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Physiologia Plantarum 139: 256–268. 2010 Copyright © Physiologia Plantarum 2010, ISSN 0031-9317

Lipoic acid and redox status in barley plants subjectedto salinity and elevated CO2

Usue Perez-Lopeza,∗, Anabel Robredoa, Maite Lacuestab, Cristina Sgherric, Amaia Mena-Petitea,Flavia Navari-Izzoc and Alberto Munoz-Ruedaa

aDepartamento de Biologıa Vegetal y Ecologıa, Facultad de Ciencia y Tecnologıa, Universidad del Paıs Vasco/EHU, Apdo. 644, E-48080 Bilbao, SpainbDepartamento de Biologıa Vegetal y Ecologıa, Facultad de Farmacia, Universidad del Paıs Vasco/EHU, P◦ de la Universidad 7, 01006 Vitoria-Gasteiz,SpaincDipartimento di Chimica e Biotecnologie Agrarie, Universita di Pisa, via del Borghetto 80, 56124 Pisa, Italia

Correspondence*Corresponding author,e-mail: [email protected]

Received 20 November 2009;revised 27 January 2010

doi:10.1111/j.1399-3054.2010.01361.x

Future environmental conditions will include elevated concentrations of saltin the soil and an elevated concentration of CO2 in the atmosphere. Becausethese environmental changes will likely affect reactive oxygen species (ROS)formation and cellular antioxidant metabolism in opposite ways, we analyzedchanges in cellular H2O2 and non-enzymatic antioxidant metabolite [lipoicacid (LA), ascorbate (ASA), glutathione (GSH)] content induced by salt stress(0, 80, 160 or 240 mM NaCl) under ambient (350 μmol mol−1) or elevated(700 μmol mol−1) CO2 concentrations in two barley cultivars (Hordeumvulgare L.) that differ in sensitivity to salinity (cv. Alpha is more sensitivethan cv. Iranis). Under non-salinized conditions, elevated CO2 increased LAcontent, while ASA and GSH content decreased. Under salinized conditionsand ambient CO2, ASA increased, while GSH and LA decreased. At 240 mMNaCl, H2O2 increased in Alpha and decreased in Iranis. When salt stresswas imposed at elevated CO2, less oxidative stress and lower increases inASA were detected, while LA was constitutively higher. The decrease inoxidative stress could have been because of less ROS formation or to a higherconstitutive LA level, which might have improved regulation of ASA and GSHreductions. Iranis had a greater capacity to synthesize ASA de novo and hadhigher constitutive LA content than did Alpha. Therefore, we conclude thatelevated CO2 protects barley cultivars against oxidative damage. However,the magnitude of the positive effect is cultivar specific.

Introduction

Soil salinity is one of the major environmental constraintslimiting plant cultivation. Moreover, salinity-affectedareas are rapidly increasing because of (1) faulty irriga-tion systems and poor quality water, and (2) temperature

Abbreviations – APX, ascorbate peroxidase; ASA, ascorbate; DHA, oxidized ascorbate; DHAR, dehydroascorbate reductase;DHLA, dihydrolipoic lipoic acid; DTNB, 5,5′-ditiobis (2-nitrobenzoic) acid; DTT, dithiotreitol; EDTA, ethylenediaminetetraaceticacid; GR, glutathione reductase; GSH, glutathione; GSSG, oxidized glutathione; HEPES, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid; HPLC, high performance liquid chromatography; LA, lipoic acid; MDHAR, monodehydroascorbatereductase; NADPH, nicotinamide adenine dinucleotide phosphate (reduced); O·−

2 , superoxide anion; OH·, hydroxyl radical;PMSF, phenylmethylsulfonyl fluoride; ROS, reactive oxygen species; SE, standard error.

increases associated with elevated plant evapotranspira-tion rates (FAO 2000).

High salinity causes both hyperionic and hyperos-motic effects in plants, leading to membrane disorgani-zation, increases in reactive oxygen species (ROS) andincreased metabolic toxicity (Perez-Lopez et al. 2009a,

256 Physiol. Plant. 139, 2010

b). Salinity reduces the supply of CO2 to leaves andfurther depresses the already low CO2/O2 ratio in chloro-plasts. The resulting accumulation of photoreducingpower causes an excess of electrochemical energy in themembrane. If this extra energy is not dissipated, it gen-erates ROS such as superoxide anion (O·−

2 ), H2O2 andthe hydroxyl radical (OH·), ultimately provoking oxida-tive stress (Dionisio-Sese and Tobita 1998, Meneguzzoet al. 1999, Sgherri et al. 1996). In addition, photores-piration, another consequence of the low CO2/O2 ratioin chloroplasts, tends to increase cellular levels of H2O2

(Hernandez et al. 2000). Although H2O2 is a componentof the intra- and intercellular signaling systems in plants(Karpinski et al. 1999, Morita et al. 1990), its concen-tration must be tightly controlled to maintain cellularhomeostasis and prevent oxidative damage.

