The mechanism(s) involved in the photoprotection of PSII at

Author's personal copy

Available online at www.sciencedirect.com

Environmental and Experimental Botany 64 (2008) 295–306

The mechanism(s) involved in the photoprotection of PSIIat elevated CO2 in nodulated alfalfa plants

Iker Aranjuelo a,b,∗, Gorka Erice b, Salvador Nogues a, Fermın Morales c,b,Juan J. Irigoyen b, Manuel Sanchez-Dıaz b

a Unitat de Fisologia Vegetal, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 645, 08028 Barcelona, Spainb Departamento de Biologıa Vegetal, Seccion Biologıa Vegetal (Unidad Asociada al CSIC, EEAD, Zaragoza),

Facultades de Ciencias y Farmacia, Universidad de Navarra, Irunlarrea no. 1, 31008 Pamplona, Spainc Departamento de Nutricion Vegetal, Estacion Experimental de Aula DEI-CSIC, Apdo. 202, 50080 Zaragoza, Spain

Received 13 November 2007; accepted 15 January 2008

Abstract

In a previous study, we found that enhanced CO2 subjected to nodulated alfalfa plants grown at different temperatures (ambient and ambient + 4 ◦C)and water availability regimes could protect PSII from photodamage. The main objective of this study was to determine the mechanism(s) involvedin the photoprotection of PSII at elevated CO2 levels in this plant. Elevated CO2 reduced carboxylation capacity-induced photosynthetic acclimationand reduced enzymatic and/or nonenzymatic antioxidant activities, suggesting that changes in electron flow did not cause any photooxidative damage(which was also confirmed by H2O2 and lipid peroxidation analyses). Enhanced nonphotochemical quenching and xanthophyll cycle pigmentsrevealed that plants grown at 700 �mol mol−1 CO2 compensated for the reduction in energy sink with a larger capacity for nonphotochemicaldissipation of excitation energy as heat, i.e., modulating the status of the VAZ components. Elevated CO2 induced the de-epoxidation of violaxanthinto zeaxanthin, facilitating thermal dissipation and protecting the photosynthetic apparatus against the deleterious effect of excess excitation energy.© 2008 Elsevier B.V. All rights reserved.

Keywords: Alfalfa; Antioxidants; Elevated CO2; Medicago sativa; Photosystem II; Photoprotection; Nonphotochemical quenching; Xanthophyll cycle

Abbreviations: A, anteraxanthin; Asat, light-saturated rate of CO2 assim-ilation; APX, ascorbate peroxidase; ASA, ascorbate; CAT, catalase; DHA,dehydroascorbate; Fv/Fm, maximal photochemical efficiency; g, leaf conduc-tance; GR, glutathione reductase; GSSG, oxidized glutathione; GSH, reducedglutathione; H2O2, hydrogen peroxide; IPCC, Intergovernmental Panel on Cli-mate Change; Jmax, electron transport rate contributing to RuBP regeneration;Je(PSII), electron transport through Photosystem II; Je(PCR), electron transportthrough photosynthetic carbon reduction; Je(PCO), electron transport throughphotorespiratory carbon oxidation; Ja, electron transport through the alternativepathway; l, stomatal limitation; PPFD, photosynthetic photon flux; PSII, Photo-system II; NPQ, nonphotochemical quenching; O2

−, superoxide anion; (OH),oxygen hydroxyl radical; OEC, oxygen evolving complex; Rd, rate of day res-piration; pO2, ambient partial pressure of O2; ROS, reactive species of oxygen;SOD, superoxide dismutase; Sr, relative specificity of Rubisco; TGT, temper-ature gradient tunnel; V, violaxanthin; VC, rate of carboxylation by Rubisco;Vcmax, maximum photosynthetic rate; Vo, rate of oxygenation by Rubisco; Z,zeaxanthin; φPSII, quantm yield of Photosystem II electron transport; θv, soilvolumetric water content.

∗ Corresponding author at: Unitat de Fisologia Vegetal, Facultat de Biolo-gia, Universitat de Barcelona, Av. Diagonal 645, 08028 Barcelona, Spain.Fax: +34 934112842.

E-mail address: [email protected] (I. Aranjuelo).

1. Introduction

The global atmospheric CO2 concentration is increasing and,according to the Intergovernmental Panel on Climate Change,it is expected to reach 700 �mol mol−1 by the end of this cen-tury (IPCC, 2001). Most experiments analysing the effect of CO2increase on vegetative processes in the context of climate changehave studied the effect of CO2 or CO2–temperature interactionunder optimal conditions of water supply. However, when study-ing the effect of climate change on Mediterranean environments,it should be kept in mind that circulation models also predictdrier conditions for the Mediterranean basin due to an increasein temperature and water deficit (IPCC, 2001; Alley et al., 2007).Furthermore, it is well known that the effect of combined stresseson plant growth causes alterations that cannot be predicted fromthe effects of the stresses alone because of synergism and antag-onism phenomena (Valladares and Pearcey, 1997). Chaves andPereira (2004) observed that, although photochemical processesare resistant to low water availability conditions, they inducedownregulation of the photosynthetic apparatus when they inter-act with elevated temperature and irradiation levels.

0098-8472/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.envexpbot.2008.01.002


296 I. Aranjuelo et al. / Environmental and Experimental Botany 64 (2008) 295–306

Although the influences of increases in CO2 levels on plantgrowth and physiology have been studied by a number ofworkers (Wolfe et al., 1998; Urban, 2003; Long et al., 2004;Aranjuelo et al., 2005a, 2006; Erice et al., 2006a), the effectof elevated CO2 on plant photochemistry and its influence onPSII have received little attention. Furthermore, the results ofprevious studies reveal significant discrepancies and variabil-ity. For example, Hymus et al. (2001) observed that plantsexposed to elevated CO2 concentrations exhibited increased(photochemical) requirements for light-saturated electron flowthrough Photosystem II, whereas Scarascia-Mugnozza et al.(1996) reported that CO2 increase had a depressive effect onplant photochemistry. According to previous studies, elevatedCO2 levels will increase the photosynthetic carbon reductioncycle (the major sink for the reducing equivalents generated bythe primary chemical reactions) and consequently the electronflow that drives it (Hymus et al., 2001). In addition, elevatedCO2 will competitively suppress the photorespiratory carbonoxidation cycle and the resulting electron flow. However, severalstudies on C3 plants grown under elevated CO2 concentrationhave observed that, after an initial stimulation of photosyn-thetic rates, the carboxylation capacity of plants decreases afterlong-term exposure (Ainsworth et al., 2004; Long et al., 2004;Aranjuelo et al., 2005b). As observed by Hymus et al. (2001),changes in C assimilation at elevated CO2 require modificationsin the partitioning of the absorbed energy between heat dissi-pation and photochemistry. When photosynthesis decreases andlight excitation energy is in excess, overexcitation of the pho-tosynthetic pigments in the antenna can occur. Impairment ofphotosynthetic function will lead to excessive excitation energyin Photosystem II (PSII), leading to an accumulation of reac-tive oxygen species (ROS) and thereby resulting in oxidativestress.

Production of ROS, including superoxide (O2−), hydro-

gen peroxide (H2O2), and the hydroxyl radical (•OH), is aninevitable consequence of life in an oxygen-rich environment(Polle et al., 1990). ROS are formed within the plant as aconsequence of (a) photochemical production of H2O2 in theatmosphere from air pollution; (b) donation of electrons directlyto oxygen during photosynthesis, especially with high light; and(c) in response to environmental stresses such as drought/heat,etc. ROS damage plant cells by oxidizing membrane lipids,including the photosynthetic apparatus (Foyer and Harbinson,1994); inhibit protoplast regeneration (Marco and Roubelakis-Angelakis, 1996); and damage proteins, chlorophyll, and nucleicacids (Foyer and Harbinson, 1994).

