Antioxidant enzyme activities and hormonal status

Physiologia Plantarum 2012

Copyright © Physiologia Plantarum 2012, ISSN 0031-9317

Antioxidant enzyme activities and hormonal status in response to Cd stress in the wetland halophyte Kosteletzkya virginica under saline conditions a b ` ´ Rui-Ming Hana , Isabelle Lefevre , Alfonso Albaceteb , Francisco Perez-Alfocea , Gregorio Barba-Esp´ınc , c a d c ´ , Pedro D´ıaz-Vivancos , Muriel Quinet , Cheng-Jiang Ruan , Jose´ Antonio Hernandez b a,∗ Elena Cantero-Navarro and Stanley Lutts a

´ etale ´ Groupe de Recherche en Physiologie veg (GRPV), Earth and Life Institute – Agronomy (ELI-A), Universite´ catholique de Louvain (UCL), Croix du Sud 5, bte L 7.07.13, B-1348, Louvain-la-Neuve, Belgium b ´ Vegetal, Centro de Edafolog´ıa y Biolog´ıa Aplicada del Segura (CEBAS), Consejo Superior de Investigaciones Cient´ıficas Departamento de Nutricion (CSIC), Campus Universitario de Espinardo, PO Box 164, E-30100, Murcia, Spain c Grupo de Biotecnolog´ıa de Frutales, Centro de Edafolog´ıa y Biolog´ıa Aplicada del Segura (CEBAS), Consejo Superior de Investigaciones Cient´ıficas (CSIC), Campus Universitario de Espinardo, PO Box 164, E-30100, Murcia, Spain d Key laboratory of Biotechnology and Bio-resources Utilisation, State Ethnic Affairs Commission and Ministry of Education, Dalian Nationalities University, 116600, Dalian, China

Correspondence *Corresponding author, e-mail: [email protected] 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 the precise impact of salinity on heavy metal toxicity for halophyte plant species remains largely unknown. Young seedlings of Kosteletzkya virginica were exposed during 3 weeks in nutrient solution to Cd 5 μM in the presence or absence of 50 mM NaCl. Cadmium (Cd) reduced growth and shoot water content and had major detrimental effect on maximum quantum efficiency (Fv /Fm ), effective quantum yield of photosystem II (Y(II)) and electron transport rates (ETRs). Cd induced an oxidative stress in relation to an increase in O2 •− and H2 O2 concentration and lead to a decrease in endogenous glutathione (GSH) and α-tocopherol in the leaves. Cd not only increased leaf zeatin and zeatin riboside concentration but also increased the senescing compounds 1aminocyclopropane-1-carboxylic acid (ACC) and abscisic acid (ABA). Salinity reduced Cd accumulation already after 1 week of stress but was unable to restore shoot growth and thus did not induce any dilution effect. Salinity delayed the Cd-induced leaf senescence: NaCl reduced the deleterious impact of Cd on photosynthesis apparatus through an improvement of Fv /Fm , Y(II) and ETR. Salt reduced oxidative stress in Cd-treated plants through an increase in GSH, α-tocopherol and ascorbic acid synthesis and an increase in glutathione reductase (EC 1.6.4.2) activity. Additional salt reduced ACC and ABA accumulation in Cd+NaCl-treated leaves comparing to Cd alone. It is concluded that salinity affords efficient protection against Cd to the halophyte species K. virginica, in relation to an improved management of oxidative stress 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, cytokinin oxidase/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 liquid chromatography; 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 its accumulation in the environment is mainly due to anthropogenic activities. High Cd concentration in plant leaves impairs photosynthesis and chlorophyll metabolism (Pietrini et al. 2003), compromises the plant water status (Perfus-Barbeoch et al. 2002) and induces numerous damages to cellular structures and membranes (Grat˜ao et al. 2009). Some of these damages could, at least partly, result from the Cd-induced synthesis of reactive oxygen species (ROS) enhancing membrane lipid peroxidation, enzyme inhibition and DNA or RNA damages (Shah et al. 2001, Asada 2006). To reduce those damages, plants possess scavenging systems consisting in non-enzymatic antioxidants such as ascorbate or glutathione (GSH), which may counteract or neutralize the harmful effects of ROS, as well as antioxidant enzymes, such as superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6) and enzymes of the ascorbate-glutathione (ASC-GSH) cycle [ascorbate peroxidase (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 strategies that plants adopt against oxidative stress induced by accumulated metal ions is the key information to further manipulate plant heavy metal tolerance. Cd may directly affect the activities of numerous antioxidative enzymes, but contrasting results have been reported in the literature: Cd reduced APX activities in Helianthus annuus (Gallego et al. 1996) but increased them in roots and leaves of Phaseolus vulgaris, as well as in suspension cultures of tobacco (Nicotiana tabacum) cells (Chaoui et al. 1997, Piqueras et al. 1999). Cdinduced oxidative stress is frequently characterized by a decrease in reduced GSH content, or in CAT and CuZnSOD activities (Romero-Puertas et al. 2007). According to Lef`evre et al. (2010), Cd tolerance in cell lines of the halophyte plant species Atriplex halimus may be related to a high content of antioxidant (GSH and ascorbic acid), a high constitutive SOD activity, and an efficient Cd-induced increase in GR and APX activities. Beside oxidative stress, inhibition of growth and development is often reported as the first symptom of Cd deleterious impact (Sanit`a di Toppi and Gabbrielli 1999). Not only plant biomass, but also plant architecture may be modified in response to this toxic element. In perennial dicotyledonous plants, growth inhibition is often more severe on ramifications than on the main stem although the concentration of Cd is lower in the former than in the latter and Cd also inhibits axillary bud

development (Lef`evre et al. 2009a, Han et al. 2012). One possible explanation is that Cd modifies plant hormone synthesis and action but data concerning the impact of this heavy metal on plant growth regulators remain scarce: most studies until now are dealing with abscisic ´ acid (ABA; Hsu and Kao 2003, Stroinski et al. 2010), commonly considered as a stress hormone, or with ethylene in relation to its putative impact on senescence processes (Yakimova et al. 2006, Liu et al. 2008, Grat˜ao et al. 2009). Only few data are available concerning Cd-induced decrease in auxin synthesis (Chaoui and El Ferjani 2005) or increase in salicylic acid (SA) synthesis (Metwally et al. 2003). Although cytokinins (CKs) assume key functions as signaling compounds in the xylem stream and act as anti-senescing agents in the leaves (Ghanem et al. 2011), to the best of our knowledge, no exhaustive data are available until now concerning Cd impact on CK synthesis and translocation. Moreover, most studies considered the Cd impact on only one or two plant growth regulators. Since different plant hormones interact among each other according to complex networks, a comprehensive approach of Cd impact on this class of compounds requires establishing a ‘hormonal profile’ through the quantification of several hormones on the same experimental system. Plant growth regulators may be involved in the management of the antioxidative status of stressed plants. Yakimova et al. (2006) and Monteiro et al. (2011) reported that Cd-induced cell mortality results directly from ethylene production which may itself trigger oxidative stress. Conversely, the SA-enhanced Cd tolerance in rice can be attributed to SA-elevated enzymatic and non-enzymatic antioxidants occurring together with SA-regulated Cd uptake, transport and distribution in plant organs (Guo et al. 2009). It has been reported in different plant species that genes coding for anti-oxidative enzymes may be induced by plant growth regulators through various transduction pathways (Foyer and Noctor 2005). The situation appears rather complex when considering that ROS may also have an impact on phytohormones synthesis in stressed plants (Mittler et al. 2010). A kinetics approach is thus required to determine the timing of Cd-induced modifications in order to clarify the complex relationship between hormonal profile and antioxidative status. To the best of our knowledge, such an approach is still missing but should afford valuable information on the sequence of events leading to the plant response during a prolonged period of exposure to Cd toxicity. Halophyte plant species are adapted to salt stress present in their native habitat and are therefore supposed to possess physiological adaptations allowing them to cope with ion toxicity, which may be useful for the Physiol. Plant. 2012

