Silvicultural management sustainsdiversity at multiple levels and thus stabilizes ecosystem ser-vices (Schwenk et al. 2012). If the challenges of growing treeson soils unsuitable for arable crop production can be

Correspondence: A. Polle. Fax: +49 551 3922705;e-mail: [email protected]

Plant, Cell and Environment (2014) doi: 10.1111/pce.12440

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overcome, a win-win situation for production and environ-mental issues will be created. In this review, we consider thepotential for expanding tree plantations and afforestation onsalt-affected soils. To situate the problem, we briefly summa-rize the definitions and terminology for salt-affected soils,provide estimates on areas potentially available for woodproduction and report the mechanisms of salt-induced injury.To assess the potential of tree species to cope with salt stress,we review the current literature on salt-tolerant tree speciesand their adaptive traits and the underlying acclimationmechanisms from the genomic to the whole-plant level.

SALINE AND SODIC SOILS –A GLOBAL PROBLEM

The semi-arid (total annual precipitation of 250 to 500 mm)and arid climate zones (50 to 250 mm per year), where eco-nomically viable agricultural productivity is achieved by irri-gation (Perry et al. 2009), account for a large proportion ofthe global land area. At the same time, inappropriate irriga-tion and fertilization practices are the major problemsleading to the loss of arable land (Pitman & Läuchli 2002).Some reasons for anthropogenic land degradation are theuse of irrigation water from municipal wastes or of ground-water charged with elevated mineral ion concentrations.Saline groundwater is often the result of deforestation thatleads to a rise in the water table, the formation of new aqui-fers and the mobilization of salt (Salama et al. 1999). In dry,hot areas, salt is then transported to the surface by evapo-transpiration, where it can accumulate to toxic concentra-tions. Furthermore, irrigation water may reach mineralstocks, dissolve the salts and transport them back to thesurface by water evaporation and plant uptake (Rengasamy2006). These scenarios lead to so-called secondary soilsalinization and result in estimated annual arable land lossesof approximately 16 000 km2 (1.6 Mha, Ghassemi et al. 1995),an area corresponding roughly to the size of Kuwait or Con-necticut. Problems related to secondary salinization mayincrease as the global climate changes because larger areas

may have to be irrigated, resulting in an increased potentialfor salinization. Estimates of land loss because of secondarysalinization range from 20 to 50% of irrigated land (Szabolcs1989; Pitman & Läuchli 2002), but these figures are one totwo decades old, and the current extent is apparentlyunknown. A model based on the FAO (2005) estimate forsecondary salinized soil of 397 Mha suggests that 17% of thesoil organic matter in these areas was lost when they becamesaline; furthermore, using a 4% annual increase in saline soils,the predicted loss until 2100 corresponds to 12.4% of all soilorganic matter lost as the consequence of anthropogenic landuse changes (Setia et al. 2013). Loss of soil carbon because ofsalinity may also have negative impact on climate feedbackprocesses.

In addition to anthropogenic soil salinity, naturally salt-affected soils exist throughout the world (Fig. 1). The largestareas are in the Middle East, Australia, North Africa andthe former Soviet Union (Fig. 1). According to recent esti-mates, salt-affected land totals 1128 Mha globally (Wickeet al. 2011). These estimates, based on the HarmonizedWorld Soil database (FAO 2008a), are higher than the pre-vious figures of 955 or 831 Mha (Szabolcs 1989; FAO2008b). The reasons for the discrepancies are assumed to bethe use of different databases and definitions of soil types(Wicke et al. 2011). The development of satellite-basedmeasurements of soil salinity is underway (Metternicht &Zinck 2003; Farifteh et al. 2007). The methods for remotesensing of saline areas, their calibration and technical pro-gress have been recently reviewed (Shoshany et al. 2013),but because of some methodological limitations, there isstill uncertainty about the extent and dynamics of salt-affected areas at the global scale.

In the strict sense, the term ‘salt-affected’ includes soilscontaining elevated concentrations of NaCl, Na2SO4,Na2CO3, NaHCO3, MgSO4, CaSO4 or CaCO3. However, it ismost commonly used to describe soils with high concentra-tions of sodium (Na+). A world map of soil types with excessNa+ concentrations, that is, saline, sodic and saline-sodic soils,has been compiled by Wicke et al. (2011) (Fig. 1). Sixty

Figure 1. Salt-affected soils around the world (reproduced from Wicke et al. 2011 with permission of the Royal Society of Chemistry).

2 A. Polle & S. Chen

© 2014 John Wiley & Sons Ltd, Plant, Cell and Environment

percent of the salt-affected soils are saline (Wicke et al.2011). Saline soils, also termed ‘Solonchaks’, contain excessconcentrations of Na+ in the soil solution and have a lowion-exchange capacity. They are generally characterized byan electrical conductivity of the soil solution greater than 2 to4 mS cm−1 (US Salinity Laboratory 1954). Electrical conduc-tivity increases with increasing NaCl concentrations, as illus-trated in Fig. 2, reaching extreme values when electricalconductivity exceeds 16 mS cm−1. Sodic soils (Solonez)exhibit high cation-exchange capacity, where > 15% isexchangeable Na+. Moderately, strongly and extremely sodicsoils have exchangeable Na+ percentages of 20 to 30%, 30 to40% and >40%, respectively (US Salinity Laboratory 1954).In sodic soils, clay or humus particles bind Na+ and becomedispersed and clogged under wet and dry conditions, respec-tively; this leads to very dense soil structures. The germina-tion and rooting of plants is difficult in these soils. Saline-sodic soils are characterized by high exchangeable Na+

percentages and high Na+ concentrations in the soilsolution.

Soil structures and properties are very important in the fieldbecause they affect the concentrations of free Na+ in the soilsolution, that is, the soil acts as an ion exchanger; when theplant takes up dissolved salt, the salty solution will berecharged. Furthermore, the salt concentrations are dynami-cally influenced by wetting-drying cycles through irrigation,rain or seasonal discharge of rivers (Salama et al. 1999). Con-sequently,salt effects on plants depend on varying interactionsin the root – soil solution – soil system. In addition, bioticcooperation in the rhizosphere, especially with mycorrhizalfungi, strongly modifies salt exposure of roots (Chen et al.2014). In controlled experiments, the complex influence of therhizosphere on plant performance is often not considered. Alarge gap exists between the tremendous increment inknowledge on the genomics and molecular biology of salttolerance (see subchapter: ″TRANSCRIPTOMIC AND

PROTEOMIC APPROACHES TO DISCLOSING THEMOLECULAR BASIS OF SALT TOLERANCE″) and theassessment of these traits under field conditions.

SALT-TOLERANT, WOODY SPECIES:OCCURRENCE AND YIELD

The existence of salt-affected habitats has led to evolutionaryadaptations by plants to these conditions.The database of theRoyal Botanic Gardens Kew (2014) lists approximately 1500salt-tolerant plant species. These species can be divided intoglycophytes, encompassing taxa that tolerate moderate saltconcentrations and halophytes, that is, plants with the ability‘to complete the life cycle in a salt concentration of at least200 mM NaCl under conditions similar to those that mightbe encountered in the natural environment’ (Flowers et al.1986). The lifestyle of the mangroves is a prominent exampleof a halophytic tree species. Mangroves comprise a numberof taxonomically unrelated plant families that occur in thetropics and subtropics at intertidal coastlines and tolerate upto 500 mM NaCl (Ball 1988). This salt concentration corre-sponds to that of seawater [3.2% salt with Na+ as the majorcation (ca. 480 mM), Harvey 1966].To cope with high salinity,mangroves excrete salt by ultrafiltration at the roots or byglands or bladders from the leaves (e.g., members of thegenera Aegiceras, Avicenna, Acanthus and Aegialitis) orsequester salt in the vacuole and use it as an osmoticum (e.g.,species of the genera Bruguiera, Kandelia and Rhizophora)(Ball 1988). Although true mangroves are not suitable forcultivation of salt-affected terrestrial land, they are interest-ing models for understanding extreme salt tolerance (Ohet al. 2012).

Highly salt-tolerant species are also known among terres-trial woody plants. For example, nitrogen-fixing trees of thegenera Casuarina, Acacia and Prosopis can grow at soilsalinities of approximately 40 mS cm−1 (Felker et al. 1981;El-Lakany & Luard 1983; Ng 1987; Rhodes & Felker 1988;Craig et al. 1990; Ahmad et al. 1994; Baker et al. 1995; Velardeet al. 2003). Tamarix spp. also tolerate high soil salinity(257 mM NaCl without and up to ca. 680 mM with slightbiomass losses) because they can excrete salt from salt glands(Kleinkopf & Wallace 1974; Carter & Nippert 2011; Ohrtman& Lair 2013). Shrubs such as Haloxylon ammodendron andReaumuria trigyna live in salt deserts under environmentalextremes (Burley et al. 1986; Dang et al. 2013).

Various tree species used for fruit, timber or fibre produc-tion have moderate to high salt tolerance. Examples includedate palms (Phoenix dactylifera), which can grow at12 mS cm−1 (Alhammadi & Kurup 2012), and olive trees(Olea europaea), which include numerous cultivars exhibit-ing salinity tolerance up to approximately 18 mS cm−1

(Benlloch et al. 1994; Chartzoulakis 2005; Kchaou et al. 2010).The family Myrtaceae comprises many important species forthe timber and paper industries (Grattapaglia et al. 2012),including Eucalyptus, with high intraspecific variation in salttolerance (Bush et al. 2013). In the United States, lists ofeconomically or horticulturally interesting trees have beencompiled, with recommendations for cultivation depending

Figure 2. Relationship between NaCl concentration andelectrical conductivity. The classification of saline soils according tothe US Salinity Laboratory (1954) is indicated. Blue = non-salineconditions.