Plants have developed three main mechanisms to diminishphotooxidation: (a) to prevent the production of ROS by dimin-ishing the electron transport chain; (b) to scavenge ROS formedby an integrated system of enzymatic and nonenzymatic antiox-idants (Asada, 1999). The ascorbate-glutathione cycle is themost important antioxidant cycle in plants (Alscher et al., 1997).The first ROS produced in the plant cells is the superoxideradical anion (O2

−), which is dismutated to H2O2 by super-oxide dismutase (SOD). The H2O2 is then reduced to H2O byascorbate peroxidase (APX). The ascorbate oxidized by APXis reduced by the reduced form of glutathione, and the glu-

tathione is yet again reduced by glutathione reductase (GR)(Alscher et al., 1997). Secondly, the chloroplastic water–watercycle, which is related to the electron flow from the water inPhotosystem II to O2 reduction in PSI without any release ofsuperoxide and hydrogen peroxide, has been proposed as aneffective mechanism to dissipate excess excitation under envi-ronmental stress (Asada, 1999; Ort and Baker, 2002; Zhou etal., 2006). (c) To diminish photooxidation through xanthopyllcycle-dependent thermal dissipation, an important photoprotec-tive process in the light-harvesting antenna of Photosystem II(Gilmore, 1997; Verhoeven et al., 1999). In this process, the for-mation of a pH gradient across the thylakoid membrane activatesthe de-epoxidation of violoxanthin (V) to zeaxanthin (Z) andanteraxanthin (A), facilitating the thermal dissipation of excessexcitation energy (Demming et al., 1987). Xanthophyll cycle-dependent energy dissipation downregulates the photochemicalefficiency of PSII, thereby protecting the reaction centres fromphotooxidation.

Several responses of photoinhibition to elevated CO2 concen-tration have been reported. In wheat plants grown under elevatedCO2 conditions, a greater proportion of the absorbed light isused in photochemistry at high light (Habash et al., 1995). Suchan increase was reflected in larger photochemical energy dissi-pation with the consequent reduction in photoinhibition. Otherauthors showed decreased photochemistry and increased pho-toinhibition in plants exposed to drought (Scarascia-Mugnozzaet al., 1996) and heat stress conditions (Roden and Ball, 1996).An increase or decrease in photochemistry and photoinhibi-tion depends on whether photosynthetic downregulation occurs(Hymus et al., 2001). In a previous study carried out by ourgroup with alfalfa plants grown under elevated CO2 conditions,it was observed that, although plants grown at 700 �mol mol−1

had photosynthetic acclimation, CO2 increase protected PSIIfrom higher excess in excitation energy as indicated by thehigher Fv/Fm (Aranjuelo et al., 2005a). Hymus et al. (2001)also observed that elevated CO2 enhanced nonphotochemicalquenching to compensate for the reduction in energy sink,thus contributing to the alleviation of excessive excitationenergy in the PSII. The influence of elevated CO2 on PSIIphotoprotection has also been highlighted by other authors(Carvalho and Amancio, 2002; Kurasova et al., 2003; Kitao etal., 2005).

In a previous study, we observed that, although elevatedCO2 resulted in significantly lower values of PSII efficiencyin the light (φPSII), higher values of Fv/Fm were measured indark-adapted leaves (Aranjuelo et al., 2005b). As a follow up,in the present work, we examine the photoprotective effect ofelevated CO2 (at different temperatures and water availabilityregimes) on PSII in nodulated alfalfa plants. This experimentwas performed in temperature gradient tunnels (TGTs) placedover plants under standard field conditions. The objective was toanalyse the mechanisms involved in CO2-induced photoprotec-tion of the photochemistry of PSII (Aranjuelo et al., 2005a) andto determine which processes could be linked to the protectionof reaction centres. To do so, the following processes associ-ated with photodamage were studied: (a) the electron transportchain; (b) scavenging of ROS by antioxidant molecules, enzy-


I. Aranjuelo et al. / Environmental and Experimental Botany 64 (2008) 295–306 297

matic and nonenzymatic; and (c) xanthopyll cycle-dependentthermal dissipation.

2. Materials and methods

2.1. Seedling growth

Alfalfa (Medicago sativa) seeds were germinated in Petridishes. One week later, they were transferred to 13-L pots (20plants per pot). According to Arp (1991), plants grown in smallpots exhibit a greater acclimation response to CO2 enrichmentthan field-grown plants. A large pot volume will avoid restrictedroot growth, which otherwise may affect the supply of water andnutrients and therefore limit the sink for assimilates. The potsubstrate was a mixture of perlite/vermiculite (2/1, v/v). Duringthe first month, plants were grown in a greenhouse, at 25/15 ◦C(day/night), with a photoperiod of 14 h under natural daylight,supplemented with fluorescent lamps (Sylvania DECOR 183,Professional-58W, Germany) providing a photosynthetic pho-ton flux density (PPFD) of ca. 400 �mol m−2 s−1. At the ageof 3, 4, and 5 weeks, plants were inoculated with Sinorhizo-bium meliloti strain 102F78 (The Nitragin Co., Milwaukee, WI,USA). When plants were 1 month old, they were transferredto TGTs located in a commercial alfalfa field at the Munovelafarm (40.95◦N, 5.50◦W, and 795 m altitude) from the Instituto deRecursos Naturales y Agrobiologıa (CSIC, Salamanca, Spain)(see below, Aranjuelo et al., 2005b).

2.2. Experimental design

The experiment was conducted during June and July in twoconsecutive years (2001 and 2002). Results presented in thispaper correspond to the mean values of data collected in the 2years.

The experimental design was a split–split–plot factorial withtwo levels of CO2 concentration, temperature, and water avail-ability, replicated in two consecutive years (July 2001 and 2002).The experiment was conducted in two TGTs. The first tunnel cor-responded to ambient CO2 conditions, where plants were grownat ca. 400 �mol mol−1 CO2. In the second tunnel, which corre-sponded to elevated CO2 concentration, plants were grown atca. 720 �mol mol−1 CO2. Each tunnel was divided into threemodules to allocate ambient and ambient + 4 ◦C temperaturetreatments. The middle module was considered a transition mod-ule and no experimental plants were included. In each tunnel, theinlet module was maintained at ambient temperature (ca. 19 ◦C)and the outlet module at ambient temperature + 4 ◦C (ca. 23 ◦C).TGTs are described in more detail by Aranjuelo et al. (2005a).

Different water availability treatments were applied at ran-dom to four of the eight pots in each temperature/CO2 subplot.Within each of the 16 main plots, 8 pots were placed in the ambi-ent temperature module, while the remaining 8 were placed inthe elevated temperature module. Within each temperature mod-ule, the tunnel was further randomly divided into 8 sub-subplots,4 of which received the full-watering treatment (control), andthe other four received the partial-watering treatment (drought).Although withholding water is the most common experimental

approach to simulate low water availability conditions, recog-nising that in nature, drought is generally not imposed so rapidly,in this experiment, a lesser-sustained stress was applied. In addi-tion, in most cases, plants grown at elevated CO2 deplete soilwater at a lower rate than plants grown at ambient CO2, due tolower stomatal conductance and lower transpiration rates, whichmeans that, in many experiments, elevated CO2 increases thetime to reach a particular level of water stress (De Luis et al.,1999). Thus, drought tolerance – i.e., the ability to maintainplant productivity under a given soil water stress (Jones, 1992)induced by elevated CO2 – remains incompletely understood.The only way to address this question is to design an experi-ment in which all treatments are subjected to the same soil watercontent. Thus, in the present experiment, fully watered plantswere irrigated until a maximum substrate volumetric water con-tent (θv) around 0.4 cm3 cm−3 was reached (field capacity). Theapplied drought level corresponded to 50% of the θv of controlplants (around 0.2 cm3 cm−3). The desired drought level wasreached around 15 days after the beginning of treatment, whenplants were 45 days old. These θv levels were then maintainedthroughout the rest of the experiment by measuring daily thetranspired water and replenishing this lost water (De Luis et al.,1999). The pots for the two water treatments were randomlyreallocated within each module each day.