phytomanagement of heavy metal contaminated area ´ (Lopez-Chuken and Young 2005, Ruan et al. 2010). Their response to osmotic and ionic constrains induced by salinity (Flowers and Colmer 2008) notably involves both phytohormones and antioxidant properties, since halophytes usually display specific adaptations in terms of oxidative status comparatively to glycophytes (Ellouzi et al. 2011). However, the impact of salinity on Cdinduced changes in phytohormone and oxidative status remains largely unknown. Several xero-halophyte plant species present putative interest for revegetation of heavy metal contaminated areas. Those species are resistant to drought and salt are, to some extent, resistant to heavy metals (Lutts et al. 2004, Lef`evre et al. 2009b, Zaier et al. 2010). According to these authors, a beneficial impact of exogenous NaCl on plant response to Cd may be, at least partly, linked to NaCl-induced growth stimulation classically reported for halophyte at moderate salinities, which therefore leads to dilution effect. Wetland halophytes comparatively received only little attention until now, despite the fact that heavy metal contamination is frequently reported in coastal areas and the coastal salt marshes are important sinks for heavy metal accumulation (Doyle and Otte 1997). Those species are not tolerant to drought and their adaptation to flooding conditions should have an impact on their behavior in terms of phytohormones synthesis and oxidative status (Jackson 1990). The impact of those adaptations on plant response to heavy metal is still unknown. Kosteletzkya virginica is a perennial halophyte native to American salt marshes and has been proposed as a promising tool for revegetation of salt-affected coastal tidal flats (Ruan et al. 2008). The response of K. virginica to Cd toxicity under saline conditions has recently been described (Han et al. 2012). Our previous works showed that: (1) Cd had a strong impact on plant architecture; (2) Cd accumulation in different shoot parts could not fully explain the pattern of growth inhibition; (3) NaCl in the absence of Cd could slightly improve plant growth and (4) NaCl significantly reduced Cd accumulation (Han et al. 2012). Until now, no information concerning the Cd-induced secondary oxidative stress and plant growth regulators which may be related to the antioxidative status in K. virginica has been reported. It is moreover hypothesized that NaCl may improve Cd resistance in halophyte independently of any dilution effect related to growth stimulation. This work therefore investigates the leaf antioxidant enzyme activities and hormonal content in response to Cd stress and the influence of low salinity on the relationship between oxidative status and hormonal profile under Cd stress. Physiol. Plant. 2012

Materials and methods Plant material and culture conditions Seeds were immersed in de-ionized water for 30 min before sowing into 85 mm diameter Petri dish with two layers of water-moistened filter paper. Petri dishes were enclosed with aluminum foils and incubated at 28◦ C for 48 h. Well germinated seeds with 2–3 cm root tips were then sowed in trays filled with artificial soil moistened regularly with water and seedlings were allowed to grow in a phytotron under a 16/8h light/dark photoperiod (mean light intensity (PAR) = 500 μmol m−2 s−1 provided by Osram Sylvania (Danvers, MA) fluorescent tubes (F36W/133-T8/CW) with 25/20◦ C day/night temperature and 65/50% atmospheric humidity). Fifteen days after sowing, seedlings were transferred into 18-l tanks and fixed on polyvinyl chloride plates floating on aerated half-strength modified Hoagland nutrient solution containing (in mM): 2.0 KNO3 , 1.7 Ca(NO3 )2 , 1.0 KH2 PO4 , 0.5 NH4 NO3 , 0.5 MgSO4 and (in μM) 17.8 Na2 SO4 , 11.3 H3 BO3 , 1.6 MnSO4 ,1 ZnSO4 , 0.3 CuSO4 , 0.03 (NH4 )6 Mo7 O24 and 14.5 Fe-EDDHA. After 10 days of acclimatization in the absence of stress (25 days after sowing), NaCl and CdCl2 were added to corresponding 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 2 days and renewed every week. The pH of solutions was set to 5.7 ± 0.02 with KOH and readjusted every day. Three replications with nine plants per replication and per treatment were used for the measurement of different parameters. The specific leaf 5 from bottom corresponding to the top-expanded leaf at the beginning of stress period, and which has reached a minimal length of 4 cm, was chosen when Cd and sodium chloride were imposed to monitor senescence and for subsequent biochemical determination. This precise leaf was exposed to Cd toxicity during the whole experimental period: leaf material from nine plants was harvested for different analyses at 1, 2 and 3 weeks of treatment. Chlorophyll fluorescence Modulated chlorophyll fluorescence was measured in tagged and dark-adapted (30 min) leaves (Ft = F0 ) in 6–10 plants per treatment, using a Imaging-PAM M-series system (OptiSciences, Herts, UK) with an excitation source intensity of 3000 μmol m−2 s−1 . The minimal fluorescence intensity (F0 ) in a dark-adapted state was measured in the presence of a background farred light to favor rapid oxidation of intersystem electron carriers. The maximal fluorescence intensities in the

dark-adapted state (Fm ) and after adaptation to white actinic light (Fm  ) were measured by 0.8 s saturating pulses (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 to measure the minimal fluorescence intensity in the lightadapted state (F0  ). The maximum quantum yield of open photosystem II (PSII) (Fv /Fm ), the effective PSII quantum yield PSII = Y(II) and the non-photochemical quenching (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 nonphotochemical energy dissipation, NPQ = Y(NPQ) and non-regulated energy dissipation, NO = Y(NO)[Y(II) + Y(NPQ) + Y(NO) = 1] were calculated as described by Bonfig et al. (2006). After kinetics and light curves were recorded, areas of interest were defined by red circles for every leaf, over which all pixel values for various fluorescence 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 . Minerals were dissolved in a spot of aqua regia and diluted with de-ionized water and then filtered. The element concentrations (Cd, Na and K) were determined using an atomic absorption spectrometer (Thermo Scientific ICE 3300; Waltham, MA) calibrated with certified standard solutions. All measurements were performed in three replicates. Oxidative stress parameters and non-enzymatic antioxidants The level of lipid peroxidation was measured as 2thiobarbituric acid-reactive substances, mainly malondialdehyde (MDA) according to Heath and Packer (1968) The MDA concentration was calculated using its molar extinction coefficient (155 mM−1 cm−1 ). Protein oxidation, given as carbonyl protein (CO-protein) content was carried out using Reznick and Packer (1994) spectrophotometric method for the detection of the reaction of diphenylhydrazine (DNPH) with carbonyl proteins to form protein hydrazones. Frozen samples were finely minced in 3 ml of homogenizing buffer (60 mM phosphate buffer, pH 7.4, containing 0.1% digitonin and antiproteases [4-(2-aminoethyl) benzenesulfonyl fluoride, bestatin, pepstatin A, leupeptin and 1,10-phenanthrolin) and 1 mM ethylenediaminetetraacetic acid (EDTA)] and were incubated for 15 min