Plants possess both enzymatic and non-enzymaticantioxidant systems that help to prevent oxidative dam-age caused by a low CO2/O2 ratio in chloroplasts andmaintain cellular homeostasis (Noctor and Foyer 1998).The non-enzymatic antioxidant system is composed oflow molecular weight antioxidant molecules such asascorbate (ASA), glutathione (GSH), α-tocopherol andLA. ASA and GSH, two water-soluble antioxidants,can directly eliminate ROS and regenerate the ROS-detoxifying enzymatic cooperative systems. Elevatedcontents of ASA and GSH and their de novo synthesis areregarded as positive responses to counteract salt stress(Meneguzzo et al. 1999, Parida and Das 2005). Usually,antioxidant substances possess antioxidant propertieswhen they are in their chemically reduced forms.LA is unique among antioxidant molecules in that itretains protective functions in both its reduced dihy-drolipoic acid (DHLA) and oxidized lipoic acid (LA)forms, although dihydrolipoic acid (DHLA) is a moreeffective antioxidant (Navari-Izzo et al. 2002). In fact,DHLA acts directly by destroying ROS such as O·−

2 andhydroperoxyl, and OH·s. Moreover, DHLA can donatean electron to the oxidized forms of GSH and ASA (Fig. 1,Biewenga et al. 1997). Because of its solubility in bothwater and lipids, DHLA connects the activity of antiox-idants in the cell membrane (α-tocopherol) to that ofantioxidants in the cytoplasm (ASA and GSH), strength-ening the antioxidant network (Navari-Izzo et al. 2002).LA also plays a pivotal role in energy metabolism. It isa component of different multienzyme complexes, suchas pyruvate dehydrogenase and glycine decarboxylasecomplexes (Gueguen et al. 2000). However, it has notbeen studied in depth because it is difficult to detect.LA has been detected in roots and leaves of wheat,potato, asparagus (Navari-Izzo et al. 2002 and refer-ences therein) and tomato (D’Amico et al. 2003, Sgherriet al. 2007, 2008). Although there are studies evaluating

the role of LA in the recycling of other antioxidants inplants exposed to stress (Sgherri et al. 2002, D’Amicoet al. 2004), no studies have evaluated the response ofLA to the interaction between salt stress and elevatedconcentrations of atmospheric CO2.

Under elevated CO2, there is a steeper CO2 con-centration gradient between the outside and inside ofthe leaf such that plants allow greater amounts of CO2

to diffuse into the leaf (Robredo et al. 2007, Sgherriet al. 2000). The increased CO2/O2 ratio at the sitesof photoreduction could increase nicotinamide adeninedinucleotide phosphate (NADPH) utilization and reducethe rate of oxygen activation and ROS formation (Halli-well and Gutteridge 1989). Consequently, there might bea less need for antioxidant upregulation (Schwanz et al.1996) under salt stress. In fact, in barley subjected tosalt stress in a CO2-enriched atmosphere, the inductionof antioxidative enzymes, ion leakage and thiobarbi-turic acid reactive substances levels were all decreased,ascribed to lower ROS formation (Perez-Lopez et al.2009b). Other studies have demonstrated the oppositeresponse as the better protection to drought and saltstress under elevated CO2 was because of the ability ofthe plants to increase the antioxidant response (Geissleret al. 2009, Schwanz and Polle 2001).

The objective of this study was to determine whetherelevated CO2 favors non-enzymatic antioxidant mecha-nisms in barley cultivars under salt stress. In particular,changes in the reduced and oxidized forms of ASA, GSHand LA, the activities of ascorbate peroxidase (APX) and

Fig. 1. Schematic illustration of the enzymatic and non-enzymaticantioxidant systems for regenerating antioxidant metabolites. APX,ascorbate peroxidase; ASA, reduced ascorbate; DHA, dehydroascorbate;DHAR, dehydroascorbate reductase; DHLA, dehydrolipoic acid, reduced;GR, glutathione reductase; GSH, reduced glutathione; GSSG, oxidizedglutathione; LA, oxidized lipoic acid; MDA, monodehydroascorbate;MDHAR; monodehydroascorbate reductase.

Physiol. Plant. 139, 2010 257

glutathione reductase (GR), and the H2O2 content wereanalyzed in two barley cultivars (Alpha and Iranis, withcv. Iranis more salt-tolerant) subjected to 0, 80, 160or 240 mM NaCl under ambient (350 μmol mol−1) orelevated (700 μmol mol−1) CO2.

Materials and methods

Plant material and experimental design

Two barley cultivars (Hordeum vulgare L.), Alpha andIranis, were used in this study. Preliminary experimentsshowed that Iranis could better tolerate elevated salinityby increasing its photosynthetic rate (Perez-Lopez et al.2008) and decreasing lipid peroxidation (Perez-Lopezet al. 2009b). Six seeds per pot, equivalent to 11.2 g m−2,were germinated in 2.5-l pots containing a mixtureof perlite: vermiculite (3:1, v/v). Plants were grown inConviron E15-controlled environment growth chambers(Conviron, Winnipeg, Manitoba, Canada) under adaily regimen of 14 h of light, an average day/nighttemperature of 24/20◦C, and a relative day/nighthumidity of 70/80%. During the entire light period, thephotosynthetic photon flux density in the chamber was400 μmol m−2 s−1. Light was provided by a combinationof incandescent bulbs and warm-white fluorescent lamps(Sylvania F48T12SHO/VHO, Sylvania, Danvers, MA).Chambers were maintained 24 h day−1 at either ambient(350 μmol mol−1) or elevated (700 μmol mol−1) CO2