2.3. Gas exchange and chlorophyll fluorescencemeasurements

Fully expanded apical leaves from 54-day-old plants wereenclosed in a gas exchange leaf chamber (1010-M, Waltz,Effeltrich, Germany), and the gas exchange rate was mea-sured with a portable photosynthesis system (HCM-1000,Waltz). The gas exchange response to CO2 was measuredfrom 0 to 1000 �mol mol−1 CO2. Measurements started at400 �mol mol−1 of CO2, decreased stepwise until 250, 100,0 �mol mol−1 and restarted at 400 and increased stepwise until700, 850, and 1000 �mol mol−1. Every year, four trials of eachof the eight treatment combinations were conducted. Light-saturated rate of CO2 assimilation (Asat) was estimated at aPPFD of 1200 �mol m−2 s−1 using equations developed by vonCaemmerer and Farquhar (1981). The Rubisco specificity fac-tor used for the von Caemmerer and Farquhar calculations wasthe one estimated for alfalfa by Keys (1986). Estimation of themaximum carboxylation velocity of Rubisco (Vcmax) and themaximum electron transport rate contributing to RuBP regen-eration (Jmax) were made by fitting a maximum likelihoodregression below and above inflexion of the A/Ci response usingthe method of Ethier and Livingston (2004). Stomatal limita-tion (l), which is the proportionate decrease in light-saturatednet CO2 assimilation attributable to stomata, was calculatedaccording to Farquhar and Sharkey (1982) as l = [(A0 − A1)/A0,where A0 is the A at Ci of 360 �mol mol−1 and A1 is A at Ca of360 �mol mol−1.

Chlorophyll fluorescence (measured in the light) was studiedafter the gas exchange analyses in the same expanded leaveswith a portable modulated fluorometer (PAM-2000) (Waltz),with the fibre optics passing through the radiation shield of the



leaf chamber at an angle of 60◦. Nonphotochemical quench-ing was calculated as (Fm/F ′

m) − 1 as described by Bilger andBjorkman (1990), at growth conditions, and with an irradianceof 250 �mol m−2 s−1 photon flux density, provided by red-light-emitting diodes (maximum at 655 nm).

2.4. Estimation of rate of alternative electron flow

The rate of electron transport through PSII [Je(PSII)] wasmeasured as described by Harley et al. (1992). The rate of oxy-genation by Rubisco (Vo) was estimated as described by vonCaemmerer and Farquhar (1981) as Vo = (Vc × pO2)/(Sr × Ci)where Vc refers to the rate of carboxylation of RuBP, pO2refers to the ambient partial pressure of O2, Sr refers to relativespecificity of Rubisco, and Ci refers to intercellular CO2 concen-tration. The rate of carboxylation by Rubisco (Vc) was estimatedas Vc = (A + Rd)/[1 − pO2/(2 × Sr × Ci)], where Rd refers to rateof day respiration (Miyake and Yokota, 2000). The electronfluxes in the two cycles, expressed as Je(PCR) = 4 × Vc andJe(PCO) = 4 × Vo, respectively (Krall and Edwards, 1992), wereconducted at growth conditions corresponding to each treatment.An alternative flux, Ja, caused by electrons that are not used bythe PCR and/or PCO cycles in the total electron flux driven byPSII, can be estimated from Je(PSII) − Je(PCR + PCO) (Miyakeand Yokota, 2000).

2.5. Hydrogen peroxide and lipid peroxidationdetermination

The hydrogen peroxide (H2O2) content was measured asdescribed in Patterson et al. (1984) with slight modifications.Three hundred milligrams of the youngest fully expanded leaveswas homogenized in a cold mortar with 5 mL 5% TCA contain-ing 0.1 g activated charcoal and 0.1% PVPP. The homogenatewas filtered and centrifuged at 18,000 × g for 10 min. The super-natant was filtered through a millipore filter (0.45 �m) andused for the assay. A 200-�L aliquot was brought to 2 mLwith 100 mM K-phosphate buffer (pH 8.4) and 1 mL colori-metric reagent was added. This reagent was made daily bymixing 1:1 (v/v) 0.6 potassium titanium oxalate and 0.6 mM 4-2 (2-pyridylazo) resorcinol (disodium salt). The samples wereincubated at 60 ◦C for 45 min and the absorbance at 508 nm wasrecorded. The blanks were made by replacing leaf extract by5% TCA. The level of lipid peroxidation in the leaves was esti-mated by measuring the content in thiobarbituric acid reactingsubstances (TBARS) as described by Dhindsa et al. (1981).

2.6. Antioxidant enzyme activities

One hundred and twenty-five milligrams of the youngest fullyexpanded developed leaves was homogenized in a mortar with5 mL 100 mM phosphate buffer (pH 7.0) containing 5 mM DTTand 50 mg PVPP. The homogenate was filtered and centrifugedat 38,000 × g for 10 min. The supernatant was separated to deter-mine the activity of antioxidant enzymes. Superoxide dismutase(SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.7)and glutathione reductase (GR, EC 1.6.4.2) activities were mea-

sured as described by Aroca et al. (2001). Catalase (CAT, EC1.11.1.6) activity was determined according to Aebi (1974).

2.7. Glutathione determination

Glutathione concentration was measured as described bySmith (1985). Five hundred milligram of apical fully expandedleaves of each treatment was homogenized in a cold mortarwith 5 mL 5% (w/v) sulfosalicylic acid; the homogenate wasfiltered and centrifuged at 1000 × g for 10 min. One milliliterof supernatant was neutralized by 1.5 mL 0.5 mM K-phosphatebuffer (pH 7.5). The standard incubation medium was a mixtureof: 0.5 mL 0.1 M sodium phosphate buffer (pH 7.5) contain-ing 5 mM EDTA, 0.2 mL 6 mM 5,5′-dithiobis-(-2-nitrobenzoicacid), 0.1 mL 2 mM NADPH, and 0.1 mL (one unit) glutathionereductase. The reaction was initiated by the addition of 0.1 mLglutathione standard or extract.

2.8. Ascorbate determination

Ascorbate (ASA) and dehydroascorbate (DHA) were assayedas described by Leipner et al. (1997). They were assayed pho-tometrically by the reduction of 2,6-dichlorophenolindophenol(DCPIP). Two hundred milligrams of apical fully expandedleaves from each treatment were homogenized in 5 mL ice-cold 2% (w/v) metaphosphoric acid in the presence of 1 gNaCl. The homogenate was filtered through a paper filter. Analiquot of 300 �L was mixed with 200 �L 45% (w/v) and100 �L 0.1% (w/v) homocysteine to reduce DHA to ASA forthe determination of the total ascorbate pool. To determineASA, the homocysteine solution was replaced by the same vol-ume of distilled water. After 15-min incubation at 25 ◦C, 1 mL2 mM citrate–phosphate buffer (pH 2.3) and 1 mL 0.003% (w/v)DCPIP were added. The absorbance at 524 nm was measuredimmediately. The content of ASA was calculated by referenceto a standard curve. The amount of DHA was obtained asthe difference between the total ascorbate pool and the ascor-bate.

2.9. Photosynthetic pigments

Leaf disks, harvested at midday and immediately plungedinto liquid nitrogen, were cut with a calibrated cork borer andwrapped in aluminum foil. Leaf pigments were extracted withacetone in the presence of Na ascorbate and stored as describedby Abadia et al. (1999). Pigment extracts were thawed onice, filtered through a 0.45-�m filter, and analysed by an iso-cratic HPLC method based on the one developed by De lasRivas et al. (1989) with some modifications. Two steps, insteadof three, were used: mobile phase A (acetonitrile: methanol,7:1, v:v) was pumped for 3.5 min, and then mobile phase B(acetonitrile:methanol:water:ethyl acetate, 7:0.96:0.04:8 by vol-ume) was pumped for 4.5 min. To both solvents, 0.7% (v:v) ofthe modified triethylamine (TEA) was added (Hill and Kind,1993) to improve pigment stability during separation. The anal-ysis time for each sample was 13 min, including equilibrationtime.



