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Environmental and Experimental Botany 78 (2012) 173– 183

Contents lists available at SciVerse ScienceDirect

Environmental and Experimental Botany

journa l h omepa g e: www.elsev ier .com/ locate /envexpbot

elative contribution of photoprotection and anti-oxidative mechanisms toifferential drought adaptation ability in grapevines

lexandros Beis, Angelos Patakas ∗

aboratory of Plant Production, School of Natural Resources and Enterprises Management, University of Ioannina, G. Seferi 2, 30 100 Agrinio, Greece

r t i c l e i n f o

rticle history:eceived 27 June 2011eceived in revised form0 December 2011ccepted 30 December 2011

eywords:ntioxidant enzymesroughtipid peroxidationhotoprotectionhotosynthesisater deficit

a b s t r a c t

A comparative study on contribution of photoprotective and anti-oxidative mechanisms to combineddrought and high radiation intensity stress adaptation was conducted on two grapevine varieties (Vitisvinifera L., cvs Sabatiano and Mavrodafni), differing in their ability for drought adaptation. Plants of bothvarieties were subjected to two different deficit irrigation regimes receiving either 50% (DI50) or 25% (DI25)of fully irrigated control plants. Sabatiano, which is considered as more drought resistant variety, main-tained significant higher values of photosynthetic rate (PN), stomatal conductance (gs) and predawn waterpotential (� PD) compared to Mavrodafni at the beginning of the drought period. Furthermore, Sabatianoshowed significantly lower electron transport rate (ETR), PSII maximum (˚exc) efficiency and signifi-cantly higher non-photochemical quenching (NPQ) values for the same values of � PD and gs respectively,which implies a more efficient dissipation of excess energy via the xanthophyll cycle. On the other hand,photorespiration was higher in Mavrodafni suggesting a possible contribution of this mechanism in thisvariety photoprotection. Drought-induced hydrogen peroxide (H2O2) concentration increased rapidly

in Mavrodafni resulting in higher lipid peroxidation. No significant differences in the up-regulation ofascorbic peroxidase (APX, EC 1.11.1.11) and superoxidase dismutase (SOD, EC 1.15.1.1) enzyme activitiesin response to drought were observed whereas catalase (CAT, EC 1.11.1.6) activity changes in responseto gs followed completely different patterns in two varieties. The similarity between nitric oxide (NO)accumulation and CAT activity increase at the early stages of drought suggests a positive role of NO incatalase up-regulation.

. Introduction

Grapevines grown in the Mediterranean zone are often exposedo a combination of drought and high light intensities (Düring,998; Rodrigues et al., 2008) due to the cloudless summer sky, highvaporative demand and low soil water availability (Patakas et al.,005). It is also well known that grapevine varieties are charac-erized by high heterogeneity concerning their adaptation abilityo such adverse environmental conditions (Schultz, 2003; Duant al., 2007). One of the first physiological responses that resultrom root sensing of soil drying is the decrease in leaf stomatalonductance (Patakas et al., 2010). Stomatal functioning appearso be regulated by multiple signals (chemical, hydraulic or evenlectric) in response to changes in both the soil and aerial environ-ent (Lovisolo et al., 2010). In previous studies it was revealed

hat there are differences in drought signaling mechanismsetween grapevine varieties which are closely related to grapevine

∗ Corresponding author. Tel.: +30 26410 74145; fax: +30 26410 74179.E-mail address: apatakas@cc.uoi.gr (A. Patakas).

098-8472/$ – see front matter © 2012 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2011.12.038

© 2012 Elsevier B.V. All rights reserved.

genotypic ability to withstand water stress conditions (Schultz,2003; Beis and Patakas, 2010).

On the other hand, stress-induced decline in stomatal and mes-ophyll conductance (Centritto et al., 2009) also results in decreasingCO2 concentration in chloroplasts, thus increasing plant’s suscep-tibility to photoinhibition. In fact, under limiting conditions of CO2fixation the rate of reducing power production can overcome therate of its use in photosynthetic electron transport chain, thusdamaging the photosynthetic apparatus. In order to prevent theproduction of excess reducing power plants have developed differ-ent protection mechanisms. Regulated thermal dissipation (NPQ)in the light-harvesting complexes, involving the xanthophyll andthe lutein cycle, is believed to safely remove excess absorbed light;thereby protecting the reaction centres from photo-oxidative dam-age (Müller et al., 2001; Li et al., 2009). Although its role is not totallyclear yet, an increase in photorespiration under drought conditionsmay also contribute in photosynthetic apparatus protection againstlight damage (Asada, 1999; Wingler et al., 1999; Ort and Baker,

2002). Moreover, a reduction in light absorbance by adjustingchlorophyll content and/or antenna size is reported as an effectiveprotection mechanism against photoinhibition (Kyparissis et al.,2000; Munne-Bosch and Alegre, 2000; Galmes et al., 2007). Since