concentrations. The pots were watered with Hoagland’ssolution (Arnon and Hoagland 1940) every 2 daysuntil the first leaf was completely expanded (14 days).The nutrient solution contained 6 mM KNO3, 4 mMCa(NO3)2, 1 mM NH4H2PO4, 2 mM MgSO4, 9 μMMnCl2, 46 μM H3BO3, 0.8 μM ZnSO4, 0.3 μM CuSO4,0.1 μM Na2MoO4 and 0.01 g l−1 Fe chelate (LibFerSP, Allied Colloids) with the pH adjusted to 5.5. Thenutrient solution was made up from deionized water.Later, seedlings were watered every 2 days with 250 mlof Hoagland’s solution supplemented with increasingconcentrations of NaCl [0 mM (2.0 dS m−1), 80 mM(9.7 dS m−1), 160 mM (17.6 dS m−1), or 240 mM(24.4 dS m−1)] for 14 days (Perez-Lopez et al. 2009a). Atthe end of the experimental period (28 days), the primaryleaf was harvested and used for all measurements.

H2O2 determination

Previously, frozen fresh leaf samples (0.15 g) werehomogenized in 0.5 ml of 1% trichloroacetic acid usinga cold mortar and pestle and centrifuged at 16 100 g for10 min at 4◦C (Mandhania et al. 2006). H2O2 contentwas measured as in Sinha (1972). Briefly, 200 μl of

supernatant were added to 2 ml of dichromate/aceticacid (5% solution of K2Cr2O7 with glacial acetic acid(1/3; v/v). The mixture was agitated vigorously andheated for 10 min in a boiling water bath. After coolingat room temperature, the optical density was measuredat 570 nm. The calibration curve was created using0–40 mM H2O2.

LA content

Reduced (DHLA) and oxidized (LA) extraction

DHLA and LA were extracted from barley leaves byacidic hydrolysis as previously reported by Sgherriet al. (2002). After hydrolysis, samples were extractedwith chloroform and the resultant organic fraction wasevaporated to dryness under vacuum and stored undernitrogen at 4◦C.

DHLA and LA determination

DHLA and LA were determined by high performanceliquid chromatography (HPLC) using an electrochemicaldetector (model 791, Metrohm, Herisan, Switzerland)equipped with a glassy carbon electrode and Millenium(Waters) software for peak integration. Chromatographicseparations were performed with a Nova-Pak C18 4-μmcolumn (3.9 × 150 mm; Waters, Milford, MA) at 25◦Cand 1.1 V, according to Teichert and Preiss (1997). Themobile phase consisted of 227.5 g acetonitrile, 31.5 g2-propanol and 674.5 g 0.05 M potassium dihydrogenphosphate adjusted to pH 2.5 with phosphoric acid. Theflow rate was 1 ml min−1. The calibration curve wasprepared using mixtures of standards of LA and DHLA(Sigma, Steinheim, Germany) in the range of 4–100 ng.

ASA and GSH content

Reduced ASA, oxidized ascorbate (DHA), reducedGSH and oxidized glutathione (GSSG) extraction

Previously frozen fresh leaf samples (0.15 g) werehomogenized in 2.25 ml of 1% HCl and 1 mM ethylene-diaminetetraacetic acid (EDTA) using a cold mortar andpestle and centrifuged at 16 100 g for 10 min. Metabo-lites were measured in a neutralized supernatant asdescribed below.

ASA and DHA determination

ASA was measured as described by Foyer et al. (1995)with some modifications. The supernatant (200 μl) wasneutralized with 200 μl of succinate buffer 1 M, 0.5 MKOH and 600 μl of water. ASA was measured by the


decrease in absorbance at 265 nm after adding 1 U ml−1

of ASA oxidase to a neutralized final volume of 1000 μl.DHA was determined by the increase in absorbance at265 nm after adding 30 μl dithiotreitol (DTT; 100 mM)to a neutralized final volume of 1000 μl.

GSH and GSSG determination

Total (GSH + GSSG) and GSSG were determined in thesupernatant of the centrifuged extract by the 5,5′-ditiobis(2-nitrobenzoic) acid-glutathione reductase (DTNB-GR)recycling procedure (Griffith 1980). The supernatant(200 μl) was neutralized with 350 μl of succinate buffer1 M, 0.5 M KOH and 350 μl of 250 mM Tris buffer (pH 7)containing EDTA (6.3 mM) and NADPH (0.9 mM). Next,100 μl of 6 mM DTNB and 0.1 U of GR were added.Changes in absorbance at 412 nm were measuredat 25◦C. Total GSH content was calculated from astandard curve in which GSH was plotted against therate of change in absorbance at 412 nm after addingGR. GSSG was determined after GSH derivatization by2-vinylpyridine. GSH was determined by subtractingGSSG from the total GSH content.