Table 1The effect of CO2, air temperature (T) and water availability (H2O) on net photosynthetic rate (Asat, �mol m−2 s−1), maximum velocity of RuBP carboxylation byRubisco (Vcmax, �mol m−2 s−1), maximum potential rate of electron transport contributed to RuBP regeneration (Jmax, �mol m−2 s−1) and stomatal limitation (l, %)in nodulated alfalfa

Treatments (H2O–CO2–T) Asat Vcmax Jmax l

Control–Amb–Amb 16.05 ± 2.22 a 73.62 ± 3.11 d 147.22 ± 20.01 bc 27.82 ± 2.99 bControl–700–Amb 10.27 ± 2.7 bc 53.65 ± 4.01 e 119.02 ± 33.54 c 23.00 ± 1.73 cControl–Amb–Amb + 4 ◦C 15.03 ± 1.8 ab 84.34 ± 1.0 c 192.42 ± 18.36 b 29.50 ± 3.54 bControl–700–Amb + 4 ◦C 12.75 ± 2.48 ab 55.47 ± 3.01 e 171.47 ± 30.01 b 31.33 ± 2.31 bDrought–Amb–Amb 12.00 ± 2.3 b 117.76 ± 3.40 a 274.60 ± 46.59 a 44.00 ± 1.00 aDrought–700–Amb 8.65 ± 1.1 c 50.55 ± 3.07 e 120.18 ± 25.71 c 41.25 ± 1.71 aDrought–Amb–Amb + 4 ◦C 14.25 ± 1.6 ab 84.72 ± 3.96 c 134.22 ± 12.16 c 16.17 ± 3.69 dDrought–700–Amb + 4 ◦C 16.22 ± 3.21 a 100.56 ± 3.96 b 176.27 ± 38.44 b 12.17 ± 0.76 d

Each value represents the mean ± S.E. (n = 8).

2.10. Statistical analyses

The experiment was carried out over two consecutive years,in almost the same growth conditions. In the second year,the growth conditions inside and between the tunnels wereexchanged. In other words, when, in a given tunnel, a microen-vironment of ambient CO2 was maintained during the first year,this was changed to a microenvironment of elevated CO2 dur-ing the second year, and vice versa. Similarly, in the modulesin which ambient temperature was maintained during the firstyear, an elevated temperature was set for the second, and viceversa. In summary, this experiment provided a total of 16 plotscomprising a combination of two different CO2 levels, two dif-ferent temperature regimes, two different conditions of wateravailability, and two repetitions.

The effect of CO2, temperature (T), and water availability(H2O) on plant development was tested by a split–split–plotmultiple analysis of variance (ANOVA) with three factorial treat-ments (CO2, temperature, and water availability) (Montgomery,1984). The CO2 treatment was the main plot factor, tempera-ture was the split–plot treatment, and water level was regardedas the split–split–plot treatment. The main plot analysis con-tained the sum of squares for CO2 divided by the main ploterror sum of squares. The split–plot test included temperatureand CO2 × T mean square and subplot error sums of square.Finally, the split–split–plot test included the mean squares cor-responding to H2O, H2O × CO2, H2O × T, and H2O × CO2 × Tdivided by the split-subplot error. The means ± S.E. was calcu-lated for each parameter. When a particular F test was significant,we compared the means using LSD multiple comparison. Theresults were accepted as significant at P < 0.05.

3. Results

Estimation of Asat revealed that, regardless of water avail-ability, elevated CO2 decreased the photosynthetic capacity ofthose plants (Table 1, F = 9.16, P = 0.02), especially under ambi-ent temperature conditions. No statistical differences associatedwith CO2 were observed in amb + 4 ◦C treatments. The resultsin Table 1 also show that Asat values were not affected bytemperature (F = 1.06, P = 0.35) or water availability (F = 1.99,P = 0.18). However, upon further analysis of water availabil-

ity effect interacting with temperature, the data showed that,although in fully watered plants, no differences related to growthtemperature were observed, in partially watered treatments,temperature enhancement increased Asat. Reduction of carboxy-lation efficiency was accompanied by decreases in the maximumcarboxylation velocity of Rubisco (Vcmax; F = 85.78, P = 0.00)and maximum electron transport rate contributing to RuBPregeneration (Jmax; F = 6.78, P = 0.04) (Table 1). The reductionof CO2 assimilation was not attributable to stomatal limitation(l), where no effect (F = 3.13, P = 0.13) associated with CO2increase, analysed separately or interacting with temperatureand water availability, was found. No effect of temperature ordrought on Vcmax and Jmax was observed. Similarly to what wasdescribed in Asat, drought effect on l was mediated by growthtemperature. Although no statistical differences were detected incontrol plants, in treatments subjected to low water availability,temperature enhancement negatively affected l.

Analyses of electron transport flow revealed that ele-vated CO2 reduced the total electron flux in PSII [Je(PSII)](F = 12.06, F = 0.03) in all treatments, except in plants grownat ambient temperature where no differences were observed(Fig. 1A). Reduction of Je(PSII) observed in plants grown under700 �mol mol−1 decreased the electron flux destined to the pho-tosynthetic carbon reduction [Je(PCR)] (F = 16.61, P = 0.01) andelectron flux for photorespiratory carbon oxidation [Je(PCO)](F = 23.64, P = 0.01) (Fig. 1B and C, respectively). Elevated CO2tended to increase the electron flux to the alternative pathway(Ja), although not in a statistical manner. This effect was mostclear in fully watered plants grown under ambient temperatureand droughty plants grown at elevated temperature (Fig. 1D).

Hydrogen peroxide determinations (Fig. 2A) revealed thatthe effect of CO2 concentration on H2O2 content was medi-ated by water availability (F = 8.95, P = 0.012). Exposure ofplants to 700 �mol mol−1 decreased H2O2 under well-wateredconditions, whereas no effect was observed under drought.Lipid peroxidation analyses, estimated by TBARS determina-tions (Fig. 2B), revealed that, regardless of water availability(F = 0.818, P = 0.383), elevated CO2 decreased TBARS con-tent (F = 12.59, P = 0.004). Although H2O2 and TBARS werenot influenced specifically by temperature increase (F = 0.06,P = 0.806; F = 5.27, P = 0.06, respectively), TBARS concentra-tions diminished when interacting with low water availability.



Fig. 1. Effect of CO2, temperature and water availability on Je(PSII), electrontransport through Photosystem II; Je(PCR), electron transport through photosyn-thetic carbon reduction; Je(PCO), electron transport through photorespiratorycarbon oxidation; Ja, electron transport through the alternative pathway in nodu-lated alfalfa. Each value represents the mean ± S.E. (n = 8). The different lettersindicate significant differences (P < 0.05) among treatments as determined byLSD

Catalase activity (Fig. 3A) was inhibited by elevated CO2(F = 58.66, P = 0.000) and elevated temperature (F = 16.44,P = 0.002). Drought enhanced CAT activity (F = 16.44,P = 0.002). Similarly, exposure to 700 �mol mol−1 diminishedSOD activity (F = 43.54, P = 0.00) (Fig. 3B), although in thiscase, no differences associated with temperature increase weredetected (F = 3.50, P = 0.110). However, Fig. 3 also revealedhow in fully watered plants, temperature enhancement increasedSOD activity. On the other hand, no CO2 effect (F = 0.70,P = 0.434) was observed on ascorbate peroxidase activity(Fig. 3C). As described in SOD activity, with plants grownunder fully watered conditions, elevated temperature enhancedAPX activity (F = 2.60, P = 0.154) independently of CO2 con-centration. Similar to the response seen for CAT and SODactivity, elevated CO2 depressed glutathione reductase activity(F = 47.74, P = 0.00) (Fig. 3D).

The glutathione (GSH) content was seen to be reduced inplants grown at elevated CO2 (F = 5.85, P = 0.032), whereasdrought (F = 67.70, P = 0.00), in both temperature and CO2 con-ditions, increased GSH content (Fig. 4A). Similarly, the oxidizedglutathione content (GSSG) also decreased in elevated CO2,especially in association with drought (F = 23.2, P = 0.00). Fur-thermore, increased CO2 reduced the values of total glutathionecontent (GSH + GSSG); (F = 41.89, P = 0.00) (Fig. 4C). Plantsgrown at 700 �mol mol−1 had higher GSH/GSSG ratios, espe-cially under drought conditions (F = 53.6, P = 0.00) (Fig. 4D).