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74 A. Beis, A. Patakas / Environmental an

xcess energy generates reactive oxygen species (ROS), drought tol-rance is expected to be positively correlated with an increasedapacity to scavenge or detoxify ROS (Smirnoff, 1993; Foyer et al.,994). Indeed, the ability for rapid up-regulation of the antioxi-ant system was found to be closely associated with decreasedxidative damage and thus drought tolerance in various speciesZhang and Kirkham, 1996; Jiang and Zhang, 2002; Lima et al.,002; Ramachandra et al., 2004; Sofo et al., 2005; Sanchez-Diazt al., 2007; Aganchich et al., 2009; Ozkur et al., 2009; Wang et al.,009). However, the role of the above-mentioned photoprotectionechanisms in determining drought adaptation in grapevines is

till unclear. In particular, the ability of grapevines to decrease lightbsorption via chlorophyll adjustment under combined droughtnd high irradiance conditions remained controversy. Chaumontt al. (1997) reported similar leaf chlorophyll content in both irri-ated and non irrigated grapevines while Maroco et al. (2002) andlexas et al. (2000) found a significant reduction of total chloro-hyll content in drought stressed grapevines. Furthermore, despitehe fact that the effect of drought on PSII photochemical efficiency,hotorespiration and thermal dissipation (NPQ) has been exam-

ned in some detail (Flexas et al., 1999, 2002; Medrano et al., 2002;aroco et al., 2002; de Souza et al., 2005), there are still contrasting

nformation concerning the relative contribution and/or coordina-ion of these photo protective mechanisms in drought adaptationn grapevines. A possible explanation of these discrepancies coulde based on differences in the degree of stress applied (Flexas et al.,002), the varieties examined (Tardieu and Simonneau, 1998), asell as the rate of drought stress imposition (Flexas et al., 1999).

n addition, no information exists concerning possible differencesn antioxidant system up-regulation patterns between varieties as

ell as their relative importance in drought tolerance.Thus, the aim of this study was to elucidate the differences in

hotoprotection mechanisms between grapevine varieties as wells to evaluate if these differences could be related to grapevine’senotypic variability in adaptation to drought.

. Materials and methods

.1. Plant material and growth conditions

The experiment was conducted during two consecutive growingeriods (2006 and 2007) at the experimental station of the Uni-ersity of Ioannina, located in Agrinio, Western Greece (38◦37′ N;1◦24′E). Three-year-old, own-rooted grapevine plants of two vari-ties (Vitis vinifera L. cvs Sabatiano and Mavrodafni) were grownnto 25 L pots under a permanent rain shelter. The selection of theserapevines varieties was based on their different ability for droughtdaptation; Sabatiano, a white wine variety, originating from morerid environment is considered as more adapted to drought, whilehe red wine variety Mavrodafni was proven to be more sensi-ive to water stress (Beis and Patakas, 2010). The pots were filledith a mixture of soil, sand, peat and vermiculite (30%, 10%, 30%

nd 30% v/v, respectively) and were wrapped in aluminium foilo minimise radiation-induced heating of the root system. Duringhe winter the plants were pruned to a single-bud spur. After budreak, the single shoot of each plant was trained vertically, while

aterals and clusters were removed immediately after formation.uring the first two months all plants were uniformly irrigated

o soil capacity on a daily basis. In order to achieve this, initiallyduring the first two days) plants were irrigated until the watertarted to leach from the holes located in the bottom of the pots.

hereafter, the daily amount of irrigation water needed to maintainoil capacity was being determined gravimetrically by estimatinghe amount of water consumed. This was calculated by weighingen randomly selected pots every evening. Then the plants of each

rimental Botany 78 (2012) 173– 183

variety were divided randomly into three uniform groups, each oneconsisting of 20 plants. Three different water regimes were appliedin each group over a 24-day period. In particular, the first groupwas irrigated to soil capacity (control plants, CON); the second andthe third group were deficit-irrigated receiving 50% (DI50) and 25%(DI25) respectively of water given to control plants. The amount ofwater given in control plants was calculated gravimetrically usingten randomly selected potted plants as described above. After the24-day drought period all stressed plants were fully irrigated to soilcapacity.

The same experiment was repeated three times during each oneof the two consecutive years (2006 and 2007). Since no significantdifferences between the two years data were observed the resultspresented here referred to the three experiments conducted during2007.

2.2. Plant water relations, gas exchange and chlorophyllfluorescence measurements

Measurements of physiological parameters were performed inall treatments, every three days, prior to irrigation. In particular,predawn water potential (� PD) was measured using a Scholanderpressure chamber (SKPD 1400, Skye Instruments, Powys, UK) onthree fully expanded leaves per treatment obtained from differ-ent plants. Stomatal conductance (gs) and photosynthetic rate (PN)were measured daily from 9 to 10 a.m. at saturating light inten-sity (PAR = 1100 �mol m−2 s−1) in ten leaves per treatment using aportable gas exchange system (LCpro+ ADC BioScientific, Herts, UK).Chlorophyll (chl) fluorescence parameters were measured witha portable pulse amplitude modulation fluorometer (PAM-2000;Walz, Effeltrich, Germany) on four leaves per treatment, every threedays. Minimum chl fluorescence (Fo) was measured using modu-lated light of sufficiently low intensity (<0.1 �mol m−2 s−1) in orderto prevent significant variable fluorescence while maximum chl flu-orescence (Fm) was determined in dark-adapted leaves followinga 0.8 s saturating light pulse at 10,000 �mol m−2 s−1. Steady-statefluorescence (Fs) was recorded after continual illumination withactinic light (1000 �mol photons m−2 s−1) while a second satu-rating pulse at 10,000 �mol m−2 s−1 was imposed to determinemaximum chl fluorescence in the light-adapted state (Fm

