Research article Effect of water deficit on leaf phenolic composition, gas exchange, oxidative damage and antioxidant activity of four Greek olive (Olea europaea L.) cultivars Antonios Petridis a, * , Ioannis Therios a , Georgios Samouris b , Stefanos Koundouras c , Anastasia Giannakoula d a Department of Horticulture, Laboratory of Pomology, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece b Laboratoryof Hygiene and Technology of Food of Animal Origin, Veterinary Research Institute of Thessaloniki, National Agricultural Research Foundation (NAGREF), 57001 Thermi- Thessaloniki, Greece c Department of Horticulture, Laboratory of Viticulture, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece d Department of Crop Production, Technological Educational Institute of Thessaloniki, 54101 Sindos-Thessaloniki, Greece article info Article history: Received 19 April 2012 Accepted 12 July 2012 Available online 2 August 2012 Keywords: Olea europaea L. Water stress Phenols Oleuropein Antioxidant activity Chlorophyll a fluorescence MDA abstract The olive tree (Olea europaea L.) is often exposed to severe water stress during the summer season. In this study, we determined the changes in total phenol content, oleuropein and hydroxytyrosol in the leaves of four olive cultivars (‘Gaidourelia’, ‘Kalamon’, ‘Koroneiki’ and ‘Megaritiki’) grown under water deficit conditions for two months. Furthermore, we investigated the photosynthetic performance in terms of gas exchange and chlorophyll a fluorescence, as well as malondialdehyde content and antioxidant activity. One-year-old self-rooted plants were subjected to three irrigation treatments that received a water amount equivalent to 100% (Control, C), 66% (Field Capacity 66%, FC 66 ) and 33% (Field Capacity 33%, FC 33 ) of field capacity. Measurements were conducted 30 and 60 days after the initiation of the experiment. Net CO 2 assimilation rate, stomatal conductance and F v /F m ratio decreased only in FC 33 plants. Photosynthetic rate was reduced mainly due to stomatal closure, but damage to PSII also contributed to this decrease. Water stress induced the accumulation of phenolic compounds, especially oleuropein, suggesting their role as antioxidants. Total phenol content increased in FC 33 treatment and oleuropein presented a slight increase in FC 66 and a sharper one in FC 33 treatment. Hydroxytyrosol showed a gradual decrease as water stress progressed. Malondialdehyde (MDA) content increased due to water stress, mostly after 60 days, while antioxidant activity increased for all cultivars in the FC 33 treatment. ‘Gaidourelia’ could be considered as the most tolerant among the tested cultivars, showing higher phenolic concentration and antioxidant activity and lower lipid peroxidation and photochemical damage after two months of water stress. The results indicated that water stress affected olive tree physiological and biochemical parameters and magnitude of this effect depended on genotype, the degree of water limitation and duration of treatment. However, the severity as well as the duration of water stress might exceed antioxidant capacity, since MDA levels and subsequent oxidative damage increased after two months of water deficit. Ó 2012 Elsevier Masson SAS. All rights reserved. 1. Introduction The olive tree, a native evergreen plant of the Mediterranean region, is often exposed to severe water deficit combined with high temperatures and high light intensities, during the summer season. Although olive withstands a high degree of drought stress [1], this characteristic is cultivar dependent and considerable genetic vari- ation exists among cultivars [2e5]. The acclimation ability of olive plants to adjust to water deficit includes both mechanisms of avoidance and tolerance [2,5]. Alter- ations at leaf level are associated with morphological, anatomical and physiological characteristics. Olive cultivars well adapted to field conditions revealed enhanced sclerophylly, with high density of foliar tissue and thick cuticle and trichome layers [2,5,6]. Cell expansion is inhibited, resulting in a limitation of the leaf area. Water stress limits not only the size of individual leaves, but also their number. Another response to water deficit is the cessation of shoot growth, while the root system is also affected [3]. Addition- ally, changes in foliar chemistry allow olive plants to tolerate temporary or prolonged periods of water shortage. Plants subjected * Corresponding author. Tel.: þ30 2310 998625; fax: þ30 2310 998674. E-mail address: [email protected](A. Petridis). Contents lists available at SciVerse ScienceDirect Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy 0981-9428/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2012.07.014 Plant Physiology and Biochemistry 60 (2012) 1e11