Salt tolerance in trees 3


on soil salinity (Table 1). However, most of these trees haveonly low salt tolerance (Table 1).

To optimize yields on salt-affected soils, species ofTamarix, Prosopis, Acacia or Eucalyptus have often beenconsidered. In unstressed environments, these speciescan produce up to 40 t ha−1 year−1 (Maguire et al. 1990; vanden Broek et al. 2001; Pasiecznik & Felker 2001). Long-termirrigation with saline water (8 to 10 mS cm−1) resulted inyields of 5.6, 4.6, 2.6 and 2 t ha−1 year−1 for Tamarix articulataand Acacia nilotica, Prosopis juliflora, Eucalyptus tereticornisand Acacia tortilis, and Cassia siamea, respectively (Tomaret al. 2003). Wicke et al. (2011) modelled the technicaland economic potentials for wood production on salt-affected soils using Acacia nilotica, Eucalyptus camaldulensisand Prosopis juliflora. Their analysis suggests mean woodyields of 3.1 t ha−1 year−1 on sodic and saline soils. If thispotential was realized on global salt-affected soils excludingprotected areas, wood production would account for a tech-nical energy potential of approximately 56 EJ year−1, corre-sponding to 11% of global primary energy use in 2010 (IEA2010).The energy potential shows strong regional differencesbut reveals realistic chances for economic revenues andincome in some areas, in addition to improved ecosystemservices, such as positive effects on soil properties and carbon

binding (Wicke et al. 2011). Despite the great opportunitiesprovided particularly by the N-fixing tree species, the physi-ology and molecular biology leading to their exceptional salttolerance has barely been studied.

To date, salt tolerance is best characterized in poplars andaspens (Populus spp.) (Chen & Polle 2010; Chen et al. 2014;Harfouche et al. 2014). Poplars are fast-growing plantationtrees, easy to propagate by cuttings and amenable to geneticengineering; they include numerous high-yielding hybridsthat can be used for the production of second-generationbiofuels (Polle et al. 2013). Among various poplar species,Populus alba and P. euphratica were found to withstand mod-erate to high salinity (7 and 14 mS cm−1, Sixto et al. 2005; 5 to10 mS cm−1, Abassi et al. 2014). P. pruinosa is a close relativeof P. euphratica (Wang et al. 2014a), found in riparian Tugaiforests (Ni et al. 2001); it can also cope with high salinity up to1% NaCl (Vtorova et al. 1993). Both P. pruinosa andP. euphratica are phreatophytic; they require access to waterto survive in desert ecosystems with annual precipitation of30 to 60 mm (Gries et al. 2003). When rivers with irregulardischarge change course, which is not uncommon in theseecosystems, mature trees of these species survive as long astheir deep rooting systems have access to ground water.P. euphratica is more widely spread than P. pruinosa and canbe found in Eurasia from the Taklimakan desert in China tothe Middle East and North Africa (Morocco) (Wang et al.1996). Approximately 60% of the stands are in China and30% in Central Asian countries (mainly Kazakhstan); theremaining 10% are scattered along the Mediterranean coast-line (Wang et al. 1996). Poplars in salt deserts are ecologicallyimportant because they stabilize the sand and protect oasesfrom wandering dunes. The standing biomass of these forestsis high (58 t ha−1) compared with other desert vegetationforms, and the productivity of 0.3 to 3 t ha−1 year−1 is in therange of other salt forests (Ni et al. 2001; Thevs et al. 2012;Buras et al. 2013). P. euphratica propagates generativelydepending on water availability or by root suckers; therefore,stands are often composed of a high number of clonal trees(Thevs et al. 2008; Wiehle et al. 2009; Eusemann et al. 2013).

Populus euphratica is an extremely valuable naturalresource for improving salt tolerance in trees. It maintainsgrowth in the presence of 200 mM NaCl and survives400 mM NaCl after salt acclimation (Watanabe et al. 2000).P. euphratica accumulates salt in the apoplast and the vacuoleand forms succulent leaves (Ottow et al. 2005a), similar to themangrove mode of life. Compared with other poplar species,it restricts salt uptake (Sixto et al. 2005), but it is neitherdrought nor heavy metal tolerant (Hukin et al. 2005; Polleet al. 2013; Sun et al. 2013a; Han et al. 2014); this indicates thatevolutionary adaptation to salinity is a highly specialized traitin this species.

MEASURES TO COPE WITH SALT STRESS ATTHE WHOLE-PLANT LEVEL

Osmotic adjustment to maintain water relations

The presence of soluble salts lowers the water potential ofthe soil solution and reduces water availability to roots. The

Table 1. Salt susceptibility of tree species recommended forcultivation in the United States

Tree species Common name

Electricconductivity

(mS cm−1)

Crataegus sp. Hawthorn 13Fraxinus pennsylvanica Green ash 12Pinus nigra Austrian pine 11Larix sibirica Siberian Larch 9Pinus sylvestris Scots pine 9Gleditsia triacanthos Honey locust 8Juniperus scopulorum Rocky mountain juniper 8Picea pungens Blue spruce 8Populus alba White cottonwood 8Robinia pseudoacacia Black locust 8Ulmus pumila Siberian elm 8Pinus ponderosa Ponderosa pine 6Prunus virginiana Chokecherry 6Abies balsamea Balsam fir 3Acer negundo Boxelder 3Juglans nigra Black walnut 3Malus sp. Crabapple 3Populus sp. Cottonwood 3Populus tremuloides Quaking aspen 3Prunus americana American plum 3Prunus sp. Cherry 3Pseudotsuga menziesii Douglas fir 3Salix pentandra Laurel willow 3Tilia cordata Little leaf Linden 3Ulmus americana American elm 3

The maximum recommended salt level is indicated according to theelectric conductivity scale by the US Salinity Laboratory (1954). Thelist was modified after USDA-NRCS (2007).



initial effect of a sudden increase in salinity is thereforesimilar to that of drought restricting water uptake and result-ing in wilting. In general, higher plants produce compatiblesolutes, lowering the cell water potential to maintain wateruptake under osmotic stress (Munns & Tester 2008; Chen &Polle 2010). The recovery of water potential is usually fasterin response to salinity than in response to drought becauseboth salt-resistant and salt-sensitive poplars immediatelytake up Na+ and other ions that act as osmolytes. Conse-quently, the leaf water potential recovers relatively quicklyunder saline conditions, as found for instance in P. euphratica,P. popularis and several poplar hybrids (Fung et al. 1998;Brinker et al. 2010). Under drought stress, organic osmolytesmust be produced to achieve osmotic adjustment, which ismore energy- and time-consuming than salt uptake. It isnotable that in P. euphratica, salt stress even causes strongdecreases in the foliar concentrations of soluble carbohy-drates such as glucose, fructose and sucrose (Ottow et al.2005a). Free amino acids increase with salinity, but their con-centrations are low, and they can therefore contribute onlymarginally to osmotic pressure adjustment in P. euphratica(Ottow et al. 2005a). Similarly, decreased organic solute con-centrations were found in Zygophyllum xanthoxylum, anextremely salt-tolerant shrub, grown under moderate saltstress (Ma et al. 2012). Model calculations revealed that salt-tolerant woody species, such as Z. xanthophyllum andP. euphratica, keep their positive turgor pressure in responseto salt mainly by increasing inorganic osmolytes, therebycompensating for the decreases in organic solutes (Ottowet al. 2005a; Janz & Polle 2012). To avoid the negativeconsequences of salt uptake for cellular metabolism,Z. xanthoxylum and P. euphratica accumulate Na+ preferen-tially in the vacuole instead of the cytosol (Ottow et al. 2005a;Wu et al. 2011). It can be speculated that the observeddecreases in soluble carbohydrates reflect an increased ener-getic need to concentrate Na+ in compartments wheredamage can be avoided. Na+ can then be used as ‘cheap’osmolytes (Janz & Polle 2012). Consequently, these plantsare able to maintain positive turgor pressure under salineconditions.

In addition to osmotic adjustment, stomatal closurereduces leaf transpiration, allowing poplars to re-establishthe balance between water gain and loss (Chen et al. 2001,2002a).Abscisic acid (ABA) concentrations in the xylem playa decisive role in the adjustment of whole-plant waterbalance. Under osmotic stresses imposed by either polyeth-ylene glycol or by NaCl, ABA concentrations in the xylemincrease (Chen et al. 2002a). In salt-exposed P. euphratica,higher ABA concentrations are maintained, allowing accli-mation (Chen et al. 2002a). There is much variation in themaintenance of water status among genotypes of the genusPopulus after salt exposure. NaCl-treated P. euphratica typi-cally exhibit higher stomatal conductance and leaf transpira-tion than salt-sensitive poplars (P. tomentosa, P. popularisand hybrid poplars, Chen et al. 2002b, 2003a). At the whole-plant level, water fluxes through P. cv. Italica and P. populariswere strongly reduced after salt treatment, whereaswhole-plant transpiration of P. euphratica did not decrease

significantly during extended exposure to salinity (Chen et al.2002b). In two non-secreting mangroves, the decrease ingas exchange was also greater in the more salt-sensitivespecies Kandelia candel than in the more tolerantBruguiera gymnorrhiza (Li et al. 2008). Overall, the ability tomaintain water status reflects the capacity of a plant speciesfor salt acclimation.