′). Max-imum quantum efficiency of PSII photochemistry was calculatedat predawn as Fv/Fm, where Fv is Fm − Fo. The PSII actual (˚PSII)and maximum (˚exc) efficiency was calculated as (Fm

′ − Fs)/Fm′

and Fv′/Fm

′ respectively (Genty et al., 1989; Harbinson et al., 1989;Baker, 2008). The fraction of light absorbed that is dissipated in thePSII antenna (1 − ˚exc) was also determined (Demmig-Adams andAdams, 1996; Morales et al., 1998). Electron transport rate (ETR)was calculated by multiplying ˚PSII by PPFD (photosynthetic pho-ton flux density incident on the leaf) by 0.5 (assuming an equaldistribution of excitation between PSI and PSII) and by 0.84 which isfoliar absorbance coefficient more common for C3 plants (Björkmanand Demmig, 1987). Non-photochemical quenching (NPQ), wascalculated using the equation: (NPQ = (Fm/Fm

′) − 1).Chlorophyll content index (CCI) was measured with Chlorophyll

Content Meter (CCM-200, Opti-Sciences, Inc 8 Winn Avenue, Hud-son USA) in forty leaves obtained from ten different plants pertreatment. These measurements were conducted in the same leavesthat were previously used for gas exchange and fluorescence anal-ysis. Photorespiration (PR) was estimated according to Valentiniet al. (1995) every three days from combined measurement of flu-orescence and gas exchange on four leaves per treatment using the

formula:

PR = 112

[ETR − 4(PN + DR)]

d Experimental Botany 78 (2012) 173– 183 175

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Fig. 1. Changes in (A) predawn leaf water potential (� PD), (B) photosynthetic rate(PN) and (C) stomatal conductance (gs), in two grapevine varieties, Mavrodafni(M) and Sabatiano (S), subjected to different irrigation treatments (CON, DI50 andDI25) over a 24-day drought period. Data shown refer to the three replicates ofthe same experiment which conducted during 2007. Each � PD value represents

A. Beis, A. Patakas / Environmental an

here DR is the rate of dark respiration which was derivedrom light response curves at PPFD = 0 �mol m−2 s−1 (Pallioti andartechini, 2001).

.3. Antioxidant enzymes

Ascorbic peroxidase (APX, EC 1.11.1.11), Guaiacol peroxi-ase (GPX, EC 1.11.1.7) and Superoxide dismutase (SOD, EC.15.1.1) enzyme activities were measured in four leaves perreatment, collected at predawn, three times during the droughteriod (days 1st, 12th and 24th), whereas Catalase (CAT, EC.11.1.6) activity was measured four times during the droughteriod (days 1st, 6th, 12th and 24th). Collected leaf samplesere immediately frozen in liquid nitrogen and then stored

t −80 ◦C.Catalase activity was determined using the method described

y Aebi (1984). The decomposition of hydrogen peroxideH2O2) was followed by the decline in absorbance at 240 nmε = 39.4 M−1 cm−1) and 25 ◦C for 1 min. The decrease in thebsorption was followed for 1 min and �mol H2O2 destroyeder min was defined as one unit of CAT. Guaiacol peroxidaseGPX) activity was determined following the method of Hemedand Klein (1990). GPX activity was calculated following the oxi-ation of guaiacol at 470 nm using an extinction coefficient of6.6 mM−1 cm−1. Enzyme activity was expressed as �moles ofuaiacol oxidized min−1 mg−1 protein. Ascorbic peroxidase (APX)ctivity was assayed by following the decrease in absorbance at90 nm (ε = 2.8 mM−1 cm−1) for 1 min (Nakano and Asada, 1981).ne enzyme unit was defined as �mol of oxidized ascorbatein−1 mg−1 protein. Superoxide dismutase (SOD) activity was

etermined by the method of Beauchamp and Fridovich (1971)y measuring the capacity of the enzyme extract to inhibit thehotochemical reduction of nitro blue tetrazolium (NBT) to blueormazan. One unit of SOD activity was defined as the amountf enzyme required to cause 50% inhibition of the rate of NBTeduction at 560 nm. The amount of soluble proteins (SP) was quan-ified using the method of Bradford (1976). Enzyme activity wasxpressed as units mg−1 protein. All spectophotometric analysesere conducted by a Hitachi U-1100 spectrophotometer at room

emperature.

.4. Lipid peroxidation assay, H2O2 and NO content

Thiobarbituric acid (TBA) and hydrogen peroxide (H2O2) con-entrations were determined in four leaves per treatment, collectedt predawn at days 1st, 12th and 24th of the drought period whereasitric oxide (NO) concentration measurements were performed

our times during each drought period (days 1st, 6th, 12th and4th). The TBA test, which determines malondialdehyde (MDA)s an end product, was used to analyse lipid peroxidation (Heathnd Packer, 1968). The MDA content was calculated using itsolar extinction coefficient of 155 mM−1 cm−1 and expressed as

mol mg−1 protein. H2O2 and NO content were determined usinghe methodology described by Zhou et al. (2005) and Loreto andelikova (2001) respectively.