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Plant Physiology and Biochemistry 60 (2012) 1e11
Contents lists available
Plant Physiology and Biochemistry
journal homepage: www.elsevier .com/locate/plaphy
Research article
Effect of water deficit on leaf phenolic composition, gas exchange, oxidativedamage and antioxidant activity of four Greek olive (Olea europaea L.) cultivars
Antonios Petridis a,*, Ioannis Therios a, Georgios Samouris b, Stefanos Koundouras c,Anastasia Giannakoula d
aDepartment of Horticulture, Laboratory of Pomology, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greeceb Laboratory of Hygiene and Technology of Food of Animal Origin, Veterinary Research Institute of Thessaloniki, National Agricultural Research Foundation (NAGREF), 57001 Thermi-Thessaloniki, GreececDepartment of Horticulture, Laboratory of Viticulture, Aristotle University of Thessaloniki, 541 24 Thessaloniki, GreecedDepartment of Crop Production, Technological Educational Institute of Thessaloniki, 54101 Sindos-Thessaloniki, Greece
a r t i c l e i n f o
Article history:Received 19 April 2012Accepted 12 July 2012Available online 2 August 2012
Keywords:Olea europaea L.Water stressPhenolsOleuropeinAntioxidant activityChlorophyll a fluorescenceMDA
0981-9428/$ e see front matter � 2012 Elsevier Mashttp://dx.doi.org/10.1016/j.plaphy.2012.07.014
a b s t r a c t
The olive tree (Olea europaea L.) is often exposed to severe water stress during the summer season. In thisstudy, we determined the changes in total phenol content, oleuropein and hydroxytyrosol in the leaves offour olive cultivars (‘Gaidourelia’, ‘Kalamon’, ‘Koroneiki’ and ‘Megaritiki’) grown under water deficitconditions for two months. Furthermore, we investigated the photosynthetic performance in terms ofgas exchange and chlorophyll a fluorescence, as well as malondialdehyde content and antioxidantactivity. One-year-old self-rooted plants were subjected to three irrigation treatments that receiveda water amount equivalent to 100% (Control, C), 66% (Field Capacity 66%, FC66) and 33% (Field Capacity33%, FC33) of field capacity. Measurements were conducted 30 and 60 days after the initiation of theexperiment. Net CO2 assimilation rate, stomatal conductance and Fv/Fm ratio decreased only in FC33
plants. Photosynthetic rate was reduced mainly due to stomatal closure, but damage to PSII alsocontributed to this decrease. Water stress induced the accumulation of phenolic compounds, especiallyoleuropein, suggesting their role as antioxidants. Total phenol content increased in FC33 treatment andoleuropein presented a slight increase in FC66 and a sharper one in FC33 treatment. Hydroxytyrosolshowed a gradual decrease as water stress progressed. Malondialdehyde (MDA) content increased due towater stress, mostly after 60 days, while antioxidant activity increased for all cultivars in the FC33
treatment. ‘Gaidourelia’ could be considered as the most tolerant among the tested cultivars, showinghigher phenolic concentration and antioxidant activity and lower lipid peroxidation and photochemicaldamage after two months of water stress. The results indicated that water stress affected olive treephysiological and biochemical parameters and magnitude of this effect depended on genotype, thedegree of water limitation and duration of treatment. However, the severity as well as the duration ofwater stress might exceed antioxidant capacity, since MDA levels and subsequent oxidative damageincreased after two months of water deficit.
� 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
The olive tree, a native evergreen plant of the Mediterraneanregion, is often exposed to severe water deficit combined with hightemperatures and high light intensities, during the summer season.Although olive withstands a high degree of drought stress [1], thischaracteristic is cultivar dependent and considerable genetic vari-ation exists among cultivars [2e5].
: þ30 2310 998674.dis).
son SAS. All rights reserved.