Exposure to 700 �mol mol−1 CO2 decreased ascorbatecontent, although the effect was limited to fully wateredtreatments (F = 94.41, P = 0.00) (Fig. 5A). Dehydroascorbatecontent was lower in elevated CO2 treatments (F = 11.54,P = 0.02), especially in plants exposed to drought (Fig. 5B).Consequently, regardless of temperature (F = 9.49, P = 0.02)or water availability (F = 45.01, P = 0.00), the total ascor-bate content (ASA + DHA) was diminished in elevated CO2treatments (F = 4.30, P = 0.080). In addition, elevated CO2reduced ASA/DHA but only in fully watered plants (F = 5.85,P = 0.032)(Fig. 5D). The temperature increase enhancedASA/DHA (F = 8.44, P = 0.025).

Elevated CO2 decreased violaxanthin concentration(F = 9.81, P = 0.014) (Fig. 6). Temperature effect on violaxan-thin was limited to fully watered plants in which temperatureenhancement negatively affected its content. In parallel to thisreduction, plants exposed to 700 �mol mol−1 CO2 showedhigher zeaxanthin concentrations (F = 14.51, P = 0.006). Theeffect of CO2 on antheraxanthin concentration was mediatedby water availability (F = 7.50, P = 0.023) and temperature(F = 6.07, P = 0.045). Elevated CO2 induced the stimulationof nonphotochemical quenching (NPQ; F = 5.84, P = 0.024)

Fig. 2. Effect of CO2, temperature and water availability on leaf hydrogen per-oxide (H2O2) and thiobarbituric acid-reacting substances (TBARS) content innodulated alfalfa. Otherwise as for Fig. 1.



Fig. 3. Effect of CO2, temperature and water availability on leaf catalase (CAT),superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathionereductase (GR) activities in nodulated alfalfa. Otherwise as for Fig. 1.

(Fig. 7A). Quantification of xanthophyll cycle pigment content(Fig. 6) indicated that, regardless of temperature (F = 2.87,P = 0.141) and water availability (F = 1.39, P = 0.262), elevatedCO2 decreased (F = 151.37, P = 0.00) the V/VAZ ratio (Fig. 7C)and altered the oxidation state of the VAZ cycle pigments.The de-epoxidated form, Z, was progressively formed at theexpense of the epoxidated form V, reaching 30% of the totalVAZ cycle (data not shown). Changes in the proportion ofde-epoxidated forms of A and Z compared with V were alsoreflected in the larger Z + A/V + A + Z ratio in elevated CO2treatments (Fig. 7B).

4. Discussion

In a previous study (Aranjuelo et al., 2005a), we showedthat, under elevated CO2 conditions, the carboxylation capac-ity of alfalfa plants decreases as a consequence of the reductionof Rubisco content and quantum efficiency (φPSII). However,chlorophyll fluorescence analyses suggested that elevated CO2

photoprotect photosynthesis apparatus in alfalfa plants. To iden-tify the primary target explaining the photoprotective role ofelevated CO2, we examined the effect of CO2 on photochem-ical reactions, alternative electron sinks, antioxidant enzymeactivities, and photosynthetic pigment contents. The A/Ci curveparameters analysed (light saturated rate of CO2 assimila-tion, Asat, maximum carboxylation velocity of Rubisco, Vcmax;and maximum electron transport rate contributing to RuBPregeneration, Jmax) confirmed that, opposite to what has beendescribed by other authors (Long et al., 2004; Idso and Kimball,1992), plants grown at 700 �mol mol−1 suffered photosyntheticacclimation. Interestingly, the data showed that the degree ofacclimation varied, depending on growth temperature. Regard-less of water availability exposure to 700 �mol mol−1, CO2decreased carboxylation capacity in ambient temperature con-ditions, whereas under elevated temperature conditions, nostatistical differences were registered. Differences in the degreeof photosynthetic acclimation could be caused by differences

Fig. 4. Effect of CO2, temperature and water availability on leaf-reducedglutathione (GSH), glutathione disulfide (GSSG), glutathione redox state(GSH/GSSG) and total glutathione (GSH + GSSG) in nodulated alfalfa. Oth-erwise as for Fig. 1.



Fig. 5. Effect of CO2, temperature and water availability on leaf-reduced ascor-bate (ASA), dehydroascorbate (DHA), ascorbate redox state (ASA/DHA) andtotal ascorbate (ASA + DHA) in nodulated alfalfa. Otherwise as for Fig. 1.

in carbon source/sink (Rogers and Ainsworth, 2006) balanceand/or nitrogen availability of those plants (Stitt and Krapp,1999; Rogers et al., 2006). Stomatal limitation (l) quantificationrevealed that the reduction in carboxylation efficiency was notcaused by stomatal closure phenomena. Photosynthetic accli-mation of those plants was caused by the reduction in Rubiscoactivity (Aranjuelo et al., 2006), which might be caused by thereduction in N availability caused by the decrease of noduleactivity (data not shown). The absence of water availabilityeffect on A/Ci curve parameters suggests that applied wateravailability was not very severe. As we previously observed,no differences were detected on water state parameters betweentreatments associated to neither CO2 concentration, tempera-ture, nor water availability (Aranjuelo et al., 2005a, 2006). Thatthere were no differences in gas exchange parameters suggeststhat plants grown at 50% of soil water content adapted theirgrowth to the available water without suffering from stressfulconditions. These findings were similar to what were earlierdescribed (Aranjuelo et al., 2005a; Erice et al., 2006a,b). Inter-estingly, Asat data also showed that, under low water availability,

elevated temperature increased the photosynthetic activity ofthose plants, probably as a consequence of their lower stomatallimitation values.

A decrease in the capacity of CO2 assimilation in the Calvincycle implies a reduction in the demand for ATP and NADPH,resulting in the reduction of electron transport to match thelower demand for ATP and NADPH. Several studies (Scarascia-Mugnozza et al., 1996; Hymus et al., 1999) carried out underfield conditions have observed that plants grown under elevatedCO2 conditions may suffer from either a decrease or an increasein photochemical requirements for light-saturated electron flowto PSII [Je(PSII)].

Our results confirmed that elevated CO2 decreases the amountof electron flow through Photosystem II [Je(PSII)]. Interestingly,as observed by other authors (Hymus et al., 2001), the reduc-tion of Je(PSII) observed in plants grown under 700 �mol mol−1

reduced the electron flux destined for photosynthetic carbonreduction [Je(PCR)] and that destined for photorespiratory car-bon oxidation [Je(PCO)]. A reduction in electron flux to PCRwould be related to the decrease of Vc,max and, consequently, thecarboxylase activity of Rubisco (Aranjuelo et al., 2005a). Thephotosynthetic acclimation blocked the short-term stimulationof Je(PCR) by atmospheric CO2 concentration (Hymus et al.,2001). Furthermore, Je(PCO) was competitively suppressed bythe increase in atmospheric CO2 concentration (Hymus et al.,2001).

Fig. 6. Effect of CO2, temperature and water availability on violaxanthin (V),antheraxanthin (A), zeaxanthin (Z) contents in nodulated alfalfa. Otherwise asfor Fig. 1.



Fig. 7. Effect of CO2, temperature and water availability on nonphotochemi-cal quenching (NPQ), de-epoxidation state of xanthophyll pool, calculated asthe ratio between antheraxanthin plus zeaxanthin contents/antheraxanthin pluszeaxanthin plus violaxanthin contents, in nodulated alfalfa. Otherwise as forFig. 1.

