Physiologia Plantarum 2012 Copyright © Physiologia Plantarum 2012, ISSN 0031-9317

Antioxidant enzyme activities and hormonal statusin response to Cd stress in the wetland halophyteKosteletzkya virginica under saline conditionsRui-Ming Hana, Isabelle Lefevrea, Alfonso Albaceteb, Francisco Perez-Alfoceab, Gregorio Barba-Espınc,Pedro Dıaz-Vivancosc, Muriel Quineta, Cheng-Jiang Ruand, Jose Antonio Hernandezc,Elena Cantero-Navarrob and Stanley Luttsa,∗

aGroupe de Recherche en Physiologie vegetale (GRPV), Earth and Life Institute – Agronomy (ELI-A), Universite catholique de Louvain (UCL), Croix duSud 5, bte L 7.07.13, B-1348, Louvain-la-Neuve, BelgiumbDepartamento de Nutricion Vegetal, Centro de Edafologıa y Biologıa Aplicada del Segura (CEBAS), Consejo Superior de Investigaciones Cientıficas(CSIC), Campus Universitario de Espinardo, PO Box 164, E-30100, Murcia, SpaincGrupo de Biotecnologıa de Frutales, Centro de Edafologıa y Biologıa Aplicada del Segura (CEBAS), Consejo Superior de Investigaciones Cientıficas(CSIC), Campus Universitario de Espinardo, PO Box 164, E-30100, Murcia, SpaindKey laboratory of Biotechnology and Bio-resources Utilisation, State Ethnic Affairs Commission and Ministry of Education, Dalian NationalitiesUniversity, 116600, Dalian, China

Correspondence*Corresponding author,e-mail: stanley.lutts@uclouvain.be

Received 7 March 2012;revised 16 May 2012

doi:10.1111/j.1399-3054.2012.01667.x

Salt marshes constitute major sinks for heavy metal accumulation but theprecise impact of salinity on heavy metal toxicity for halophyte plant speciesremains largely unknown. Young seedlings of Kosteletzkya virginica wereexposed during 3 weeks in nutrient solution to Cd 5 μM in the presence orabsence of 50 mM NaCl. Cadmium (Cd) reduced growth and shoot watercontent and had major detrimental effect on maximum quantum efficiency(Fv/Fm), effective quantum yield of photosystem II (Y(II)) and electron transportrates (ETRs). Cd induced an oxidative stress in relation to an increase in O2

•−

and H2O2 concentration and lead to a decrease in endogenous glutathione(GSH) and α-tocopherol in the leaves. Cd not only increased leaf zeatin andzeatin riboside concentration but also increased the senescing compounds 1-aminocyclopropane-1-carboxylic acid (ACC) and abscisic acid (ABA). Salinityreduced Cd accumulation already after 1 week of stress but was unable torestore shoot growth and thus did not induce any dilution effect. Salinitydelayed the Cd-induced leaf senescence: NaCl reduced the deleteriousimpact of Cd on photosynthesis apparatus through an improvement of Fv/Fm,Y(II) and ETR. Salt reduced oxidative stress in Cd-treated plants through anincrease in GSH, α-tocopherol and ascorbic acid synthesis and an increase inglutathione reductase (EC 1.6.4.2) activity. Additional salt reduced ACC andABA accumulation in Cd+NaCl-treated leaves comparing to Cd alone. It isconcluded that salinity affords efficient protection against Cd to the halophytespecies K. virginica, in relation to an improved management of oxidativestress and hormonal status.

Abbreviations – ABA, abscisic acid; ACC, 1-aminocyclopropane-1-carboxylic acid; APX, ascorbate peroxidase; AsA,ascorbate; ASC-GSH, ascorbate-glutathione; CAT, catalase; Cd, cadmium; CK, cytokinin; CKX, cytokininoxidase/dehydrogenase; DHA, dehydroascorbate; DHAR, dehydroascorbate reductase; DNPH, diphenylhydrazine;EDTA, ethylenediaminetetraacetic acid; ETR, relative rate of photosynthetic electron transport; FW, fresh weight;GR, glutathione reductase; GSH, reduced glutathione; GSSG, oxidized glutathione; HPLC, high-performance liquidchromatography; IAA, indole-3-acetic acid; MDA, malondialdehyde; MDHAR, monodehydroascorbate reductase;MS, mass spectrometry; NPQ, non-photochemical quenching; PAR, photosynthetically active radiation; POX,peroxidase; PSII, photosystem II; qP, photochemical quenching; ROS, reactive oxygen species; SA, salicylic acid;SOD, superoxide dismutase; TCA, trichloroacetic acid; Z, zeatin; ZR, zeatin riboside.

Physiol. Plant. 2012

Introduction

Cadmium (Cd) is a widespread pollutant and itsaccumulation in the environment is mainly due toanthropogenic activities. High Cd concentration inplant leaves impairs photosynthesis and chlorophyllmetabolism (Pietrini et al. 2003), compromises the plantwater status (Perfus-Barbeoch et al. 2002) and inducesnumerous damages to cellular structures and membranes(Gratao et al. 2009). Some of these damages could, atleast partly, result from the Cd-induced synthesis ofreactive oxygen species (ROS) enhancing membranelipid peroxidation, enzyme inhibition and DNA or RNAdamages (Shah et al. 2001, Asada 2006). To reduce thosedamages, plants possess scavenging systems consistingin non-enzymatic antioxidants such as ascorbate orglutathione (GSH), which may counteract or neutralizethe harmful effects of ROS, as well as antioxidantenzymes, such as superoxide dismutase (SOD; EC1.15.1.1), catalase (CAT; EC 1.11.1.6) and enzymes ofthe ascorbate-glutathione (ASC-GSH) cycle [ascorbateperoxidase (APX; EC 1.11.1.11) glutathione reductase(GR; EC 1.6.4.2), dehydroascorbate reductase (DHAR;EC 1.8.5.1) and monodehydroascorbate reductase(MDHAR; EC 1.6.5.4)] (Foyer and Noctor 2005,Asada 2006). Understanding the detoxification strategiesthat plants adopt against oxidative stress induced byaccumulated metal ions is the key information to furthermanipulate plant heavy metal tolerance.

Cd may directly affect the activities of numerousantioxidative enzymes, but contrasting results have beenreported in the literature: Cd reduced APX activities inHelianthus annuus (Gallego et al. 1996) but increasedthem in roots and leaves of Phaseolus vulgaris, as well asin suspension cultures of tobacco (Nicotiana tabacum)cells (Chaoui et al. 1997, Piqueras et al. 1999). Cd-induced oxidative stress is frequently characterized by adecrease in reduced GSH content, or in CAT and CuZn-SOD activities (Romero-Puertas et al. 2007). Accordingto Lefevre et al. (2010), Cd tolerance in cell lines of thehalophyte plant species Atriplex halimus may be relatedto a high content of antioxidant (GSH and ascorbicacid), a high constitutive SOD activity, and an efficientCd-induced increase in GR and APX activities.

Beside oxidative stress, inhibition of growth anddevelopment is often reported as the first symptom of Cddeleterious impact (Sanita di Toppi and Gabbrielli 1999).Not only plant biomass, but also plant architecturemay be modified in response to this toxic element.In perennial dicotyledonous plants, growth inhibition isoften more severe on ramifications than on the mainstem although the concentration of Cd is lower in theformer than in the latter and Cd also inhibits axillary bud

development (Lefevre et al. 2009a, Han et al. 2012). Onepossible explanation is that Cd modifies plant hormonesynthesis and action but data concerning the impactof this heavy metal on plant growth regulators remainscarce: most studies until now are dealing with abscisicacid (ABA; Hsu and Kao 2003, Stroinski et al. 2010),commonly considered as a stress hormone, or withethylene in relation to its putative impact on senescenceprocesses (Yakimova et al. 2006, Liu et al. 2008, Grataoet al. 2009). Only few data are available concerningCd-induced decrease in auxin synthesis (Chaoui andEl Ferjani 2005) or increase in salicylic acid (SA)synthesis (Metwally et al. 2003). Although cytokinins(CKs) assume key functions as signaling compounds inthe xylem stream and act as anti-senescing agents inthe leaves (Ghanem et al. 2011), to the best of ourknowledge, no exhaustive data are available until nowconcerning Cd impact on CK synthesis and translocation.Moreover, most studies considered the Cd impact ononly one or two plant growth regulators. Since differentplant hormones interact among each other accordingto complex networks, a comprehensive approach of Cdimpact on this class of compounds requires establishinga ‘hormonal profile’ through the quantification of severalhormones on the same experimental system.

Plant growth regulators may be involved in themanagement of the antioxidative status of stressedplants. Yakimova et al. (2006) and Monteiro et al.(2011) reported that Cd-induced cell mortality resultsdirectly from ethylene production which may itselftrigger oxidative stress. Conversely, the SA-enhancedCd tolerance in rice can be attributed to SA-elevatedenzymatic and non-enzymatic antioxidants occurringtogether with SA-regulated Cd uptake, transport anddistribution in plant organs (Guo et al. 2009). It has beenreported in different plant species that genes coding foranti-oxidative enzymes may be induced by plant growthregulators through various transduction pathways (Foyerand Noctor 2005). The situation appears rather complexwhen considering that ROS may also have an impact onphytohormones synthesis in stressed plants (Mittler et al.2010). A kinetics approach is thus required to determinethe timing of Cd-induced modifications in order to clarifythe complex relationship between hormonal profile andantioxidative status. To the best of our knowledge, suchan approach is still missing but should afford valuableinformation on the sequence of events leading to theplant response during a prolonged period of exposure toCd toxicity.

