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Journal of Plant Physiology 201 (2016) 1–8

Contents lists available at ScienceDirect

Journal of Plant Physiology

journa l h om epage: www.elsev ier .com/ locate / jp lph

hysiology

cclimation improves salt stress tolerance in Zea mays plants

amilla Pandolfia,b,∗, Elisa Azzarellob, Stefano Mancusob, Sergey Shabalaa

School of Land and Food, University of Tasmania, Private Bag 54, Hobart, Tas 7001, AustraliaDepartment of Agrifood and Environmental Science, University of Florence, Viale delle Idee 30, 50019 Sesto Fiorentino, FI, Italy

r t i c l e i n f o

rticle history:eceived 6 March 2016eceived in revised form 15 June 2016ccepted 16 June 2016vailable online 23 June 2016

eywords:cclimation

on channelsriming salinity

a b s t r a c t

Plants exposure to low level salinity activates an array of processes leading to an improvement of plantstress tolerance. Although the beneficial effect of acclimation was demonstrated in many herbaceousspecies, underlying mechanisms behind this phenomenon remain poorly understood. In the present studywe have addressed this issue by investigating ionic mechanisms underlying the process of plant acclima-tion to salinity stress in Zea mays. Effect of acclimation were examined in two parallel sets of experiments:a growth experiment for agronomic assessments, sap analysis, stomatal conductance, chlorophyll con-tent, and confocal laser scanning imaging; and a lab experiment for in vivo ion flux measurements fromroot tissues. Being exposed to salinity, acclimated plants (1) retain more K+ but accumulate less Na+ inroots; (2) have better vacuolar Na+ sequestration ability in leaves and thus are capable of accumulating

+

ea maysacuolar sequestration

larger amounts of Na in the shoot without having any detrimental effect on leaf photochemistry; and(3) rely more on Na+ for osmotic adjustment in the shoot. At the same time, acclimation affect was notrelated in increased root Na+ exclusion ability. It appears that even in a such salt-sensitive species asmaize, Na+ exclusion from uptake is of a much less importance compared with the efficient vacuolar Na+

sequestration in the shoot.

. Introduction

Salt stress in plants is one of the main causes limiting agricul-ural productivity in the world’s irrigated land. A way to tackle theroblem is to try to enhance plant tolerance to salt stress by under-tanding basic natural mechanisms that naturally occur in plantsnder changing environmental conditions. As such, acclimation toxternal environmental changes occurs in plants thanks to inter-al adjustments within tissues and cells, enabling cell metabolismo proceed under these somewhat altered conditions (Demmig-dams et al., 2008). It was reported that salt tolerance of manylant species can be increased by previous exposure to a low

evel of stress for a certain period of time (Amzallag et al., 1990;

ethke and Drew, 1992; Umezawa et al., 2000; Silveira et al., 2001;janaguiraman et al., 2006). Reported beneficial effects included

mproved survival, growth rate and yield (Amzallag et al., 1990;

Abbreviations: DW, dry weight; EK, equilibrium potential; FW, fresh weight; Gs,tomatal conductance; KOR, outwardly rectifying potassium channel; MIFE, micro-lectrode ion flux estimation technique; NHX, vacuolar Na+/H+ antiporters; NSCC,on-selective cation; SOS1, salt overlay sensitive antiporters 1; PM, plasma mem-rane; PCD, programmed cell death.∗ Corresponding author at: Department of Agrifood and Environmental Science,niversity of Florence, Viale delle Idee 30, Sesto Fiorentino, FI, 50019, Italy

E-mail address: camilla.pandolfi@unifi.it (C. Pandolfi).

ttp://dx.doi.org/10.1016/j.jplph.2016.06.010176-1617/© 2016 Elsevier GmbH. All rights reserved.

© 2016 Elsevier GmbH. All rights reserved.