In the present study, the previously mentioned lack of tem-perature and water availability effect on A/Ci curve parameters(also reflected on electron flux sinks) was accompanied byminor effects on electron flux sinks. The slight increase inelectron flux to the alternative pathway (Ja) under elevatedCO2 conditions was similar to that observed by other authorsunder mild water or temperature stress conditions (Bartak etal., 1996; Hymus et al., 1999, 2001). Other studies on plantsexposed to more severe stressful growth conditions (chill-ing, elevated temperature, and low water availability) andexposed to such conditions for longer periods of time haveobserved an electron flow from water through the oxygen-evolving complex (OEC) in PSII to water in PSI (Asada,1999).

On the basis of previous studies, the reduction of CO2 assim-ilation capacity and electron flow to PSII was expected to causephotooxidative damage, especially in high-irradiance condi-tions. When photosynthetically active photon flux density isin excess of that required for CO2 assimilation (which is thecase in plants grown under elevated CO2 conditions), elec-tron carriers are over reduced and the photon energy excesscan generate ROS if the scavenging capacity of the plants isexceeded. Interestingly, elevated CO2 prevented photodamage

in plants and photooxidative damage was only observed inplants grown at 350 �mol mol−1. Plants scavenge ROS throughthe antioxidant system (enzymatic and nonenzymatic). Twocommon parameters used to estimate the degree of oxidativestress are the measurements of H2O2 and lipid peroxidation(Dat et al., 2000). We estimated lipid peroxidation by measur-ing the TBARS (Dhindsa et al., 1981). Absence or inhibitoryeffect of elevated CO2 on H2O2 and TBARS revealed that theexcess photon energy did not generate ROS. Furthermore, ele-vated CO2 protected those plants from photooxidative stress.In agreement with previous findings, the absence of droughtand temperature effect on TBARS and H2O2 confirmed thatthose plants adapted their development to the available soilwater content. Furthermore, droughty plants grown at elevatedtemperature (at both CO2 concentrations) had lower TBARSvalues.

Among the photoprotective mechanism, two biochemicalprocesses have received attention over the last 15 years. Oneis the photoreduction of oxygen and the subsequent detoxifi-cation of the ROS by an integrated system of enzymatic andnonenzymatic antioxidants. The second is xanthophyll cycle-dependent thermal dissipation (Demming-Adams and Adams,1992). Enzymes involved in ROS scavenging to minimize thecellular oxidative damage plant produce include catalase (CAT),superoxide dismutase (SOD), ascorbate peroxidase (APX),and glutathione reductase (GR). In agreement with H2O2 andTBARS results, the analyses of antioxidant enzymes revealedthat, except in the case of APX activity where no clear responsepattern was detected, elevated CO2 decreased CAT, SOD, andGR activities. In general, the status of the plant’s antioxidantsystem is believed to be a function of oxidative stress (Polleet al., 1997). Therefore, the observed reduction in antioxidantactivities may reflect a reduction of oxidative stress resultingfrom growth in CO2-enriched environments (Polle et al., 1997;Pritchard et al., 2000).

In addition to the enzymatic antioxidants, the plants alsohave nonenzymatic antioxidants. Most importantly, reduced glu-tathione (GSH) and reduced ascorbate (ASA) can be involvedin the deactivation of the ROS (Demming-Adams and Adams,1992). Under normal physiological conditions, glutathione andascorbate are largely found as GSH and ASA, which areregenerated by the reduction of GSSG and ASA (oxidizedforms of glutathione and ascorbate, respectively). Both reac-tions are extremely efficient processes, catalysed by GR andAPX, that dissipate energy and contribute to the adjustment ofATP/NADPH ratios when CO2 fixation is limited (Foyer et al.,1984). Our results revealed that GSH and ASA concentrations,together with total glutathione and ascorbate concentrations,decreased under elevated CO2, indicating that, in agreementwith the enzymatic antioxidant system results, the antioxidantsystem of those plants was not enhanced because those plantsdid not suffer from significant photooxidative damage. The the-sis that the plants did not experience photooxidative stress wasalso confirmed by the increased ASA/DHA and especially by theGSH/GSSG ratio. Foyer et al. (1995) observed that overexpres-sion of GR in poplar plants increased antioxidative capacity andimproved the tolerance for photoinhibition by raising GSH levels



and the GSH/GSSG ratio. Similarly, nonenzymatic antioxidantsrevealed that no statistical differences were observed associatedwith water treatment, corroborating their ability to grow underwater-limited conditions.

According to Demming-Adams and Adams (1992), thedecrease in efficiency of photosynthetic energy conversion (i.e.,photodamage) can result not only in some form of “damage” toPSII but also an increase in thermal energy dissipation (a pho-toprotective process that does not represent damage) that occursprior to the occurrence of any photooxidative process. Nonpho-tochemical quenching enhancement detected in elevated CO2treatments revealed that, regardless of water availability and tem-perature, plants grown at 700 �mol mol−1 had a greater capacityfor nonradioactive dissipation of excitation energy as heat. Assuggested by other authors (Hymus et al., 2001; Kitao et al.,2005), NPQ increase would protect the reaction centres fromphotoinactivation and damage under elevated CO2 treatmentswhere the rate of excitation of PSII was in excess of the rate ofphotochemistry. Thermal dissipation involves a transthylakoidpH difference and the xanthophyll cycle (Demming-Adams andAdams, 1992). In this process, the formation of a pH gradi-ent across the thylakoid membrane activates the de-epoxidationof violaxanthin (V) to zeaxanthin (Z) and antheraxanthin (A),facilitating the thermal dissipation of excess excitation energy.Closer examination of the xanthophyll pigments showed that ele-vated CO2 decreased violaxanthin (V) concentration, whereasantheraxanthin (A) and especially zeaxanthin (Z) concentra-tions were enhanced. This means that the conversion stateof the xanthophyll is increased, indicating de-epoxidation ofvioloxanthin. (A + Z)/(V + A + Z) and the V/(V + A + Z) con-firmed that plants grown under elevated CO2 concentrationshave a greater thermal dissipation capacity (Kurasova et al.,2003). This implies that changes in CO2 assimilation observedat elevated CO2 required a modification in the partitioningof absorbed energy between photochemistry in the thylakoidmembrane and heat dissipation as observed by Hymus et al.(1999).

In conclusion, our results confirmed that elevated CO2induced photosynthetic acclimation caused by reduced Rubiscocontent quantum efficiency. However, we would like to mentionthat the degree of CO2 effect on the carboxylation capacity dif-fered, depending on growth temperature. Interestingly, our dataalso showed that CO2 effect did not interact with water availabil-ity. Lack of statistical differences on gas exchange parameters(together with the antioxidant enzymatic and nonenzymaticmeasurements) highlighted the fact that partially watered plantsadapted their metabolism to available soil water content. Ele-vated CO2 also protected Photosystem II from photodamage bydelivering the excess energy through the modulation of the VAZstatus components. Elevated CO2 induced the depo-oxidationof violaxanthin to zeaxanthin, thus facilitating the thermal dis-sipation that protects the photosynthetic apparatus from thedeleterious effect of excess excitation energy. The analyses ofH2O2, TBARS, and enzymatic and nonenzymatic parametersrevealed that the photoprotective effect of elevated CO2 wasnot caused by improvements in the antioxidant system. Fur-thermore, under elevated CO2 conditions, the rate of superoxide

production decreased and, consequently, the antioxidant defencesystem also diminished.

Acknowledgements

This work was supported by the Spanish Science andTechnology Ministry (BFI2000-0154), the Spanish Scienceand Education Ministry (BFU-2004-05096/BFI and AGL2004-00194/AGR), Fundacion Caja Navarra and the FundacionUniversitaria de Navarra. This work was supported in part bythe European Project PERMED (INCO-CT-2004-509140). Thetemperature gradient tunnels used in this study were funded bythe Spanish Commission of Science and Technology (AMB96-0396). The valuable comments of Paul Lazzeri in reviewingthis manuscript are acknowledged. The valuable help of RafaelMartınez-Carrasco (IRNA-CSIC, Salamanca), Ruth Sagardoy,and Inaki Tacchini (Estacion Experimental de Aula Dei-CSIC,Zaragoza, Spain) is greatly appreciated.