ASA peroxidase and GSH reductase activity

The results on ASA peroxidase and GSH reductasewere obtained from a repetition of a previous experi-ment (Perez-Lopez et al. 2009b) being the method asdescribed below. All operations were carried out at0–4◦C. Aliquots of leaf tissue (0.15 g fresh weight) wereground in a cold mortar using a specific buffer (3 ml)for each enzyme extraction. The homogenates weresqueezed through two layers of muslin, centrifuged at16 100 g for 25 min, and assayed as described below.Conditions for all assays were chosen so that the rate ofreaction was constant for the entire experimental periodand proportional to the amount of enzyme added.

GSH reductase

GR (EC 1.6.4.2) was extracted with 50 mM Tris-HCl(pH 7.8), 0.1 mM EDTA, 0.2% Triton X-100, 1 mMphenylmethylsulfonyl fluoride (PMSF) and 2 mM DTT.The supernatant was gel-filtered over Shepadex G-25columns (NAPTM-10, Amersham Biosciences, Uppsala,Sweden) that had been equilibrated with 50 mM Tris-HCl (pH 7.8), 0.1 mM EDTA and 0.2% (v/v) Triton X-100.GR activity was measured according to Edwards et al.(1990). The GSSG-dependent oxidation of NADPH wasmonitored by the decrease in absorbance at 340 nm at25◦C. Briefly, the assay mixture (final volume, 300 μl)

contained 100 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES; pH 7.8), 1 mM EDTA, 3 mMMgCl2 and 0.5 mM GSSG. The reaction was initiatedby the addition of NADPH (0.2 mM) and supernatant(30 μl). Corrections were made for the non-enzymaticreduction of GSSG by NADPH.

ASA peroxidase

The extraction of APX (EC 1.11.1.11) was performed in50 mM (pH 7.8) potassium phosphate buffer containing2% PVPP, 0.1 mM EDTA and 5 mM cysteine. ASA(2 mM) was added to the medium to avoid inactivationof APX during extraction and assay. APX activitywas assayed by measuring the oxidation of ASA at290 nm according to Hossain and Asada (1984). Briefly,the reaction mixture (300 μl final volume) contained0.25 mM ASA, 50 mM HEPES (pH 6.6) and 10 μl ofsupernatant. The oxidation rate of ASA, measured asthe decline in absorbance at 290 nm, was estimatedbetween 1 and 60 s after starting the reaction with theaddition of 0.1 mM H2O2 (5 μl). Corrections were madefor the non-enzymatic oxidation of ASA by H2O2 andfor the oxidation of ASA in the absence of H2O2.

Statistical analysis

Results are reported as the mean ± standard error(SE) of three independent experiments, measuring threedifferent plants in each experiment (n = 9). The SE

was calculated directly from crude data. Statisticalanalyses were carried out using the SPSS 16.0 (Chicago,IL) software package. Data were subjected to two-way analysis of variance (ANOVA) to determine themain effects of salt and CO2 concentrations on alldependent variables. Means were compared usingDuncan’s multiple range test. P-values ≤0.05 wereconsidered statistically significant.

Results

H2O2 content

Under non-salinized conditions (controls), elevated CO2

did not cause a significant change in H2O2 contentsin Alpha (Fig. 2A), whereas a significant increase wasobserved in Iranis (Fig. 2B). Overall, at ambient CO2,plants subjected to 240 mM NaCl showed a 28%increase in H2O2 in Alpha and a 9% decrease inIranis as compared with controls, being H2O2 contentsalways statistically lower in Iranis (Fig. 2B) than in Alpha(Fig. 2A). On the other hand, under elevated CO2, thesalt stress did not modify the H2O2 content.


Fig. 2. Effects of salinity (0, 80, 160 and 240 mM NaCl) and CO2 (350 μmol mol−1, white bars and 700 μmol mol−1, black bars) treatment on H2O2

content in leaves (A, Alpha; B, Iranis). Each value is the mean ± SE of at least three independent experiments, with three replicates per experiment.Lowercase and uppercase letters refer to the significance of differences between NaCl treatments at ambient or elevated CO2, respectively. Foreach cultivar within a salt concentration, the same letter indicates no significant differences between CO2 treatments (P > 0.05). Within a CO2

concentration, the same letter indicates no significant differences between salt treatments. * refers to a significant difference between cultivars foreach salt treatment and for each CO2 concentration.

DHLA and LA contents

In the control plants at ambient CO2, DHLA and LAwere present in both Alpha and Iranis cultivars. BothDHLA and LA were significantly influenced by salt stress(Fig. 3). More DHLA was present in Iranis than in Alpha(P < 0.05), with control plants of Iranis having twice asmuch DHLA as Alpha [3.7 μg g−1 DW in Alpha (Fig. 3A)and 7.7 μg g−1 DW in Iranis (Fig. 3B)]. On the con-trary, control Alpha plants contained twice as much LAthan did control Iranis plants [0.6 μg g−1 DW in Alpha(Fig. 3C) and 0.3 μg g−1 DW in Iranis (Fig. 3D)]. Thus,in Alpha, 86% of total lipoic acid (DHLA + LA) was inthe reduced form (DHLA) as compared with 96% in Ira-nis. Under elevated CO2 and non-salinized conditions,DHLA increased by 43% in Alpha and by 39% in Iranis.With salt treatment and at ambient CO2, DHLA con-tent reduced by 94, 96 and 99% in Alpha (Fig. 3A) andby 83, 100 and 100% in Iranis (Fig. 3B) at 80, 160, and240 mM NaCl, respectively. However, LA showed differ-ent trends between cultivars. In Alpha, LA decreased by77, 40 and 64% at 80, 160 and 240 mM NaCl (Fig. 3C).In Iranis, LA showed three- and six-fold increases relativeto the control value when treated with 80 and 160 mMNaCl, respectively, and returned to the control value at240 mM NaCl (Fig. 3D).