Morphological and anatomical plasticity reflectplant adjustment to salt stress

In the dry, hot regions that are typical habitats of salt-adapted species, a number of morphological and anatomicalfeatures help plants address restricted water availability.The xylem anatomy of woody plants in arid climates is char-acterized by thick cell walls, small vessel lumina and highvessel numbers (Tyree & Ewers 1991; Sperry et al. 2008).These properties decrease the risk of cavitation (Tyree &Sperry 1989; Sperry et al. 2008). The xylem anatomy ofpoplar demonstrates high plasticity; vessel numbers andlumina adjust to the prevailing water availability (Beniwalet al. 2010). Exposure to salt stress causes alterations to thestructure of the hydraulic system of the salt-sensitive poplarspecies P. × canescens that are similar to those found afterdrought (Junghans et al. 2006; Janz et al. 2012). The reasonsfor decreased vessel expansion in P. × canescens are theNa+-induced decrease in K+ and the decline in the growthhormone auxin (Junghans et al. 2006; Escalante-Pérez et al.2009). A decrease in active auxin in response to salt wasalso localized in the xylem by the beta-glucuronidase(GUS) reporter gene under the control of an auxin-responsive promoter (Teichmann et al. 2008). Comparedwith P. × canescens, P. euphratica exhibits smaller vessellumina in the absence of salt stress; moderate salt stress inthis species results neither in changes in the xylem anatomynor in a decline in auxin (Junghans et al. 2006), confirmingpre-adaptation of the hydraulic system of P. euphratica tosaline conditions.

The transcriptional regulation underlying salt-inducedalterations in the xylem anatomy has been studied inP. × canescens (Janz et al. 2012). The developing xylemundergoing hydraulic acclimation exhibits decreased abun-dances of transcripts of genes responsible for tension woodformation, particularly of genes encoding FASCICLIN-likearabinogalactan proteins (FLAs) (Janz et al. 2012). FLAs areinvolved in various processes of xylem differentiation, forexample, cell division, adhesion and microfibril orientation(Humphrey et al. 2007; Seifert & Blaukopf 2010). Further-more, the expression of orthologs of genes involved in cellu-lose biosynthesis in Arabidopsis (Lukowitz et al. 2001; Brownet al. 2005) is suppressed and the ratio of lignin to cellulose isenhanced (Janz et al. 2012). Collectively, these findingssuggest that the increased osmotic pressure imposed by salin-ity results in adjustments of cell wall composition to stress,resembling compression wood in gymnosperms (Janz et al.2012). Cell wall composition also varies in wood formedduring drought (Arend & Fromm 2007). Therefore,mechanosensing may exist in the cambium to adjust wood



structural development and wall composition to varyinghydraulic requirements. This hypothetical mechanism wouldbe useful for woody species exposed to large changes inenvironmental conditions, such as riparian poplars that haveto cope with varying water tables and fluctuating concentra-tions of solutes in the stream flow. In contrast, inP. euphratica, a species that occurs in ecosystems with highpersistent salt loads, flexibility appears to be less important; itforms small vessels and exhibits correspondingly low radialgrowth, even under favourable conditions.

A striking morphological alteration that is beneficial forsalt tolerance in P. euphratica is the development of leaf suc-culence during prolonged periods of salt exposure (Ottowet al. 2005a). Succulence is a general anatomical feature ofhalophytic plants (Hameed et al. 2010) because it leads to thedilution of salt, thereby avoiding excessive concentrations inthe tissues (Albert 1975). A recent study shows that the cellwall enzymes xyloglucan endotransglucosylase/hydrolases(XTHs) are likely involved in salt-elicited leaf succulence inhigher plants (Han et al. 2013). With their transglucosylase/hydrolase activities (Thompson & Fry 2001; Rose et al. 2002),XTHs catalyze the splitting and/or reconnection ofxyloglucan cross-links in the cellulose-hemicellulose frame-work of cell walls (Fry et al. 1992; Nishitani & Tominaga1992). Arabidopsis plants overexpressing XTHs show ana-tomical alterations (Cho et al. 2006; Shin et al. 2006). In theXTH multigene family of P. euphratica, one member wasup-regulated in leaves of salinized plants (Han et al. 2013).Tobacco plants overexpressing this PeXTH exhibit higherwater content per unit leaf area and a higher ratio of fresh todry weight of the leaves, a hallmark of succulence (Han et al.2013). Morphometric studies revealed that succulent leavesof P. euphratica also exhibit an increased number of celllayers (Ottow et al. 2005a). Similarly, two to three layers ofpalisade parenchyma cell were found in PeXTH-expressinglines, compared with one layer of palisade parenchyma cellsin the wild type (Han et al. 2013). Moreover, PeXTH-transgenic plants exhibited an increased number of spongymesophyll cells, although they were smaller in size (Han et al.2013). Similarly, the overexpression of pepper CaXTH3 or ofBrassica campestris BcXTH in Arabidopsis altered leaf mor-phology (Cho et al. 2006; Shin et al. 2006), but this effect wasapparently not observed in CaXTH3-expressing tomato(Choi et al. 2011). CaXTH3 overexpression was previouslyshown to improve salt tolerance in Arabidopsis and tomatoplants (Cho et al. 2006; Choi et al. 2011), but these findingshave not been linked with leaf morphological changes. Choet al. (2006) suggested that CaXTH3 is involved in cell wallremodelling to strengthen the wall layers, participating in theprotection of mesophyll cells against water deficit and highsalinity. Choi et al. (2011) showed that detached leaves fromCaXTH3-transgenic plants exhibited lower water loss thanthe wild type (Choi et al. 2011). They supposed that theincreased cell wall remodelling activity of CaXTH3 in guardcells reduces transpiration water loss in response to dehydra-tion stress. In PeXTH-transgenic tobacco, the increased suc-culence diluted Na+ and Cl− concentrations at the tissue andcellular levels, precluding the accumulation of toxic

concentrations (Han et al. 2013). Therefore, we suggestthat leaf succulence caused by XTH overexpression isbeneficial for plants dealing with prolonged periods of saltstress.

SALT SIGNALLING, CELLULAR HOMEOSTASISAND DEFENCE

Keeping salt under control

Salt-exposed plants accumulate high levels of Na+ in rootsand leaves, irrespective of whether they are salt-resistant orsalt-sensitive species (Chen & Polle 2010). A hallmark ofresistant tree species is their better ability to control Na+ netuptake and to mediate a favourable Na+/K+ ratio (Maathuis& Amtmann 1999). For example, P. euphratica exhibits agreater capacity than salt-sensitive poplars to restrict theuptake and transport of Na+ (Chen et al. 2002b, 2003a).The effective avoidance of excessive Na+ uptake is likely theresult of a highly active Na+ extrusion system in the plasmamembrane (PM, Sun et al. 2009a). Janz et al. (2010) showedthat transporters including members of the Na+/H+ antiporterfamily were constitutively overexpressed in P. euphraticacompared with P. × canescens. Under salt stress, P. euphraticamaintains higher expression levels of Na+/H+ antiporters(e.g., the Na+/H+ antiporters SOS1, NHD2, NaHD1) and H+

pumps (e.g., PM H+-ATPase) than do salt-sensitive poplars(Ottow et al. 2005b; Ding et al. 2010). In the salt-sensitivepoplars, salt persistence can be increased by root interactionswith mycorrhizae because the interaction greatly increasesNa+ efflux (Li et al. 2012a). High Na+ efflux was also observedin the halophytic species of mangroves (Lu et al. 2013). Fluxdata indicate that the ability to maintain Na+ homeostasis inK. candel and B. gymnorrhiza roots is the result of enhancedNa+/H+ antiporter activities under NaCl stress (Lu et al.2013).

The functioning of Na+/H+ antiporters requires the main-tenance of a proton gradient across the membrane. Thisgradient is established by P-type ATPases (Fig. 3). Notably,the gene family encoding P-type H+-ATPases has morecopies in the P. euphratica than in the P. trichocarpagenome (Ma et al. 2013). Significant differences in PMATPase activities were also observed between salt-sensitiveand salt-tolerant genotypes of P. alba (Beritognolo et al.2007), supporting the significance of PM ATPases for Na+

extrusion.Furthermore, high-affinity K+ (HKT) transporters play key

roles in limiting Na+ xylem loading in Arabidopsis thaliana(Davenport et al. 2007). Four members of HKT1 weredetected in the P. euphratica genome, compared with onlyone member in the P. trichocarpa genome (Ma et al. 2013).These transporters may account for the reduced transloca-tion of Na+ to leaves of P. euphratica (Chen et al. 2001, 2002b,2003a; Ma et al. 2013).Taken together, these data suggest thatthe expansion of gene families and enhanced activities ofHKT and Na+/H+ transporters as well as of P-type ATPasesconstitute an important molecular basis for increasedNa+ tolerance. The plant is thus able to master the critical



balance between Na+ as a useful osmolyte or an injuriouscompound that disrupts ion homeostasis.

Salt signalling for ion homeostasis involvescalcium and the salt overly sensitive(SOS) pathway

Trees must be able to sense and signal varying external saltconditions to trigger acclimatory responses to fluctuatingenvironmental stimuli. There is now evidence that numerouscompounds, such as extracellular ATP (eATP), hydrogen

peroxide (H2O2), nitric oxide (NO), Ca2+, salicylic acid andothers are involved in salt-stress signalling (Zhang et al. 2007;Chen et al. 2010; Pandolfi et al. 2010; Sun et al. 2010a,b, 2012;Jayakannan et al. 2013; Pottosin et al. 2014). Currently, Ca2+ asa second messenger for stress responses and its role in theSOS pathway are among the best-studied salt signallingmechanisms in plants (Zhu 2001, 2002, 2003). Ca2+ concen-trations in the cytosol ([Ca2+]cyt) are usually very low, which isa precondition for Ca2+ acting as a signalling compound inplant cells (Clarkson & Hanson 1980; Knight & Knight 2001;Cramer 2002). The plants’ first response to changing Na+

concentrations is the enhancement of [Ca2+]cyt in outer rootcells (Sanders et al. 1999; Hasegawa et al. 2000; Munns &Tester 2008). This signal is sensed by the SOS pathway, regu-lates the SOS3-SOS2 complex [a calcineurin B-like (CBL)interacting protein kinase and CBL4, a calcineurin-like phos-phatase] and enhances SOS1, a Na+/H+ antiporter (Zhu 2001,2002, 2003; Quintero et al. 2011) (Fig. 3). The components ofthe SOS pathway, PtSOS1, PtSOS2 and PtSOS3 were alsoidentified in P. trichocarpa (Tang et al. 2010). Heterologousexpression of PtSOS1, PtSOS2 and PtSOS3 rescues salt-sensitive Arabidopsis sos deletion mutants (Tang et al. 2010).This finding implies that there is strong functional conserva-tion of the SOS pathway across plant species (Tang et al.2010).