Halophyte plant species are adapted to salt stresspresent in their native habitat and are therefore supposedto possess physiological adaptations allowing them tocope with ion toxicity, which may be useful for the

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phytomanagement of heavy metal contaminated area(Lopez-Chuken and Young 2005, Ruan et al. 2010).Their response to osmotic and ionic constrains inducedby salinity (Flowers and Colmer 2008) notably involvesboth phytohormones and antioxidant properties, sincehalophytes usually display specific adaptations in termsof oxidative status comparatively to glycophytes (Ellouziet al. 2011). However, the impact of salinity on Cd-induced changes in phytohormone and oxidative statusremains largely unknown. Several xero-halophyte plantspecies present putative interest for revegetation of heavymetal contaminated areas. Those species are resistantto drought and salt are, to some extent, resistant toheavy metals (Lutts et al. 2004, Lefevre et al. 2009b,Zaier et al. 2010). According to these authors, abeneficial impact of exogenous NaCl on plant responseto Cd may be, at least partly, linked to NaCl-inducedgrowth stimulation classically reported for halophyte atmoderate salinities, which therefore leads to dilutioneffect. Wetland halophytes comparatively received onlylittle attention until now, despite the fact that heavymetal contamination is frequently reported in coastalareas and the coastal salt marshes are important sinksfor heavy metal accumulation (Doyle and Otte 1997).Those species are not tolerant to drought and theiradaptation to flooding conditions should have an impacton their behavior in terms of phytohormones synthesisand oxidative status (Jackson 1990). The impact of thoseadaptations on plant response to heavy metal is stillunknown.

Kosteletzkya virginica is a perennial halophyte nativeto American salt marshes and has been proposed as apromising tool for revegetation of salt-affected coastaltidal flats (Ruan et al. 2008). The response of K. virginicato Cd toxicity under saline conditions has recently beendescribed (Han et al. 2012). Our previous works showedthat: (1) Cd had a strong impact on plant architecture; (2)Cd accumulation in different shoot parts could not fullyexplain the pattern of growth inhibition; (3) NaCl in theabsence of Cd could slightly improve plant growth and(4) NaCl significantly reduced Cd accumulation (Hanet al. 2012). Until now, no information concerning theCd-induced secondary oxidative stress and plant growthregulators which may be related to the antioxidativestatus in K. virginica has been reported. It is moreoverhypothesized that NaCl may improve Cd resistance inhalophyte independently of any dilution effect relatedto growth stimulation. This work therefore investigatesthe leaf antioxidant enzyme activities and hormonalcontent in response to Cd stress and the influence of lowsalinity on the relationship between oxidative status andhormonal profile under Cd stress.

Materials and methods

Plant material and culture conditions

Seeds were immersed in de-ionized water for 30 minbefore sowing into 85 mm diameter Petri dish with twolayers of water-moistened filter paper. Petri dishes wereenclosed with aluminum foils and incubated at 28◦C for48 h. Well germinated seeds with 2–3 cm root tips werethen sowed in trays filled with artificial soil moistenedregularly with water and seedlings were allowed to growin a phytotron under a 16/8h light/dark photoperiod(mean light intensity (PAR) = 500 μmol m−2 s−1 providedby Osram Sylvania (Danvers, MA) fluorescent tubes(F36W/133-T8/CW) with 25/20◦C day/night temperatureand 65/50% atmospheric humidity). Fifteen daysafter sowing, seedlings were transferred into 18-ltanks and fixed on polyvinyl chloride plates floatingon aerated half-strength modified Hoagland nutrientsolution containing (in mM): 2.0 KNO3, 1.7 Ca(NO3)2,1.0 KH2PO4, 0.5 NH4NO3, 0.5 MgSO4 and (in μM)17.8 Na2SO4, 11.3 H3BO3, 1.6 MnSO4,1 ZnSO4, 0.3CuSO4, 0.03 (NH4)6Mo7O24 and 14.5 Fe-EDDHA.

After 10 days of acclimatization in the absence of stress(25 days after sowing), NaCl and CdCl2 were added tocorresponding containers to create four treatments: (1)control; (2) 5 μM Cd; (3) 50 mM NaCl; (4) 5 μM Cd+ 50 mM NaCl. Solutions were readjusted every 2days and renewed every week. The pH of solutionswas set to 5.7 ± 0.02 with KOH and readjusted everyday. Three replications with nine plants per replicationand per treatment were used for the measurement ofdifferent parameters. The specific leaf 5 from bottomcorresponding to the top-expanded leaf at the beginningof stress period, and which has reached a minimallength of 4 cm, was chosen when Cd and sodiumchloride were imposed to monitor senescence andfor subsequent biochemical determination. This preciseleaf was exposed to Cd toxicity during the wholeexperimental period: leaf material from nine plants washarvested for different analyses at 1, 2 and 3 weeks oftreatment.

Chlorophyll fluorescence

Modulated chlorophyll fluorescence was measured intagged and dark-adapted (30 min) leaves (Ft = F0)in 6–10 plants per treatment, using a Imaging-PAMM-series system (OptiSciences, Herts, UK) with anexcitation source intensity of 3000 μmol m−2 s−1. Theminimal fluorescence intensity (F0) in a dark-adaptedstate was measured in the presence of a background far-red light to favor rapid oxidation of intersystem electroncarriers. The maximal fluorescence intensities in the

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dark-adapted state (Fm) and after adaptation to whiteactinic light (Fm

′) were measured by 0.8 s saturatingpulses (3000 μmol m−2 s−1). After the Fm

′ measurement,the actinic light (400 μmol m−2 s−1) was switched off,and the far-red light was applied for 3 s in order tomeasure the minimal fluorescence intensity in the light-adapted state (F0

′). The maximum quantum yield ofopen photosystem II (PSII) (Fv/Fm), the effective PSIIquantum yield �PSII = Y(II) and the non-photochemicalquenching (NPQ) were calculated as(Fm − F0)/Fm, (Fm

′

− Ft)/Fm′ and (Fm − Fm

′)/Fm′, respectively. At a known

flux of incident photosynthetically active radiation (PAR)the relative rate of photosynthetic electron transport(ETR), the effective PSII quantum yields of regulated non-photochemical energy dissipation, �NPQ = Y(NPQ) andnon-regulated energy dissipation, �NO = Y(NO)[Y(II) +Y(NPQ) + Y(NO) = 1] were calculated as described byBonfig et al. (2006). After kinetics and light curves wererecorded, areas of interest were defined by red circlesfor every leaf, over which all pixel values for variousfluorescence parameters were averaged.

Ion concentration

For each sample, digestion of dry matter (50–100 mg)was accomplished at 80◦C in 67% (v/v) HNO3. Mineralswere dissolved in a spot of aqua regia and dilutedwith de-ionized water and then filtered. The elementconcentrations (Cd, Na and K) were determined using anatomic absorption spectrometer (Thermo Scientific ICE3300; Waltham, MA) calibrated with certified standardsolutions. All measurements were performed in threereplicates.

Oxidative stress parametersand non-enzymatic antioxidants

The level of lipid peroxidation was measured as 2-thiobarbituric acid-reactive substances, mainly malon-dialdehyde (MDA) according to Heath and Packer(1968) The MDA concentration was calculated usingits molar extinction coefficient (155 mM−1 cm−1). Pro-tein oxidation, given as carbonyl protein (CO-protein)content was carried out using Reznick and Packer(1994) spectrophotometric method for the detection ofthe reaction of diphenylhydrazine (DNPH) with car-bonyl proteins to form protein hydrazones. Frozensamples were finely minced in 3 ml of homogeniz-ing buffer (60 mM phosphate buffer, pH 7.4, containing0.1% digitonin and antiproteases [4-(2-aminoethyl) ben-zenesulfonyl fluoride, bestatin, pepstatin A, leupeptinand 1,10-phenanthrolin) and 1 mM ethylenediaminete-traacetic acid (EDTA)] and were incubated for 15 min

at room temperature. Samples were centrifuged (6000 gduring 10 min at room temperature); 600 μl of 10 mMDNPH (dissolved in 2.5 M HCl) were added to 150 μMof the supernatant. After 1 h incubation at room tem-perature, 750 μl of 20% trichloroacetic acid (TCA) (w/v)were added and samples were thereafter kept on ice for10 min and centrifuged at 10 000 g for 5 min at 4◦C.Supernatants were discarded and pellets were washedthree times with 400 μl ethanol-ethyl acetate (1:1) (v/v)to remove the free DNPH and lipid contaminants. Thefinal precipitates were dissolved in 500 μl of 6 M guani-dine hydrochloride solution and shaken for 10 min at37◦C. Insoluble material was removed by additionalcentrifugation (6000 g; 1 min at room temperature). Car-bonyl content was calculated using the peak absorbance(spectra at 320–390 nm) and a 2.5 M HCl-treated samplewithout DNPH as a blank.