at room temperature. Samples were centrifuged (6000 g during 10 min at room temperature); 600 μl of 10 mM DNPH (dissolved in 2.5 M HCl) were added to 150 μM of the supernatant. After 1 h incubation at room temperature, 750 μl of 20% trichloroacetic acid (TCA) (w/v) were added and samples were thereafter kept on ice for 10 min and centrifuged at 10 000 g for 5 min at 4◦ C. Supernatants were discarded and pellets were washed three times with 400 μl ethanol-ethyl acetate (1:1) (v/v) to remove the free DNPH and lipid contaminants. The final precipitates were dissolved in 500 μl of 6 M guanidine hydrochloride solution and shaken for 10 min at 37◦ C. Insoluble material was removed by additional centrifugation (6000 g ; 1 min at room temperature). Carbonyl content was calculated using the peak absorbance (spectra at 320–390 nm) and a 2.5 M HCl-treated sample without DNPH as a blank. For hydrogen peroxide quantification, frozen fresh leaves (0.5 g) were ground to powder in the presence of 5 ml 5% TCA and 0.15 g activated charcoal. The mixture was centrifuged at 10 000 g for 20 min at 4◦ C. The supernatant was adjusted to pH 8.4 with 17 M ammonia solution and then filtered. The filtrate was divided into aliquots of 1 ml. To one of these (the blank) 8 μg of CAT (10 000 U mg−1 ) were added and samples were then kept at room temperatures for 10 min. To both aliquots (with and without CAT), 1 ml of colorimetric reagent was added. The reaction solution was incubated for 10 min at 30◦ C. Absorbance at 505 nm was determined spectrophotometrically. The colorimetric reagent contained 10 mg of 4-aminoantipyrine, 10 mg of phenol and 5 mg of peroxidase (POX) (150 U mg−1 ) dissolved in 50 ml of 100 mM acetic buffer (pH 5.6) (Zhou et al. 2006). Superoxide ions (O2 •− ) were measured according to Elstner and Heupel (1976) by monitoring nitrate formation from hydroxylamine. The concentrations of α-tocopherol and its oxidation product α-tocopherol quinone were quantified according to Munn´e-Bosch et al. (2007). Leaf samples were extracted four times with ice-cold n-hexane containing 1 ppm butylated hydroxytoluene using ultrasonication. Tocopherols were separated on a Partisil 10 ODS-3 column at a flow rate of 1 ml min−1 . The solvents consisted of (A) methanol/water (95:5, v/v) and (B) methanol. The gradient used was: 0–10 min 100% A, 10–20 min decreasing to 0% A, 20–25 min 0% A, 25–28 min increasing to 100% A and 28–33 min 100% A. The α-tocopherol and α-tocopherol quinone were quantified by their absorbance at 283 and 265 nm, respectively. GSH was assayed by the enzymatic recycling procedure in which it is sequentially oxidized by 5,5 dithiobis (2-nitrobenzoic acid) and reduced by NADPH Physiol. Plant. 2012

in the presence of GR according to Griffith (1980) and modified by Lutts et al. (2004). Briefly, extraction was performed in 5% sulphosalicylic acid on ca. 1 g fresh weight (FW) ground tissue and the extent of 2-nitro-5thiobenzoic acid formation was monitored at 412 nm for GSH plus GSSG evaluation. For determination of GSSG alone, the extract was pre-treated with 2-vinylpyridine to scavenge GSH by derivatization. For ascorbate extraction, frozen tissues were homogenized in ice cold 5% metaphosphoric acid solution (1:5, w/v) and then centrifuged at 20 000 g and 4◦ C for 10 min. Total ascorbate (AsA + DHA) contents were determined according to Wang et al. (1991) on the basis of Fe3+ –Fe2+ reduction by ascorbate in acid solution. Fe2+ forms a red chelate with bathophenanthroline absorbing at 534 nm. The ascorbate (reduced form) assay mixture contained 0.1 ml of the extract, 0.5 ml of absolute ethanol, 0.6 M TCA, 3 mM bathophenanthroline, 8 mM H3 PO4 and 0.17 mM FeCl3 . The final total volume was 1.5 ml and the mixture was allowed to stand at 30◦ C for 90 min. The absorbance of the colored solution was read at 534 nm. The total ascorbate assay mixture contained 0.1 ml of the sample, 0.15 ml of 3.89 mM dithiothreitol and 0.35 ml of absolute ethanol in a total volume of 0.6 ml. Then, the reaction mixture was left standing at room temperature for 10 min. After reduction of dehydroascorbate to ascorbate, 0.15 ml of 20% TCA was added and the color was developed by adding 0.15 ml of 0.4% (v/v) H3 PO4 ethanol, 0.3 ml of 0.5% (w/v) bathophenantrolineethanol and 0.15 ml of 0.03% (w/v) FeCl3 -ethanol. Dehydroascorbate concentrations were estimated from the difference of ‘total ascorbate’ and ascorbate concentration. Standard curve in the range 0–10 μmol ascorbate was used. Antioxidant enzymes activities All operations were performed at 0–4◦ C. Leaves (around 0.5 g fresh mass) were homogenized with 2 ml of an ice-cold buffer containing 50 mM Tris–acetate buffer (pH 6.0), 0.1 mM EDTA, 5 mM cysteine, 2% (w/v) insoluble polyvinylpolypyrrolidone, 0.1 mM phenylmethane-sulphonyl fluoride and 0.2% (v/v) Triton X-100. For the APX activity assay, 20 mM Na-ascorbate was added to the extraction medium. The extracts were filtered through two layers of nylon cloth and centrifuged at 8000 g for 20 min, at 4◦ C. The supernatant fractions were then filtered on Sephadex G-25 NAP columns (GE Healthcare, Madrid, Spain) equilibrated with the extraction buffer. The APX, DHAR, MDHAR, GR, POX (EC1.11.1.7), CAT and total SOD activities and proteins contents Physiol. Plant. 2012

were assayed as described in Clemente-Moreno et al. (2010). Enzyme activities were corrected for nonenzymatic rates and for interfering oxidation. For APX, the oxidation rate of ascorbate was estimated between 1.0 and 60 s after starting the reaction by the addition of H2 O2 . Correction was made for the low non-enzymatic oxidation of ascorbate by H2 O2 . To determine MDHAR activity, monodehydroascorbate was generated by the ascorbate/ascorbate oxidase system. The rate of monodehydroascorbate-independent NADH oxidation (without ascorbate and ascorbate oxidase) was subtracted from the initial monodehydroascorbatedependent NADH oxidation rate (with ascorbate and ascorbate oxidase). For DHAR activity, the reaction rate was corrected for the non-enzymatic reduction of DHA by GSH. A 2% contribution to the absorbance by GSSG was also taken into account. Values of GR activity were corrected for the small, nonenzymatic oxidation of NADPH by GSSG (Jim´enez et al. 1998). Hormone extraction and analysis CKs [zeatin (Z) and zeatin riboside (ZR)], indole-3-acetic acid (IAA) and ABA were extracted on 1 g leaf material in methanol/water/formic acid (15/4/1, v/v/v, pH 2.5) and purified as previously described (Ghanem et al. 2008) according to the method of Dobrev and Kam´ınek (2002). Pooled supernatants were passed through Sep-Pak Plus® C18 cartridge (SepPak Plus, Waters, Milford, MA) to remove interfering lipids and part of plant pigments and evaporated either to near dryness or until organic solvent was removed. The residue was dissolved in 5 ml 1 M formic acid and applied to Oasis MCX mixed mode (cation-exchange and reverse phase) column (150 mg; Waters) pre-conditioned with 5 ml of methanol followed by 5 ml of 1 M formic acid. To separate different CKs (nucleotides, bases, ribosides and glucosides) from IAA and ABA, the column was washed and eluted stepwise with the different appropriate solutions indicated in Dobrev and Kam´ınek (2002). ABA and IAA were analyzed in the same fraction. Samples then dissolved in mobile phase A, consisting of water/acetonitrile/formic acid (94.9:5:0.1 v/v) mixture for high-performance liquid chromatography/mass spectrometry (HPLC)/mass spectrometry (MS) analysis. The analysis were carried out on a HPLC/MS system consisting of an Agilent 1100 Series HPLC (Agilent Technologies, Santa Clara, CA) equipped with a μ-wellplate autosampler and a capillary pump, and connected to an Agilent Ion Trap XCT Plus mass spectrometer (Agilent Technologies) using an electrospray interface. Previous to injection, 100 μl of each fraction extracted from leaf tissues were filtered