Djanaguiraman et al., 2006). However, the physiological mech-anisms beyond this acquired resistance have not been clearlyelucidated. Umezawa et al. (2000) related the better performanceof acclimated soybean to a reduced accumulation of Na+ in plantleaves, whereas Saha et al. (2010) and Ottow et al. (2005) related itto an improvement in the ability to withstand osmotic stress. At acellular level, salinity stress can be distinguished between its ionicand osmotic component thanks to the work of Munns (1993) whodeveloped a model for the whole-plant level. In our recent workon peas (Pandolfi et al., 2012) we have shown that acclimation innon-ionic (ie polyethylene glycol) isotonic media was not as effi-cient as in NaCl, suggesting that acclimation to salinity is relatedto the ion-specific rather than the osmotic component. Further-more, metabolic acclimation via previous exposure to a low levelof salinity was induced primarily in roots and was related to a betterregulation of xylem ion loading (Pandolfi et al., 2012).

One of the hallmarks of detrimental effects of salinity at thetissue level is K+ efflux from plant roots (Shabala and Cuin, 2008;Cuin et al., 2012; Wu et al., 2013) via both depolarization-activatedoutward-rectifying K+ (KOR; Chen et al., 2007) and ROS-activatednon-selective cation (NSCC; Bose et al., 2014) channels. This efflux

disturbs cytosolic K+ homeostasis (Cuin et al., 2003), with majorimplications to cell metabolism and its fate (e.g. transition to pro-grammed cell death; Shabala et al., 2007; Shabala and Pottosin,2014). For this reason, a strong correlation between plant’s K+

2 Plant Physiology 201 (2016) 1–8

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Fig. 1. Experimental procedure for the acclimation experiment. Seedlings wereestablished under control conditions (no salt) until 10 days old; then a four potswere irrigated with a 25 mM NaCl solution for one week (A25) and after one week,

C. Pandolfi et al. / Journal of

etention ability and salt tolerance has been observed in both rootChen et al., 2007; Cuin et al., 2008; Smethurst et al., 2008) and leafWu et al., 2013) tissues in several species. Another key determi-ant of salinity tolerance is Na+ exclusion from the cytosol. Na+/H+

ntiporters are thought to drive the active transport of Na+ outf plant cells (Apse and Blumwald, 2007), either back to externaledia, or into vacuole. Overexpression of the plasma membranea+/H+ antiporter SOS1 has been found to reduce Na+ accumula-

ion and improve salinity tolerance in transgenic Arabidopsis (Shit al., 2003), while efficient sequestration of Na+ in the vacuoles byeans of Na+/H+ antiporters from the NHX family was also essential

o confer salinity tolerance in a range of plant species (Apse et al.,999). In the latter case, in addition to avoiding accumulation ofoxic Na+ in the cytosol, vacuolar Na+ sequestration also contributeso the turgor maintenance (Zhang et al., 2001; Yokoi et al., 2002).ompartmentalization of sodium in the vacuole has been reporteds one of the clue to salt adaptation (Munns and Tester, 2008). Vac-olar NHX proteins (NHX1 and NHX2; NHX = Na+/H+ exchanger)re considered the main players in sodium compartmentalizationn the vacuoles (Apse et al., 1999, 2003; Blumwald, 2000). Moreecently, vesicle trafficking has been described as a contributor forodium compartmentation (Liu et al., 2007; Hamaji et al., 2009;iu, 2012). Control of Na+-permeable slow (SV) and fast (FV) vac-olar channels is also essential for effective Na+ retention in vacuoleBonales-Alatorre et al., 2013).

The aim of this study was to reveal the role and relative contribu-ion of the ionic mechanisms that play a role in plant acclimation toalinity. This was achieved by a whole-plant physiological assess-ent of plants pre-treated with NaCl and by studying patterns of

on flux across cellular membranes in salt-exposed acclimated andon-acclimated roots. In addition, we aimed to see if acclimationffect reported earlier for C3 Pisum sativum species (Pandolfi et al.,012) could be also observed in more tolerant C4 Zea mays plants,here Na+ is considered to be a beneficial nutrient (Subbarao et al.,

003). Our results suggest that exposing Zea mays to moderatealinity activates a set of physiological adjustments enabling plantso withstand severe saline conditions, and that it is the acclimationo the ion toxicity component of salt stress that play a major rolen plant acclimation. This acclimation takes place in both root andhoot tissues. At a root level, it involves better potassium retentionnd, as a result, a better control of intracellular K/Na ratio. In leaves,cclimation results in a better sequestration of sodium in the vac-oles. The implications for this results will be discussed the in theollowing paragraphs.