For hydrogen peroxide quantification, frozen freshleaves (0.5 g) were ground to powder in the presenceof 5 ml 5% TCA and 0.15 g activated charcoal. Themixture was centrifuged at 10 000 g for 20 min at 4◦C.The supernatant was adjusted to pH 8.4 with 17 Mammonia solution and then filtered. The filtrate wasdivided into aliquots of 1 ml. To one of these (the blank)8 μg of CAT (10 000 U mg−1) were added and sampleswere then kept at room temperatures for 10 min. To bothaliquots (with and without CAT), 1 ml of colorimetricreagent was added. The reaction solution was incubatedfor 10 min at 30◦C. Absorbance at 505 nm wasdetermined spectrophotometrically. The colorimetricreagent contained 10 mg of 4-aminoantipyrine, 10 mgof phenol and 5 mg of peroxidase (POX) (150 U mg−1)dissolved in 50 ml of 100 mM acetic buffer (pH5.6) (Zhou et al. 2006). Superoxide ions (O2

•−) weremeasured according to Elstner and Heupel (1976) bymonitoring nitrate formation from hydroxylamine.

The concentrations of α-tocopherol and its oxida-tion product α-tocopherol quinone were quantifiedaccording to Munne-Bosch et al. (2007). Leaf sam-ples were extracted four times with ice-cold n-hexanecontaining 1 ppm butylated hydroxytoluene using ultra-sonication. Tocopherols were separated on a Partisil10 ODS-3 column at a flow rate of 1 ml min−1. Thesolvents consisted of (A) methanol/water (95:5, v/v)and (B) methanol. The gradient used was: 0–10 min100% A, 10–20 min decreasing to 0% A, 20–25 min0% A, 25–28 min increasing to 100% A and 28–33 min100% A. The α-tocopherol and α-tocopherol quinonewere quantified by their absorbance at 283 and 265 nm,respectively.

GSH was assayed by the enzymatic recyclingprocedure in which it is sequentially oxidized by 5,5′-dithiobis (2-nitrobenzoic acid) and reduced by NADPH

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in the presence of GR according to Griffith (1980) andmodified by Lutts et al. (2004). Briefly, extraction wasperformed in 5% sulphosalicylic acid on ca. 1 g freshweight (FW) ground tissue and the extent of 2-nitro-5-thiobenzoic acid formation was monitored at 412 nm forGSH plus GSSG evaluation. For determination of GSSGalone, the extract was pre-treated with 2-vinylpyridineto scavenge GSH by derivatization.

For ascorbate extraction, frozen tissues were homog-enized in ice cold 5% metaphosphoric acid solution(1:5, w/v) and then centrifuged at 20 000 g and 4◦C for10 min. Total ascorbate (AsA + DHA) contents weredetermined according to Wang et al. (1991) on thebasis of Fe3+ –Fe2+ reduction by ascorbate in acid solu-tion. Fe2+ forms a red chelate with bathophenanthrolineabsorbing at 534 nm. The ascorbate (reduced form)assay mixture contained 0.1 ml of the extract, 0.5 mlof absolute ethanol, 0.6 M TCA, 3 mM bathophenan-throline, 8 mM H3PO4 and 0.17 mM FeCl3. The finaltotal volume was 1.5 ml and the mixture was allowedto stand at 30◦C for 90 min. The absorbance of thecolored solution was read at 534 nm. The total ascor-bate assay mixture contained 0.1 ml of the sample,0.15 ml of 3.89 mM dithiothreitol and 0.35 ml of abso-lute ethanol in a total volume of 0.6 ml. Then, thereaction mixture was left standing at room temperaturefor 10 min. After reduction of dehydroascorbate to ascor-bate, 0.15 ml of 20% TCA was added and the color wasdeveloped by adding 0.15 ml of 0.4% (v/v) H3PO4-ethanol, 0.3 ml of 0.5% (w/v) bathophenantroline-ethanol and 0.15 ml of 0.03% (w/v) FeCl3-ethanol.Dehydroascorbate concentrations were estimated fromthe difference of ‘total ascorbate’ and ascorbate concen-tration. Standard curve in the range 0–10 μmol ascorbatewas used.

Antioxidant enzymes activities

All operations were performed at 0–4◦C. Leaves (around0.5 g fresh mass) were homogenized with 2 ml ofan ice-cold buffer containing 50 mM Tris–acetatebuffer (pH 6.0), 0.1 mM EDTA, 5 mM cysteine,2% (w/v) insoluble polyvinylpolypyrrolidone, 0.1 mMphenylmethane-sulphonyl fluoride and 0.2% (v/v) TritonX-100. For the APX activity assay, 20 mM Na-ascorbatewas added to the extraction medium. The extracts werefiltered through two layers of nylon cloth and centrifugedat 8000 g for 20 min, at 4◦C. The supernatant fractionswere then filtered on Sephadex G-25 NAP columns(GE Healthcare, Madrid, Spain) equilibrated with theextraction buffer.

The APX, DHAR, MDHAR, GR, POX (EC1.11.1.7),CAT and total SOD activities and proteins contents

were assayed as described in Clemente-Moreno et al.(2010). Enzyme activities were corrected for non-enzymatic rates and for interfering oxidation. ForAPX, the oxidation rate of ascorbate was estimatedbetween 1.0 and 60 s after starting the reaction bythe addition of H2O2. Correction was made for thelow non-enzymatic oxidation of ascorbate by H2O2. Todetermine MDHAR activity, monodehydroascorbate wasgenerated by the ascorbate/ascorbate oxidase system.The rate of monodehydroascorbate-independent NADHoxidation (without ascorbate and ascorbate oxidase)was subtracted from the initial monodehydroascorbate-dependent NADH oxidation rate (with ascorbate andascorbate oxidase). For DHAR activity, the reactionrate was corrected for the non-enzymatic reduction ofDHA by GSH. A 2% contribution to the absorbanceby GSSG was also taken into account. Values ofGR activity were corrected for the small, non-enzymatic oxidation of NADPH by GSSG (Jimenezet al. 1998).

Hormone extraction and analysis

CKs [zeatin (Z) and zeatin riboside (ZR)], indole-3-aceticacid (IAA) and ABA were extracted on 1 g leaf material inmethanol/water/formic acid (15/4/1, v/v/v, pH 2.5) andpurified as previously described (Ghanem et al. 2008)according to the method of Dobrev and Kamınek (2002).Pooled supernatants were passed through Sep-Pak Plus®

C18 cartridge (SepPak Plus, Waters, Milford, MA) toremove interfering lipids and part of plant pigments andevaporated either to near dryness or until organic solventwas removed. The residue was dissolved in 5 ml 1 Mformic acid and applied to Oasis MCX mixed mode(cation-exchange and reverse phase) column (150 mg;Waters) pre-conditioned with 5 ml of methanol followedby 5 ml of 1 M formic acid. To separate different CKs(nucleotides, bases, ribosides and glucosides) from IAAand ABA, the column was washed and eluted stepwisewith the different appropriate solutions indicated inDobrev and Kamınek (2002). ABA and IAA wereanalyzed in the same fraction. Samples then dissolved inmobile phase A, consisting of water/acetonitrile/formicacid (94.9:5:0.1 v/v) mixture for high-performanceliquid chromatography/mass spectrometry (HPLC)/massspectrometry (MS) analysis. The analysis were carriedout on a HPLC/MS system consisting of an Agilent1100 Series HPLC (Agilent Technologies, Santa Clara,CA) equipped with a μ-wellplate autosampler and acapillary pump, and connected to an Agilent Ion TrapXCT Plus mass spectrometer (Agilent Technologies) usingan electrospray interface. Previous to injection, 100 μlof each fraction extracted from leaf tissues were filtered

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through 13 mm diameter Millex filters with 0.22 μmpore size nylon membrane (Millipore, Bedford, MA):8 μl of each sample, dissolved in mobile phase A,was injected onto a Zorbax SB-C18 HPLC column(5 μm, 150 × 0.5 mm; Agilent Technologies), heldat 40◦C, and eluted at a flow rate of 10 μl min−1.Mobile phase A, consisting of water/acetonitrile/formicacid (94.9:5:0.1), and mobile phase B, consisting ofwater/acetonitrile/formic acid (10:89.9:0.1), were usedfor the chromatographic separation with the gradientelution described by Ghanem et al. (2008). The UVchromatogram was recorded at 280 nm with the DADmodule (Agilent Technologies). The mass spectrometerwas operated in the positive mode with a capillary sprayvoltage of 3500 V, and a scan speed of 22 000 (m/z)/sfrom 50 to 500 m/z. The nebulizer gas (He) pressure wasset to 30 psi, whereas the drying gas was set to a flow of6 l min−1 at a temperature of 350◦C. Mass spectra wereobtained using the DATA ANALYSIS PROGRAM FOR LC/MSDTRAP Version 3.2 (Bruker Daltonik GmbH, Bremen,Germany). For quantification of Z, ZR, ABA and IAA,calibration curves were constructed for each analyzedcomponent (0.05, 0.075, 0.1, 0.2 and 0.5 mg l−1) andcorrected for 0.1 mg l−1 internal standards: [2H5]trans-Z, [2H5]trans-ZR, [2H6]cis,trans-abscisic acid (OlcheminLtd, Olomouc, Czech Republic) and [13C6]indole-3-acetic acid (Cambridge Isotope Laboratories Inc.,Andover, MA). Recovery percentages ranged between92 and 95%.