When salt stress was imposed under elevated CO2,DHLA decreased suddenly (Fig. 3A and B). Regarding

LA, in Alpha, at elevated CO2, it decreased with salin-ity by 15, 32 and 40% at 80, 160 and 240 mM NaCl,respectively (Fig. 3C). In Iranis, LA increased up to fivetimes the control value at 160 mM NaCl (Fig. 3D), withvalues 55% greater under elevated CO2 than underambient CO2. A two-way interaction indicated that CO2

concentration and salt stress modified each other’s effecton the LA content. In both cultivars and under both CO2

concentrations, salt stress reduced the DHLA/LA ratio(Table 1).

ASA and DHA contents

ASA was significantly influenced by CO2 and salt,separately (Fig. 4). In the control plants and at ambientCO2, total ASA was 13.3 and 12.0 μmol g−1 DWfor Alpha (Fig. 4A) and Iranis (Fig. 4B), respectively,although the ASA/DHA ratio was two times greater inAlpha than in Iranis (Table 1). Under ambient CO2,salinity enhanced total and reduced ASA content in bothcultivars. In Alpha, total ASA increased by 26, 46 and36% at 80, 160 and 240 mM NaCl, respectively, relativeto control plants (Fig. 4A). In Iranis, the total ASA contentincreased by 63 and 76% at 160 and 240 mM NaCl,respectively (Fig. 4B). In Alpha, ASA followed the samepattern as total ASA, increasing by about 23, 42 and 30%(Fig. 4C) at 80, 160 and 240 mM NaCl, respectively.


Fig. 3. Effects of salt (0, 80, 160 and 240 mM NaCl) and CO2 (350 μmol mol−1, white bars and 700 μmol mol−1, black bars) treatments on reducedlipoic acid (DHLA; A, Alpha; B, Iranis) and oxidized lipoic acid (LA; C, Alpha; D, Iranis) content in leaves. Statistical analyses as in Fig. 2. Note thatY-axis intervals are not the same for A and B panels and C and D panels.

DHA was affected to a greater extent, as it increasedby 40, 70 and 100%. Consequently, the ASA/DHA ratiodecreased as the salinity increased, reaching 14.2 at240 mM NaCl (Table 1).On the contrary, in Iranis, ASAcontent increased more than DHA content did. ASAincreased by about 28, 64 and 77% (Fig. 4D), whereas

DHA increased by 9, 19 and 20% at 80, 160 and240 mM NaCl, respectively. Thus, the ASA/DHA ratioincreased by 12, 38 and 49% at 80, 160 and 240 mMNaCl, respectively (Table 1). Exposure to elevatedCO2 under non-salinized conditions resulted in aremarkably lower total ASA (8–12%; Fig. 4A and B) and

Table 1. Ratios of ascorbate (ASA)/dehydroascorbate (DHA), glutathione (GSH)/oxidized glutathione (GSSG) and dehydrolipoic acid (DHLA)/lipoic acid(LA) in Hordeum vulgare (cvs. Alpha and Iranis) leaves after 14 days of salt (0, 80, 160 and 240 mM NaCl) and 28 days of CO2 (350 μmol mol−1 and700 μmol mol−1) treatments. Statistical analysis is as in Fig. 2. Lowercase and uppercase letters refer to the significance of differences between NaCltreatment at ambient or elevated CO2, respectively. For each cultivar within a salt concentration, the same letter indicates no significant differencesbetween CO2 treatments (P > 0.05). Within a CO2 concentration, the same letter indicates no significant differences between salt treatments. *refersto a significant difference between cultivars for each treatment and for each CO2 concentration

0 mM 80 mM 160 mM 240 mM

350 700 350 700 350 700 350 700

ASA/DHA Alpha 21.9 ± 3.1b 15.0 ± 1.8A 19.2 ± 2.4b 13.6 ± 1.9A 18.2 ± 1.8b 13.0 ± 1.7A 14.2 ± 1.82a 14.1 ± 1.9A

Iranis 11.5 ± 1.2a∗ 9.2 ± 1.0A 12.9 ± 1.4a∗ 11.3 ± 1.0A 15.8 ± 3.1ab 13.2 ± 1.7B 17.1 ± 2.13c∗ 13.2 ± 2.4B

GSH/GSSG Alpha 25.2 ± 1.4b 21.2 ± 1.5A 25.9 ± 1.4b 20.0 ± 1.7A 24.4 ± 1.3b 21.9 ± 1.6A 26.0 ± 1.71b 21.6 ± 2.0A

Iranis 28.0 ± 3.0d 21.9 ± 1.2A 25.2 ± 3.6c 18.7 ± 1.9A 24.4 ± 3.3c 18.9 ± 0.8A 21.9 ± 2.18ab 20.9 ± 1.2A