. Materials and methods

.1. Growth experiment

Maize plants (Zea mais L cv B73; a kind gift of Dr Trevor Gar-ett, Univ Adelaide) were grown from seeds between Novembernd December 2010. Seeds were placed in 4 l plastic pots, 4 seedsor each pot, in a standard potting mixture (70% composted pineark; 20% coarse sand; 10% sphagnum peat; Limil at 18 kg m−3; andolomite at 18 kg m−3). Plant nutrient balance was maintained bydding the slow release Osmocote PlusTM fertilizer (at 6 kg m−3)lus ferrous sulphate (at 500 g m−3). Plants were grown undermbient light in a temperature-controlled glasshouse (day/nightemperature 26 ◦C/19 ◦C; average humidity at 65%) at the Univer-ity of Tasmania (Hobart, Australia). Plants were hand wateredn a daily basis to achieve full water-holding capacity and leach

ut any possible salt accumulating in root rhizosphere to ensureniform and constant EC values in soil solution (tested by peri-dic measurements of soil electric conductivity; data not shown).eedlings were established under control conditions (no salt) until

were irrigated with a solution containing 100 mM NaCl, for two weeks, alongsidewith non-acclimated pots (NA). The remaining pots were irrigated daily with waterand used as control.

10 days old; then 4 of the 12 pots were irrigated with a 25 mMNaCl solution for one week (Fig. 1). These plants are referred as“acclimated” in this study. After one week of acclimation period,these pots were irrigated with a solution containing 100 mM NaCl,for two weeks, alongside with 4 non- acclimated pots. The plantswere irrigated with the final concentration of 100 mM NaCl with-out any progressive increments, to mimic conditions observed inthe field brought by raising saline water tables. The remainingpots were irrigated daily with water and used as control. At thebeginning of acclimation plants’ height was 12–15 cm, and theyhad three fully developed leaves. The three different sets of plantswere termed as follow: Control (non-acclimated, non-stressed), NA(non-acclimated, stressed), A(25) (acclimated, stressed). The salin-ity levels were chosen on the base of the following consideration.Pre-treatment with 25 mM was selected to exclude the possibilityof (i) a strong reduction of growth during the acclimation period, inorder to have acclimated and not-acclimated plants of comparablesizes at the start of the salinity treatment; and (ii) on the basis of ourprevious experiment of the salt-sensitive Pisum sativum (Pandolfiet al., 2012), in which two pre-treatments were tested (10 mM and25 mM) and only the lower one triggered a beneficial reaction in theacclimated plants. The final salinity treatment was set at 100 mM toensure a significant reduction of the growth as reported in previousexperiments (e.g. Rodríguez et al., 1997).

2.2. Agronomical assessment

Eight plants were harvested for each treatment at the end ofacclimation (day 17) and at the end of NaCl stress period (day 31).Plants were divided into leaves and roots, and their fresh weight(FW) was measured. Samples were then dried at 70 ◦C for 72 h, andtheir dry weight (DW) then determined.

2.3. Sap analysis for K+, Na+ and osmolarity

For each plant, the third from the bottom (fully expanded but notsenescing) leaf was collected at the end of acclimation and treat-ment periods. Root samples were also collected by rinsing themthoroughly in 10 mM CaCl2 for 2 min to remove apoplastic NaCland then blotting them dry with paper towels. Samples were col-lected in Falcon tubes and stored at −20 ◦C. Leaf and root sap was

extracted using the freeze-thaw method as described in (Cuin et al.,2010) and its osmolarity was determined using a vapour pressureosmometer (Vapro, Wescor Inc Logan, Utah, USA). For the deter-mination of Na+ and K+ contents, samples were diluted 1:50 and

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easured using a flame photometer (PFP7, Jenway, Felsted Dun-ow, Essex, England). The contribution of compatible solutes was

stimated by calculating the contribution of three major inorganicsmolytes (Na+, Cl−, and K+) measured in direct experiments, andhen subtracting this value from the measured overall osmolality,s described elsewhere (Shabala et al., 2012).