ACC (1-aminocyclopropane-1-carboxylic acid) wasdetermined after conversion into ethylene by gaschromatography using an activated alumina column anda FID detector (Cromatix-KNK-2000; Konik, Barcelona,Spain). ACC was extracted with 80% (v/v) ethanol andassayed by degradation with alkaline hypochlorite in the

presence of 5 mM HgCl2 according to the descriptionof Ghanem et al. (2008). A preliminary purificationstep was performed by passing the extract through aDowex 50W-X8, 50–100 mesh, H+-form resin and laterrecovered with 0.1N NH4OH. The conversion efficiencyof ACC into ethylene was calculated separately byusing a replicate sample containing 2.5 nmol of ACCas internal standard and used for correction of data.

Statistical analysis

Data were subjected to an ANOVA II using the SPSS software(IBM® SPSS® software, version 16.0.0), with the natureof stress and the duration of stress considered as themain factors. The statistical significance of the resultswas analyzed by the Student–Newman–Keuls test at the5% level.

Results

Shoot dry weight and leaf water content

The presence of NaCl had no impact on the shootdry weight while Cd induced an obvious decrease inshoot growth (Fig. 1A). The addition of NaCl to the Cdsolution was unable to reduce the deleterious impactof the heavy metal on shoot dry weight. Salinity didnot affect the shoot water content (Fig. 1B). In contrast,5 μM Cd significantly reduced this parameter, alreadyafter 1 week of treatment. The presence of NaCl partlymitigated the impact of Cd on the leaf water content,especially after 2 weeks of treatment. The expansion ofleaf 5 was slightly inhibited in the presence of Cd: atthe end of the stress period, average leaf surface reached194 and 214 cm2 in controls and NaCl-treated plants vs

Fig. 1. Evolution of the shoot dry weight (A) and water content (B) of Kosteletzkya virginica seedlings exposed to 5 μM Cd during 3 weeks in thepresence or absence of 50 mM NaCl. Data points and vertical bars represent means of five individual plants (n = 5) and SE, respectively. For a givenduration of treatment, means with different letters are significantly different at P < 0.05.

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Fig. 2. Evolution of chlorophyll fluorescence parameters in leaf 5 of Kosteletzkya virginica seedlings exposed to 5 μM Cd during 3 weeks in thepresence or absence of 50 mM NaCl. Images of maximum PS II quantum yield, Fv/Fm, effective PS II quantum yield, Y(II) and the NPQ are shown after2 and 3 weeks (A, B) of treatment with an Imaging-PAM M-series system. The false-color code depicted between the two images ranged from 0.000(black) to 1.000 (purple). Quantitative analyses of the aforementioned parameters are shown in C–E. Data points and vertical bars represent means(n = 3) and SE, respectively. For a given duration, different indices indicate significant difference according to Student–Newman–Keuls test at the 5%level.

154 and 152 cm2 in Cd and Cd+NaCl-treated plants,respectively (detailed data not shown).

Chlorophyll fluorescence and leafsenescence-related parameters

The images of leaf 5 obtained using imaging-PAMfluorometer and the evolution of chlorophyll fluores-cence and senescence-related parameters are shown inFig. 2. From false-color images, changes in photosyn-thetic activities could be readily discerned. The highestnegative effects occurred in response to Cd treatment,whereas NaCl alone did not or only slightly affected these

parameters. The mixed treatment showed intermediateeffects (Fig. 2A, B).

In leaf 5 of control plants, the maximum quantumefficiency of PSII (Fv/Fm) was almost constant duringthe growing period, whereas in Cd-stressed plants, thesevalues sharply decreased below those of control plants.Sodium treatment had no impact on Fv/Fm, whereasthe addition of NaCl to Cd significant increased thesevalues compared to Cd alone (Fig. 2C). The effectivequantum yield of PSII (Y(II)) decreased during the wholestress period and exhibited the lowest value in responseto Cd treatment. In NaCl-treated plants, the Y(II) valueshowed no difference with those of control at week 1

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and remained almost constant thereafter. Due to anincrease in control plants during the last 2 weeks, theY(II) in NaCl-treated plants became significantly lowerthan those of control. In the mixed treatment, Y(II) alsodecreased after 2 weeks of stress but less than in responseto Cd alone (Fig. 2D).

In control plants, NPQ decreased as the leavesaged, whereas in Cd-treated plants, NPQ decreased inweek 2, reaching a minimum by day 14 before stronglyincreasing during the last week. After 1 week treatmentwith NaCl alone, NPQ was lower than that of control andwas constant during week 2 before slightly decreasingthereafter. The mixed treatment decreased NPQ steadilyfrom a same level comparatively to NaCl-treated plantsafter 1 week and even to a significant lower level after 2and 3 weeks (Fig. 2E).

For further analysis, light response curves of therelative ETR were examined. Cd application decreasedETR in the presence or absence of NaCl. The effectof NaCl either alone or in the mixed treatment wasnot obvious during week 1, but it decreased ETR byitself and mitigated the Cd-induced decline during theweek 2 and 3. When PAR value was below 50 μmolm−2 s−1, the light response curve of the relative ETRvalue in treated and untreated groups was similar (Fig. 3).This result showed that NaCl alleviated Cd toxicityeffect on electron-transfer system and consequentlyreduced the poisoning effect of this heavy metal onK. virginica.

Our results assessed by the imaging-PAM chlorophyllfluorometer showed that the Cd-induced decreases inY(II) were paralleled by the increase in Y(NPQ) andY(NO), indicating that photosynthesis was inhibited.However, the addition of NaCl reduced the declineof Y(II) and therefore alleviated the Cd toxicity(Fig. 4).

Cd, sodium and potassium concentration

Cd was accumulated to a significantly higher level inresponse to Cd stress alone than in response to themixed treatment (Fig. 5A). Sodium was accumulated toa similar extent (300–320 μmol g−1 DM) during the first2 weeks in the presence of NaCl alone and under themixed treatments, but a strong increase (up to 450 μmolg−1 DM) occurred at the end of the third week in leavesof plants exposed to Cd+NaCl (Fig. 5B). Salinity induceda quick and strong reduction in K concentration, whereasCd induced a strong increase in this parameter (Fig. 5C).In the mixed treatment, the K concentration increasedto a maximum value after 2 weeks of treatment beforedeclining to reach a value similar to NaCl-treated plantsat the third week.

Fig. 3. Light response curves of the relative photosynthetic ETR in leaf5 of Kosteletzkya virginica seedlings exposed to 5 μM Cd after 1 (A),2 (B) and 3 (C) weeks in the presence or absence of 50 mM NaCl. Datapoints and vertical bars represent means (n = 3) and SE, respectively.

Oxidative stress parameters and antioxidativemetabolism

Cd toxicity induced an oxidative stress in leaves fromK. virginica plants, as observed by the increase insome oxidative stress-related parameters. In this way,a significant increase in lipid peroxidation and proteinoxidation was observed (Table 1). In addition, anaccumulation of ROS occurred, mainly O2

•−, whoselevels increased near sixfold in Cd-treated plants(Table 1). In NaCl-treated plants, no important damageto membranes or proteins was detected, although anincreased O2

•− content was observed (Table 1). Thepresence of NaCl partially reduced the oxidative stress-induced damage in Cd-treated plants that was manifestedby lower increases in lipid peroxidation and proteinoxidation. Surprisingly, in this case no accumulation ofH2O2 or O2

•− was observed (Table 1).

Physiol. Plant. 2012

Fig. 4. Time-dependent changes in excitation flux at PS II in leaf 5 of Kosteletzkya virginica seedlings exposed to 5 μM Cd after 1 (A), 2 (B) and3 (C) weeks in the presence or absence of 50 mM NaCl. Complementary changes in the effective PS II quantum yield Y(II) (white), quantum yield ofnon-regulated energy dissipation, Y(NO) (coarse), and regulated energy dissipation and Y(NPQ) (fine) were assessed with an Imaging-PAM M-seriessystem as described in Materials and methods section. Experiments were repeated three times.

Fig. 5. Evolution of Cd (A), sodium (B) and potassium (C) concentrations in leaf 5 of Kosteletzkya virginica seedlings exposed to 5 μM Cd during 3weeks in the presence or absence of 50 mM NaCl. Data points and vertical bars represent means (n = 3) and SE, respectively. For a given duration,asterisks and different indices indicate significant difference according to Student–Newman–Keuls test at the 5% level.

Table 1. MDA (nmol g−1 FW), carbonyl (nmol mg−1 protein), H2O2

(nmol g–1 FW) and superoxide radical (O2•−; in nmol g−1 FW)

concentrations in leaves of Kosteletzkya virginica seedlings exposedto 5 μM Cd in the absence or presence of 50 mM NaCl for 2 weeks.Values represent means ± SE (n = 5) and different letters within a linedenote significant difference from the respective controls at the 5%level.