Furthermore, several members of the calcium-sensingprotein family – CBL proteins – are sensors in stress sig-nalling pathways (Luan et al. 2002). Transcript analysis ofCBLs in P. euphratica suggests that seven genes of thisfamily play an important role in regulating the plants’response to abiotic stress (Zhang et al. 2008). For instance,the calcium sensor PeCBL1 interacts with the CBL-interacting protein kinases (CIPKs), PeCIPK24/25 andPeCIPK26, and regulates K+/Na+ homeostasis inArabidopsis (Zhang et al. 2013a). The importance of Ca2+

for mediating K+/Na+ homeostasis was also demonstrated inpoplar (Sun et al. 2009b, 2010a,b, 2012) and mangrovespecies (Lu et al. 2013). Flux measurements revealed thatthe maintenance of the Ca2+ level is essential for restrictingK+ efflux from poplar roots, enhancing Na+ exclusion andsustaining membrane selectivity, thereby mediating salt tol-erance (Sun et al. 2009b).

Calmodulin (CaM), another sensor of [Ca2+]cyt (Knight &Knight 2001; Hu et al. 2007), is also involved in salt-stressresponses in woody plants (Chang et al. 2006; Li et al. 2009a).However, its role appears to be complex; it was activatedduring recovery from salt stress in P. euphratica (Gu et al.2004) but not after short-term salt shock or long-term saltacclimation (Ottow et al. 2005a). In poplar species of varyingsalt tolerance, CaM increased most in P. euphratica leavesand less in salt-sensitive poplars (P. popularis and P. cv.Italica, Chang et al. 2006). After continuous salt exposure,CaM levels declined in all poplar species. The decline inP. euphratica, however, was lower than in the other twospecies (Chang et al. 2006). These findings suggest thatP. euphratica may have a greater capacity for CaMmaintenance under salt stress, contributing to its higher salttolerance.

Figure 3. A schematic model showing a multiple signallingnetwork in the response of Populus euphratica cells to NaCl stress.This model is modified from the salt signalling network proposedby Sun et al. (2010b, 2012). In brief, NaCl salinity depolarizes theplasma membrane (PM), causing Na+ entry into the cell and K+

loss through depolarization-activated channels such asoutward-rectifying K+ channels (KORCs) and non-selective cationchannels (NSCCs). The PM H+-coupled ion transporters, forexample, a H+-ATPase (HA1), a Na+/H+ antiporter (SOS1) and achloride symporter (Cl−/2H+ symporter) sense the ion-specificeffect of NaCl and trigger H+ fluxes across the PM. The pHalterations at the apoplastic and cytosolic sides activate PMNADPH oxidases and lead to H2O2 production. Superoxideproduced by NADPH oxidases is converted to H2O2 by superoxidedismutases (SODs). In addition, NaCl stress also induces a releaseof intracellular ATP (iATP) resulting in a transient increase in theextracellular ATP (eATP) concentrations. The salt-induced eATPis sensed by purinoceptors, for example, purinoceptor 2 receptorkinase 1 (P2K1) in the PM, leading to the induction ofdownstream signals, for example, H2O2. The salt-elicited H2O2

stimulates Ca2+ entry via Ca2+-permeable channels and theelevated [Ca2+]cyt stimulates the PM Na+/H+ antiporters throughthe salt overly sensitive (SOS) signalling pathway. Inside the cell,H2O2 leads to transcriptional activation of a heat-shocktranscription factor (HSF), which initiates transcription of targetgenes encoding antioxidant enzymes, for example, ascorbateperoxidase (APX), contributing to ROS homeostasis. Moreover,H2O2 induces a Ca2+-dependent increase in PM H+-ATPaseactivity. The up-regulated H+-pumps can sustain an H+ gradient todrive the Na+/H+ antiport across the PM and preserve aless-depolarized membrane potential, restricting K+ efflux throughdepolarization-activated KORCs and NSCCs. As a result, cellularK+/Na+ homeostasis is maintained in salinized P. euphratica cells.



Reactive oxygen species (ROS) as signals forion homeostasis and activators ofantioxidative defences

ROS are important salt-stress signalling molecules (Chen &Polle 2010). They trigger the increase in [Ca2+]cyt by the acti-vation of non-selective cation channels that mediate Ca2+

entry into the cytosol (Demidchik et al. 2003). P. euphratica,but not the salt-sensitive P. popularis, respond to salt withrapid H2O2 production (Sun et al. 2010a). Exogenous H2O2

application enhanced the Na+/H+ exchange (Sun et al.2010b). Pharmacological experiments with the ROS scaven-ger 1,3-dimethyl-2-thiourea (DMTU) and the PM NADPHoxidase inhibitor diphenylene iodonium (DPI) clearly indi-cated that the NaCl-induced Na+/H+ antiport was restrictedwhen H2O2 was not present; consequently, Na+ extrusionfrom the salinized cells declined (Sun et al. 2010b). Zhanget al. (2007) reported that the H2O2 signal is responsible forthe up-regulation of PM H+-ATPases, whose activities arerequired to control the cellular K+/Na+ homeostasis underNaCl stress. The activated PM H+-ATPases limit NaCl-induced K+ efflux via depolarization-activated channels, thatis, the outward-rectifying K+ channels (KORCs) and the non-selective cation channels (NSCCs) in poplars (Sun et al.2009b) (Fig. 3). The importance of H2O2 for the regulation ofion homeostasis is also evident because salt-sensitive poplarspecies do not increase H2O2 in response to NaCl shock andcannot prevent excess Na+ accumulation (Sun et al. 2010a).Clearly, H2O2-mediated K+/Na+ homeostasis through the PMNa+/H+ antiporter system and K+ channels helpsP. euphratica manage NaCl stress. Similarly, H2O2 is involvedin mediating K+/Na+ homeostasis in salt-stressed non-secreting mangroves (Lu et al. 2013).

The H2O2-triggered K+/Na+ homeostasis in NaCl-stressedcells is Ca2+-dependent (Sun et al. 2010b). Exogenous H2O2

causes a net Ca2+ influx and subsequent elevation of Ca2+ inthe cytosol (Sun et al. 2010b). The elevated [Ca2+]cyt stimu-lates the PM-localized Na+/H+ antiporters through the SOSsignalling pathway (Zhu 2001, 2002, 2003). Furthermore,H2O2 mediates increased SOS1 mRNA stability inArabidopsis and may therefore contribute to cellular Na+

protection (Chung et al. 2008).NADPH oxidases located at the PM are important

sources of H2O2. NADPH oxidases catalyze the productionof superoxide that is converted to H2O2 by superoxidedismutases (SOD, Foreman et al. 2003; Kwak et al. 2003;Mittler et al. 2004; Sagi & Fluhr 2006). The increase in salt-triggered H2O2 is inhibited by DPI, supporting the involve-ment of NADPH oxidase (Sun et al. 2010a,b). Furthermore,the inhibition of PM H+-coupled ion transporters supports arole for NaCl-induced NADPH oxidases and H2O2 produc-tion in salt signalling (Sun et al. 2010b). The PM H+-ATPasemay function as an ionic sensor to induce an early H2O2

burst in P. euphratica cells (Zhang et al. 2007; Sun et al.2010a,b). To clarify the roles of the PM H+-ATPase in saltsensing and acclimation, the PM H+-ATPase gene PeHA1was isolated from P. euphratica and introduced intoA. thaliana (Wang et al. 2013b). In PeHA1-expressing

Arabidopsis roots, H2O2 production was higher undercontrol conditions and increased more rapidly than in thewild type when the plants were subjected to NaCl treatment(Wang et al. 2013b). In salinized Arabidopsis, ectopic expres-sion of PeHA1 remarkably enhanced the capacity to controlthe homeostasis of ions and ROS (Wang et al. 2013b). Fluxdata from salinized roots showed that transgenic plantsexhibited stronger Na+/H+ antiporter activities and lowerreduction of K+ influx than the wild type (Wang et al.2013b). Furthermore, the PeHA1-expressing Arabidopsisplants were unable to control K+/Na+ homeostasis when thesalt-induced H2O2 production was inhibited by DPI (Wanget al. 2013b). These observations suggest that in Arabidopsis,PeHA1 accelerates salt acclimation partially through rapidH2O2 production upon salt treatment, triggering adjustmentsin K+/Na+ homeostasis and antioxidant defence. The PeHA1-expressing Arabidopsis plants also exhibited enhanced H+

extrusion, a precondition for rapid H2O2 production (Wanget al. 2013b). The facilitation of H2O2 production by protonshas also been shown in other systems. For example, H+

extrusion is essential for H2O2 formation in the hypersensi-tive response of barley to powdery mildew (Beffagna et al.2005) and an acidic medium increases H2O2 production byArabidopsis roots (Wang et al. 2013b). In conclusion, salttolerance in P. euphratica involves an enhanced H+ effluxdriven by PM H+-ATPase, causing a transient apoplast acidi-fication under NaCl. The pH decrease is thought to beresponsible for the activation of NADPH oxidases, leadingto H2O2 production that then stimulates Na+/H+ antiportthrough the Ca2+-SOS pathway (Fig. 3).