through 13 mm diameter Millex filters with 0.22 μm pore 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), held at 40◦ C, and eluted at a flow rate of 10 μl min−1 . Mobile phase A, consisting of water/acetonitrile/formic acid (94.9:5:0.1), and mobile phase B, consisting of water/acetonitrile/formic acid (10:89.9:0.1), were used for the chromatographic separation with the gradient elution described by Ghanem et al. (2008). The UV chromatogram was recorded at 280 nm with the DAD module (Agilent Technologies). The mass spectrometer was operated in the positive mode with a capillary spray voltage of 3500 V, and a scan speed of 22 000 (m/z)/s from 50 to 500 m/z. The nebulizer gas (He) pressure was set to 30 psi, whereas the drying gas was set to a flow of 6 l min−1 at a temperature of 350◦ C. Mass spectra were obtained using the DATA ANALYSIS PROGRAM FOR LC/MSD TRAP Version 3.2 (Bruker Daltonik GmbH, Bremen, Germany). For quantification of Z, ZR, ABA and IAA, calibration curves were constructed for each analyzed component (0.05, 0.075, 0.1, 0.2 and 0.5 mg l−1 ) and corrected for 0.1 mg l−1 internal standards: [2 H5 ]transZ, [2 H5 ]trans-ZR, [2 H6 ]cis,trans-abscisic acid (Olchemin Ltd, Olomouc, Czech Republic) and [13 C6 ]indole3-acetic acid (Cambridge Isotope Laboratories Inc., Andover, MA). Recovery percentages ranged between 92 and 95%. ACC (1-aminocyclopropane-1-carboxylic acid) was determined after conversion into ethylene by gas chromatography using an activated alumina column and a FID detector (Cromatix-KNK-2000; Konik, Barcelona, Spain). ACC was extracted with 80% (v/v) ethanol and assayed by degradation with alkaline hypochlorite in the

presence of 5 mM HgCl2 according to the description of Ghanem et al. (2008). A preliminary purification step was performed by passing the extract through a Dowex 50W-X8, 50–100 mesh, H+ -form resin and later recovered with 0.1N NH4 OH. The conversion efficiency of ACC into ethylene was calculated separately by using a replicate sample containing 2.5 nmol of ACC as 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 nature of stress and the duration of stress considered as the main factors. The statistical significance of the results was analyzed by the Student–Newman–Keuls test at the 5% level.

Results Shoot dry weight and leaf water content The presence of NaCl had no impact on the shoot dry weight while Cd induced an obvious decrease in shoot growth (Fig. 1A). The addition of NaCl to the Cd solution was unable to reduce the deleterious impact of the heavy metal on shoot dry weight. Salinity did not affect the shoot water content (Fig. 1B). In contrast, 5 μM Cd significantly reduced this parameter, already after 1 week of treatment. The presence of NaCl partly mitigated the impact of Cd on the leaf water content, especially after 2 weeks of treatment. The expansion of leaf 5 was slightly inhibited in the presence of Cd: at the end of the stress period, average leaf surface reached 194 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 the presence or absence of 50 mM NaCl. Data points and vertical bars represent means of five individual plants (n = 5) and SE, respectively. For a given duration of treatment, means with different letters are significantly different at P < 0.05.

Physiol. Plant. 2012

Fig. 2. Evolution of chlorophyll fluorescence parameters in leaf 5 of Kosteletzkya virginica seedlings exposed to 5 μM Cd during 3 weeks in the presence 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 after 2 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 leaf senescence-related parameters The images of leaf 5 obtained using imaging-PAM fluorometer and the evolution of chlorophyll fluorescence and senescence-related parameters are shown in Fig. 2. From false-color images, changes in photosynthetic activities could be readily discerned. The highest negative effects occurred in response to Cd treatment, whereas NaCl alone did not or only slightly affected these Physiol. Plant. 2012

parameters. The mixed treatment showed intermediate effects (Fig. 2A, B). In leaf 5 of control plants, the maximum quantum efficiency of PSII (Fv /Fm ) was almost constant during the growing period, whereas in Cd-stressed plants, these values sharply decreased below those of control plants. Sodium treatment had no impact on Fv /Fm , whereas the addition of NaCl to Cd significant increased these values compared to Cd alone (Fig. 2C). The effective quantum yield of PSII (Y(II)) decreased during the whole stress period and exhibited the lowest value in response to Cd treatment. In NaCl-treated plants, the Y(II) value showed no difference with those of control at week 1

and remained almost constant thereafter. Due to an increase in control plants during the last 2 weeks, the Y(II) in NaCl-treated plants became significantly lower than those of control. In the mixed treatment, Y(II) also decreased after 2 weeks of stress but less than in response to Cd alone (Fig. 2D). In control plants, NPQ decreased as the leaves aged, whereas in Cd-treated plants, NPQ decreased in week 2, reaching a minimum by day 14 before strongly increasing during the last week. After 1 week treatment with NaCl alone, NPQ was lower than that of control and was constant during week 2 before slightly decreasing thereafter. The mixed treatment decreased NPQ steadily from a same level comparatively to NaCl-treated plants after 1 week and even to a significant lower level after 2 and 3 weeks (Fig. 2E). For further analysis, light response curves of the relative ETR were examined. Cd application decreased ETR in the presence or absence of NaCl. The effect of NaCl either alone or in the mixed treatment was not obvious during week 1, but it decreased ETR by itself and mitigated the Cd-induced decline during the week 2 and 3. When PAR value was below 50 μmol m−2 s−1 , the light response curve of the relative ETR value in treated and untreated groups was similar (Fig. 3). This result showed that NaCl alleviated Cd toxicity effect on electron-transfer system and consequently reduced the poisoning effect of this heavy metal on K. virginica. Our results assessed by the imaging-PAM chlorophyll fluorometer showed that the Cd-induced decreases in Y(II) were paralleled by the increase in Y(NPQ) and Y(NO), indicating that photosynthesis was inhibited. However, the addition of NaCl reduced the decline of Y(II) and therefore alleviated the Cd toxicity (Fig. 4). Cd, sodium and potassium concentration Cd was accumulated to a significantly higher level in response to Cd stress alone than in response to the mixed treatment (Fig. 5A). Sodium was accumulated to a similar extent (300–320 μmol g−1 DM) during the first 2 weeks in the presence of NaCl alone and under the mixed treatments, but a strong increase (up to 450 μmol g−1 DM) occurred at the end of the third week in leaves of plants exposed to Cd+NaCl (Fig. 5B). Salinity induced a quick and strong reduction in K concentration, whereas Cd induced a strong increase in this parameter (Fig. 5C). In the mixed treatment, the K concentration increased to a maximum value after 2 weeks of treatment before declining to reach a value similar to NaCl-treated plants at the third week.

Fig. 3. Light response curves of the relative photosynthetic ETR in leaf 5 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. Data points and vertical bars represent means (n = 3) and SE, respectively.

Oxidative stress parameters and antioxidative metabolism Cd toxicity induced an oxidative stress in leaves from K. virginica plants, as observed by the increase in some oxidative stress-related parameters. In this way, a significant increase in lipid peroxidation and protein oxidation was observed (Table 1). In addition, an accumulation of ROS occurred, mainly O2 •− , whose levels increased near sixfold in Cd-treated plants (Table 1). In NaCl-treated plants, no important damage to membranes or proteins was detected, although an increased O2 •− content was observed (Table 1). The presence of NaCl partially reduced the oxidative stressinduced damage in Cd-treated plants that was manifested by lower increases in lipid peroxidation and protein oxidation. Surprisingly, in this case no accumulation of H2 O2 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) and 3 (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 of non-regulated energy dissipation, Y(NO) (coarse), and regulated energy dissipation and Y(NPQ) (fine) were assessed with an Imaging-PAM M-series system 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 3 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, 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), H2 O2 (nmol g –1 FW) and superoxide radical (O2 •− ; in nmol g−1 FW) concentrations in leaves of Kosteletzkya virginica seedlings exposed to 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 line denote 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