The acclimation ability of olive plants to adjust to water deficitincludes both mechanisms of avoidance and tolerance [2,5]. Alter-ations at leaf level are associated with morphological, anatomicaland physiological characteristics. Olive cultivars well adapted tofield conditions revealed enhanced sclerophylly, with high densityof foliar tissue and thick cuticle and trichome layers [2,5,6]. Cellexpansion is inhibited, resulting in a limitation of the leaf area.Water stress limits not only the size of individual leaves, but alsotheir number. Another response to water deficit is the cessation ofshoot growth, while the root system is also affected [3]. Addition-ally, changes in foliar chemistry allow olive plants to toleratetemporary or prolonged periods of water shortage. Plants subjected
Table 1Midday leaf water potential of the four olive cultivars under contrasting waterregimes (C, control; FC66, field capacity 66%; FC33, field capacity 33%). Valuesrepresent means of 3 replications. Means followed by different letters within a lineare significantly different (p � 0.05).
Leaf water potential (MPa)
C FC66 FC33
15/5/2010Gaidourelia’ �1.45 a �2.38 b �3.45 cKalamon’ �1.39 a �2.32 b �3.28 cKoroneiki’ �1.30 a �2.10 b �3.26 cMegaritiki’ �1.20 a �2.17 b �3.15 c
15/6/2010Gaidourelia’ �2.14 a �2.91 b �3.71 cKalamon’ �2.01 a �3.02 b �3.82 cKoroneiki’ �2.06 a �2.80 b �3.70 cMegaritiki’ �2.01 a �2.87 b �3.65 c
A. Petridis et al. / Plant Physiology and Biochemistry 60 (2012) 1e112
to water deficit may synthesize and accumulate amino acids,proteins, sugars, methylated quaternary ammonium compoundsand organic acids [7]. These physiological responses permit olivetrees to lower their osmotic potential facilitating water flow intotheir roots and leaves, thus, maintaining cell turgor [8]. Thesesolutes, also sequester water molecules, protect cell membranesand protein complexes and allow the metabolic machinery tocontinue functioning [9]. Carbohydrates are the most commonsolutes accumulated in olive tree tissue under water deficitconditions [2,10]. Ben Ahmed et al. [11] found that proline wasaccumulated in olives under water deficit conditions. Furthermore,a close relationship between net photosynthetic rate and prolinecontent was recorded, pointing the important role of this osmolytein the maintenance of photosynthetic activity.
Under mild and moderate water stress, photosynthetic ratedecreases in olive plants mostly due to stomatal closure [12].However, as water stress becomes severe, the inactivation ofphotosynthetic activity could be ascribed not only to stomatalrestrictions, but also to non-stomatal factors related to inhibition ofprimary photochemistry and electron transport in chloroplasts[13]. Frequently, under water stress the rate of light absorptionexceeds the capacity for photosynthesis. This often results ina repression of photosynthesis e a phenomenon known as photo-inhibition [14].
According to Smirnoff [15] low water availability is often asso-ciated with increased levels of reactive oxygen species (ROS), suchas superoxide anion (O2
��), hydrogen peroxide (H2O2), hydroxylradical (HO�) and singlet oxygen (1O2). Briefly, low internal CO2concentration results in a reduction of oxidized NADPþ pool, as anelectron acceptor. Therefore, the light energy absorbed is not fullyused by photosynthesis, photorespiration or heat generation and isdiverted to molecular oxygen, which is abundant in the chloroplast[9]. ROS are highly reactive species and seriously disrupt normalmetabolism of the plant. Plants are endowed with a complexantioxidant system to cope with ROS [15]. The major enzymaticscavengers of ROS are superoxide dismutase (SOD), ascorbateperoxidase (APX) and catalase (CAT), as well as several enzymesinvolved inmaintaining reduced antioxidant pools [16]. In addition,plants contain several low molecular weight antioxidants such asascorbate, glutathione and phenolic compounds, which are watersoluble, and a-tocopherol and carotenoids, which are lipid soluble[14].
Malondialdehyde (MDA), a decomposition product of poly-unsaturated fatty acids hydroperoxides, has been utilized veryoften as a suitable biomarker of lipid peroxidation [17,18]. None-theless, lipids are not the only targets for MDA action, since itdamages DNA, forming adducts to deoxyguanosine and deoxy-adenosine [18,19].