DHLA/LA Alpha 6.9 ± 1.4b 43.7 ± 15.4C 1.3 ± 1.0a 1.8 ± 0.6A 0.4 ± 0.3a 2.5 ± 0.7A 0.2 ± 0.06a –Iranis 31.2 ± 6.0b∗ 16.5 ± 12.1B 1.5 ± 0.6a 5.3 ± 1.2A – – – 4.6 ± 0.9A


Fig. 4. Effects of salinity (0, 80, 160 and 240 mM NaCl) and CO2 (350 μmol mol−1, white bars and 700 μmol mol−1, black bars) treatments on totalascorbate content [ascorbate (ASA) + dehydroascorbate (DHA); A, Alpha; B. Iranis], ASA content (C, Alpha; D, Iranis), and ascorbate peroxidase (APX)activity (E, Alpha; F, Iranis) in leaves. Statistical analyses as in Fig. 2.

lower ASA/DHA redox ratios (20–30%; Table 1). Thecombination of elevated CO2 and salt stress provoked anincrease in ASA and DHA in both Alpha and Iranis, butlower than at ambient CO2. In fact, total ASA increasedby 22 and 30% in Alpha (Fig. 4A) and by 38 and 47% inIranis (Fig. 4B) at 160 and 240 mM NaCl, respectively.

APX activity

In the control plants of Alpha, APX activity was consider-ably greater than in Iranis (Fig. 4E and F). Elevated CO2

alone did not change total APX activity in Alpha (Fig. 4E)

or in Iranis (Fig. 4F). Moreover, under salt stress andambient CO2, APX activity remained constant in Iranis,whereas it increased (27%) in Alpha at 240 mM NaCl.When barley plants of both cultivars were subjected tosalt stress at elevated CO2, APX activity did not changesignificantly, although the values were always lower inIranis than in Alpha.

GSH and GSSG contents

Total GSH was influenced by CO2 and salt stress,separately. At ambient CO2, control plants of both


Fig. 5. Effects of salinity (0, 80, 160 and 240 mM NaCl) and CO2 (350 μmol mol−1, white bars and 700 μmol mol−1, black bars) treatments ontotal glutathione content [glutathione (GSH) + oxidized glutathione (GSSG); A, Alpha; B, Iranis], GSH content (C, Alpha; D, Iranis) and glutathionereductase (GR) activity (E, Alpha; F, Iranis) in leaves. Statistical analyses as in Fig. 2.

cultivars contained similar amounts of total GSH(Fig. 5A and B). Comparing with plants grown underambient CO2, at high CO2, total GSH contents and theGSH/GSSG ratios of both cultivars were always lower(P < 0.05; Fig. 5A and B; Table 1). On the other hand,at ambient CO2, increased salinity decreased total GSHcontent in both cultivars. In Alpha, total GSH contentsdecreased by 18% when the salinity treatment was moresevere (Fig. 5A). Similar patterns were observed for both

GSH (Fig. 5C) and GSSG, so that the GSH/GSSG ratioremained constant during the entire experimental period(Table 1). In Iranis, the decrease in total GSH intensifiedas the severity of salt stress intensified, decreasing 11,17 and 27% at 80, 160 and 240 mM NaCl, respectively(Fig. 5B). Because this decrease was mainly the result ofthe decline in GSH (Fig. 5D), the GSH/GSSG ratio alsodecreased with the increase in salinity (Table 1). In Iranis,when salt stress was imposed under elevated CO2, both


the total and the reduced GSH showed smaller declinesthan at ambient CO2 (Fig. 5D), whereas in Alpha, bothtotal and reduced GSH contents dropped by similarpercentages as they did at ambient CO2 (Fig. 5C).

GR activity

GR activity was influenced by CO2 and salt stress,separately. An interaction between CO2 and salt stresswas also detected in Iranis (Fig. 5). In control plants,the activity of GR was higher in Alpha than in Iranis(P < 0.05) under ambient CO2. Moreover, high CO2

alone caused a decrease in GR activity in Alpha (Fig. 5E)but not in Iranis (Fig. 5F). At 240 mM NaCl and ambientCO2, total GR activity increased by 7% in Alphaand 21% in Iranis (Fig. 5E and F), relative to controlplants. Exposing barley leaves to elevated salinity underelevated CO2 increased GR activity in both cultivars(Fig. 5E and F). In fact, GR activity increased by 13, 16and 16% in Alpha and by 39, 44 and 45% in Iranis at80, 160 and 240 mM NaCl, respectively.

Discussion

Responses to salt stress under ambient CO2

Previously, we showed that cv. Iranis was more salt-tolerant than cv. Alpha, with Iranis possessing higherconstitutive superoxide dismutase (SOD) and catalase(CAT) activities than Alpha (Perez-Lopez et al. 2009b).The present study confirms the existence of differences inthe antioxidant metabolism between Iranis and Alpha,which makes Iranis more tolerant to increasing NaClconcentrations. Indeed, higher constitutive amounts ofDHLA were detected in Iranis than in Alpha (Fig. 3).