References

Abadia, J., Morales, F., Abadia, A., 1999. Photosystem II efficiency in lowchlorophyll, iron deficient leaves. Plant Soil 215, 183–192.

Aebi, H., 1974. Catalase. In: Bergmeyer, H.V. (Ed.), Methods of EnzymaticAnalysis, vol. 2. Academic Press, New York, ISBN 3-527-25596-6, pp.673–684.

Ainsworth, E.A., Rogers, A., Nelson, R., Long, S.P., 2004. Testing the “source-sink” hypothesis of down-regulation of photosynthesis in elevated [CO2]in the field with single gene substitutions in Glicine max. Agric. ForestMeteorol. 122, 85–94.

Alley, R., Berntsen, T., Bindoff, N.L., Chen, Z., Chidthaisong, A., Friedlingstein,P., Gregory, J., Hegerl, G., Heimann, M., Hewitson, B., Hoskins, B., Joos,F., Jouzel, J., Kattsov, V., Lohmann, U., Manning, M., Matsuno T., Molina,M., Nicholls, N., Overpeck, J., Qin, D., Raga, G., Ramaswamy, V., Ren, J.,Rusticucci, M., Solomon, S., Somerville, R., Stocker, T.F., Stott, P., Souf-fer, R.J., Whetton, P., Wood R.A., Wratt, D., 2007. Climate Change 2007:The Physical Science Basis. Summary of Policymakers. Fourth AssessmentReport of Working Group I. Intergovernmental Panel on Climate Change.Geneva, Switzerland.

Alscher, R.G., Donahue, J.H., Cramer, C.L., 1997. Reactive oxygen species andantioxidants: relationships in green cells. Physiol. Plant. 100, 224–233.

Aranjuelo, I., Irigoyen, J.J., Perez, P., Martınez-Carrasco, R., Sanchez-Dıaz,M., 2005a. The use of temperature gradient greenhouses for studying thecombined effect of CO2, temperature and water availability in N2 fixingalfalfa plants. Ann. Appl. Biol. 146, 51–60.

Aranjuelo, I., Perez, P., Hernandez, L., Irigoyen, J.J., Zita, G., Martınez-Carrasco, R., Sanchez-Dıaz, M., 2005b. The response of nodulated alfalfato water supply, temperature and elevated CO2: photosynthetic down-regulation. Physiol. Plant. 123, 348–358.

Aranjuelo, I., Irigoyen, J.J., Perez, P., Martınez-Carrasco, R., Sanchez-Dıaz,M., 2006. Response of nodulated alfalfa to water supply, temperature andelevated CO2: productivity and water relations. Environ. Exp. Bot. 55,130–141.

Aroca, R., Irigoyen, J.J., Sanchez-Dıaz, M., 2001. Photosynthetic characteris-tics and protective mechanisms against oxidative stress during chilling andsubsequent recovery in two maize varieties differing in chilling sensitivity.Plant Sci. 161, 719–726.

Arp, W.J., 1991. Effect of source-sink relations on photosynthetic acclimationto elevated CO2. Plant, Cell Environ. 14, 869–875.

Asada, K., 1999. The water–water cycle in chloroplast: scavenging of activeoxygens and dissipation of excess photons. Annu. Rev. Plant Physiol. PlantMol. Biol. 50, 601–639.

Bartak, M., Nijs, I., Impens, I., 1996. The effect of long-term exposure of Loliumperenne L. plants to elevated CO2 and/or elevated air temperature on quan-



tum yield of photosystem 2 and net photosynthesis. Photosynthetica 32,549–562.

Bilger, W., Bjorkman, O., 1990. Role of the xanthophylls cycle in photopro-tection elucidated by measurements of light-induced absorbance changes,fluorescence and photosynthesis in leaves of Hedera canariensis. Photo-synth. Res. 25, 173–185.

Carvalho, L.C., Amancio, S., 2002. Antioxidant defense system in plantletstransferred from in vitro to ex vitro: effects of increasing light intensity andCO2 concentration. Plant Sci. 162, 33–40.

Chaves, M.M, Pereira, J.S., 2004. Respuestas de la estabilidad de las plantas alestres multiple y la habilidad de enfrentarse a un ambiente cambiante. In:Reigosa, M.J., Pedrol, N., Sanchez, A. (Eds.), La Ecofisiologıa Vegetal, pp.577–602.

Dat, J., Vandenabeele, S., Vranova, E., Van Montagu, M., Inze, D., VanBreusegem, F., 2000. Dual action of the active oxygen species during plantstress responses. Cell Mol. Life Sci. 57, 779–795.

De las Rivas, J., Abadıa, A., Abadıa, J., 1989. A new reversed phase HPLCmethod resolving all major higher plant photosynthetic pigments. PlantPhysiol. 91, 190–192.

De Luis, I., Irigoyen, J.J., Sanchez-Dıaz, M., 1999. Elevated CO2 enhancesplant growth in droughted N2-fixing alfalfa without improving water status.Physiol. Plant. 107, 84–89.

Demming, B.K., Winter, A., Kruger, A., Czygan, F.C., 1987. Photoinhibitionand zeaxanthin formation in intact leaves. A possible role of the xantho-phylls cycle in the dissipation of excess light energy. Plant Physiol. 84,218–224.

Demming-Adams, B., Adams, W.W., 1992. Photoprotection and anotherresponse of plants to high light stress. Annu. Rev. Plant Physiol. Plant Mol.Biol. 48, 599–626.

Dhindsa, R.S., Plumb-Dhindsa, P., Thorpe, T.A., 1981. Leaf senescence: is corre-lated with increased levels of membrane permeability and lipid peroxidation,and decreased levels of superoxide dismutase and catalase. J. Exp. Bot. 32,93–101.

Erice, G., Irigoyen, J.J., Perez, P., Martınez-Carrasco, R., Sanchez-Dıaz, M.,2006a. Effect of elevated CO2, temperature and drought on photosynthesisof nodulated alfalfa during a cutting regrowth cycle. Physiol. Plant. 126 (3),458–468.

Erice, G., Irigoyen, J.J., Perez, P., Martınez-Carrasco, R., Sanchez-Dıaz, M.,2006b. Effect of elevated CO2, temperature and drought on dry matter par-titioning and photosynthesis before and after cutting of nodulated alfalfa.Plant Sci. 170 (6), 1059–1067.

Ethier, G.J., Livingston, N.J., 2004. On the need to incorporate sensitivity toCO2 transfer conductance into the Farquhar-von Caemmerer-Berry leafphotosynthesis model. Plant, Cell Environ. 27, 137–153.

Farquhar, G.D., Sharkey, T.D., 1982. Stomatal conductance and photosynthesis.Annu. Rev. Plant Physiol. 33, 317–345.

Foyer, C.H., Harbinson, J.C., 1994. Oxygen metabolism and the regulation ofphotosynthetic electron transport. In: Foyer, C.H., Mullineaux, P.M. (Eds.),Causes of Photooxidative Stress and Amelioration of Defence Systems inPlant. CRC, Boca Raton, FL, pp. 1–42.

Foyer, C.H., Anderson, J., Walker, D.A., 1984. Light-dependent reduction ofhydrogen peroxide via the ascorbate-gluthatione cycle in intact spinachchloroplast. In: Sysbema, C. (Ed.), Advances in Photosynthesis Research III,vol. 7. Martinus Nijhoff/Dr. W. Junk, The Hague, The Netherlands, pp. 689–692.

Foyer, C.H., Souriau, N., Perret, S., Lelandais, M., Kunert, K., Pruvost, C.,Jouanin, L., 1995. Overexpression of glutathione reductase but not glu-tathione synthetase leads to increase in antioxidant capacity and resistanceto photoinhibition in poplar trees. Plant Physiol. 109, 1047–1057.

Gilmore, A.M., 1997. Mechanistic aspects of xanthopyll cycle-dependent pho-toprotection in higher plant chloroplasts and leaves. Physiol. Plant. 99,197–209.