Parameter Control 5 μM Cd 50 mM NaCl Cd+Na

MDA 61.4 ± 5.2 a 89.5 ± 7.8 b 66.7 ± 4.3 a 75.6 ± 11.5 ab

Carbonyl 44.7 ± 5.2 a 147.4 ± 21.4 c 51.2 ± 3.8 a 74.1 ± 3.2 b

H2O2 14.7 ± 1.2 a 42.3 ± 0.9 b 17.8 ± 2.4 a 16.3 ± 2.7 a

O2•− 2.5 ± 0.3 a 13.9 ± 2.0 c 4.2 ± 0.2 b 3.6 ± 1.0 ab

The Cd-induced oxidative stress was paralleled bydecreases in reduced GSH and α-tocopherol levels(Table 2). However, a rise in both reduced (AsA) and

oxidized (DHA) ascorbate took place, leading to adecrease in the redox state of ascorbate. In contrast, NaCltreatment increased GSH, whereas no changes in ascor-bate or α-tocopherol were observed (Table 2). WhenCd-treated plants were grown in the presence of 50 mMNaCl, an increase in the non-enzymatic antioxidantswas recorded comparatively to plants exposed to Cdstress only (Table 2). Specifically, significant increasesin GSH (63%), AsA (twofold) and α-tocopherol (40%)comparing to the controls were noticed in this case(Table 2).

The evaluation of enzymatic activities in responseto treatments exhibited similar trends after 1, 2 and3 weeks and are therefore presented after 2 weeks oftreatment for the sake of clarity (Table 3). NaCl alonehad no effects on tested enzyme activities, except a slightpromotion in total SOD and APX activities. In contrast,

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Table 2. Concentration of endogenous antioxidant [reduced (GSH)and oxidized GSH (GSSG) in nmol g−1 FW; ascorbate (AsA) anddehydroascorbate (DHA); in nmol g−1 FW), α-tocopherol (α-toc; innmol g−1 FW)] in leaves of Kosteletzkya virginica seedlings exposed to5 μM Cd in the absence or presence of 50 mM NaCl for 2 weeks.Values represent means ± SE (n = 5) and different letters within aline denote significant difference from the respective controls at the5% level.

Parameter Control 5 μM Cd 50 mM NaCl Cd+NaCl

GSH 55.2 ± 4.1 b 38.4 ± 2.1 a 77.9 ± 3.8 c 89.7 ± 7.7 c

GSSG 8.3 ± 0.4 ab 6.5 ± 1.2 a 12.4 ± 1.7 b 8.4 ± 2.3 ab

AsA 106.2 ± 8.1 a 171.9 ± 9.4 b 95.4 ± 0.9 a 222.6 ± 12.5 c

DHA 22.6 ± 1.4 a 51.3 ± 4.3 b 23.4 ± 3.1 a 18.7 ± 1.0 a

α-toc 184.1 ± 11.1 b 121.3 ± 2.8 a 202.4 ± 15.4 b 256.9 ± 7.5 c

Table 3. Specific activities of SOD (in U mg−1 protein), APX, MDHAR,DHAR, GR (in nmol min−1 mg−1 protein), CAT and POX (in μmol min−1

mg−1 protein) in soluble fractions from leaf 5 of Kosteletzkya virginicaseedlings exposed to 5 μM Cd in the absence or presence of 50 mMNaCl for 2 weeks. Values represent means ± SE (n = 6) and differentletters within a line denote significant difference from the respectivecontrols at the 5% level.

Enzyme Control 5 μM Cd 50 mM NaCl Cd+NaCl

SOD 55.7 ± 1.2 a 105.3 ± 25.5 a 74.5 ± 11.4 a 96.1 ± 25.4 a

APX 731.4 ± 39.9 b 478.2 ± 73.8 ab 953.1 ± 139.7 b 374.2 ± 41.2 a

MDHAR 342.4 ±74.2 a 291.7 ± 36.9 a 445.7 ± 52.0 a 358.0 ± 30.8 a

DHAR 54.5 ± 7.9 a 132.8 ± 13.2 b 52.8 ± 3.0 a 78.5 ± 4.2 a

GR 29.2 ± 3.3 a 62.3 ± 7.9 b 47.3 ± 0.4 ab 68.8 ± 7.0 b

CAT 32.5 ± 8.7 b 10.0 ± 2.5 a 47.2 ± 5.1 b 5.4 ± 1.3 a

POX 340.5 ± 52.2 a 635.3 ± 96.4 b 261.7 ± 42.1 a 347.3 ± 7.8 a

the treatment with Cd produced changes in some ofthe H2O2-scavenging enzymes activities. Specifically, aslight decrease in APX (35%) and a dramatic drop inCAT (3.3-fold) were observed, whereas POX exhibitedan 87% increase. We also observed a strong increase inDHAR and GR (2.5- and 2-fold, respectively) activitiesthat correlated with the mentioned increase in reducedAsA. In plants subjected to both treatments (Cd+NaCl)significant decreases in APX (49%) and CAT were alsoobserved. In this case, CAT activity showed a sixfolddecrease comparatively to controls. On the other hand,increases in SOD (73%) and GR (2.4-fold) were alsoproduced.

Leaf hormonal profiling

In leaf 5, initial hormonal content at the time of stressimposition was similar to that determined at week 1for control plants, whatever the considered compound(detailed data not shown). In control plants, Z and ZRconcentration steadily increased by 82 and 72% duringthe growth period, respectively, in relation to the normal

expansion of the considered organ (Fig. 6A, C). After1 week of treatment, both Z and ZR concentrations werehigher in response to Cd alone than in other treatments.Although Cd increased Z and Z+ZR concentration byaround 45% after 2 weeks of stress, the values remainedconstant for Z and slightly decreased for Z+ZR duringthe third week. Salinity and Cd+NaCl treatment didnot affect either Z or Z+ZR content during the first2 weeks. However, during the third week, salinityinduced a decline in Z concentrations while Cd induceda decrease in ZR, only. The mixed treatment induced astrong increase in Z concentrations. It was noteworthythat ZR levels in all the treatment remained low andchanged within a narrow range (23–33 ng g−1 FW),which consequently composed a much lower proportion(not higher than 20%) than Z in the total CK content.

After 1 and 2 weeks of treatment, the concentrationof the ethylene precursor ACC was lower in mixed(Cd+NaCl) than in the other treatments (Fig. 7A).It slowly decreased in control leaves during leafdevelopment while Cd induced an increase during thesecond week of stress before a rapid decrease occurringduring the third week. NaCl alone induced a similardecrease in ACC as observed in control plants duringthe second week of treatment and even a higher rate ofdecrease during the third week. Leaf ABA concentrationswere similar for all treatments after 1 week of treatmentand increased thereafter. After 2 weeks, the highestleaf ABA concentration was recorded in Cd-treatedplants while values recorded for the mixed treatment(Cd+NaCl) remained similar to control (Fig. 7B). Anobvious increase in leaf ABA concentration was recordedduring the third week of exposure to NaCl, maximalvalues being recorded for Cd alone and NaCl alone atthe end of the treatment.

After 1 week of treatment, the leaf IAA concentrationwas the highest in Cd-treated plants and the lowest inNaCl and in Cd+NaCl-treated plants (Fig. 7C). Leaf IAAconcentration decreased in all plants during the secondweek and then remained constant in control, Cd or themixed treatment. At the end of the experiment, NaClalone induced an increase in IAA concentration whilethe lowest value was recorded for the mixed treatment.After 1 week of treatment, the highest SA concentrationswere recorded for control plants while the lowest valuewas recorded for NaCl-treated plants (Fig. 7D). SAin controls then linearly decreased until the end ofthe treatment. In contrast, SA concentrations increasedduring the second week of treatment in response to Cd,NaCl or Cd+NaCl. After 3 weeks, SA concentrationswere the lowest in controls and the highest in Cd+NaCl-treated plants.

Physiol. Plant. 2012

Fig. 6. Evolution of zeatin (Z) (A), zeatin-riboside (ZR) (B) and total CKs (Z+ZR) (C) contents in leaf 5 of Kosteletzkya virginica seedlings exposed to5 μM Cd during 3 weeks in the presence or absence of 50 mM NaCl. Data points and vertical bars represent means (n = 3) and SE, respectively. For agiven duration, different indices indicate significant difference according to Student–Newman–Keuls test at the 5% level.

Fig. 7. Evolution of ACC (A), ABA (B), IAA (C) and SA (D) concentrations in leaf 5 of Kosteletzkya virginica seedlings exposed to 5 μM Cd during3 weeks in the presence or absence of 50 mM NaCl. Data points and vertical bars represent means (n = 3) and SE, respectively. For a given duration,different indices indicate significant difference according to Student–Newman–Keuls test at the 5% level.