.4. SPAD and Gs measurements

Leaf chlorophyll content was measured indirectly usingPAD-502 chlorophyll meter (Minolta Camera Co Ltd, Japan). Mea-urements were taken from the second topmost fully expandedeaves of all the plants at weekly intervals. Concurrently, stomatalonductance (Gs) was measured using a Delta-T MK3 porometerDelta-T devices, Cambridge, UK).

.5. Growth condition for laboratory experiments

A separate set of seedlings was grown for ion flux measurementsnder laboratory conditions. Seeds were surface sterilized with 3%2O2 for 10 min and thoroughly rinsed with distilled water. Seedsere germinated in a dark growth cabinet at 24 ◦C. Uniformly ger-inated seedlings were selected and grown in hydroponics in three

lastic containers located in the same growth cabinet. Seedlingsere suspended on a plastic grid so that their roots were completely

mmersed in one of the following solutions: (1) control solution0.5 mM KCl and 0.1 mM CaCl2); (2) acclimation solution (05 mMCl; 01 mM CaCl2; 25 mM NaCl); and (3) saline solution (0.5 mMCl; 0.1 mM CaCl2; 100 mM NaCl); Aeration was provided by thequarium air pumps via flexible plastic tubing. Seedlings were accli-ated for either one (abbreviated A(1D)) or three (A(3D)) days. All

he seedlings were 6 days old at the time of the measurements.

.6. Ion flux measurements

Net K+, Na+ and H+ fluxes were measured using the non-invasiveicroelectrode ion flux estimation (MIFE) technique (UTas Inno-

ation Ltd, Hobart, Tasmania) as described elsewhere (Chen et al.,007; Bose et al., 2014). The electrode travel range was 100 �m,etween 50 and 150 �m from the root surface. During experiments,aize seedlings were placed in a 10 ml measuring chamber. Their

oots were immobilized in a horizontal position as described else-here (Chen et al., 2007; Cuin et al., 2011) and pre-incubated in aSM solution (0.5 mM KCl and 0.1 mM CaCl2) for 1 h. The measuringhamber was transferred into the Faraday cage and immobilized onhe computer-driven 3D hydraulic manipulator. Electrodes wereositioned near the root surface, and net fluxes of specific ionsere measured for about 10 min. Then 100 mM NaCl treatmentas given, followed by another 25 min of recording. Measurementsere performed in the mature zone (20 mm from the root apex) of

ntact roots.

.7. Measuring net Na+ efflux in “recovery” experiments

Roots of five days-day-old seedlings were exposed to 100 mMaCl for 24 h. One hour prior to measurement, a seedling was

ransferred to a 10 ml measuring chamber containing the bathingedium, still in the presence of 100 mM NaCl After 1 h, this solu-

ion was poured off and the root was quickly rinsed three timesn 10 mM CaCl2 to remove surface NaCl. The chamber was thenlled with the standard bathing medium, minus NaCl, and net+ and Na+ fluxes were monitored concurrently for up to 30 min.

he first 20 min of measurements were discarded to account forossible apoplastic contribution (see Cuin et al., 2011 for all theethodological aspects and validation of the protocol). The mea-

ured net Na+ efflux reflected the functional activity of SOS1-like

Physiology 201 (2016) 1–8 3

Na+/H+ exchanger, as proven in direct pharmacological and geneticexperiments using Arabidosis sos mutants (Cuin et al. 2011).

2.8. K+ leakage in 24 h

Acclimated (A(3D)) and non-acclimated seedlings (NA) uniformseedlings were grouped into two groups (5 seedlings each) andtransferred in two Falcon tubes containing 7 ml of 50 mM of NaCl.Five more non-acclimated seedlings were transferred into distilledwater as additional control. After the 24 h exposure to saline solu-tion, solution was sampled and K+ concentration was assessed bythe flame photometer as described before.

2.9. Confocal laser scanning imaging

Confocal imaging was performed using an upright Leica LaserScanning Confocal Microscope SP5 (Leica Microsystems, Germany)equipped with a 40 oil immersion objective essentially as describedin Cuin et al. (2011). Leaf disks 5 mm in diameters were incubated inEppendorf tubes in 500 ml of the 10 mM Corona Green (MolecularProbes, USA). After 2 h of incubation, the samples were rinsed ina buffered MES solution and examined using confocal microscopyfollowing the standard protocol (Cuin et al., 2011). The excitationwavelength was set at 488 nm, and the emission was detected at510 − 520 nm.