Habash, D., Parry, P.M., Parry, M.A.J., Keys, A.J., Lawlor, D.W., 1995. Increasedcapacity for photosynthesis in wheat grown at elevated CO2: the relationshipbetween electron transport and carbon metabolism. Planta 197, 482–489.

Harley, P.C., Loreto, F., Marco, G.D., Sharkey, T.D., 1992. Theoretical consider-ations when estimating the mesophyll conductance to CO2 flux by analysisof the response of photosynthesis to CO2. Plant Physiol. 98, 1429–1436.

Hill, D.W., Kind, A.J., 1993. The effect of type B silica and triethylamineon the retention of drugs in silica based reverse phase high performancechromatography. J. Liquid Chromatogr. 16, 3941–3964.

Hymus, G.J., Ellsworth, D.S., Baker, N.R., Long, S.P., 1999. Does free-air car-bon dioxide enrichment affect photochemical energy use by evergreen treesin different seasons? A chlorophyll fluorescence study of mature loblollypine. Plant Physiol. 120, 1183–1191.

Hymus, G.J., Baker, N.R., Long, S.P., 2001. Growth in elevated CO2 can bothincrease and decrease photochemistry and photoinhibition of photosynthesisin a predictable manner. Dactylis glomerata grown in two levels of nitrogennutrition. Plant Physiol. 127, 1204–1211.

Idso, S.B., Kimball, B.A., 1992. Effects of atmospheric CO2 enrichment onphotosynthesis, respiration and growth of four orange trees. Plant Physiol.99, 341–343.

IPCC, 2001. In: Watson, R., Houghton, J.T., Yihui, D. (Eds.), The ScientificBasis. Third Assessment Report of Working Group I. Cambridge UniversityPress, Cambridge.

Jones, H.G., 1992. Plants and Microclimate: A Quantitative Approach to Envi-ronmental Plant Physiology. Cambridge University Press, Cambridge, UK.

Keys, A.J., 1986. Rubisco: its role in the photorespiration. Physiol. Trans. R.Soc. Lond. 313, 325–336.

Kitao, M., Koike, T., Tobita, H., Maruyama, Y., 2005. Elevated CO2 and limitednitrogen nutrition can restrict excitation energy dissipation in photosystem IIof Japanese white birch (Betula platyphyllos var. japonica) leaves. Physiol.Plant. 125, 64–73.

Krall, J.P., Edwards, G.E., 1992. Relationship between photosystem II activityand CO2 fixation in leaves. Physiol. Plant. 86, 180–187.

Kurasova, I., Kalina, J., Urban, O., Stroch, M., Spunda, V., 2003. Acclimationof two distinct plant, spring barley and Norway spruce, to combined effectof various irradiance and CO2 concentration during cultivation in controlledenvironment. Photosynthetica 41 (4), 513–523.

Leipner, J., Fracheboud, Y., Stamp, P., 1997. Acclimation by suboptimal temper-ature diminishes photooxidative damage in maize leaves. Plant Cell Environ.20, 366.372.

Long, S.P., Ainsworth, E.A., Rogers, A., Ort, D.R., 2004. Rising atmosphericcarbon dioxide: plants FACE the future. Annu. Rev. Plant Biol. 55, 591–628.

Marco, A., Roubelakis-Angelakis, K.A., 1996. The complexity of enzymaticcontrol of hydrogen peroxide concentration may affect the regenerationpotential of plant protoplasts. Plant Physiol. 110, 137–145.

Miyake, C., Yokota, A., 2000. Determination of the rate of photoreduction ofO2 in the water–water cycle in watermelon leaves and enhancement of therate by limitation of photosynthesis. Plant Cell Physiol. 41, 335–343.

Montgomery, D.C., 1984. Designs and Analyses of Experiments, 2nd edition.John Wiley & Sons, New York.

Ort, D.R., Baker, N.R., 2002. A photoprotective role for O2 as an alternativeelectron sink in photosynthesis. Curr. Opin. Plant Biol. 5, 193–198.

Patterson, B.D., MacRae, E.A., Ferguson, I.B., 1984. Estimation of hydrogenperoxide in plant extracts using titanium (IV). Ann. Biochem. 139, 487–492.

Polle, A., Chakrabarti, K., Schumermann, W., Rennenberg, H., 1990. Com-position and properties of hydrogen peroxide decomposing systems inextracellular and total extracts from needles of Norway spruce (Picea AbiesL., Karst.). Plant Physiol. 94, 312–319.

Polle, A., Eiblmeier, M., Sheppard, L., Murray, M., 1997. Response of antioxida-tive enzymes to elevated CO2 in leaves of beech (Fagus sylvatica) seedlingsgrown under a range of nutrient regimes. Plant, Cell Environ. 20, 1317–1321.

Pritchard, S.G., Ju, Z., van Santen, E., Qiu, J., Weaver, D.B., Prior, S.A., Rogers,H.H., 2000. The influence of elevated CO2 on the activities of antioxidantenzymes in two soybean genotypes. Austr. J. Plant Physiol. 27, 1061–1068.

Roden, J., Ball, M., 1996. Growth and photosynthesis of two eucalyptus speciesduring high temperature stress under ambient and elevated [CO2]. GlobalChange Biol. 2, 115–128.

Rogers, A., Ainsworth, E.A., 2006. The response of foliar carbohydrates to ele-vated carbon dioxide concentration. In: Nosberger, J., Long, S.P., Norby,R.J., Stitt, M., Hendrey, G.R., Blum, H. (Eds.), Managed Ecosystems andCO2. Case Studies, Processes and Perspectives. Springer-Verlag, Heidel-berg, Berlin, pp. 293–308.

Rogers, A., Gibon, Y., Stitt, M., Morgan, P.B., Bernacchi, C.J., Ort, D.R., Long,S.P., 2006. Increased C availability at elevated carbon dioxide concentra-



tion improves N assimilation in legume. Plant, Cell Environ. 29, 1651–1658.

Scarascia-Mugnozza, G., De Angelis, P., Matteuci, G., Valentın, R., 1996. Longterm exposure to elevated CO2 in a natural Quercus ilex L. community: netphotosynthesis and photochemical efficiency of PSII at different levels ofwater stress. Plant Cell Environ. 19, 643–654.

Smith, I.K., 1985. Stimulation of glutathione synthesis in photorespiring plantsby catalase inhibitors. Plant Physiol. 79, 1044–1047.

Stitt, M., Krapp, A., 1999. The interaction between elevated carbon dioxide andnitrogen nutrition: the physiological and molecular background. Plant, CellEnviron. 22, 583–621.

Urban, O., 2003. Physiological impacts of elevated CO2 concentration rangingfrom molecular to whole plant responses. Photosynthetica 41 (1), 9–20.

Valladares, F., Pearcey, R.W., 1997. Interactions between water stresses, sun-shade acclimation, heat tolerance and photoinhibition in the sclerophyllHeteromeles arbutifoliar. Plant, Cell Environ. 20, 25–36.

Verhoeven, A.S., Adams III, W.W., Demmig-Adams, B., Croce, R., Bassi, R.,1999. Xanthophyll cycle pigment localization and dynamics during exposureto low temperatures and light stress in Vinca major. Plant Physiol. 120,727–737.

von Caemmerer, S., Farquhar, G.D., 1981. Some relationships between the bio-chemistry of photosynthesis and the gas exchange of leaves. Planta 153,376–387.

Wolfe, D.W., Gifford, R.M., Hilbert, D., Luo, Y., 1998. Integration of photosyn-thetic acclimation to CO2 at the whole-plant level. Global Change Biol. 4,879–893.

Zhou, Y.H., Yu, J.Q., Mao, W.H., Huang, L.F., Song, X.S., Nogues, S., 2006.Genotypic variation of Rubisco expression, photosynthetic electron flow andantioxidant metabolism in the chloroplast of chill-exposed cucumber plants.Plant Cell Physiol. 47 (2), 234–240.

Documents

The mechanism(s) involved in the photoprotection of PSII at