H2 O2

14.7 ± 1.2 a

42.3 ± 0.9 b

17.8 ± 2.4 a

16.3 ± 2.7 a

2.5 ± 0.3 a

13.9 ± 2.0 c

4.2 ± 0.2 b

O2 •−

3.6 ± 1.0 ab

The Cd-induced oxidative stress was paralleled by decreases in reduced GSH and α-tocopherol levels (Table 2). However, a rise in both reduced (AsA) and Physiol. Plant. 2012

oxidized (DHA) ascorbate took place, leading to a decrease in the redox state of ascorbate. In contrast, NaCl treatment increased GSH, whereas no changes in ascorbate or α-tocopherol were observed (Table 2). When Cd-treated plants were grown in the presence of 50 mM NaCl, an increase in the non-enzymatic antioxidants was recorded comparatively to plants exposed to Cd stress only (Table 2). Specifically, significant increases in 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 response to treatments exhibited similar trends after 1, 2 and 3 weeks and are therefore presented after 2 weeks of treatment for the sake of clarity (Table 3). NaCl alone had no effects on tested enzyme activities, except a slight promotion in total SOD and APX activities. In contrast,

Table 2. Concentration of endogenous antioxidant [reduced (GSH) and oxidized GSH (GSSG) in nmol g−1 FW; ascorbate (AsA) and dehydroascorbate (DHA); in nmol g−1 FW), α-tocopherol (α-toc; in nmol g−1 FW)] in leaves of Kosteletzkya virginica seedlings exposed to 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 line denote significant difference from the respective controls at the 5% level. Parameter GSH GSSG AsA

Control 55.2 ± 4.1 b 8.3 ± 0.4 ab 106.2 ± 8.1 a

5 μM Cd

50 mM NaCl

Cd+NaCl

38.4 ± 2.1 a

77.9 ± 3.8 c

89.7 ± 7.7 c

6.5 ± 1.2 a

12.4 ± 1.7 b

8.4 ± 2.3 ab

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 virginica seedlings exposed to 5 μM Cd in the absence or presence of 50 mM NaCl for 2 weeks. Values represent means ± SE (n = 6) and different letters within a line denote significant difference from the respective controls at the 5% level. Enzyme

Control

5 μM Cd

50 mM NaCl

Cd+NaCl

74.5 ± 11.4 a

96.1 ± 25.4 a

SOD

55.7 ± 1.2 a

105.3 ± 25.5 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

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

POX

340.5 ± 52.2 a

635.3 ± 96.4 b

261.7 ± 42.1 a

358.0 ± 30.8 a

5.4 ± 1.3 a 347.3 ± 7.8 a

the treatment with Cd produced changes in some of the H2 O2 -scavenging enzymes activities. Specifically, a slight decrease in APX (35%) and a dramatic drop in CAT (3.3-fold) were observed, whereas POX exhibited an 87% increase. We also observed a strong increase in DHAR and GR (2.5- and 2-fold, respectively) activities that correlated with the mentioned increase in reduced AsA. In plants subjected to both treatments (Cd+NaCl) significant decreases in APX (49%) and CAT were also observed. In this case, CAT activity showed a sixfold decrease comparatively to controls. On the other hand, increases in SOD (73%) and GR (2.4-fold) were also produced. Leaf hormonal profiling In leaf 5, initial hormonal content at the time of stress imposition was similar to that determined at week 1 for control plants, whatever the considered compound (detailed data not shown). In control plants, Z and ZR concentration steadily increased by 82 and 72% during the growth period, respectively, in relation to the normal

expansion of the considered organ (Fig. 6A, C). After 1 week of treatment, both Z and ZR concentrations were higher in response to Cd alone than in other treatments. Although Cd increased Z and Z+ZR concentration by around 45% after 2 weeks of stress, the values remained constant for Z and slightly decreased for Z+ZR during the third week. Salinity and Cd+NaCl treatment did not affect either Z or Z+ZR content during the first 2 weeks. However, during the third week, salinity induced a decline in Z concentrations while Cd induced a decrease in ZR, only. The mixed treatment induced a strong increase in Z concentrations. It was noteworthy that ZR levels in all the treatment remained low and changed 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 concentration of the ethylene precursor ACC was lower in mixed (Cd+NaCl) than in the other treatments (Fig. 7A). It slowly decreased in control leaves during leaf development while Cd induced an increase during the second week of stress before a rapid decrease occurring during the third week. NaCl alone induced a similar decrease in ACC as observed in control plants during the second week of treatment and even a higher rate of decrease during the third week. Leaf ABA concentrations were similar for all treatments after 1 week of treatment and increased thereafter. After 2 weeks, the highest leaf ABA concentration was recorded in Cd-treated plants while values recorded for the mixed treatment (Cd+NaCl) remained similar to control (Fig. 7B). An obvious increase in leaf ABA concentration was recorded during the third week of exposure to NaCl, maximal values being recorded for Cd alone and NaCl alone at the end of the treatment. After 1 week of treatment, the leaf IAA concentration was the highest in Cd-treated plants and the lowest in NaCl and in Cd+NaCl-treated plants (Fig. 7C). Leaf IAA concentration decreased in all plants during the second week and then remained constant in control, Cd or the mixed treatment. At the end of the experiment, NaCl alone induced an increase in IAA concentration while the lowest value was recorded for the mixed treatment. After 1 week of treatment, the highest SA concentrations were recorded for control plants while the lowest value was recorded for NaCl-treated plants (Fig. 7D). SA in controls then linearly decreased until the end of the treatment. In contrast, SA concentrations increased during the second week of treatment in response to Cd, NaCl or Cd+NaCl. After 3 weeks, SA concentrations were the lowest in controls and the highest in Cd+NaCltreated 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 to 5 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 a given 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 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 a given duration, different indices indicate significant difference according to Student–Newman–Keuls test at the 5% level.

Discussion Salinity reduces Cd accumulation independently of growth stimulation As a halophyte plant species, K. virginica is able to cope with high concentrations of salt in its natural environment (Ruan et al. 2008). It is therefore not surprising that 50 mM NaCl had no detrimental effect Physiol. Plant. 2012

on plant growth. It has been reported that in numerous halophyte species, low to moderate doses of NaCl may even improve the plant growth (Flowers and Colmer 2008, Ruan et al. 2010). Growth stimulation in the aerial part may lead to a dilution effect of toxic elements (Lef`evre et al. 2009b, Zaier et al. 2010): for a given rate of ion translocation from root-to-shoot, concentration

expressed on a dry weight basis may decrease as a result of growth stimulation. In K. virginica, 100 mM NaCl was shown to increase shoot growth (Ghanem et al. 2010). The purpose of this work, however, was to assess the putative impact of NaCl on the plant resistance to Cd, independently of growth stimulation. Hence, 50 mM NaCl was chosen in as much as this salinity does not improve growth in K. virginica (Han et al. 2012). In this study, 50 mM NaCl did not improve shoot growth and had no beneficial impact on the leaf expansion process. It is noteworthy that exogenous concentrations of 50 mM NaCl reduced Cd accumulation by 50% in the studied leaf of K. virginica exposed to 5 μM Cd. This data suggest that salinity may reduce Cd absorption and/or translocation from root-to-shoot in some halophyte plant species and that such an impact could lead to lower shoot Cd concentration occurring in the absence of growth stimulation. According to Lef`evre et al. (2009b), chloride ions may form CdCl+ complexes in solution which are less efficiently absorbed by the roots (thus explaining the recorded decrease in Cd accumulation) but may also improve tissular Cd tolerance in halophyte species through an increase in the synthesis of protective compounds. Our data suggest that such a protection may involve both the management of oxidative stress and the regulation of hormonal status leading to a delay in stress-induced leaf senescence. The onset of Cd-induced leaf senescence was delayed by salt This study focuses on a specific leaf initiated before stress imposition. Although physiological behavior of this precise leaf is not necessarily relevant from the whole shoot behavior, quantification of senescencerelated parameters in Cd- and in Cd+NaCl-treated plants allowed us to analyze NaCl impact on Cd-induced leaf senescence. Clearly, Cd stress decreased PSII efficiency as well as the PSII quantum yield and correlated with the ETR decrease [photochemical quenching (qP) also decreases according to data not shown]. This indicates lower photosynthetic rates, and probably a leakage of electron to oxygen, producing O2 •− and H2 O2 which may have been responsible for oxidative stress as suggested by MDA and carbonyl accumulation. The decreases in these parameters (Fv /Fm and Y(II)) could have been caused by a loss of qP or by an increase in the NPQ. A decrease in qP suggests an increased production of ROS, such as 1 O2 . NPQ increase indicated an enhancement of mechanisms of energy dissipation, as was recorded here in response to Cd stress alone. The maintenance of NPQ values under stress situations has been associated with a capacity to dissipate light energy