ROS are also a potential threat because they can injuremembranes by lipid peroxidation, can disturb the redox regu-lation of proteins and may even cause DNA breaks (Mittler2002). Antioxidant systems keep ROS under control (Mittler2002). Interestingly, ROS also act as second messengers thatinduce antioxidant defences in herbaceous species (Jiang &Zhang 2002; Hu et al. 2007) and trees (Wang et al. 2006, 2007,2008; Shen et al. 2013). Both poplars and mangroves detoxifyROS to alleviate salt-induced oxidative stress byup-regulation of antioxidant enzymes and increased levels oflow molecular weight antioxidant compounds (Takemuraet al. 2002; Parida et al. 2004; Wang et al. 2007, 2008). Geno-typic differences in ROS detoxification among poplars havebeen previously reviewed (Chen & Polle 2010). Briefly, salt-tolerant poplars have a higher capacity to scavenge ROSthan salt-sensitive poplars (Wang et al. 2007, 2008) andcontain a higher abundance of transcripts encoding antioxi-dant enzymes than sensitive poplars (Ottow et al. 2005a; Dinget al. 2010). The observation that gene families encodingcatalases (CAT) and glutathione reductase 1 (GR1) areexpanded in the P. euphratica compared with theP. trichocarpa genome supports the significance ofantioxidative protection for enhanced salt tolerance (Maet al. 2013). In mangroves (B. parviflora, B. gymnorrhiza),salt exposure also elicits increased activities of antioxidantenzymes, such as guaiacol peroxidase (GPX), ascorbateperoxidase (APX) and GR, to detoxify elevated ROS levelsin leaves (Takemura et al. 2002; Parida et al. 2004).Therefore,



the up-regulation of antioxidant defences is crucial forglycophyte and halophyte species to adjust to long-term saltstress.

There is now emerging evidence that a heat-shock tran-scription factor (HSF) can sense H2O2 and then activate anti-oxidant enzymes to detoxify ROS in plant cells under saltstress (Shen et al. 2013). HSF was shown to be a direct sensorof H2O2 in Drosophila and mammalian cells (Zhong et al.1998; Ahn & Thiele 2003). PeHSF expression is induced byNaCl in P. euphratica leaves and callus cultures but not whenthe H2O2 scavenger DPI is also present (Shen et al. 2013).This ties HSF into the salt signalling cascade. In Arabidopsis,HSF–heat shock element (HSE) binding activation is H2O2-dependent and Class A HSF (AtHSFA1a or AtHSFA1b) issubject to oxidative stress activation (Volkov et al. 2006).Furthermore, HSFA4a is a candidate sensor molecule forH2O2 perception in Arabidopsis (Davletova et al. 2005).Genes encoding antioxidant enzymes appear to be down-stream targets of HSF because PeHSF overexpression intobacco enhances the activities of APX, glutathioneperoxidase and GR (Shen et al. 2013). In Arabidopsis,AtHsfA2 modulates APX1 and APX2 expression andenhances tolerance to heat, oxidative and salt/osmoticstresses (Panchuk et al. 2002; Li et al. 2005; Ogawa et al.2007). It is likely that the salt-induced H2O2 leads to tran-scriptional activation of PeHSF, initiating the transcription ofgenes encoding antioxidant enzymes and contributing toROS homeostasis under saline conditions (Fig. 3).

Salt exposure also leads to increases in ABA, CaM andNO, which are likely to be involved in the rapidup-regulation of antioxidant defences (Guan & Scandalios1998; Chen et al. 2002a,b; Jiang & Zhang 2002; Chang et al.2006). Similar to P. euphratica (Chen et al. 2002a, 2003b),the leaf ABA in K. candel increases rapidly after salt stress,reaching its peak after 4 h; the maximum activation of anti-oxidant enzymes occurs after 8 h (Li et al. 2009a). InB. gymnorrhiza leaves, increases in SOD and peroxidase(POD) activities correlate with the increase in CaM duringsalt exposure (Li et al. 2009a). CaM stimulates CAT activ-ities, thereby accelerating H2O2 removal (Yang & Poovaiah2002). The role of NO has been studied by comparing thesalt responses of P. euphratica and P. popularis cells (Sunet al. 2010a). A rapid increase of NO was accompanied byincreases in SOD, APX, GR and CAT only in P. euphratica.The stimulation of antioxidant enzymes was inhibitedwhen Nɷ -nitro-L-arginine methyl ester hydrochloride(L-NAME, an inhibitor of NO synthetase) and 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO, a NOscavenger) were employed (Sun et al. 2010a). Theseresults suggest that salt-elicited NO contributes to theup-regulation of antioxidant defences.

eATP signalling is a novel player insalt-stress acclimation

eATP serves as an important signalling agent for plantgrowth and defence responses to environmental stimuli.eATP mediates the growth of cotton fibres, root hairs and

pollen tube (Kim et al. 2006; Reichler et al. 2009; Clark et al.2010) and affects auxin transport and root gravitropism(Tang et al. 2003), stomatal movement (Clark et al. 2011; Haoet al. 2012), membrane potential (Lew & Dearnaley 2000)and gene expression (Jeter et al. 2004; Chivasa et al. 2005;Song et al. 2006). Several excellent reviews have summarizedeATP signalling in plant growth and development (Roux &Steinebrunner 2007; Clark & Roux 2009, 2011; Tanaka et al.2010).

Recently, eATP was shown to be involved in plantresponses to abiotic and biotic stress (Jeter et al. 2004;Chivasa et al. 2009; Kim et al. 2009). The contribution ofelevated eATP to salt acclimation has been explored in salt-resistant P. euphratica. P. euphratica callus cells exhibit atransient elevation of eATP upon NaCl stress (Sun et al.2012). When eATP was removed, for example by P2 recep-tor antagonists [suramin, pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS)] or by an ATP trap systemcomposed of hexokinase-glucose (H-G trap), the viability ofthe cells strongly declined under salt stress, suggesting thateATP mediates acclimation of P. euphratica to salt stress(Sun et al. 2012). A series of experiments showed that eATPregulates a wide range of cellular processes required for saltadjustment. When eATP signalling was blocked withthe H-G trap system or by P2 receptor antagonists,P. euphratica cells were unable to trigger salt acclimationprocesses, such as K+ homeostasis; Na+/H+ exchange acrossthe PM; vacuolar Na+ compartmentalization; ROS regula-tion and salt-responsive gene expression including AHA(PM H+-ATPase), mitogen-activated protein kinase (MPK),SOS1 (PM Na+/H+ antiporter) and vacuolar H+-ATPase-subunit c (VHA-c; Sun et al. 2012). Interestingly, exogenousATP application rescued the H-G-mediated inhibition ofsalt adaptation but not in suramin- or PPADS-treated cells(Sun et al. 2012). This result implies that eATP can besensed by PM purinergic receptors, such as P2K1 (Choi et al.2014), and then initiate the downstream signallingcomponents.

In P. euphratica cells, eATP signalling of salt stress occursupstream of H2O2 and Ca2+ signalling. eATP and the non-hydrolysable ATP analogue ATPλS induce a rapid increase inH2O2 (Sun et al. 2012), similar to that found after NaCl treat-ment (Sun et al. 2010a,b). The elevation of [Ca2+]cyt inP. euphratica cells is dependent on the presence of eATP atthe beginning of salt stress (Sun et al. 2012). Transient Ca2+

kinetics reveal that the salt-elicited [Ca2+]cyt results fromCa2+ entry (Sun et al. 2012). The NaCl-induced H2O2 burst,Ca2+ influx and [Ca2+]cyt elevation in P. euphratica cells areblocked by GdCl3, suramin, PPADS or H-G (Sun et al. 2012).After the application of ATP, the cytosolic Ca2+ influx isresumed in H-G pre-treated, salt-exposed cells, but not insuramin- or PPADS-treated cells (Sun et al. 2012). Theseresults imply that eATP signalling is mediated by PMpurinoceptors and contributes to the rapid H2O2 burst andelevated [Ca2+]cyt triggered by NaCl stress (Fig. 3). Conse-quently, the salt-elicited eATP-H2O2-[Ca2+]cyt cascadeup-regulates genes required for K+/Na+ homeostasis andplays a crucial role in salt tolerance.



TRANSCRIPTOMIC AND PROTEOMICAPPROACHES TO DISCLOSING THEMOLECULAR BASIS OF SALT TOLERANCE

With the advent of sequencing techniques,a rapidly increasingnumber of publications are using omics techniques to uncovermolecular mechanisms of salt adaptation. Current studiesencompass EST (expressed sequence tag) sequencing, oftenof cDNA libraries enriched in salt-responsive genes, oligo-and whole-genome microarrays and next-generationsequencing (NGS) of whole-genome transcriptomes. Thelatter technique is also suitable for non-model plants whosegenomes have not been sequenced. Table 2 compiles thetranscriptomic studies on woody plants and salt stress thatappeared in the last decade (2004–2014). The majority ofresearchers investigated poplars; a few fruit trees (olives,apple and lemon) and a number of mangroves were alsostudied (Table 2). The reason for this bias is that theP. trichocarpa genome, which is relatively closely related toP. euphratica (Ma et al. 2013), was the first sequenced treegenome; numerous molecular tools therefore became avail-able for this genus (Tuskan et al. 2006).Transcriptomic studieson the salt response of a number of economically importanttree species, especially the extremely salt-tolerant N-fixingspecies,are lacking,with the exception of one study conductedwith Tamarix (Gao et al. 2008).With the availability of NGS, itis expected and hoped that this gap will be filled soon.