LA is an antioxidant molecule that may contributeto the non-enzymatic regeneration of GSH and ASA(Fig. 1), and in this study we demonstrated the presenceof this important antioxidant both in the reduced (DHLA)and oxidized (LA) forms in barley (Fig. 3). The almostcomplete disappearance of DHLA in leaves of bothbarley cultivars under salinized conditions (Fig. 3) wasalso found in wheat by D’Amico et al. (2004) whosuggested that it could be transferred to roots, the organdirectly exposed to the saline treatment. Besides, itcould be functioning to reduce DHA to ASA in leaves,with a higher contribution in Iranis, protecting the plantagainst damaging forms of activated oxygen generated byoxidative stress. ASA is a powerful metabolic antioxidantthat functions during salt stress (Noctor and Foyer1998, Sairam et al. 2005). When ASA is used todestroy H2O2 via APX, ASA is oxidized. Dependingon the species and the severity of the treatment,

DHA is re-reduced by monodehydroascorbate reductase(MDHAR; Mittova et al. 2000, Shalata et al. 2001) anddehydroascorbate reductase (DHAR; Hernandez et al.2000, Meneguzzo et al. 1999), and/or by non-enzymaticmechanisms (Noctor and Foyer 1998). MDHAR andDHAR activities have been shown to increase under saltstress (Perez-Lopez et al. 2009b) and were greater inAlpha than in Iranis. However, under increased salinity,the ASA/DHA ratio decreased in Alpha and increased inIranis. These opposing effects could be because of thefact that in Iranis, in which the constitutive content ofDHLA is higher than in Alpha, DHA is also being reducednon-enzymatically via DHLA without the involvement ofreducing equivalents, owing to the redox potential of theLA/DHLA pair (Navari-Izzo et al. 2002). Moreover, inIranis, LA responded positively to salt stress, increasingat 80 and 160 mM NaCl (Fig. 3C) but decreasing inAlpha at all salt concentrations (Fig. 3D). This fact couldalso be beneficial for Iranis, as LA is the unique form ofLA able to react with singlet oxygen (Borbe and Ulrich1989).

On the other hand, ASA, besides being enzymaticallyand non-enzymatically regenerated, was also synthe-sized de novo as the total content of ASA plus DHAincreased in both cultivars. ASA increases have beenrelated with salt tolerance. In fact, some authors havecorrelated higher ASA content with greater salt tolerancein different cultivars of watermelon (Ashraf and Harris2004) and cotton (Yasar et al. 2006). In Iranis plantsgrown under ambient CO2, total ASA increased with theintensity of the stress (Fig. 4B). However, in Alpha, totalASA increased as salinity increased up to 160 mM NaCl,but remained constant thereafter (Fig. 4A). These resultssuggest that in Alpha plants, a threshold NaCl concentra-tion of 160 mM NaCl exists above which an imbalancebetween ROS generating and scavenging systems occurs(Fig. 2A), similar to that described for other plants (Cha-parzadeh et al. 2004, Hernandez et al. 2000). On theother hand, in Iranis, ASA increases in response to saltstress were progressive and no increases in H2O2 weredetected (Fig. 2B). These results support that the synthesisof ASA and LA together with adequate APX activity andDHLA content might exert positive influences for main-taining redox homeostasis under salt stress, especially inIranis which has higher constitutive DHLA content andthe ability to synthetise LA and ASA progressively to saltstress, which Alpha does not.

GSH is also an important antioxidant metabolite thatacts as (1) a direct antioxidant, (2) a substrate in theASA–GSH cycle and (3) a regulator of protein thiol-disulphide redox status (Noctor and Foyer 1998). Thegenerated GSSG is usually reduced by GR. The greaterGR activity in Alpha (Fig. 5E and F) could have been


responsible for better turnover of GSSG to GSH inAlpha than in Iranis, explaining the maintenance of theGSH/GSSG ratio in Alpha. However, as long as salt stressincreased, the produced GSSG could have been alsoreduced by DHLA without the involvement of reducingequivalents (Fig. 1; Haenen and Bast 1989, Navari-Izzoet al. 2002). In fact, D’Amico et al. (2004) proposed thatthe LA/DHLA pair, together with GR activity, contributedto the reduction of GSSG in wheat irrigated with 20% seawater, generating a noticeable response of these plantsto salt stress. Despite this possible regeneration, totalabsolute GSH content decreased (Fig. 5A and B). GSHreductions were also detected in cotton by Gossett et al.(1996) and in wheat by Meneguzzo et al. (1999). Theseresults are in agreement with those obtained by Noctorand Foyer (1998), who suggested that the oxidation ofGSH is accompanied by net degradation.