safely, and it can be seen as a protective response in order to avoid photoinhibitory damage to the reaction center (Rahoutei et al. 2000). In this experiment, NaCl contributed to avoid NPQ increase in the Cd-treated plants. However, after 1 week of treatment, we recorded an obvious decrease in NPQ. This situation could reflect a diminished capacity for the safe dissipation of excess light energy, and therefore does not avoid the production of harmful species, such as 1 O2 (Fryer et al. 2002). In K. virginica, however, MDA and carbonyl content were lower in plants exposed to the mixed treatment than in those exposed to Cd alone, therefore suggesting that recorded decrease in NPQ was not responsible for major oxidative stress in plants exposed to the mixed treatment. A burst occurring in the NPQ of Cd-treated plants at the end of the treatment may be considered as a consequence of senescence processes hastened through oversynthesis of the senescing compounds ACC (acting as a precursor of ethylene) and ABA, while such increases did not occur to similar extent in plants exposed to the mixed treatment Cd+NaCl. Salt ameliorates Cd-induced oxidative damage by modulating oxidants and antioxidative enzyme metabolism Cd-treatment induced an oxidative stress as observed by the increase in oxidative stress parameters, affecting essential macromolecules such as lipids and proteins, and producing damage to membranes. A strong accumulation in ROS was produced, especially O2 •− . The strong increases in H2 O2 and O2 •− could be produced by different sources such as the imbalance of the electron transport chains both in chloroplasts and mitochondria (Hern´andez et al. 1995), or by the activation of some ROS-generating enzymes (NADPH oxidase or extracellular POXs) (Bolwell and Wojtaszek 1997). In BY-2 tobacco cells, Cd induced a rapid H2 O2 generation, located in the plasma membrane. Treatments with diphenyleneiodonium and imidazole, prevented the H2 O2 generation induced by Cd, suggesting the involvement of an NADPH oxidase-like enzyme, leading to the H2 O2 production through the O2 •− dismutation by SOD (Olmos et al. 2003). This suggestion agrees with the twofold increase in SOD activity that we found in the Cd-treated plants. In this fact, we must take into account that SOD activity transforms one ROS (O2 •− ) into another (H2 O2 ), contributing to the accumulation of H2 O2 in Cd-treated plants. However, the possible involvement of a NADH-POX in the H2 O2 generation cannot be ruled out (D´ıaz-Vivancos et al. 2006). In this work, a significant increase in POX activity was observed in Cd-treated plants. Physiol. Plant. 2012

As far as non-enzymatic antioxidants are concerned, Cd appeared to mainly affect GSH synthesis since no significant GSSG accumulation was recorded. Increased AsA concentration correlated with the increase in DHAR and GR activities in Cd-treated plants. These data suggest that AsA was efficiently recycled by DHAR using GSH as reducing power. It is noteworthy that NaCl increased GSH concentration in the presence or in the absence of Cd, and that, in both cases an increase in GR was observed. These results are different to those observed in NaCl-tolerant pea plants (cv. Puget) (Hern´andez et al. 2000), where a progressive decrease in GSH was produced with the NaCl concentration used, accompanied by an accumulation in GSSG, producing a drop in the redox state of GSH. It could thus be hypothesized that physiological strategies used by halophyte to cope with oxidative stress may differ from the strategies adopted by resistant glycophyte. Lef`evre et al. (2010) already reported that GR activities constitute a major component of stress tolerance in the xero-halophyte A. halimus and that it is directly involved in cell lines responses to Cd toxicity. Indeed, Cd toxicity is known to produce important disturbances in the antioxidative metabolism of plants as well as increased ROS production (Romero-Puertas et al. 2007). On the other hand, Cd induces damage to membranes, protein oxidation and changes in enzyme activity and disruption of electron transport (Chen et al. 2003). In this sense lipid peroxidation and protein oxidation could also be partially attributed to elevated activities of ROS-generating enzymes such as lipoxygenases, SODs, exocellular POXs, oxalate oxidases and amine oxidase or NADPH oxidase (Bolwell and Wojtaszek 1997). In addition, the observed decreases in APX and CAT can facilitate the H2 O2 accumulation. The Cd-induced decrease in CAT has been also reported in other plant species such as rice or Arabidopsis (Cho and Seo 2004, Kuo and Kao 2004). It is also known that salinity induced an oxidative stress at subcellular level (Hern´andez et al. 1995, 2001). In this case, the treatment of plants with 50 mM NaCl produced an increased accumulation of O2 •− 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 a simple passive consequence of NaCl-induced decrease in Cd accumulation: indeed, for both GSH, AsA and α-tocopherol content, plants exposed to Cd+NaCl did not exhibit an intermediate behavior between control and Cd-treated plants but rather showed a higher concentration than plants exposed to other treatments, thus confirming that they were able to actively adjust their metabolism to this precise environmental condition. Physiol. Plant. 2012

Hormone involvement in delaying leaf senescence in response to Cd toxicity The stress-induced changes in hormone concentrations have to be analyzed in relation to the modification in the leaf mineral content. As far as Cd is concerned, salinity reduced its accumulation after already 1 week of treatment and the difference between Cd- and Cd+NaCltreated leaves thereafter remained constant in terms of accumulated Cd. The fact that Z and ZR concentrations were higher in Cd-treated leaves than in those exposed to mixed treatment is puzzling. Indeed, those compounds are known to act as anti-senescing agents, helping to delay chlorophyll breakdown, as well as cell membrane and protein degradation (Ananieva et al. 2008, Ghanem et al. 2008, 2011, S´ykorov´a et al. 2008). Our data obtained for MDA and carbonyl concentrations suggest that senescence was not delayed in Cd-treated leaves, despite CKs accumulation. The presence of NaCl in a Cd-containing solution decreased CK concentration but also paradoxically delayed senescence in terms of MDA and carbonyl accumulation. This leads us to conclude that CKs were not, under our experimental conditions, the major determinant of Cd-induced senescence in K. virginica. Senescence is a complex process, which is only partly under hormonal control. The availability of carbon relative to nitrogen was not considered in this study while it may directly influence the tissular response to CK levels (S´ykorov´a et al. 2008). Leaf senescence may also involve different phases which could be differentially regulated. There is no evidence that the increase in Z which was observed during the first week in Cd-treated plants and during the third week in Cd+NaCl-treated ones occurred through similar modalities. Ananieva et al. (2008) suggested that modifications in cytokinin oxidase/dehydrogenase (CKX) activity may be involved in the control of endogenous CK levels and that CKX signaling could be a possible regulatory mechanism controlling senescence. It could therefore not be excluded that Cd induced an inhibition of CKX activity rather than an increase in Z synthesis. The senescing hormone ABA was produced to higher extent in Cd-treated leaves than in Cd+NaCl-exposed plants during the second and the third week of treatment. The ABA concentration was however similar for all treatments at week 1, at a time when symptoms of senescence were already recorded in Cd-treated plants. Conversely, the precursor of ethylene ACC was lower in Cd+NaCl-treated plants than in plants exposed to Cd only. In salt-treated tomato, ACC was showed to increase in leaf tissue concomitantly with the onset of oxidative damage and the decline in chlorophyll fluorescence (Ghanem et al. 2008). This, however, was probably not