To study global salt responses, mangroves and other halo-phytic trees were most commonly exposed to 500 mMNaCl, whereas salt-tolerant glycophytes were investigatedafter exposure to 200 mM or lower NaCl concentrations(Table 2). In most cases, the strategy to reveal salt-responsive genes was to expose non-salt-stressed plants tosaline conditions and to investigate the transcriptomicchanges during salt acclimation. The studies lasted fromonly a few hours up to 1 d. Long-term studies for weeks ormonths are less common (Table 2) and field studies havebeen conducted only in very few cases (Brosché et al. 2005;Nguyen et al. 2006). The problem with field studies is thatproper controls are missing and stress responses to salinityare therefore difficult to disentangle from other environ-mental constraints. For example, Brosché et al. (2005)uncovered only a very small number of genes (22 of 6340distinct genes) that displayed significantly different tran-script levels in leaves of P. euphratica trees grown undersaline-field conditions compared with trees of similar agethat were regularly irrigated with fresh non-saline water.These genes were mainly from the category ‘oxidativestress’ (increased transcript levels of a lipid transfer protein,an asparagine synthase, aldehyde dehydrogenase, etc.) and‘signalling’ (receptor-like Ser/Thr kinase, cyclic nucleotideand CaM-regulated ion channels and a phospholipase C)and decreased levels of aquaporins and RD22 (Broschéet al. 2005). Nguyen et al. (2006) extracted RNA from leavesof seawater-grown mangroves and investigated the resultingcomposition of a cDNA library. They also identified a highpercentage of genes related to oxidative stress defence andsignalling. In addition, osmolyte biosynthesis, transporters

and transcription factors were important categories presentin the cDNA library (Nguyen et al. 2006).

Controlled short-term salt exposure experiments andgene ontology (GO) term annotation of the resultingtranscriptomes identified antioxidative defence, transporters,signal transduction and osmoregulation as typical categoriesthat were significantly enriched, regardless whetherglycophytes or halophytes were investigated (Table 2).Genes that were often identified were related to salt exclu-sion, such as HKT, Na+/H+ antiporters, the CIPK/SOSpathway, ABA signalling (AP2/EREB), oxidative stressdefence, osmolyte production and acclimation of photosyn-thesis in leaves (Nanjo et al. 2004; Fu et al. 2005; Mehta et al.2005; Terol et al. 2007; Dassanayake et al. 2009; Yamanakaet al. 2009; Brinker et al. 2010; Ding et al. 2010; Beritognoloet al. 2011; Chen et al. 2012; Huang et al. 2012; Janz et al. 2012;Dang et al. 2013; Li et al. 2013; Ma et al. 2013; Zhang et al.2013c). The activation of a special protein (bg 70) with anunknown function was reported only in mangroves (Miyamaet al. 2006; Dassanayake et al. 2009).

Proteomic studies of salt responses of tree species arescarce and have been mainly conducted with mangroves(Table 3). They revealed that photosynthesis and energymetabolism were major salt-responsive categories. Mainproteins identified in the category ‘stress’ were ascorbateperoxidase and glutathione transferase (Wang et al. 2013a).Overall, the number of regulated proteins identified aftersalt stress is still very small. The main categories identified inmangroves, such as photosynthesis and energy metabolism(Table 3), also formed the main categories in the proteomeof drought-stressed P. euphratica (Bogeat-Triboulot et al.2007). Therefore, global conclusions on protein setsspecifically required for salt adaptation are currently notpossible.

Transcriptional activation of distinct salt-stress pathwayswas identified by comparing the responses of tolerant andsusceptible trees (Dassanayake et al. 2009; Ding et al. 2010;Beritognolo et al. 2011). However, these comparisons are dif-ficult because salt-sensitive tree species usually show higherNa+ uptake than salt-tolerant species; therefore, thetranscriptomes after distinct salt exposure times are notrelated to the same tissue stress. Large differences between asalt-tolerant and a salt-sensitive poplar in osmotic adjust-ment and Na+ accumulation throughout the plant body havebeen demonstrated by Janz et al. (2012). They showed thatP. euphratica largely excludes Na+ from the developingxylem, whereas P. × canescens accumulates Na+ in this tissue.P. × canescens acclimates to Na+-induced osmotic stress byforming more vessels than non-stressed controls with smallerlumina. In contrast, salt uptake and osmotic adjustment islow in P. euphratica (Janz et al. 2012). With the exceptionof a few cell wall-related genes (β-amylase, xyloglucanendotransglucosylase, pectin methylesterase peroxidase,COBRA-like extracellular glycosyl-phosphatidyl inositol-anchored protein family and FLA), no significant changes inthe transcriptome of the developing xylem were detected inthe salt-tolerant poplar (Janz et al. 2012). Nevertheless, underunstressed conditions, the vessel lumina of P. euphratica are



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much smaller than those of P. × canescens, indicating an evo-lutionary adaptation of the xylem structure to osmotic stressin this species.

Although transcriptome analyses suggest convergentresponses in evolutionarily distant salt-tolerant tree species(Dassanayake et al. 2009; Zhang et al. 2013c), the questionremains which of the identified pathways or genes are crucialfor coping with high salt levels. In an increasing number ofstudies, poplars were transformed with candidate genes andtested for their performance under salt stress see subchapter:″IMPROVEMENT OF TREE SALT TOLERANCE BYGENETIC ENGINEERING″). However, only two studiesdirectly used candidates from lists of salt-responsive genes todevelop novel strategies to enhance our understanding ofsalt tolerance (Yamanaka et al. 2009; Brinker et al. 2010).Yamanaka et al. (2009) transformed Agrobacteriumtumefaciens with 28 candidate genes from salt-exposedB. gymnorrhiza roots. Those genes that improved salt toler-ance of Agrobacterium were transferred to Arabidopsisthaliana, and three of them (a lipid transfer protein, a zincfinger transcription factor ZAT, an ankyrin repeat protein)were found to enhance Arabidopsis salt resistance(Yamanaka et al. 2009).Brinker et al. (2010) reasoned that lowsalt tolerance might be due to restrictions in adaptiveresponses of some key genes that are present but insufficientlyactivated in salt-sensitive species.Among candidate genes thatwere up-regulated in P. euphratica leaves after salt acclima-tion, in silico analysis identified some orthologous genes thatwere not salt responsive in Arabidopsis (Brinker et al. 2010).Knock-out mutants of these genes, a temperature-inducedlipocalin (TIL) and a salt-induced serine rich protein, ren-dered Arabidopsis more salt sensitive,supporting the idea thatgenes not regulated in response to salt may, nevertheless, becrucial for salt tolerance (Brinker et al. 2010). Arabidopsisknock-out mutants of TIL accumulated more salt than thewild type in chloroplasts, suggesting that TIL protected pho-tosynthetic integrity from excessive salt.The mechanism is stillunknown because TIL is localized to the PM and translocatedto the cytosol, not to the chloroplasts, during salt stress(Abo-Ogiala et al. 2014). These studies show that after theidentification of candidate genes in poplar, Arabidopsis is avaluable tool for proof-of-concept. However, this approachwill only lead to the discovery and refinement of our under-standing of common salt-tolerant mechanisms.

Unfortunately, large-scale mutant collections of seeds(SALK Institute of Genomic Analysis Laboratory or othergermplasm stock centres), which have served as criticalresources for the discovery of genes controlling traits of agro-nomic interest in the model plant Arabidopsis are not avail-able for poplars or halophytic tree species. Mutantpopulations of Populus created by activation tagging andtransposon-based activation systems also exist, but theyrequire outdoor growth in areas with special permission forthe cultivation of transgenic plants or time-consuming andmanpower-intense maintenance in tissue cultures (Busovet al. 2003, 2011; Fladung 2011; Fladung & Polak 2012).There-fore, the access to these resources is less easy than that toArabidopsis T-DNA lines and there are no examples yet forTa

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the successful identification of novel genes for salinity toler-ance in these collections.

The possibility of sequencing whole transcriptomes andgenomes has now opened new avenues for understandingsalt tolerance of non-model plants. Ma et al. (2013)sequenced the P. euphratica genome and found an expansionof gene families related to salt tolerance. For example, HKT1,H+-ATPase, CAT and GR and genes involved in ABA sig-nalling, heat-shock response and osmolyte production(galactinol synthase and betaine aldehyde dehydrogenase)were expanded. Members of salt signalling pathways(CIPK1, PSD1), transcription factors (HB40, bHLH87, AP2/ERF) and oxidoreductases, such as peroxidases, were amongthe genes discovered to undergo positive selection inP. euphratica (Ma et al. 2013). Notably, the expanded genefamilies in P. euphratica belonged mainly to the categories‘carbohydrate metabolism’, ‘ATPase activity’, ‘ion transport’,‘oxidoreductase’ and ‘cell wall organisation’ (Ma et al. 2013).These categories have previously been identified as beingconstitutively up-regulated in the P. euphratica comparedwith the P. × canescens transcriptome under non-stressedconditions (Janz et al. 2010). Furthermore, genes involved insalt export such as the NHD members were found to main-tain high transcript levels under saline stress, whereas adecline was found in salt-susceptible poplars (Ottow et al.2005b; Ma et al. 2013). Overall, the recent genomic studiessupport the conclusion that salt tolerance implies perma-nently high expression of a set of stress-related genes (Janzet al. 2010). These genes cannot be detected by conventionalapproaches exposing plants to elevated salinity because theirexpression levels are already high and are not increased uponsalt stress. Future studies should therefore focus on the analy-sis of constitutively up-regulated pathways to increase ourknowledge of salt tolerance.