.5. Statistical analyses

All data were analysed by using two way analysis of varianceANOVA) to test the effect of different water regimes and varieties

n physiological and biochemical parameters. Duncan’s multipleange test (P < 0.05) were carried out to test the significance of dif-erences between treatments means using SPSS version 17.0 for

indows (SPSS Inc., Chicago, IL, USA).

the mean ± S.E. of 12 measurements (4 leaves × 3 replicates); PN and gs data aremeans ± S.E. of 30 measurements (10 leaves × 3 replicates). For each day, differentletters indicate statistically significant differences between treatments at p < 0.05.

3. Results

Predawn leaf water potential (� PD) decreased progressivelyin deficit irrigated compared to control treatments in both vari-eties throughout the drought period (Fig. 1A). As it was expected,

176 A. Beis, A. Patakas / Environmental and Experimental Botany 78 (2012) 173– 183

Fig. 2. Relationship between stomatal conductance (gs) and photosynthetic rate(PN) in the two grapevine varieties, Mavrodafni (M) and Sabatiano (S), subjectedto different irrigation treatments (CON, DI50 and DI25). Data are means ± S.E. forbdi

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Fig. 3. Changes in (A) PSII actual efficiency (˚PSII), (B) electron transport rate(ETR) and (C) non photochemical quenching (NPQ) in the two grapevine varieties,Mavrodafni (M) and Sabatiano (S), subjected to different irrigation treatments (CON,DI50 and DI25) over a 24 days drought period. Data shown refer to the three replicatesof the same experiment which conducted during 2007. Each value represents themean ± S.E of 12 measurements (4 leaves × 3 replicates). Different letters indicate

oth x and y-axes of 30 measurements (10 × 3 replicates). Probability (p-values)etermined by ANCOVA for each main effect (Treatment and gs) and their interaction

s shown.

ignificant differences in � PD between DI50 and DI25 treatments inoth varieties were observed. Mavrodafni stressed plants exhibited

steeper decline in � PD compared to Sabatiano irrespective theater regime applied. On the contrary, Sabatiano stressed plants

eemed to maintain higher values of photosynthetic rate (PN) andtomatal conductance (gs) compared to Mavrodafni at the begin-ing of the drought period (days 0–12) while no differences PN ands between the two varieties were evident as water stress becameevere (Fig. 1B and C). The relationship between PN and gs revealedo significant differences between the two varieties (Fig. 2).

PSII actual efficiency (˚PSII) and electron transport rate (ETR)ecreased progressively during the drought period in both varietiesFig. 3A and B) whereas an opposite trend was observed regardingon-photochemical quenching (NPQ) (Fig. 3C). However, Sabatianoxhibited significantly higher NPQ and lower ETR values respec-ively for the same value of leaf � PD compared to Mavrodafni inoth water regimes (Fig. 4). Similar results were also obtained inI50 irrigation treatment when stomatal conductance was used asrought stress indicator (Fig. 5A and C). On the contrary, no signif-

cant differences in ETR and NPQ values in response to gs changesetween varieties occurred in DI25 treatment (Fig. 5B and D). Fur-hermore, the fraction of light absorbed that is dissipated in theSII antenna (1 − ˚exc) was significant higher in Sabatiano stressedlants (Fig. 6).

Drought did not affect chlorophyll content index (CCI) in Saba-iano (Table 1). By contrast, chlorophyll content in Mavrodafniecreased significantly at the end of drought period (day 24th)

n DI25 treatment. Changes in predawn Fv/Fm ratio in all treat-ents were similar to those of chlorophyll content with onlyavrodafni plants to exhibit significant lower values of Fv/Fm, inI25 treatment at the end of the drought period. Dark respiration

DR) increased in both varieties under drought conditions (Table 1).abatiano exhibited a steeper decline in photorespiration result-ng in significant lower values at the end of the drought periodompared to Mavrodafni. Both ETR/PN and ETR/(PN + DR + PR) ratiosncreased more markedly in Mavrodafni in response to drought.

n the contrary, Sabatiano ETR/(PN + DR + PR) ratio remained almostnaffected during the drought period.

Hydrogen peroxide (H2O2) content increased in both varieties inesponse to drought (Fig. 7A). However, this increase was higher in

statistically significant differences between treatments each day at p < 0.05.

Mavrodafni plants resulting in significantly higher levels of H2O2concentration in both DI50 and DI25 treatments at the end of thedrought period compared to Sabatiano (Fig. 7A). Malondialdehyde

(MDA) content changes in relation to the drought period followedalmost similar patterns with that of H2O2 concentration (Fig. 7B).