the case in K. virginica since both Cd-treated plants and NaCl-treated ones exhibited similar ACC values than controls at week 1, while ACC concentration was the highest in controls at week 3. The hypothesis that this high concentration of ACC in controls could be a consequence of a lower rate of ACC conversion to ethylene still has to be tested. According to Rodr´ıguez-Serrano et al. (2006), an increase in SA concentration in Cd-treated plants should be regarded as an attempt to regulate the cellular response in order to cope with damages imposed by Cd. In this work, SA concentration progressively decreased in control plants, probably as a consequence of the normal leaf expansion occurring in leaf 5. In contrast, SA concentration increased in the presence of Cd after 2 weeks of treatment. According to Zhang and Chen (2011), SA could prevent Cd-induced photosynthetic damage and cell death through the inhibition of ROS overproduction. We however noticed that SA concentration was higher in Cd- than in Cd+NaCltreated plants after 1 week of stress and that it remained similar in the two treatments thereafter. The beneficial effect of NaCl on ROS production by Cd-treated plants could thus not be explained by a higher synthesis of SA signaling molecule. Beside their involvement in the regulation of leaf senescence, some phytohormones such as CKs, ABA and ACC also act as major components of root-to-shoot communication. Han et al. (2012) recently showed that Cd accumulated to higher concentration in the roots than in the shoots of K. virginica but that, in contrast to its impact on the shoot, NaCl had only a minor impact on the root Cd concentration. Nevertheless, it is still possible that the presence of salt has an impact on root hormone synthesis through a modulation of genes encoding key enzymes involved in ABA, CKs and ACC synthesis, or on the loading of these hormonal compounds in the xylem of Cd-treated plants. Kosteletzkya virginica is a typical wetland species well adapted to waterlogging and flooded conditions. According to Jackson (1990), these species may exhibit specific hormonal features in relation to their specific habitat. Further studies are thus required to decipher the putative impact of NaCl on Cd resistance in this plant species.

Conclusion The result clearly showed that NaCl afforded a partial protection against the Cd toxicity, as observed by the lower damage to membranes. This partial protection seems 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 the maintenance of POX, MDHAR and DHAR activities. The delay of senescence induced by NaCl on Cdtreated leaf could be, at least partly, related to a lower production of ABA and ethylene precursor ACC while CKs accumulated in plants exposed to Cd in the absence of NaCl but appeared unable to efficiently reduce senescence in those organs. Acknowledgements – We thank Dr Alejandro Torrecillas (CEBAS, Murcia) for valuable help in analyzing hormonal contents and Dr Hong Wang (CEBAS, Murcia) for stimulating discussion of many aspects covered. This work was supported by the Belgian Science Policy (BELSPO): Chinese-Belgian Cooperation Project (2004411505) and by the Fonds de la Recherche Scientifique of Belgium (FNRS): ‘Cr´edit Bref S´ejour a` l’´etranger’ [2010/V 3/5/215 – IB/JN – 10190]. R-M. H. is grateful to the FNRS for the award of a research fellowship, and especially to the Universit´e catholique de Louvain (UCL) for his PhD grant. This paper is dedicated to the memory of Prof. Luc Waterkeyn.

References Ananieva K, Ananiev ED, Doncheva S, Georgieva K, Tzvetkova N, Kam´ınek M, Motyka V, Dobrev P, Gajdoˇsov´a S, Malbeck J (2008) Senescence progression in a single darkened cotyledon depends on the light status of the other cotyledon in Cucurbita pepo (zucchini) seedlings: potential involvement of cytokinins and cytokinin oxidase/dehydrogenase activity. Physiol Plant 134: 609–623 Asada K (2006) Production and scavenging of reactive oxygen species in chloroplasts and their functions. Plant Physiol 141: 391–396 Bolwell GP, Wojtaszek P (1997) Mechanisms for the generation 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. syringae strains differentially affects photosynthesis and sink metabolism in Arabidopsis leaves. Planta 225: 1–12 Chaoui A, El Ferjani E (2005) Effects of cadmium and copper on antioxidant capacities, lignifications and auxin 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 and effects 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 to cadmium. Chemosphere 50: 789–793

Physiol. Plant. 2012

Cho UH, Seo NH (2004) Oxidative stress in Arabidopsis thaliana exposed to Cd is due to hydrogen peroxide accumulation. Plant Sci 168: 113–120 Clemente-Moreno MJ, D´ıaz-Vivancos P, Barba-Esp´ın G, Hern´andez JA (2010) Benzothiadiazole and L-2-oxothiazolidine-4-carboxylic acid reduced the severity of Sharka symptoms in pea leaves: effect on the antioxidative metabolism at subcellular level. Plant Biol 12: 88–97 D´ıaz-Vivancos P, Rubio M, Mesonero V, Periago PM, Ros ´ Barcelo´ A, Mart´ınez-Gomez P, Hern´andez JA (2006) The apoplastic antioxidant system in Prunus: response to plum pox virus. J Exp Bot 57: 3813–3824 Dobrev PI, Kam´ınek M (2002) Fast and efficient separation of cytokinins from auxin and abscisic acid and their purification using mixed-mode solid-phase extraction. J Chromatogr A950: 21–29 Doyle MO, Otte ML (1997) Organism-induced accumulation of iron, zinc and arsenic in wetland soils. Environ Pollut 96: 1–11 Ellouzi H, Ben-Hamed KB, Cela J, Munn´e-Bosch S, Abdelly C (2011) Early effects of salt stress on the physiological and antioxidative status of Cakile maritima (halophyte) and Arabidopsis thaliana (glycophyte). Physiol Plant 142: 128–143 Elstner EF, Heupel A (1976) Inhibition of nitrate formation from hydroxyl ammonium chloride: a simple assay for superoxide dismutase. Anal Biochem 70: 616–620 Flowers TJ, Colmer TD (2008) Salinity tolerance in halophytes. New Phytol 179: 945–963 Foyer CH, Noctor G (2005) Oxidant and antioxidant signalling in plants: a re-evaluation of the concept of oxidative stress in physiological context. Plant Cell Environ 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, Benav´ıdes MP, Tomaro ML (1996) Effect of heavy metal ion excess on sunflower leaves: evidence for involvement of oxidative stress. Plant Sci 121: 151–159 Ghanem ME, Albacete A, Mart´ınez-Andjar C, Acosta M, Romero-Aranda R, Dodd IC, Lutts S, P´erez-Alfocea F (2008) Hormonal changes during salinity induced leaf senescence in tomato (Solanum lycopersicum L.). J Exp Bot 59: 3039–3050 Ghanem ME, Han RM, Classen B, Quetin-Leclercq J, Mahy G, Ruan CJ, Qin P, P´erez-Alfocea F, Lutts S (2010) Mucilage and polysaccharides in the halophyte plant species Kosteletzkya virginica: localization and composition in relation to salt stress. J Plant Physiol 167: 382–392 Ghanem ME, Albacete A, Smigocki AC, Fr´ebort I, ´ Posp´ısˇilov´a H, Mart´ınez-Andujar C, Acosta M, S´anchez-Bravo J, Lutts S, Dodd IC, P´erez-Alfocea F