IMPROVEMENT OF TREE SALT TOLERANCE BYGENETIC ENGINEERING

Novel methods for genome-wide identification ofgenes for tolerance

Whole-genome sequencing, single nucleotide polymorphism(SNP) discovery, next-generation RNA sequencing andgenomic selection (GS) are new technologies to identify genesinvolved in stress protection (Harfouche et al. 2014). Forexample, genome sequencing of P. trichocarpa (Tuskan et al.2006) has advanced surveys of gene families conferring stresstolerance such as homeodomain-leucine zipper (HD-ZIP, Huet al. 2012), late embryogenesis abundant (LEA, Lan et al.2013), WRKY (He et al. 2012) and HSF (Zhang et al. 2013b).As outlined earlier, the whole-genome sequence information,now available for the salt-resistant P. euphratica (Ma et al.2013), is a valuable resource to identify sequence variationand traits that can be used for GS for salinity tolerance.

Quantitative-trait loci (QTL) mapping has been applied toelucidating traits for abiotic stress tolerance in forest trees;next-generation RNA sequencing provides new insights intothe regulatory mechanisms of the expression underlying theQTLs (Harfouche et al. 2014). The strength of this approach

was recently demonstrated when a novel ion transporter,GmCHX1, was discovered in soybean by a combination of denovo sequencing, high-density marker QTL mapping andresequencing data as well as functional analysis (Qi et al.2014). Moreover, genome-wide analysis of alternative splic-ing of pre-mRNA can explore the relationship between theexpression of the alternatively spliced transcripts and stressconditions (Iida et al. 2004). In forest trees, the application ofthese technologies can serve as an effective strategy touncover novel information to facilitate tree improvement.

Genetic engineering for tree salttolerance improvement

Genomic selection, QTL mapping, epigenetic analysis andnext-generation ecotilling are not only new technologies todiscover key genes and molecular mechanism in stressresponse in forest trees, but also accelerating traditionalbreeding programmes (Marroni et al. 2011; Resende et al.2012; Vanholme et al. 2013; Harfouche et al. 2011; 2014).Marker-based breeding requires in-depth knowledge of thetarget genes and their metabolic pathways, but has not yetbeen adapted to improve tree salinity tolerance. In recentyears, the main strategy was to identify candidate genes insalt screens or by comparison of salt-tolerant with salt-sensitive trees and to use them for poplar transformation.Poplars were chosen because transformation systems areavailable for various species in the genus and because theirrapid growth renders them interesting for biomass produc-tion on salt-affected soils (Polle & Douglas 2010; Polle et al.2013). Here, we compiled the available data on transgenicpoplar that exhibit increased salt tolerance. The genes thatwere successfully tested could be classified into the maincategories controlling ion homeostasis, counteracting ROSformation, and improving stress signalling and transcrip-tional activation of salt target genes.

Strategies to improve ion homeostasis and tocombat oxidative stressSalt tolerance can be improved by transformation withselected candidate genes. Examples have been compiled inTable 4. A major challenge for salt-stressed trees is to keepNa+ and Cl− under control. Therefore, transformation withNa+/H+ antiporter genes was tested. Overexpression of theArabidopsis Na+/H+ antiporter gene (AtNHX1) enhancedsalt tolerance in hybrid poplars P. × euramericana ‘Neva’(Jiang et al. 2011, 2012) and P. deltoides CL × P. euramericanaCL ‘NL895’ (Qiao et al. 2011). Positive effects were alsoobserved when OsNHX1 (Na+/H+ antiporter gene of Oryzasativa L.) was introduced into Poplar 84K (Wang et al. 2005).Under saline conditions, growth, photosynthetic capacity andchlorophyll content were enhanced in the NHX1-transgenicpoplars compared with the wild-type poplars (Table 4). TheNHX1-transgenic plants accumulated even more Na+ inroots and leaves during NaCl exposure than the wild type,implying that the transgenic plants increased the vacuolarcompartmentalization of Na+. Na+ sequestration in the



Tab

le4.

Tran

sfor

mat

ion

ofca

ndid

ate

gene

sfo

rim

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ing

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ity

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ranc

ein

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Gen

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arR

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ence

AtN

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1(v

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lar

Na+

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anti

port

erge

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vacuoles reduces the toxic effects of salt, evident fromdecreases in the oxidative stress marker malondialdehyde(MDA) relative to levels in wild-type poplars. The transfor-mation of PeNhaD1 (putative Na+/H+ antiporter gene fromP. euphratica) into P. tomentosa reduced the inhibitoryeffects of NaCl on photosynthesis and antioxidant enzymes(Chen 2007). PeNhaD1-expressing P. tomentosa lines dis-played an enhanced capacity for Na+ extrusion from roots,which is favourable for the plants because it lowers the risk ofexcess ROS production and lipid peroxidation.

The soybean CLC1 encodes a vacuolar Cl− transporterprotein that translocates ions from the cytoplasm to thevacuole (Sun et al. 2013b). GmCLC1-overexpressing hybridpoplars (P. deltoides × P. euramericana ‘Nanlin895’) sufferedless defoliation under salt stress than the wild-type plants.Under NaCl stress, chlorophyll contents and the activity ofantioxidant enzymes (POD and SOD) were higher inGmCLC1-expressing plants than in the wild type, and theMDA concentration was significantly lower. Apparently, theoverexpression of GmCLC1 can diminish salt damage tomembrane structures, presumably because of osmotic adjust-ment and up-regulation of antioxidative enzymes.

Studies of the overexpression of genes encodingantioxidative enzymes to improve salinity tolerance inpoplars are also relatively common. The transformation ofTaMnSOD (MnSOD gene from Tamarix androssowii) intoP. davidiana × P. bolleana resulted in a relatively higherweight gain in the transgenic than in the wild-type plantsafter NaCl stress (Wang et al. 2010). TaMnSOD-transgenicpoplar also exhibited lower MDA content than the wild typeafter being exposed to NaCl stress. High SOD activityenables the transgenic plants to better control ROS homeo-stasis. Symptoms of oxidative stress, such as membraneleakage, enhanced MDA and chlorophyll loss, were alsoameliorated in salt-stressed P. tomentosa overexpressingA. thaliana phospholipase Dα (AtPLDα) (Zhang et al. 2008).The transgenic poplars exhibited increased antioxidantenzyme activity (SOD, POD and CAT); additionally, rootgrowth of salt-stressed transgenic plants was less suppressedthan in the wild type.

LEA proteins are known to improve plant desiccation tol-erance (Ingram & Bartels 1996). The transformation ofP. simonii × P. nigra with TaLEA, a gene encoding a LEAprotein from Tamarix androssowii, increased their stress tol-erance under salt and drought conditions (Gao et al. 2013).Constitutive expression of TaLEA in poplars increasedheight growth and decreased the number of wilted or injuredleaves. Because the MDA content and relative electrolyteleakage were significantly lower in transgenic lines than inwild-type plants under stress conditions, TaLEA can protectcell membranes from the salt- and drought-induced oxidativedamage (Gao et al. 2013). Similarly, heterologous expressionof ApDnaK, the heat-shock protein Hsp70 from thehalophilic cyanobacterium Aphanothece halophytica, dimin-ished salt-induced injury in P. alba (Takabe et al. 2008). Heatshock proteins (HSPs) serve as molecular chaperones toprevent protein misfolding and aggregation under conditionsof high salinity.

Because salinity-stressed trees also suffer osmotic stress,Hu et al. (2005) overexpressed mtlD (a gene encoding amannitol-1-phosphate dehydrogenase, from Escherichia coli)in P. tomentosa. The transformed poplars survived a higherlevel of NaCl (75 mM) than the wild-type poplar plants(25 mM NaCl) during a 40 d hydroponic experiment (Huet al. 2005). The beneficial effect of mtlD most likely resultedfrom increases in compatible solutes, which improved thecapacity for osmotic adjustment in transgenic poplars.

Signalling and transcriptional regulation ofsalt responsivenessSignalling of salt stress involves the SOS pathway, inwhich SOS2 and SOS3 are required for the transductionof the signal (Zhu 2001). SOS2 genes fromP. trichocarpa overexpressed in the poplar clone T89(P. tremula × P. tremuloides Michx clone T89) promotedgrowth under salinity stress (Zhou et al. 2014). Comparedwith wild-type plants, the PtSOS2-transgenic lines PtSOS2.1,PtSOS2.2 and PtSOS2.3 maintained increased concentra-tions of proline and photosynthetic pigments, higher relativewater content and activities of antioxidant enzymes andlower MDA concentrations under salinity. Positive effects ongrowth and biomass production were also observed in thehybrid poplar clone Shanxin (P. davidiana × P. bolleana)transformed with a PtSOS3 gene (Tang et al. 2014).The trans-genic plants accumulated less Na+ and retained higher K+

concentrations in the roots than the wild type (Tang et al.2014). Apparently, PtSOS2 and PtSOS3 improve the salt tol-erance of poplars by enhancing osmotic adjustment andreducing oxidative stress.