A. Beis, A. Patakas / Environmental and Experimental Botany 78 (2012) 173– 183 177

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ig. 4. Changes in electron transport rate (ETR) (A, B), and non-photochemical quenarieties, Mavrodafni (M) and Sabatiano (S), subjected to two deficit irrigation treaturing 2007. Best-fit regressions (r2) and p-values determined by ANCOVA for each

Under mild drought conditions (0.10 < gs < 0.30 mol H2O m−2 s−1

ovisolo et al., 2010) catalase (CAT) activity increased significantlyn Sabatiano plants whereas in Mavrodafni CAT activity responseso decreasing leaf stomatal conductance followed the oppositeattern (Fig. 8A). However, under severe drought conditionsgs < 0.05 mol H2O m−2 s−1) no significant differences betweenhe two varieties regarding CAT activity were observed. Guaiacoleroxidase (GPX) activity was inversely related to drought stress

ntensity in both grapevine varieties, showing a gradual decreasender prolonged drought (Fig. 8B). On the other hand, both ascor-ic peroxidase (APX) and superoxide dismutase (SOD) activities

ncreased markedly at mild drought stress; this increase beingore pronounced under severe drought stress in both varieties

Fig. 8C and D). The relationship between stomatal conductancend nitric oxide production indicated a more rapid increase in NOn Sabatiano compared to Mavrodafni at the onset of the droughttress (Fig. 9).

. Discussion

The significant differences in ETR depression rate and thermalissipation (NPQ) increase in response to � PD between the twoarieties (Fig. 4) suggest that occurrence of different mechanismsegarding their adaptation to drought. In particular, Sabatianoxhibited an earlier and steeper decrease in ETR and a more rapid

ncrease in NPQ under prolonged stress compared to Mavrodafni.onsequently, in Sabatiano these two procedures seem to respondore quickly and directly to restriction in CO2 availability in the

hloroplast, caused by stomatal closure, a fact that implies that in

(NPQ) (C, D), in relation to predawn leaf water potential (� PD) in the two grapevine (DI50) (A–C) and (DI25) (B–D). Data represent the three replicates which conducted

effect (Treatment and � PD) and their interactions are shown.

this variety the drought-induced dawn regulation of ETR is com-pensated for by increased thermal dissipation. An increase in NPQcould lead in the reduction of the quantum yield of PSII photochem-istry thus maintaining a balance with electron flow requirementsfor carbon metabolism. The higher genotypic xanthophyll depen-dent photo-protective capacity has already been reported to bepositively related to adaptation ability to drought in grapevinesvarieties (Medrano et al., 2002). This is consistent with our previousresults, indicating greater drought resistance ability in Sabatianocompared to Mavrodafni (Beis and Patakas, 2010). However, theability of predawn water potential to accurately describe grapevinewater status have been questioned mainly due to already knowngenotypic differences in leaf water potential responses to drought(Beis and Patakas, 2010). The latter resulted in grapevine varietyclassification as either isohydric or anisohydric (Schultz, 2003).It was suggested that stomatal conductance could be more reli-able and thus preferable indicator of plant water status conferringthe capability to overcome the above-mentioned genotypic differ-ences (Lovisolo et al., 2010). Using this drought stress indicator,the fraction of light absorbed that is dissipated in the PSII antenna(1 − ˚exc) was significant higher in Sabatiano than Mavrodafni irre-spective the water regime applied (Fig. 6), a fact that confirmsthe occurrence of different strategies of photoprotection in thetwo varieties. As far as the influence of different water regimesin discrimination ability of photoprotection mechanisms is con-

cerned, our data revealed similar patterns of ETR and NPQ changesin response to gs with those in response to � PD only at gradualimposition of plant water stress (DI50 treatment) (Figs. 4 and 5).On the other hand, when drought intensity increased rapidly (DI25

178 A. Beis, A. Patakas / Environmental and Experimental Botany 78 (2012) 173– 183

Fig. 5. Changes in electron transport rate (ETR) (A, B) and non-photochemical quenching (NPQ) (C, D) in response to stomatal conductance (gs) in the two grapevine varieties,M I50) (A2 and g

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avrodafni (M) and Sabatiano (S), subjected to two deficit irrigation treatments (D007. Probability (p-value) determined by ANCOVA for each main effect (treatment

reatments) the differences in photoprotective behaviour betweenhe two varieties tend to eliminate indicating that the rate ofrought stress imposition might affects the discrimination abil-

ty of plants adaptation mechanisms. These results might explainhe controversial results obtained in the literature regarding the

echanisms of drought adaptation in grapevine varieties (Chavest al., 2010; Lovisolo et al., 2010). From the methodological point ofiew it seems that the gradual imposition of stress might be consid-red as more suitable to elucidate possible differences in droughtdaptation strategies.

Sabatiano exhibited significantly higher NPQ values comparedo Mavrodafni at the end of drought period (day 24) in DI25 treat-

ent while no differences in ETR and actual PSII efficiency (˚PSII)alues were revealed (Fig. 3). A possible explanation for this dis-repancy could be based on the significant reduction in chlorophyllontent observed in leaves of Mavrodafni plants (Table 1) at the endf drought period which reduces the amount of photons absorbedy leaves. These differences in chlorophyll content might also coin-ide with the significant lower predawn Fv/Fm ratios observed inavrodafni plants under severe drought conditions (Table 1). How-

ver, increasing evidences tend to confirm that changes in lightarvesting through chlorophyll degradation plays only a minor role

n photoprotection (Baroli et al., 2003) and that chlorophyll con-entration changes significantly affect light absorption only whenhlorophyll loss is very important (Morales et al., 1991; Abadía etl., 1999). In our results, despite its significant reduction, Fv/Fm ratio