Discussion

Salinity reduces Cd accumulation independentlyof growth stimulation

As a halophyte plant species, K. virginica is able tocope with high concentrations of salt in its naturalenvironment (Ruan et al. 2008). It is therefore notsurprising that 50 mM NaCl had no detrimental effect

on plant growth. It has been reported that in numeroushalophyte species, low to moderate doses of NaCl mayeven improve the plant growth (Flowers and Colmer2008, Ruan et al. 2010). Growth stimulation in theaerial part may lead to a dilution effect of toxic elements(Lefevre et al. 2009b, Zaier et al. 2010): for a given rateof ion translocation from root-to-shoot, concentration

Physiol. Plant. 2012

expressed on a dry weight basis may decrease as a resultof growth stimulation. In K. virginica, 100 mM NaCl wasshown to increase shoot growth (Ghanem et al. 2010).The purpose of this work, however, was to assess theputative impact of NaCl on the plant resistance to Cd,independently of growth stimulation. Hence, 50 mMNaCl was chosen in as much as this salinity does notimprove growth in K. virginica (Han et al. 2012). Inthis study, 50 mM NaCl did not improve shoot growthand had no beneficial impact on the leaf expansionprocess. It is noteworthy that exogenous concentrationsof 50 mM NaCl reduced Cd accumulation by 50% in thestudied leaf of K. virginica exposed to 5 μM Cd. This datasuggest that salinity may reduce Cd absorption and/ortranslocation from root-to-shoot in some halophyte plantspecies and that such an impact could lead to lowershoot Cd concentration occurring in the absence ofgrowth stimulation. According to Lefevre et al. (2009b),chloride ions may form CdCl+ complexes in solutionwhich are less efficiently absorbed by the roots (thusexplaining the recorded decrease in Cd accumulation)but may also improve tissular Cd tolerance in halophytespecies through an increase in the synthesis of protectivecompounds. Our data suggest that such a protectionmay involve both the management of oxidative stressand the regulation of hormonal status leading to a delayin stress-induced leaf senescence.

The onset of Cd-induced leaf senescencewas delayed by salt

This study focuses on a specific leaf initiated beforestress imposition. Although physiological behavior ofthis precise leaf is not necessarily relevant from thewhole shoot behavior, quantification of senescence-related parameters in Cd- and in Cd+NaCl-treated plantsallowed us to analyze NaCl impact on Cd-induced leafsenescence. Clearly, Cd stress decreased PSII efficiencyas well as the PSII quantum yield and correlated withthe ETR decrease [photochemical quenching (qP) alsodecreases according to data not shown]. This indicateslower photosynthetic rates, and probably a leakage ofelectron to oxygen, producing O2

•− and H2O2whichmay have been responsible for oxidative stress assuggested by MDA and carbonyl accumulation. Thedecreases in these parameters (Fv/Fm and Y(II)) couldhave been caused by a loss of qP or by an increasein the NPQ. A decrease in qP suggests an increasedproduction of ROS, such as 1O2. NPQ increase indicatedan enhancement of mechanisms of energy dissipation,as was recorded here in response to Cd stress alone. Themaintenance of NPQ values under stress situations hasbeen associated with a capacity to dissipate light energy

safely, and it can be seen as a protective response inorder to avoid photoinhibitory damage to the reactioncenter (Rahoutei et al. 2000). In this experiment, NaClcontributed to avoid NPQ increase in the Cd-treatedplants. However, after 1 week of treatment, we recordedan obvious decrease in NPQ. This situation could reflecta diminished capacity for the safe dissipation of excesslight energy, and therefore does not avoid the productionof harmful species, such as 1O2 (Fryer et al. 2002).In K. virginica, however, MDA and carbonyl contentwere lower in plants exposed to the mixed treatmentthan in those exposed to Cd alone, therefore suggestingthat recorded decrease in NPQ was not responsible formajor oxidative stress in plants exposed to the mixedtreatment. A burst occurring in the NPQ of Cd-treatedplants at the end of the treatment may be considered as aconsequence of senescence processes hastened throughoversynthesis of the senescing compounds ACC (actingas a precursor of ethylene) and ABA, while such increasesdid not occur to similar extent in plants exposed to themixed treatment Cd+NaCl.

Salt ameliorates Cd-induced oxidative damageby modulating oxidants and antioxidativeenzyme metabolism

Cd-treatment induced an oxidative stress as observedby the increase in oxidative stress parameters, affectingessential macromolecules such as lipids and proteins,and producing damage to membranes. A strongaccumulation in ROS was produced, especially O2

•−.The strong increases in H2O2 and O2

•− could beproduced by different sources such as the imbalanceof the electron transport chains both in chloroplastsand mitochondria (Hernandez et al. 1995), or by theactivation of some ROS-generating enzymes (NADPHoxidase or extracellular POXs) (Bolwell and Wojtaszek1997). In BY-2 tobacco cells, Cd induced a rapid H2O2

generation, located in the plasma membrane. Treatmentswith diphenyleneiodonium and imidazole, preventedthe H2O2 generation induced by Cd, suggesting theinvolvement of an NADPH oxidase-like enzyme, leadingto the H2O2 production through the O2

•− dismutationby SOD (Olmos et al. 2003). This suggestion agrees withthe twofold increase in SOD activity that we found inthe Cd-treated plants. In this fact, we must take intoaccount that SOD activity transforms one ROS (O2

•−)into another (H2O2), contributing to the accumulationof H2O2 in Cd-treated plants. However, the possibleinvolvement of a NADH-POX in the H2O2 generationcannot be ruled out (Dıaz-Vivancos et al. 2006). In thiswork, a significant increase in POX activity was observedin Cd-treated plants.

Physiol. Plant. 2012

As far as non-enzymatic antioxidants are concerned,Cd appeared to mainly affect GSH synthesis since nosignificant GSSG accumulation was recorded. IncreasedAsA concentration correlated with the increase in DHARand GR activities in Cd-treated plants. These datasuggest that AsA was efficiently recycled by DHARusing GSH as reducing power. It is noteworthy thatNaCl increased GSH concentration in the presenceor in the absence of Cd, and that, in both cases anincrease in GR was observed. These results are differentto those observed in NaCl-tolerant pea plants (cv. Puget)(Hernandez et al. 2000), where a progressive decreasein GSH was produced with the NaCl concentrationused, accompanied by an accumulation in GSSG,producing a drop in the redox state of GSH. It couldthus be hypothesized that physiological strategies usedby halophyte to cope with oxidative stress may differfrom the strategies adopted by resistant glycophyte.Lefevre et al. (2010) already reported that GR activitiesconstitute a major component of stress tolerance inthe xero-halophyte A. halimus and that it is directlyinvolved in cell lines responses to Cd toxicity. Indeed,Cd toxicity is known to produce important disturbancesin the antioxidative metabolism of plants as well asincreased ROS production (Romero-Puertas et al. 2007).On the other hand, Cd induces damage to membranes,protein oxidation and changes in enzyme activity anddisruption of electron transport (Chen et al. 2003).In this sense lipid peroxidation and protein oxidationcould also be partially attributed to elevated activities ofROS-generating enzymes such as lipoxygenases, SODs,exocellular POXs, oxalate oxidases and amine oxidaseor NADPH oxidase (Bolwell and Wojtaszek 1997).In addition, the observed decreases in APX and CATcan facilitate the H2O2 accumulation. The Cd-induceddecrease in CAT has been also reported in other plantspecies such as rice or Arabidopsis (Cho and Seo 2004,Kuo and Kao 2004). It is also known that salinity inducedan oxidative stress at subcellular level (Hernandez et al.1995, 2001). In this case, the treatment of plants with50 mM NaCl produced an increased accumulation ofO2

•− that correlated with a rise in SOD.An improved ability to cope with oxidative stress in

response to mixed treatment should not be regarded as asimple passive consequence of NaCl-induced decreasein Cd accumulation: indeed, for both GSH, AsA andα-tocopherol content, plants exposed to Cd+NaCldid not exhibit an intermediate behavior betweencontrol and Cd-treated plants but rather showed ahigher concentration than plants exposed to other treat-ments, thus confirming that they were able to activelyadjust their metabolism to this precise environmentalcondition.

Hormone involvement in delaying leaf senescencein response to Cd toxicity

The stress-induced changes in hormone concentrationshave to be analyzed in relation to the modification inthe leaf mineral content. As far as Cd is concerned,salinity reduced its accumulation after already 1 week oftreatment and the difference between Cd- and Cd+NaCl-treated leaves thereafter remained constant in terms ofaccumulated Cd.

The fact that Z and ZR concentrations were higherin Cd-treated leaves than in those exposed to mixedtreatment is puzzling. Indeed, those compounds areknown to act as anti-senescing agents, helping to delaychlorophyll breakdown, as well as cell membrane andprotein degradation (Ananieva et al. 2008, Ghanemet al. 2008, 2011, Sykorova et al. 2008). Our dataobtained for MDA and carbonyl concentrations suggestthat senescence was not delayed in Cd-treated leaves,despite CKs accumulation. The presence of NaCl in aCd-containing solution decreased CK concentration butalso paradoxically delayed senescence in terms of MDAand carbonyl accumulation. This leads us to concludethat CKs were not, under our experimental conditions,the major determinant of Cd-induced senescence inK. virginica. Senescence is a complex process, whichis only partly under hormonal control. The availabilityof carbon relative to nitrogen was not considered inthis study while it may directly influence the tissularresponse to CK levels (Sykorova et al. 2008). Leafsenescence may also involve different phases whichcould be differentially regulated. There is no evidencethat the increase in Z which was observed duringthe first week in Cd-treated plants and during thethird week in Cd+NaCl-treated ones occurred throughsimilar modalities. Ananieva et al. (2008) suggested thatmodifications in cytokinin oxidase/dehydrogenase (CKX)activity may be involved in the control of endogenousCK levels and that CKX signaling could be a possibleregulatory mechanism controlling senescence. It couldtherefore not be excluded that Cd induced an inhibitionof CKX activity rather than an increase in Z synthesis.