2.10. Statistical analysis

Statistical analysis of data was processed using analysis of vari-ance t-test and one-way ANOVA and differences between columnswere assessed using Tukey’s Multiple Comparison Test with thesoftware Graph-Pad Prism (Ver 50a for MAC OS X). Differencesbetween treatments were considered significant at P < 0.05.

3. Results

3.1. Plant growth

One week of acclimation had no significant (P < 0.05) effect onfresh and dry weights of acclimated plants or in the biomass distri-bution between root and shoot (data not shown). No changes werealso recorded in the root and shoot K content, stomatal conductance(Gs), and SPAD units (data not shown). Therefore, in physiologicalterms the acclimated plants were comparable with untreated ones,before the onset of the salt stress. One week of acclimation in 25 mMNaCl has significantly reduced detrimental effects of salt. As a result,fresh weight of acclimated (A(25)) plants was comparable to con-trols after plant’s exposure to 100 mN NaCl for 2 weeks (Table 1),and whereas the shoots of both stressed plants (NA and A(25)) werecomparable, root apparatus was more developed in A(25) than inNA. The 100 mM NaCl stress caused a decrease in SPAD units in not-acclimated (NA) plants, whereas this decline was less pronouncedin A(25) plants. The same trend was observed for stomatal conduc-tance (Gs) (Table 1). The root/shoot ratio of acclimated plants wasalso significantly (P < 0.05) higher than both NA and control plants.These results are consistent with our previous findings on Pisumsativum for which acclimation mechanisms are mainly noticed inroots (Pandolfi et al., 2012).

3.2. Effects of acclimation on plant ionic relations and osmolarity

One week of acclimation treatment significantly increased theroot and shoot Na+ content (Fig. 2C, I) and osmolarity (Fig. 2E, K).At the same time, no significant (at P < 0.05) changes in tissue K+

content was detected in acclimated plants. Salinity stress (100 mM

4 C. Pandolfi et al. / Journal of Plant Physiology 201 (2016) 1–8

Table 1Indirect measure of chlorophyll content (measured by chlorophyll meter SPAD), stomatal conductance (Gs), root to shoot ratio, water contents (WC), fresh and dry weightsafter 2 weeks of salt stress Mean ± SE (n = 6).

Treatment Chlorophyll Gs Root/Shoot WC Fresh Weight (g)

(SPAD units) nmol m−2 s −1 Ratio (%) Shoot Root Total

Control 360 ± 159a 758± 842a 054 ± 006b 804 ± 048 ns 242 ± 12 a 131 ± 16 ab 373 ± 22 aNA 272 ± 086c 416± 357b 054 ± 011b 783 ± 134 ns 147 ± 09 b 78 ± 14 b 225 ± 18 bA(25) 314 ± 081b 646 ± 825ab 085 ± 005a 814 ± 086 ns 169 ± 148 b 144 ± 111 a 312 ± 239 a

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ig. 2. Na+ and K+ concentrations and osmolarity of leaf and root sap, measured at t00 mM) (n = 6).

aCl for two weeks) has further increased root and shoot Na+ con-ent (Fig. 2D, J) and osmolarity (Fig. 2F,L). No significant effect ofcclimation was reported in either organ (Fig. 2F, L). At the sameime, acclimated plants accumulated less Na+ in roots comparedith non-acclimated ones (Fig. 2 D) but more in the shoot tissue

Fig. 2J) under salt stress conditions Both differences are significantt P < 0.05. Acclimated plants also retained more K+ in their rootsompared with NA ones after 2 weeks of 100 mM NaCl exposureFig. 2B).

The osmolarity of stressed plants (acclimated and non-cclimated) was comparable, however, potassium and sodium totalontent differs (Fig. 3). In shoots, inorganic ions accounted for 75%f tissue osmolarity in NA plants but to 95% in acclimated A(25)lants (Fig. 3A) In roots, the ameliorating effect of acclimation isroved by higher K/Na ratio (Fig. 3B).