–C) and (DI25) (B–D). Data represent the three replicates which conducted durings) and their interactions is shown.

remained relatively high in absolute values in stressed Mavrodafniplants (0.78). Flexas et al. (2001) reported a curvilinear relationshipbetween Fv/Fm and the actual number of functional PSII units sug-gesting that a decrease in this ratio could result in a negligible photoinactivation of functional PSII units. Taking into consideration thatstressed plants were able to fully recover normal physiologicalparameters of well irrigated conditions only a few hours after irriga-tion (data not shown), it can be concluded that the predawn Fv/Fm

decrease in Mavrodafni vines could not be considered as perma-nent damage to photosynthetic apparatus and consequently couldnot be attributed to chlorophyll degradation. This is consistent withothers results indicating that permanent photoinhibition is rare ingrapevines, even under severe drought (Flexas et al., 2002; Marocoet al., 2002; de Souza et al., 2003). Thus, our results suggest signifi-cant differences regarding the dissipation ability between the twovarieties. Sabatiano seemed to dissipate more efficiently the excessenergy via the xanthophyll cycle while in Mavrodafni the possibleexistence of an additional protective mechanism mediated Fv/Fm

reduction under severe drought conditions remained to be elu-cidated. Evidences suggesting the occurrence of such mechanismin some other Mediterranean species have already been reportedrecently (Peguero-Pina et al., 2009).

Taking into consideration the differences in NPQvalues between the two varieties, the higher ETR inMavrodafni under moderate water stress conditions (0.05 molH2O m−2 s−1 < gs < 0.3 mol H2O m−2 s−1) (Fig. 5A and C) could be

A. Beis, A. Patakas / Environmental and Experimental Botany 78 (2012) 173– 183 179

Table 1Changes in chlorophyll content index (CCI), maximal photochemical efficiency (Fv/Fm), dark respiration (DR), photorespiration (PR), electron transport rate (ETR) and net CO2

assimilation ratio (ETR)/(PN) and (ETR)/(PN + DR + PR) ratio, in the two grapevines varieties Mavrodafni and Sabatiano subjected to different irrigation treatments (CON, DI50

and DI25).

Variety Water regime Chl (CCI) Fv/Fm Dark respiration(�mol CO2 m−2 s−1)

Photorespiration(�mol CO2 m−2 s−1)

ETR/Pn ETR/(Pn + DR + PR)

Day 0Mavrodafni CON 26.54 ± 1.36a 0.823 ± 0.0014a 0.55 ± 0.076a 12.12 ± 0.33a 12.05 7.30Sabatiano CON 25.33 ± 1.16a 0.823 ± 0.0013a 0.60 ± 0.073a 11.25 ± 0.24a 12.31 7.48

Day 12Mavrodafni CON 28.06 ± 1.86a 0.833 ± 0.0032 0.54 ± 0.075a 11.35 ± 0.67bc 12.23 7.37

DI50 28.20 ± 1.37a 0.833 ± 0.0036 0.73 ± 0.120ab 10.46 ± 1.23bc 22.37 9.22DI25 24.66 ± 1.30b 0.836 ± 0.0031 0.96 ± 0.113b 14.39 ± 0.30a 27.84 9.10

Sabatiano CON 27.12 ± 1.77a 0.823 ± 0.0072 0.63 ± 0.035ab 11.81 ± 0.63bc 13.51 7.53DI50 26.92 ± 1.70a 0.831 ± 0.0024 0.91 ± 0.074ab 10.22 ± 0.25c 15.34 7.96DI25 25.26 ± 1.01a 0.830 ± 0.0015 0.97 ± 0.083b 12.32 ± 0.51b 24.87 8.79

Day 24Mavrodafni CON 27.83 ± 1.89a 0.829 ± 0.0038a 0.55 ± 0.035a 12.81 ± 0.64a 12.78 7.29

DI50 27.45 ± 1.69a 0.817 ± 0.0013a 1.16 ± 0.106bc 7.37 ± 0.08b 18.24 7.79DI25 19.30 ± 1.31b 0.785 ± 0.0022b 0.78 ± 0.083ab 5.08 ± 0.15c 37.13 9.22

Sabatiano CON 26.08 ± 0.76a 0.826 ± 0.0021a 0.79 ± 0.084ab 12.75 ± 0.43a 13.80 7.56DI50 26.66 ± 1.22a 0.825 ± 0.0036a 1.24 ± 0.113c 3.97 ± 0.42d 9.39 6.29DI25 25.10 ± 1.35a 0.824 ± 0.0028a 0.91 ± 0.091abc 3.88 ± 0.20d 15.04 7.40

Two-way ANOVA (p-values)Day 12Varieties (V) 0.350 0.064 0.329 0.251Watering regime (W) 0.134 0.345 0.019 0.001V × W 0.529 0.555 0.770 0.140

Day 24Varieties (V) 0.410 0.009 0.132 <0.001Watering regime (W) 0.001 0.005 0.002 <0.001V × W 0.037 0.010 0.795 0.001