Salt tolerance was also improved by the overexpression ofkinases. In P. × euramericana ‘Neva’, the expression of Nico-tiana tabacum histidine kinase 1 (NtHK1) improved chloro-phyll retention, increased K+ content and decreasedmembrane leakage compared with wild-type plants undersalt stress (Zhang et al. 2011). Moreover, the Na+ concentra-tion in the transgenic plants was much higher than in the wildtype, suggesting enhanced vacuolar Na+ sequestration(Zhang et al. 2011). AtGSK1-transgenic P. alba × P. tremulavar. glandulosa (AtGSK1 is a glycogen synthase kinase3/shaggy-like kinase gene from A. thaliana) maintained vig-orous root growth in the presence of 125 mM NaCl and trans-formed calli survived 150 mM NaCl (Han et al. 2013). Thetransgenic poplars showed higher photosynthetic rates,stomatal conductance and evaporation under NaCl treat-ment than the wild type.The chloride content and membraneleakage of the transgenic poplars were lower than in wild-type poplars, although leaf Na+ increased dramatically andleaf K+ decreased in transgenic poplars (Han et al. 2013).

Further attempts were made to improve salinity toleranceby the overexpression of salt-responsive transcriptionfactors, such as CBLs. Selected CBL genes from P. euphraticawere introduced into the triploid high-yielding white poplarP. tomentosa ‘YiXianCiZhu B385’ (Li et al. 2012b). Trans-genic poplars overexpressing PeCBL6 or PeCBL10 exhibitedstronger height growth and fewer wilted leaves under high



salt than the wild type. It was suggested that theoverexpression of CBL improves calcium signalling andameliorates acclimation to salt stress. The PtCBL genesPtCBL10A and PtCBL10B from P. trichocarpa wereoverexpressed in P. davidiana × P. bolleana (Tang et al.2014). PeCBL10A- and PeCBL10B-transgenic lines alsoshowed significantly greater shoot height and biomass thanthe wild-type plants under salt stress. The Na+ content ofleaves in the transgenic plants, but not of roots, was signifi-cantly lower than that in the wild type. Transgenic and wild-type plants accumulated equivalent levels of K+ during saltstress, but their Na+/K+ ratio was greatly improved because ofdecreased Na+ in the transgenic lines.

Dehydration-responsive element-binding protein (DREB)like transcription factors have been implicated in droughttolerance. P. tomentosa cannot survive NaCl concentrationsabove 100 mM, but its survival rate is greatly increased aftertransformation with AhDREB1, a DREB-like transcriptionfactor from Atriplex hortensis (Du et al. 2012). This observa-tion suggests that this transcription activates a spectrum ofstress defences.

The balance between primary and secondary metabolisminvolves regulation via jasmonate- and elicitor-responsivepromoter elements (JERE). A JERE gene was transferredinto the hybrid poplar P. alba × P. berolinensis (Li et al.2009b). At NaCl concentrations of up to 300 mM, the extentof salt damage, reduction in leaf water content and suppres-sion of shoot growth were less pronounced in the transgenicthan in wild-type plants. The transgenic plants accumulatedhigher proline and higher Na+ concentrations than the wildtype (Li et al. 2009b). Field tests in a coastal area, where thetotal soil salt concentration was 0.3%, showed that 3-year-oldtransgenic plants displayed more vigorous growth than wild-type plants (Li et al. 2009b).

Multiple gene transformation for salt tolerance

Salinity tolerance is a multigenic trait.Therefore, long-lastingimprovement of salt tolerance in trees may require modifica-tion with a number of different genes to improve the signallingand activation of stress responses on the one hand and toenable morphological adjustment on the other hand. Genestacking has shown to be able to maximize salinity stresstolerance in model plants and crop species (Naqvi et al. 2009;Yang et al. 2009; Que et al. 2010; Storer et al. 2012). However,these approaches are still in their infancy in forest trees.Thereis only one published study that attempted to improve treetolerance for multiple stresses by simultaneously transferringfive target genes. Using biolistic bombardment-mediatedco-transformation, the target genes were introduced into theP. × euramericana hybrid, ‘Guariento’ (Su et al. 2011). Themultigene-transformed poplars contained vgb, encoding theaerobe Vitreoscilla haemoglobin; SacB, encoding levansucrase involved in fructan biosynthesis in Bacillus subtilis;BtCry3A, encoding an endotoxin from Bacillus thuringiensis;OC-I, an insect-resistance gene encoding the proteinaseinhibitor oryzacystatin I from rice;and JERF36,a tomato geneencoding a jasmonate/ethylene-responsive factor.The perfor-

mance of the multigene-transformed poplars in response todrought, salinity,waterlogging and insects was tested in green-house experiments and field trials (Su et al. 2011). In green-house studies, the multigene-transformed lines showedgreater stem heights, basal diameter increments and totalbiomass in response to salt stress than the wild-type plants.The improved growth was primarily attributed to higher chlo-rophyll concentrations and instantaneous water-use efficiencyin the transgenic trees. In field trials, the average height anddiameter at breast height of 2.5-year-old transgenic treesgrowing in naturally saline soil were slightly greater than thoseof the wild-type trees.The improved plant performance undersalinity and other stress factors (drought, waterlogging, resist-ance to the leaf beetle Plagiodera versicolora) suggests thatmultiple-stress resistance can be achieved in tree species bysimultaneous multigene transformation. However, theinteraction between each transgene in the multigenetransformants is largely unknown. It is of great interest toimprove poplar salt tolerance by simultaneously transferringmultiple salt-related genes, but the potential metabolic trade-offs that may result for instance in biomass reductions need tobe avoided.Therefore, further studies are needed to assess thestability of the transformants across different environmentsand during the typical life times of poplar plantations.

CONCLUSIONS AND RESEARCH NEEDS

This review addresses the perspectives of using salt-affectedland for biomass production by developing and growing salt-tolerant tree species. Expanding tree plantations and affor-estation in a large proportion of arid areas that have salineground water may be an option for increasing the productionof feedstocks for non-food goods. Estimates suggest that sig-nificant areas are available for this purpose if tree tolerance isimproved. Because areas have to be set aside for protection ofthe vegetation and the conservation of naturally saline habi-tats, utilization of trees is an important option for therecultivation of secondary salinized areas and for soil recla-mation of degraded, former agricultural areas. In addition totheir economic potential, tree plantations and afforestation insodic and salt-affected land areas provide significant ecosys-tem services,such as increasing carbon sequestration,prevent-ing erosion and improving the microclimate.Despite attemptsto catalogue areas of secondary salinization,our knowledge ofthe global extent and current dynamics of saline areas is verylimited. Therefore, improving survey methods for detectingand recording soil salinity is an important ongoing task.

Salinized soils are usually located in dry, hot areas. There-fore, tree species that can cope with strong osmotic and ionicstress are required for cultivation of salt-affected land.Nitrogen-fixing tree species of the genera Casuarina,Prosopisand Acacia naturally occur on salinized soils in dry climates,but the molecular and physiological mechanisms underlyingtheir salt tolerance have barely been studied. This is a pro-found research gap that should be filled in the near future;understanding the mechanistic basis of the extraordinarystress tolerance of these species may drastically accelerate theselection and breeding of high-yielding, stress-resistant trees.



In the last decade, significant progress has been made inunderstanding salt adaptation and acclimation in model treespecies. The stress responses of halophytic and glycophytictree species have been extensively investigated at thegenomic, molecular, cellular and whole-plant level. The saltsignalling networks in water, ionic and ROS homeostasishave been of particular interest. Whole transcriptome andgenome sequencing of salt-tolerant and salt-sensitive modeltrees has uncovered specific and genotypic differences in salttolerance. Evolutionary adaptation involves the expansion ofgene families required for salt signalling and ion transport tocontrol Na+ accumulation. Transcriptional levels of genes inenergy metabolism, ROS and ion homeostasis are constitu-tively higher in salt-tolerant than in salt-sensitive species. Atthe anatomical and morphological level, plant traits such ashydraulic adaptedness and plasticity and the formation ofleaf succulence have been identified, but their molecularbases need to be further studied. A limitation to our under-standing of salt tolerance is that key mechanisms have beenidentified almost exclusively after treatment with NaCl undercontrolled conditions. Understanding how trees acclimate tosodic and saline-sodic soils and to natural fluctuations inenvironmental stress requires further research. Therefore,field trials in saline and sodic areas are needed to investigatethe long-term effects of varying water supply, radiation inten-sity and temperature on tree performance.

Genetic engineering has been used for improving salt tol-erance of economically important trees. Currently, single can-didate genes for desirable traits can be introduced andexpressed efficiently in model woody plants, for example,poplars. However, salt tolerance is a multigene trait. There-fore, an improvement of valuable biomass or fruit trees thatcan cope with multiple aspects of salt stress, such as saltextrusion, ion compartmentalization, osmolyte synthesis andanatomical adjustments, will require the simultaneous intro-duction of multiple target genes and/or modified expressionof a set of stress-related genes. Stable expression and know-ledge of the interactions among the transgenes in themultigene transformants are key issues that need to beresolved before transgenic trees can be used commercially.Special attention should also be paid to transgene confine-ment to avoid ecological risks.

ACKNOWLEDGMENTS

The research in the authors’ laboratories is financiallysupported by the German Science Foundation (DFG), theGuest Lecturer Scheme of Georg-August UniversitätGöttingen (Germany), the Alexander von Humboldt-Stiftung (Germany), travel grants by the Bundesministeriumfür Ernährung, Landwirtschaft und Verbraucherschutz(BMELV), and by the National Natural Science Foundationof China (grant no. 31270654), the Research Project of theChinese Ministry of Education (grant no. 113013A), theProgram for Changjiang Scholars and Innovative ResearchTeams in University (grant no. IRT13047), the key project forOversea Scholars by the Ministry of Human Resources andSocial Security of China (grant no. 2012001) and the Program

of Introducing Talents of Discipline to Universities (111Project, grant no. B13007). The authors declare no conflict ofinterest.

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On the salty side of life: molecular, physiological and anatomical adaptation and acclimation of trees to extreme habitats