The senescing hormone ABA was produced to higherextent in Cd-treated leaves than in Cd+NaCl-exposedplants during the second and the third week of treatment.The ABA concentration was however similar for alltreatments at week 1, at a time when symptoms ofsenescence were already recorded in Cd-treated plants.Conversely, the precursor of ethylene ACC was lowerin Cd+NaCl-treated plants than in plants exposed to Cdonly. In salt-treated tomato, ACC was showed to increasein leaf tissue concomitantly with the onset of oxidativedamage and the decline in chlorophyll fluorescence(Ghanem et al. 2008). This, however, was probably not

Physiol. Plant. 2012

the case in K. virginica since both Cd-treated plantsand NaCl-treated ones exhibited similar ACC valuesthan controls at week 1, while ACC concentration wasthe highest in controls at week 3. The hypothesis thatthis high concentration of ACC in controls could bea consequence of a lower rate of ACC conversion toethylene still has to be tested.

According to Rodrıguez-Serrano et al. (2006), anincrease in SA concentration in Cd-treated plants shouldbe regarded as an attempt to regulate the cellularresponse in order to cope with damages imposed by Cd.In this work, SA concentration progressively decreasedin control plants, probably as a consequence of thenormal leaf expansion occurring in leaf 5. In contrast,SA concentration increased in the presence of Cd after2 weeks of treatment. According to Zhang and Chen(2011), SA could prevent Cd-induced photosyntheticdamage and cell death through the inhibition ofROS overproduction. We however noticed that SAconcentration was higher in Cd- than in Cd+NaCl-treated plants after 1 week of stress and that it remainedsimilar in the two treatments thereafter. The beneficialeffect of NaCl on ROS production by Cd-treated plantscould thus not be explained by a higher synthesis of SAsignaling molecule.

Beside their involvement in the regulation of leafsenescence, some phytohormones such as CKs, ABAand ACC also act as major components of root-to-shootcommunication. Han et al. (2012) recently showed thatCd accumulated to higher concentration in the rootsthan in the shoots of K. virginica but that, in contrast toits impact on the shoot, NaCl had only a minor impact onthe root Cd concentration. Nevertheless, it is still possiblethat the presence of salt has an impact on root hormonesynthesis through a modulation of genes encoding keyenzymes involved in ABA, CKs and ACC synthesis, oron the loading of these hormonal compounds in thexylem of Cd-treated plants. Kosteletzkya virginica is atypical wetland species well adapted to waterloggingand flooded conditions. According to Jackson (1990),these species may exhibit specific hormonal features inrelation to their specific habitat. Further studies are thusrequired to decipher the putative impact of NaCl on Cdresistance in this plant species.

Conclusion

The result clearly showed that NaCl afforded a partialprotection against the Cd toxicity, as observed by thelower damage to membranes. This partial protectionseems to be related with a decrease in Cd absorption,increase in some antioxidant molecules, such as GSH,AsA and α-tocopherol levels, as well as with the increase

in some antioxidant enzymes such SOD and GR and themaintenance of POX, MDHAR and DHAR activities.The delay of senescence induced by NaCl on Cd-treated leaf could be, at least partly, related to alower production of ABA and ethylene precursor ACCwhile CKs accumulated in plants exposed to Cd inthe absence of NaCl but appeared unable to efficientlyreduce senescence in those organs.

Acknowledgements – We thank Dr Alejandro Torrecillas(CEBAS, Murcia) for valuable help in analyzing hormonalcontents and Dr Hong Wang (CEBAS, Murcia) forstimulating discussion of many aspects covered. This workwas supported by the Belgian Science Policy (BELSPO):Chinese-Belgian Cooperation Project (2004411505) and bythe Fonds de la Recherche Scientifique of Belgium (FNRS):‘Credit Bref Sejour a l’etranger’ [2010/V 3/5/215 – IB/JN– 10190]. R-M. H. is grateful to the FNRS for the awardof a research fellowship, and especially to the Universitecatholique de Louvain (UCL) for his PhD grant. This paperis dedicated to the memory of Prof. Luc Waterkeyn.

References

Ananieva K, Ananiev ED, Doncheva S, Georgieva K,Tzvetkova N, Kamınek M, Motyka V, Dobrev P,Gajdosova S, Malbeck J (2008) Senescence progressionin a single darkened cotyledon depends on the lightstatus of the other cotyledon in Cucurbita pepo(zucchini) seedlings: potential involvement of cytokininsand cytokinin oxidase/dehydrogenase activity. PhysiolPlant 134: 609–623

Asada K (2006) Production and scavenging of reactiveoxygen species in chloroplasts and their functions. PlantPhysiol 141: 391–396

Bolwell GP, Wojtaszek P (1997) Mechanisms for thegeneration of reactive oxygen species in plant defence –a broad perspective. Physiol Mol Plant Pathol 51:347–366

Bonfig KB, Schreiber U, Gabler A, Roitsch T, Berger S(2006) Infection with virulent and avirulent P. syringaestrains differentially affects photosynthesis and sinkmetabolism in Arabidopsis leaves. Planta 225: 1–12

Chaoui A, El Ferjani E (2005) Effects of cadmium andcopper on antioxidant capacities, lignifications andauxin degradation in leaves of pea (Pisum sativum L.)seedlings. C R Biol 328: 23–31

Chaoui A, Mazhoudi S, Ghorbal MH, El Ferjani E (1997)Cadmium and zinc induction of lipid peroxidation andeffects on antioxidant enzyme activities in bean(Phaseolus vulgaris L.). Plant Sci 127: 139–147

Chen YX, He YF, Luo YM, Yu YL, Lin Q, Wong MH (2003)Physiological mechanism of plant roots exposed tocadmium. Chemosphere 50: 789–793

Physiol. Plant. 2012

Cho UH, Seo NH (2004) Oxidative stress in Arabidopsisthaliana exposed to Cd is due to hydrogen peroxideaccumulation. Plant Sci 168: 113–120

Clemente-Moreno MJ, Dıaz-Vivancos P, Barba-Espın G,Hernandez JA (2010) Benzothiadiazole andL-2-oxothiazolidine-4-carboxylic acid reduced theseverity of Sharka symptoms in pea leaves: effect on theantioxidative metabolism at subcellular level. Plant Biol12: 88–97

Dıaz-Vivancos P, Rubio M, Mesonero V, Periago PM, RosBarcelo A, Martınez-Gomez P, Hernandez JA (2006)The apoplastic antioxidant system in Prunus: response toplum pox virus. J Exp Bot 57: 3813–3824

Dobrev PI, Kamınek M (2002) Fast and efficient separationof cytokinins from auxin and abscisic acid and theirpurification using mixed-mode solid-phase extraction.J Chromatogr A950: 21–29

Doyle MO, Otte ML (1997) Organism-inducedaccumulation of iron, zinc and arsenic in wetland soils.Environ Pollut 96: 1–11

Ellouzi H, Ben-Hamed KB, Cela J, Munne-Bosch S,Abdelly C (2011) Early effects of salt stress on thephysiological and antioxidative status of Cakile maritima(halophyte) and Arabidopsis thaliana (glycophyte).Physiol Plant 142: 128–143

Elstner EF, Heupel A (1976) Inhibition of nitrate formationfrom hydroxyl ammonium chloride: a simple assay forsuperoxide dismutase. Anal Biochem 70: 616–620

Flowers TJ, Colmer TD (2008) Salinity tolerance inhalophytes. New Phytol 179: 945–963

Foyer CH, Noctor G (2005) Oxidant and antioxidantsignalling in plants: a re-evaluation of the concept ofoxidative stress in physiological context. Plant CellEnviron 28: 1056–1071

Fryer MJ, Oxborough K, Mullineaux PM, Baker NR (2002)Imaging of photo-oxidative stress responses in leaves.J Exp Bot 53: 1249–1254

Gallego SM, Benavıdes MP, Tomaro ML (1996) Effect ofheavy metal ion excess on sunflower leaves: evidencefor involvement of oxidative stress. Plant Sci 121:151–159

Ghanem ME, Albacete A, Martınez-Andjar C, Acosta M,Romero-Aranda R, Dodd IC, Lutts S, Perez-Alfocea F(2008) Hormonal changes during salinity induced leafsenescence in tomato (Solanum lycopersicum L.). J ExpBot 59: 3039–3050

Ghanem ME, Han RM, Classen B, Quetin-Leclercq J, MahyG, Ruan CJ, Qin P, Perez-Alfocea F, Lutts S (2010)Mucilage and polysaccharides in the halophyte plantspecies Kosteletzkya virginica: localization andcomposition in relation to salt stress. J Plant Physiol 167:382–392

Ghanem ME, Albacete A, Smigocki AC, Frebort I,Pospısilova H, Martınez-Andujar C, Acosta M,Sanchez-Bravo J, Lutts S, Dodd IC, Perez-Alfocea F

(2011) Root-synthesized cytokinins improve shootgrowth and fruit yield in salinized tomato (Solanumlycopersicum L.) plants. J Exp Bot 62: 125–140

Gratao PL, Monteiro CC, Rossi ML, Martinelli AP, PeresLEP, Medici LO, Lea PJ, Azevedo RA (2009) Differentialultrastructural changes in tomato hormonal mutantsexposed to cadmium. Environ Exp Bot 67: 387–394

Griffith OW (1980) Determination of glutathionedisulphide using glutathione reductase and2-vinylpyridine. Anal Biochem 106: 207–212