All data are means ± S.E. obtained from 12 (4 leaves × 3 replicates) measurements for Fv/Fm, PR and DR and from 30 (10 leaves × 3 replicates) measurements for CCI. Meansf an’s td

afcb(oeptaimtmvcouctabdorcprp

tM

Hanaux et al., 2005; Alboresi et al., 2009), malondialdehyde (MDA)concentration was significantly higher in Mavrodafni compared toSabatiano plants (Fig. 7A). In order to cope with oxidative dam-age, plants have developed an antioxidant system that includes

Fig. 6. Relationship between stomatal conductance (gs) and the fraction of light

ollowed by different letters are significantly different (p < 0.05) according to a Duncrought period.

ttributed to either higher photosynthetic rate or to higher abilityor photorespiration. However, the relationship between stomatalonductance and photosynthetic rate revealed no differencesetween the two varieties concerning photosynthetic efficiencyFig. 2). Since no differences in PN between the two varietiesccurred, it would be expected that Mavrodafni plants wouldxhibit significantly higher photorespiration. Indeed, photores-iration rate in Mavrodafni was significantly higher comparedo Sabatiano (Table 1) resulting in maintenance of higher ETRnd ˚PSII (Fig. 3A and B). The lower rate of photorespirationn Sabatiano implies that this mechanism seems to play only a

inor role in photoprotection of PSII in this variety. However,he relative significance of photorespiration as a photoprotective

echanism could be more accurately evaluated by estimating thealues of ETR/PN ratio. Higher values of this ratio in Mavrodafniompared to Sabatiano seem to confirm the predominant rolef photorespiration in this variety (Table 1). Furthermore, thenbalance between electrons generated photosynthetically andonsumed in photosynthesis, dark respiration and photorespira-ion was more evident in Mavrodafni plants suggesting that anlternative sink for electrons, such as the Mehler reaction, maye more important in this variety especially under more severerought stress conditions. Thus, it would be expected a higher riskf oxidative damage in this variety since electrons in excess couldeact with O2 generating ROS (Lawlor, 2002). On the contrary theonstancy of ETR/(PN + DR + PR) ratio at the end of the droughteriod in Sabatiano implies that the sum of photosynthesis, darkespiration and photorespiration fully accounted for the electrons

roduced photosynthetically (Valentini et al., 1995).

A higher photorespiration rate is known to increase oxida-ive load in tissues, since this process generates ROS. Indeed,

avrodafni exhibited higher H2O2 values compared to Sabatiano

est. Probability (p-value) of two way analysis is shown for 12th and 24th day of the

at the end of drought period (Fig. 7B). Since cell membranes arethe first targets of ROS (Smirnoff, 1993; Zhang and Kirkham, 1996;

absorbed that is dissipated in the PSII antenna (1 − ˚exc) in the two grapevine vari-eties subjected to different irrigation treatments (CON, DI50 and DI25). Data shownrefer to the three replicates which conducted during 2007. Best-fit regressions (r2)and p-values determined by ANCOVA for each main effect (variety and gs) and theirinteraction are shown.

180 A. Beis, A. Patakas / Environmental and Experimental Botany 78 (2012) 173– 183

Fig. 7. Changes in (A) malondialdehyde (MDA) and (B) hydrogen peroxide (H2O2)concentrations in grapevine varieties Mavrodafni (M) and Sabatiano (S) subjectedto different irrigation treatments (CON, DI50 and DI25). Vertical bars indicatemfp

bnmraitbiitaeiaaeeis

Fig. 8. Changes in (A) catalase (CAT), (B) guaiacol peroxidase (GPX), (C) ascorbicperixidase (APX) and (D) superoxidase dismutase (SOD) activities in response tostomatal conductance (gs), in the two grapevines varieties subjected to differentirrigation treatments (CON, DI and DI ). Data are means ± S.E for both x and y-

eans ± S.E. of 12 measurements (4 leaves × 3 replicates). At each sampling day, barsollowed by different letters denote statistical differences between all treatments at

< 0.05.

oth enzymatic (CAT, GPX, APX, SOD) (Foyer and Noctor, 2005) andon-enzymatic compounds (Apel and Hirt, 2004). The capacity toaintain higher levels of antioxidants and/or the capacity for up-

egulation of antioxidant enzymes is closely related to genotypicbility for adaptation to various environmental stresses includ-ng drought (Ramachandra et al., 2004). In our results an oppositerend in catalase (CAT) activity in response to drought intensityetween the two grapevine varieties was revealed (Fig. 8A). CAT

s known to be primarily localized in the peroxisomes where it isnvolved in removing the bulk H2O2 generated by photorespira-ion. Thus, the reduction in CAT activity in Mavrodafni might bessociated with the more rapid accumulation of H2O2 in this vari-ty (Fig. 7B). Accumulating evidence suggest that H2O2 implicatedn redox signaling, acting as secondary message in hormonal medi-ted stomatal responses to drought. A pronounced effect of H2O2ccumulation on guard cells was first reported in Vicia Faba where

xogenous application of H2O2 induced stomatal closure (Zhangt al., 2001). Taking into account the above-mentioned role of H2O2n stomatal closure it would be expected significant differences intomatal responses to drought between the two varieties to occur.

50 25

axes of 12 measurements (4 leaves × 3 replicates). Probability (p-values) determinedby ANCOVA for each main effect (Variety and gs) and their interactions is shown.