Responses to salt stress under elevated CO2

Under non-salinized conditions, regarding H2O2 con-tent, there seem to be clear differences between cultivarsin response to the increase in CO2. Thus, although inAlpha there was a balance between H2O2 productionand the supply of ASA for scavenging it, the ASA levelsin Iranis were not enough to destroy all the H2O2 pro-duced and, although the APX activity measured underoptimal conditions was not different from that measuredat ambient CO2 (Fig. 4B), the actual APX activity wasnot able to consume all the H2O2. Consequently, lessH2O2 was destroyed, so less LA was consumed forthe regeneration of DHA. On the other hand, underelevated CO2, both cultivars generally showed lowercontent of total GSH and reduced GSH/GSSG ratiosthan at ambient CO2, regardless of salinity. This couldbe explained by the fact that exposure to elevated CO2

represses the photorespiratory cycle (Ogren 1984) bywhich cysteine and glycine are synthesized. Similarresults were observed by Robinson and Sicher (2004)in barley, suggesting that the lower levels of GSH atelevated CO2 were the result of lower photorespirationrates. Moreover, lower constitutive contents of ASA werealso detected in both cultivars under elevated CO2, inagreement with findings by Robinson and Sicher (2004)in barley, and Schwanz and Polle (1998) and Badi-ani et al. (1998) in oak species. However, the processlimiting ASA generation at elevated CO2 must still bedetermined, since contrasting data have been reported(Sanita di Toppi et al. 2002, Sgherri et al. 1998). Inboth control cultivars, the decrease in the GSH/GSSGand ASA/DHA ratios under elevated CO2 could havebeen because of lower demands for GSH and ASA asincreasing CO2 enhances utilization of photoreducing

power, reducing the risk of oxyradical formation (Halli-well and Gutteridge 1989). Thus, the reductions in totalASA and GSH responsible for the changes in GSH/GSSGand ASA/DHA could be adaptive responses to divertmetabolic energy for the plant growth rather than forplant protection. In contrast to the behavior of ASA andGSH under non-salinized conditions and elevated CO2,the content of LA was greater than that under ambientCO2 (Fig. 3). This could be considered an advantagethat would allow barley plants to cope with oxidativedamage when ASA and GSH decreased. Under elevatedCO2, greater metabolic activity in response to higherrates of photosynthesis (Perez-Lopez et al. 2008) couldallow greater LA synthesis, likely explaining its increase.

Under elevated CO2 in both cultivars, not only werethe constitutive levels of ASA lower, but also were lowerthe relative increases in ASA because of salt stresscomparing to ambient CO2 conditions. Because ASAis a metabolite responsible for the elimination of someROS generated by electron leakage from chloroplasts,the increase in the pCO2/pO2 ratio at the sites ofphotoreduction under high CO2 conditions might havereduced the basal rate of oxygen activation (Sgherri et al.2000) and, consequently, the need of ASA synthesis.This is in agreement with a previous report in whichlower activity of antioxidant enzymes was explainedby decreased generation of ROS (Perez-Lopez et al.2009b, Schwanz et al. 1996). Besides, ASA/DHA ratiowas constant overall the salt treatment under elevatedCO2, confirming the maintenance of the redox status.On the other hand, in Iranis the decreases in GSH withsalinity were smaller than at ambient CO2. Moreover,the facts that under elevated CO2 both cultivars hadhigher constitutive levels of DHLA and, in particular,Iranis could increase the level of LA at 160 mM NaClindicate that plants grown at elevated CO2 possessincreased metabolic reserves for LA synthesis, and thusmight be more capable of responding to oxidative stress,maintaining higher GSH levels, as previously suggestedby Schwanz et al. (1996). These results support theassertion that elevated CO2 enables plants to remainturgid and functional for a longer period and at a highersalt concentration than at ambient CO2 (Perez-Lopezet al. 2009a). Some authors have suggested that plantsunder elevated CO2 seemed to use additional energysupply for increasing the investment in antioxidantenzymes such as superoxide dismutase rather than inantioxidant metabolites (Geissler et al. 2009, Schwanzand Polle 2001). The discrepancy between studies wouldindicate that the response under elevated CO2 is not onlycultivar specific but also species specific.


Conclusions

The present study highlights the presence of LA in barleyleaves in both reduced and oxidized forms under non-salinized and salinized conditions. Under non-salinizedconditions, LA was present mainly as DHLA, which,being the most effective antioxidant form, might partlyexplain the great tolerance of barley to elevated salinity.In Iranis, the higher constitutive amounts of DHLAcomparing to Alpha, as well as a greater capacity toincrease ASA and LA with increasing salt stress, couldhave strengthened its antioxidant network, as LA seemsto be functioning in the re-reduction of DHA and GSSG(Fig. 1). However, more research about the interactionbetween LA and DHA and GSSG regeneration wouldbe desirable. On the other hand, the effects of saltstress were mitigated by elevated CO2 likely because ofdecreased ROS formation, but also because of a higherconstitutive level of LA, which would have improvedregeneration of DHA, and GSSG under salt stress. Theseeffects are not only species specific but are cultivarspecific, suggesting the possibility of selecting cultivarswith high probabilities of optimal performance in anenvironment characterized by elevated CO2 and highsoil salinity.

Acknowledgements – This project was supported by grantsMEC-BFU2007-60523/BFI, UNESCO 07/02, ETORTEK07/44, GRUPO UPV-GIU 07/43, and by the Universityof Pisa (Fondi di Ateneo, 2006–2007). During her stayin the Dipartimento di Chimica e Biotecnologie Agrariedell’Universita di Pisa, U. Perez-Lopez was the recipient ofa grant from Departamento de Educacion, Universidades eInvestigacion del Gobierno Vasco (Spain).

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