Guo B, Liang YC, Zhu YG (2009) Does salicylic acidregulate antioxidant defense system, cell death,cadmium uptake and partitioning to acquire cadmiumtolerance in rice? J Plant Physiol 166: 20–31

Han RM, Lefevre I, Ruan CJ, Qin P and Lutts S (2012) NaCldifferently interferes with Cd and Zn toxicities in thewetland halophyte species Kosteletzkya virginica (L.)Presl. Plant Growth Regul, in pressdoi: 10.1007/s10725-012-9697-z

Heath RL, Packer L (1968) Photoperoxidation inisolated chloroplasts. I. Kinetics and stoichiometry offatty acid peroxidation. Arch Biochem Biophys 125:189–198

Hernandez JA, Olmos E, Corpas FJ, Sevilla F, del Rıo LA(1995) Salt-induced oxidative stress in chloroplast of peaplants. Plant Sci 105: 151–167

Hernandez JA, Jimenez A, Mullineaux P, Sevilla F (2000)Tolerance of pea (Pisum sativum L.) to long-term saltstress is associated with induction of antioxidantdefences. Plant Cell Environ 23: 853–862

Hernandez JA, Ferrer MA, Jimenez A, Ros-Barcelo A,Sevilla F (2001) Antioxidant systems and O2

.– /H2O2

production in the apoplast of Pisum sativum L. leaves:its relation with NaCl-induced necrotic lesions in minorveins. Plant Physiol 127: 817–831

Hsu YT, Kao CH (2003) Role of abscisic acid in cadmiumtolerance of rice (Oryza sativa L.) seedlings. Plant CellEnviron 56: 867–874

Jackson MB (1990) Hormones and developmental changesin plants subjected to submergence or soil waterlogging.Aquat Bot 38: 49–72

Jimenez A, Hernandez JA, Pastori G, del Rıo LA, Sevilla F(1998) Role of the ascorbate-glutathione cycle ofmitochondria and peroxisomes in the senescence of pealeaves. Plant Physiol 118: 1327–1335

Kuo MC, Kao CH (2004) Antioxidant enzyme activities areupregulated in response to cadmium in sensitive, but notin tolerant rice (Oryza sativa L.) seedlings. Bot Bull AcadSin 45: 291–299

Lefevre I, Correal E, Faz-Cano A, Zanuzzi A, Lutts S(2009a) Structural development, water status, pigmentconcentrations, and oxidative stress of Zygophyllumfabago seedlings in relation to cadmium distribution inthe shoot organs. Int J Plant Sci 170: 226–236

Physiol. Plant. 2012

Lefevre I, Marchal G, Meerts P, Correal E, Lutts S (2009b)Chloride salinity reduces cadmium accumulation by theMediterranean halophyte species Atriplex halimus L.Environ Exp Bot 64: 145–152

Lefevre I, Marchal G, Ghanem ME, Correal E, Lutts S(2010) Cadmium has contrasting effects on polyethyleneglycol-sensitive and resistant cell lines in theMediterranean halophyte species Atriplex halimus L.J Plant Physiol 167: 365–374

Liu KL, Shen L, Sheng JP (2008) Improvement in cadmiumtolerance of tomato seedlings with an antisense DNA for1-aminocyclopropane-1-carboxylate synthase. J PlantNutr 31: 809–827

Lopez-Chuken UJ, Young SD (2005) Plant screening ofhalophyte species for cadmium phytoremediation.Z Naturforsch 60: 236–243

Lutts S, Lefevre I, Delperee C, Kivits S, Dechamps C,Robledo A, Correal E (2004) Heavy metal accumulationby the halophyte species Mediterranean saltbush.J Environ Qual 33: 1271–1279

Metwally A, Finkemeier I, Georgi M, Dietz KJ (2003)Salicylic acid alleviates the cadmium toxicity in barleyseedlings. Plant Physiol 132: 272–281

Mittler R, Vanderauwera S, Suzuki N, Miller G, TognettiVB, Vandepoele K, Gollery M, Shulaev V, VanBreusegem F (2010) ROS signalling: the new wave?Trends Plant Sci 16: 300–309

Monteiro CC, Carvalho RF, Gratao PL, Carvalho G,Tezotto T, Medici LO, Peres LEP, Azevedo RA (2011)Biochemical responses of the ethylene-insensitive Neverripe tomato mutant subjected to cadmium and sodiumstresses. Environ Exp Bot 71: 306–320

Munne-Bosch S, Weiler EW, Alegre L, Muller M, DuchtingP, Falk J (2007) α-Tocopherol may influence cellularsignalling by modulating jasmonic acid levels in plants.Planta 225: 681–691

Olmos E, Martınez-Solano JR, Piqueras A, Hellın E (2003)Early steps in the oxidative burst induced by cadmium incultured tobacco cells (BY-2 line). J Exp Bot 54:291–301

Perfus-Barbeoch L, Leonhardt N, Vavasseur A, Forestier C(2002) Heavy metal toxicity: cadmium permeatesthrough calcium channels and disturbs the plant waterstatus. Plant J 32: 539–548

Pietrini F, Iannelli MA, Pasqualini S, Massacci A (2003)Interaction of cadmium with glutathione andphotosynthesis in developing leaves and chloroplasts ofPhragmites australis (Cav.) Trin. ex Steudel. PlantPhysiol 133: 829–837

Piqueras A, Olmos E, Martınez-Solano JR, Hellın E (1999)Cd-induced oxidative burst in tobacco BY2 cells: timecourse, subcellular location and antioxidant response.Free Radic Res 31: S33–S38

Rahoutei J, Garcıa-Luque I, Baron M (2000) Inhibition ofphotosynthesis by viral infection: effect on PSII structureand function. Physiol Plant 110: 286–292

Reznick AZ, Packer L (1994) Oxidative damage toproteins: spectrophotometric method for carbonyl assay.Method Enzymol 233: 357–363

Rodrıguez-Serrano M, Romero-Puertas MC, Zabalza A,Corpas FJ, Gomez M, Del Rıo LA, Sandalio LM (2006)Cadmium effect on oxidative metabolism of pea (Pisumsativum L.) roots. Imaging of reactive oxygen speciesand nitric oxide accumulation in vivo. Plant CellEnviron 29: 1532–1544

Romero-Puertas MC, Corpas FJ, Rodrıguez-Serrano M,Gomez M, del Rıo LA, Sandalio LM (2007) Differentialexpression and regulation of antioxidative enzymesby cadmium in pea plants. J Plant Physiol 164:1346–1357

Ruan CJ, Li H, Guo YQ, Qin P, Gallagher JL, Seleskar DM,Lutts S, Mahy G (2008) Kosteletzkya virginica, anagroecoengineering halophytic species for alternativeagricultural production in China’s east coast:ecological adaptation and benefits, seed yield, oilcontent, fatty acid and biodiesel properties. Ecol Eng 32:320–328

Ruan CJ, Teixeira da Silva JA, Mopper S, Qin P, Lutts S(2010) Halophyte improvement for a salinized world.Crit Rev Plant Sci 29: 329–359

Sanita di Toppi L, Gabbrielli R (1999) Responses tocadmium in higher plants. Environ Exp Bot 41:105–130

Shah K, Kumar RG, Verma S, Dubey RS (2001) Effect ofcadmium on lipid peroxidation, superoxide aniongeneration and activities of antioxidant enzymes ingrowing rice seedlings. Plant Sci 161: 1135–1144

Stroinski A, Chadzinikolau T, Gizewska K, Zielezinska M(2010) ABA or cadmium induced phytochelatinsynthesis in potato tubers. Biol Plant 54:117–120

Sykorova B, Kuresova G, Daskalova S, Trckova M,Hoyerova K, Raimanova I, Motyka V, Travnıckova A,Elliott MC, Kamınek M (2008) Senescence-inducedectopic expression of the A. tumefaciens ipt genein wheat delays leaf senescence, increasescytokinin content, nitrate flux, and nitrate reductaseactivity, but does not affect grain yield. J Exp Bot 59:377–387

Wang SY, Jiao HJ, Faust M (1991) Changes in ascorbate,glutathione, and related enzyme activities duringthidiazuron-induced bud break of apple. Physiol Plant82: 231–236

Yakimova ET, Kapchina-Toteva VM, Laarhoven LJ, HarrenFM, Woltering EJ (2006) Involvement of ethylene andlipid signalling in cadmium-induced programmed celldeath in tomato suspension cells. Plant Physiol Biochem44: 581–589

Physiol. Plant. 2012

Zaier H, Ghnaya T, Lakhdar A, Baioui R, Ghabriche R,Mnasri M, Lutts S, Abdelly C (2010) Comparative studyof Pb-phytoextraction potential in Sesuviumportulacastrum and Brassica juncea: tolerance andaccumulation. J Hazard Mater 183: 609–615

Zhang WN, Chen WL (2011) Role of salicylic acid inalleviating photochemical damage and autophagic cell

death induction of cadmium stress in Arabidopsisthaliana. Photochem Photobiol Sci 10:947–955

Zhou B, Wang J, Guo Z, Tan H, Zhu X (2006) A simplecolorimetric method for determination of hydrogenperoxide in plant tissues. Plant Growth Regul 49:113–118

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Physiol. Plant. 2012