A. Beis, A. Patakas / Environmental and Expe

Fig. 9. Changes in leaf nitric oxide (NO) content in relation to stomatal conductance(gs) in the two grapevine varieties subjected to different irrigation treatments (CON,Dsf

IdsadaucbbmlanrawaitPa

dirsctppticCiii

I50 and DI25). Each point represents the mean ± S.E for both x and y-axes of 12 mea-urements (4 leaves × 3 replicates). Probability (p-values) determined by ANCOVAor each main effect (Variety and gs) and their interactions is shown.

ndeed, Mavrodafni exhibited a steeper decline in stomatal con-uctance compared to Sabatiano during the first stages of droughttress (Fig. 1C). In more susceptible to water stress varieties, suchs Mavrodafni, this mechanism of rapid decrease in stomatal con-uctance and thus in transpirational water losses might considereds more efficient for survival under drought conditions. However,nder severe drought condition, when H2O2 and thus oxidative riskonsiderably increased, the up-regulation of CAT activity seems toe adequate for effectively reducing ROS in this variety as indicatedy the sharp decrease in H2O2 concentration observed in DI25 treat-ent at the end of drought period (Fig. 7). On the other hand, the

ower photorespiration rate in relation to earlier increase in CATctivity in response to drought in Sabatiano coincides with the sig-ificant lower H2O2 production and accumulation rate (Fig. 7B). Theapid scavenging of reactive oxygen species in this variety mightlso serve in maintaining electron flow from PSII to PSI via theater–water cycle which is known to generate a �pH gradient

cross the thylakoid (Asada, 1999). The later is considered of greatmportance in order to sustain xanthophyll cycle/�pH-dependenthermal energy dissipation in the antenna pigment complexes ofSII (Chen and Cheng, 2003) which was found to be the preponder-nt photo protective mechanism in this variety.

Up-regulation of antioxidant enzymes activity in response torought is reported to be a complex process mediated by several

ntermediates. Recently Zhang et al. (2003) and Farooq et al. (2009)evealed NO contribution in increasing CAT activity in rice leavesubjected to osmotic stress. In our results the similarity in both NOoncentration increase (Fig. 9) and CAT up-regulation (Fig. 8A) pat-erns at the beginning of drought period (gs > 0.2 mol H2O m−2 s−1),rovide evidences of a positive role of NO in CAT up-regulation. Inarticular, the earlier rapid increase in NO concentration in Saba-iano at the beginning of drought cycle (Fig. 9) coincides with thencrease in CAT activity while in Mavrodafni the lack of signifi-ant increase in NO seemed to correspond to the relative stable

AT activity under mild drought conditions. However, under more

ntense drought conditions (gs < 0.2 mol H2O m−2 s−1) the increasen NO accumulation seemed not to be adequate to sustain anncrease in CAT activity. A possible explanation could be based on

rimental Botany 78 (2012) 173– 183 181

the already proven CAT sensitivity to photoinactivation. Reductionin CAT activity is greatly enhanced under more intense stress condi-tions such as prolonged drought via protein synthesis suppression(Zhang and Kirkham, 1996; Sofo et al., 2005).

Peroxidases in grapevines are characterized by a high degree ofpolymorphism (Ros Barcelo et al., 2003), the extent varying withthe genotype, plant organ and even with the developmental stage(Perez and Burgos, 2004). The fact that the guaicol peroxidase (GPX)activity tends to decline with drought in both varieties (Fig. 8B)suggests that this enzyme have a negligible antioxidative func-tion in grapevines, which is consistent with the results reportedin previous studies (Ros Barcelo et al., 2003). On the other hand,superoxidase dismutase (SOD) activity significantly increased inboth varieties in response to drought intensity (Fig. 8D). Since SODis located in mitochondria as well as in chloroplasts it can be spec-ulated that the increase in SOD activity may be related not only tothe observed increase in dark respiration (Table 1) but also to anincreased rate of photo-reduction of oxygen in chloroplasts. Theobserved significant increase in ascorbic oxidase (APX) activity inboth varieties might also contribute in the effective detoxificationof reactive oxygen species in chloroplasts.

5. Conclusion

Our results revealed significant differences in the mechanismsof drought adaptation between the two varieties examined. Inmore drought resistant Sabatiano, photoprotection seemed to beattained by enhancing thermal dissipation and by a rapid up-regulation of the antioxidant defence system. In contrary, in themore susceptible to drought genotype, photoprotection in the earlystages of drought is mainly based on the activation of alternate sinksof excessive excitation such as photorespiration. In this variety adelicate regulation of H2O2 production via CAT activity modula-tion might contribute to rapid decrease in stomatal conductancepreventing excessive water loss. However, under more intensedrought conditions the increase in NPQ in relation to the activationof antioxidant enzymes constitutes the preponderant mechanismfor preserving photochemistry apparatus.

Acknowledgements

This work was part of the 03ED480 research project, imple-mented within the framework of the ‘Reinforcement Programmeof Human Research Manpower’ (PENED) and co-financed byNational and community Funds (20% from the Greek Ministry ofDevelopment-General Secretariat of Research and Technology and80% from EU-European Social Fund).

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