.3. Acclimation improves root K+ retention ability under salineonditions

Roots K+ retention ability was assessed by measuring net K+

fflux triggered by NaCl treatment using the microelectrode ionux estimation (MIFE) technique 100 mM NaCl treatment induced

of acclimation period (1 wk 25 mM) and after 2 weeks of the salinity stress (2 wks

a significant K+ efflux from epidermal cells in the mature regionof maize root seedlings (Fig. 4A). This salt-induced K+ efflux wasinstantaneous, reaching peak values immediately after the treat-ment. Neither one day – A(1D) – or three days – A(3D) – ofacclimation altered the peak K+ efflux observed within the firstminutes after sudden salt exposure (Fig. 4A). This massive K+ leakfollowed by the gradual recovery which was different betweenacclimated and non-acclimated plants. Regardless of the lengthof acclimation (1 or 3 days), acclimated roots showed ∼50% lessK+ efflux after 20 min of stress onset (Fig. 4A), showing ∼30%higher overall K+ retention ability over the first 20 min (insert inFig. 4A). Consistent with these results, the amount of K+ leakedover the 24 h period was significantly (P < 005) less in acclimatedroots (Fig. 4B).

3.4. Root Na+ efflux ability was not affected by acclimation

Root Na+ efflux ability was evaluated (see Materials and Meth-

ods for details) by transferring salt-treated roots in Na+-free mediaand measuring the magnitude of Na+ efflux. As shown in pharma-cological and loss-of-a function Arabidopsis transport mutants, themeasured Na+ efflux reflects the activity of the plasma membrane

C. Pandolfi et al. / Journal of Plant Physiology 201 (2016) 1–8 5

Fig. 3. Relative contribution of ions (K+, Na+ and Cl−) and compatible solutes to the total osmolarity, and K/Na ratio in shoots and roots at the end of the experiment.

Fig. 4. (A) Transient K+ fluxes measured from maize roots in response to 100 mMNaCl treatment from control and 1 and 3 day acclimated samples Means ± SE. Thesign convention is “efflux negative” for all MIFE measurements (n = 6–8 roots). Thet + +

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Fig. 5. Sodium efflux from maize roots measured immediately after the removal of100 mM NaCl. Six-day-old seedlings were treated with 100 mM NaCl for 24 h before

7 days in rice (Djanaguiraman et al., 2006) to 20 days in Sorghum

otal K leaked from the root in 20 min. (B) The total K leaked from the root in0 mM of NaCl in 24 h treatment Mean ± SE (n = 4–7).

OS1 Na+/H+ exchanger (Cuin et al., 2011). Three days of acclima-

ion has no significant (P < 0.05) effect on SOS1-like activity in maizeoots, with both NA and A(3D) plants showing net Na+ efflux ofround −100 nmol m−2 s−1 (Fig. 5).

its removal, and the resultant net Na+ fluxes measured. Mean SE (n = 6 seedlings).

3.5. Acclimation improves vacuolar Na+ sequestration in leafepidermal cells

Imaging profiles showed that Na+ specific fluorescence occurredboth in acclimated and not acclimated plants. Interestingly, Na+

localisation was mainly in the cytosol in the NA leaves whereas inA(25) Na+ was mainly confined in vacuolar regions (Fig. 6). Underno-salt stress, CoroNa-Green fluorescence was almost undetectablein the leaves due to low Na+ content (data not shown).

4. Discussion

Plants exposure to low level salinity activates an array of pro-cesses leading to an improvement of plant stress tolerance. This hasalready been demonstrated for different herbaceous species such assoybean, rice, sorghum and pea (Amzallag et al., 1990; Umezawaet al., 2000; Djanaguiraman et al., 2006; Pandolfi et al., 2012). Inthe literature the timing of pre-treatment is very different, from

bicolor (Amzallag et al., 1990), and it appears that the length of thepre-treatment is strongly related to the plant species. Umezawaet al. (2000) addressed specifically this issue, and reported that a

6 C. Pandolfi et al. / Journal of Plant Physiology 201 (2016) 1–8

Fig. 6. Laser scanning confocal images of maize leaves. Leaf segments were cut and labelled with 10 mM Corona Green dye for 1 h before the confocal images were taken.( ted an +

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A–B) One (of six) typical leaf segment taken for each treatment (NA-Not Acclimaontent ratio in epidermal leaf cells of the two treatments was done. The Na+ contenshowed in arbitrary units).

re-treatment shorter than 10 days is unable to trigger a beneficialffect in soybean, whereas in our previous work on Pisum sativum,

days were enough to see visible changes (Pandolfi et al., 2012),nd we retained the same protocol for this experiment.