Physiol. Plant. 2012

(2011) Root-synthesized cytokinins improve shoot growth and fruit yield in salinized tomato (Solanum lycopersicum L.) plants. J Exp Bot 62: 125–140 Grat˜ao PL, Monteiro CC, Rossi ML, Martinelli AP, Peres LEP, Medici LO, Lea PJ, Azevedo RA (2009) Differential ultrastructural changes in tomato hormonal mutants exposed to cadmium. Environ Exp Bot 67: 387–394 Griffith OW (1980) Determination of glutathione disulphide using glutathione reductase and 2-vinylpyridine. Anal Biochem 106: 207–212 Guo B, Liang YC, Zhu YG (2009) Does salicylic acid regulate antioxidant defense system, cell death, cadmium uptake and partitioning to acquire cadmium tolerance in rice? J Plant Physiol 166: 20–31 Han RM, Lef`evre I, Ruan CJ, Qin P and Lutts S (2012) NaCl differently interferes with Cd and Zn toxicities in the wetland halophyte species Kosteletzkya virginica (L.) Presl. Plant Growth Regul, in press doi: 10.1007/s10725-012-9697-z Heath RL, Packer L (1968) Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125: 189–198 Hern´andez JA, Olmos E, Corpas FJ, Sevilla F, del R´ıo LA (1995) Salt-induced oxidative stress in chloroplast of pea plants. Plant Sci 105: 151–167 Hern´andez JA, Jim´enez A, Mullineaux P, Sevilla F (2000) Tolerance of pea (Pisum sativum L.) to long-term salt stress is associated with induction of antioxidant defences. Plant Cell Environ 23: 853–862 Hern´andez JA, Ferrer MA, Jim´enez A, Ros-Barcelo´ A, Sevilla F (2001) Antioxidant systems and O2 .– /H2 O2 production in the apoplast of Pisum sativum L. leaves: its relation with NaCl-induced necrotic lesions in minor veins. Plant Physiol 127: 817–831 Hsu YT, Kao CH (2003) Role of abscisic acid in cadmium tolerance of rice (Oryza sativa L.) seedlings. Plant Cell Environ 56: 867–874 Jackson MB (1990) Hormones and developmental changes in plants subjected to submergence or soil waterlogging. Aquat Bot 38: 49–72 Jim´enez A, Hern´andez JA, Pastori G, del R´ıo LA, Sevilla F (1998) Role of the ascorbate-glutathione cycle of mitochondria and peroxisomes in the senescence of pea leaves. Plant Physiol 118: 1327–1335 Kuo MC, Kao CH (2004) Antioxidant enzyme activities are upregulated in response to cadmium in sensitive, but not in tolerant rice (Oryza sativa L.) seedlings. Bot Bull Acad Sin 45: 291–299 Lef`evre I, Correal E, Faz-Cano A, Zanuzzi A, Lutts S (2009a) Structural development, water status, pigment concentrations, and oxidative stress of Zygophyllum fabago seedlings in relation to cadmium distribution in the shoot organs. Int J Plant Sci 170: 226–236

Lef`evre I, Marchal G, Meerts P, Corr´eal E, Lutts S (2009b) Chloride salinity reduces cadmium accumulation by the Mediterranean halophyte species Atriplex halimus L. Environ Exp Bot 64: 145–152 Lef`evre I, Marchal G, Ghanem ME, Correal E, Lutts S (2010) Cadmium has contrasting effects on polyethylene glycol-sensitive and resistant cell lines in the Mediterranean halophyte species Atriplex halimus L. J Plant Physiol 167: 365–374 Liu KL, Shen L, Sheng JP (2008) Improvement in cadmium tolerance of tomato seedlings with an antisense DNA for 1-aminocyclopropane-1-carboxylate synthase. J Plant Nutr 31: 809–827 ´ Lopez-Chuken UJ, Young SD (2005) Plant screening of halophyte species for cadmium phytoremediation. Z Naturforsch 60: 236–243 Lutts S, Lef`evre I, Delp´er´ee C, Kivits S, Dechamps C, Robledo A, Correal E (2004) Heavy metal accumulation by 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 barley seedlings. Plant Physiol 132: 272–281 Mittler R, Vanderauwera S, Suzuki N, Miller G, Tognetti VB, Vandepoele K, Gollery M, Shulaev V, Van Breusegem F (2010) ROS signalling: the new wave? Trends Plant Sci 16: 300–309 Monteiro CC, Carvalho RF, Grat˜ao PL, Carvalho G, Tezotto T, Medici LO, Peres LEP, Azevedo RA (2011) Biochemical responses of the ethylene-insensitive Never ripe tomato mutant subjected to cadmium and sodium stresses. Environ Exp Bot 71: 306–320 ¨ ¨ Munn´e-Bosch S, Weiler EW, Alegre L, Muller M, Duchting P, Falk J (2007) α-Tocopherol may influence cellular signalling by modulating jasmonic acid levels in plants. Planta 225: 681–691 Olmos E, Mart´ınez-Solano JR, Piqueras A, Hell´ın E (2003) Early steps in the oxidative burst induced by cadmium in cultured 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 permeates through calcium channels and disturbs the plant water status. Plant J 32: 539–548 Pietrini F, Iannelli MA, Pasqualini S, Massacci A (2003) Interaction of cadmium with glutathione and photosynthesis in developing leaves and chloroplasts of Phragmites australis (Cav.) Trin. ex Steudel. Plant Physiol 133: 829–837 Piqueras A, Olmos E, Mart´ınez-Solano JR, Hell´ın E (1999) Cd-induced oxidative burst in tobacco BY2 cells: time course, subcellular location and antioxidant response. Free Radic Res 31: S33–S38

´ M (2000) Inhibition of Rahoutei J, Garc´ıa-Luque I, Baron photosynthesis by viral infection: effect on PSII structure and function. Physiol Plant 110: 286–292 Reznick AZ, Packer L (1994) Oxidative damage to proteins: spectrophotometric method for carbonyl assay. Method Enzymol 233: 357–363 Rodr´ıguez-Serrano M, Romero-Puertas MC, Zabalza A, ´ Corpas FJ, Gomez M, Del R´ıo LA, Sandalio LM (2006) Cadmium effect on oxidative metabolism of pea (Pisum sativum L.) roots. Imaging of reactive oxygen species and nitric oxide accumulation in vivo. Plant Cell Environ 29: 1532–1544 Romero-Puertas MC, Corpas FJ, Rodr´ıguez-Serrano M, ´ Gomez M, del R´ıo LA, Sandalio LM (2007) Differential expression and regulation of antioxidative enzymes by 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, an agroecoengineering halophytic species for alternative agricultural production in China’s east coast: ecological adaptation and benefits, seed yield, oil content, 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 Sanit`a di Toppi L, Gabbrielli R (1999) Responses to cadmium in higher plants. Environ Exp Bot 41: 105–130 Shah K, Kumar RG, Verma S, Dubey RS (2001) Effect of cadmium on lipid peroxidation, superoxide anion generation and activities of antioxidant enzymes in growing rice seedlings. Plant Sci 161: 1135–1144 ´ ´ Stroinski A, Chadzinikolau T, Gi˙zewska K, Zielezinska M (2010) ABA or cadmium induced phytochelatin synthesis in potato tubers. Biol Plant 54: 117–120 S´ykorov´a B, Kureˇsova G, Daskalova S, Trˇckov´a M, Hoyerov´a K, Raimanov´a I, Motyka V, Tr´avn´ıcˇ kova A, Elliott MC, Kam´ınek M (2008) Senescence-induced ectopic expression of the A. tumefaciens ipt gene in wheat delays leaf senescence, increases cytokinin content, nitrate flux, and nitrate reductase activity, 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 during thidiazuron-induced bud break of apple. Physiol Plant 82: 231–236 Yakimova ET, Kapchina-Toteva VM, Laarhoven LJ, Harren FM, Woltering EJ (2006) Involvement of ethylene and lipid signalling in cadmium-induced programmed cell death in tomato suspension cells. Plant Physiol Biochem 44: 581–589

Physiol. Plant. 2012

Zaier H, Ghnaya T, Lakhdar A, Baioui R, Ghabriche R, Mnasri M, Lutts S, Abdelly C (2010) Comparative study of Pb-phytoextraction potential in Sesuvium portulacastrum and Brassica juncea: tolerance and accumulation. J Hazard Mater 183: 609–615 Zhang WN, Chen WL (2011) Role of salicylic acid in alleviating photochemical damage and autophagic cell

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Physiol. Plant. 2012

death induction of cadmium stress in Arabidopsis thaliana. Photochem Photobiol Sci 10: 947–955 Zhou B, Wang J, Guo Z, Tan H, Zhu X (2006) A simple colorimetric method for determination of hydrogen peroxide in plant tissues. Plant Growth Regul 49: 113–118