In the present study we investigated in depth the mechanismsehind plant acclimation to salinity stress using a Zea mays as a rep-esentative salt-sensitive glycophyte species. The major findings ofhis work can be summarised as follows. Being exposed to salinity,cclimated plants (1) retain more K+ but accumulate less Na+ inoots; (2) have better vacuolar Na+ sequestration ability in leavesnd thus are capable of accumulating larger amounts of Na+ in thehoot without having any detrimental effect on leaf photochem-stry; and (3) rely more on Na+ for osmotic adjustment in the shoot.t the same time, acclimation affect was not related in increasedoot Na+ exclusion ability. The physiological rationale behind thesebservations is discussed below.

In our experiments, no reduction in roots biomass was reported;either during the acclimation phase nor after the main salt treat-ent (Table 1). Interestingly biomass accumulation was equal to

ontrol despite A(25) plants had higher Na+ content in shoots at the

nd of the 2 weeks of salt treatment (Fig. 2). This points out thatcclimated plants possessed highly efficient mechanisms for Na+

equestration in the shoot. Experiments with fluorescent CoroNa

d A25-acclimated) is shown. (C–E) Quantification of the cytosolic to vacuolar Naach cell compartment is proportional to the intensity of Corona Green fluorescence

Green dye (Fig. 6) strongly suggest that improved vacuolar Na+

sequestration was behind this phenomenon.The transport of Na+ into the vacuoles is mediated by a tono-

plast Na+/H+ antiporter encoded by NHX genes that were found tobe present and operate in both root and leaf cells (Zhang et al.,2001; Yokoi et al., 2002). Identified first in Arabisopsis (Gaxiolaet al., 1999), homologous NHX transporters were identified in >60plant species (Pardo et al., 2006) including maize (Zorb et al.,2005). Contrary to halophytes (Shabala and Mackay, 2011), tono-plast antiporters are not constitutively expressed in glycophytes(Zhang and Blumwald, 2001). Instead, salt-stressed can induce NHXactivity in glycophytes (Garbarino and Dupont, 1988; Apse et al.,1999). Maize is classified as a salt-sensitive species and, hence, isexpected to have rather low levels of NHX transcripts that are notsufficient to cope with salinity. These comments are consistent withreports of Zorb et al. (2005) that the expression signal of ZmNHXwas very weak (at the detection limit of the autoradiography) andvisible only after prolonged plant exposure to salinity. Also, a sig-nificant up-regulation of the ZmNHX in leaves of the salt-resistanthybrids SR 03 and SR 05 was reported by Pitann et al. (2013). In

the light of above, it appears that in our experiments one weekacclimation in 25 mM NaCl was sufficient to induce higher NHXactivity (at either transcriptional or post-translational levels), as

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vident from our Na+ sequestration data (Fig. 6). Overexpressionf NHX antiporters has been used to improve salt tolerance in sev-ral plant species (Apse et al., 1999; Zhang and Blumwald, 2001;hang et al., 2001; Li et al., 2011), with all transformed plants show-ng improved plant survival and increased shoot growth over theontrol lines under salt stress − results similar to our observationseported here.

In our work, differences in ion accumulation also varied inoots, where acclimated plants showed a higher capacity to excludeodium and retain potassium. Hence beneficial effect of acclima-ion in maize is achieved by two separate mechanisms acting athoot and root level, and not mainly at the root level as reportedor peas (Pandolfi et al., 2012). These results are also indicativehat acclimated plants use Na+ and Cl− accumulated in shoots as

cheap osmoticum reducing the energetic investment to produceompatible solutes. Indeed, inorganic ions accounted for 75% of tis-ue osmolarity in NA plants but to 95% in acclimated A(25) plantsFig. 3A). Given the high cost of osmolyte production (between 50o 70 mol ATP per one mole of compatible solute; Raven, 1995;habala and Shabala, 2011), acclimated plants are capable to redi-ect more carbohydrate reserves towards other energy-demandingrocesses. One of these processes is membrane potential mainte-ance to ensure K+ homeostasis under stressed conditions. Highytosolic K+ levels that are required for optimal cell metabolismre achieved primarily by the maintenance of a large (−120 to180 mV) negative voltage difference across the plasma membrane

PM) (Shabala and Pottosin, 2014) This resting potential is set byhe plasma membrane H+-ATPase and is normally kept close tohe equilibrium potential for K+, EK (Hirsch et al., 1998). A shiftn membrane potential values positive of EK leads to substantial+ leak through the outward-rectifying K+ (KOR) channels, result-

ng in a disturbance to cytosolic K+ homeostasis and a possibilityf triggering programmed cell death (PCD) in roots resulting fromow K+-induced stimulation of proteases and endonucleases (Petersnd Chin 2007; Shabala et al., 2007). The number of cells under-oing PCD in Arabidopsis gork1-1mutants plants lacking functionalOR channels was about 4-fold lower compared with wild type

Demidchik et al., 2010). Thus, the better K+ retention in acclimatedoots (Fig. 4) may be attributed to the ability of A(25) plants to allo-ate more ATP for membrane potential maintenance. Although thebovementioned experiments were performed on small seedlings,t is known that K+ efflux measurements correlates with maturelants’ homeostasis of cytosolic K+ and Na+, a key determinantf plant salinity tolerance (Chen et al., 2007). It should be alsoommented that MIFE technique measures net fluxes of the ionf interest, e.g. in this case a balance between K+ uptake mediatedy both low- and high-affinity transport systems and K+ mediatedy efflux channels such as GORK or NSCC. It remains to be inves-igated which of this components is most affected by salinity andcclimation.

Sodium exclusion from uptake is often named as a most crucialrait contributing to salinity tolerance in glycophytes (Munns andester, 2008). Thermodynamically, Na+ extrusion from the cyto-ol to the external medium under saline conditions is an active,nergy-consuming process that is mediated by plasma membranea+/H+ exchangers fuelled by the existence of sharp H+ gradientst both sides of the plasma membrane (Apse and Blumwald, 2007).n Arabidopsis, a Na+/H+ antiporter function has been attributed toOS1 gene (Shi et al., 2000; Qiu et al., 2003). Experimental evi-ence for the presence of SOS1-homologues has been shown forther species, both glycophytes (Mullen et al., 2007; Cuin et al.,011) and halophytes (Chen et al., 2010), and over-expression of

OS1 has been found to reduce Na+ accumulation and improvealinity tolerance in transgenic Arabidopsis (Shi et al., 2003). How-ver, it appears that acclimation to salinity is not attributed toetter ability of maize roots to exclude Na+, given the lack of any

Physiology 201 (2016) 1–8 7

significant difference in net Na+ fluxes between acclimated andnon-acclimated roots (Fig. 5). These findings are in a full agree-ment that acclimated plants accumulated more Na+ in the shootcompared with non-acclimated ones (Fig. 2J). Although furtherinvestigation is needed in order to unravel a clear picture of theionic component of the acclimation mechanisms at the molecularlevel, the reported results allow us to suggest that the involvementof the root SOS1 plasma membrane transporters in this process isrelatively minor, and instead points out at the important role ofvacuolar compartmentation of Na+ as a component of acclimationmechanism.

In conclusion, despite Na+ exclusion from uptake has alwaysbeen named as a central component of plant adaptive responsesto salinity (Munns and Tester, 2008), it appears that it is not themechanism that is “targeted” by acclimation. Rather, improved vac-uolar Na+ sequestration in the shoots appears to play a dominantrole in this process. These findings not only caution against thevalidity of breeding strategies aimed at reduction of Na+ uptake byplants, but also highlight the need to focus on shoot tissue toler-ance mechanisms (and, specifically, vacuolar Na+ sequestration) asa more promising approach in the production of tolerant plants (eg.Shabala, 2013).

Acknowledgments

This research was supported by the Australian Research Counciland Grain Research and Development Corporation grants to SergeyShabala and by an Endeavour Research Fellowship and a MarieCurie IEF Fellowship to Camilla Pandolfi Marie Curie IEF Fellowshipto Camilla Pandolfi.

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