Although studies on the enzymatic antioxidant system of theolive tree under water deficit have demonstrated that antioxidantenzymes play a major role in protecting olive leaf tissue againstoxidative stress [11,18,20], limited attention has been given to theeffect of phenolic compounds on the olive tree water stress toler-ance. Phenolic compounds are constitutively expressed in all higherplants. However, phenylpropanoid metabolism is often inducedwhen plants are exposed to a wide range of environmental stresses[21].
Oleuropein and hydroxytyrosol (30,40-dihydroxyphenylethanol)are two important phenolic compounds present in the olive tree.These compounds are responsible for crucial nutritional andorganoleptic properties of olives and olive oil and are also involvedin plant defense mechanisms towards pathogens and herbivores[22]. Oleuropein is a heterosidic ester of b-glucosylated elenolicacid and hydroxytyrosol and it can be easily transformed by theendogenous and exogenous b-glucosidase into glucose and
oleuropein aglycon [23]. Hydroxytyrosol is a phenolic alcoholpresent in the Olea europaea, but, unlike oleuropein, it is notconfined only to this botanical family [24].
Changes in oleuropein and hydroxytyrosol levels during fruitdevelopment and maturation of olive trees have been reportedadequately [25e28], however, the involvement of thesecompounds in environmental stress tolerance and particularlydrought has received limited attention. Remorini et al. [29]studying the interaction between root-zone salinity, resulting inwater stress, and solar irradiance on olive noted that sunlight andnot salinity altered polyphenol metabolism and that significantsalinity � light interactive effects were observed for total poly-phenol as well as for secoiridoid concentration, which eitherdecreased at the shaded or increased at the sun exposed side of thetree.
The aim of the present work was to determine the changes intotal phenol content, oleuropein and hydroxytyrosol of four olivecultivars grown under water deficit conditions. We hypothesizedthat, due to water stress, an up-regulation of the phenylpropanoidmetabolism may alter and equip leaves with an arsenal of defensecompounds, capable of protecting leaves. In addition, consideringthat a possible increase in phenolic compound concentration inresponse to water stress should be accompanied by an adequatephysiological function of plants, we investigated the photosyn-thetic performance in terms of gas exchange parameters andchlorophyll a fluorescence. Finally, we measured MDA levels, inorder to assess the effect of water stress on oxidative damage, aswell as antioxidant activity. We used four olive cultivars, namely‘Gaidourelia’, ‘Kalamon’, ‘Koroneiki’ and ‘Megaritiki’. ‘Koroneiki’ isconsidered to be a drought-tolerant cultivar, whereas there are nodata for the other three widely planted cultivars.
2. Results
2.1. Changes in leaf water potential, gas exchange parameters andchlorophyll a fluorescence indices
Midday leaf water potential (Jw) was affected significantly bywater stress and varied from �1.20 MPa in the C to �3.82 MPa inthe FC33 treatment (Table 1). After one month of water stress, Jwwas more negative in all cultivars. No significant differences amongcultivars were recorded (data not shown).
Water stress also affected photosynthetic performance (A, gs, Eand Ci) of the olive tree plants (Fig. 1). For all cultivars and bothdates, the net CO2 assimilation rate (A) was lower in FC33 plants,while FC66 did not differ from C plants. The reduction of A in FC33 ascompared to C was 85.9%, 96.4%, 95.9% and 93.7% for ‘Gaidourelia’,‘Kalamon’, ‘Koroneiki’ and ‘Megaritiki’, respectively (Fig. 1A).
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15/6/2010
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15/5/2010
15/5/2010 15/6/2010
15/5/2010 15/6/2010
A
B
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D
Fig. 1. Leaf net photosynthetic rate (A), stomatal conductance (B), transpiration (C) and internal CO2 concentration (D) of four olive cultivars (G, Gaidourelia; Ka, Kalamon; Ko,Koroneiki; M, Megaritiki) under contrasting water regimes (C, control; FC66, field capacity 66%; FC33, field capacity 33%). Vertical bars represent means of 3 replications � S.E(p � 0.05).
A. Petridis et al. / Plant Physiology and Biochemistry 60 (2012) 1e11 3
Likewise, stomatal conductance (gs) decreased significantly inall cultivars, under FC33 conditions, but it remained at C levels inFC66 conditions (Fig. 1B). The strong reduction of gs at FC33 treat-ment was obvious in both dates of measurement and it was 91.3%,95.8%, 94.7% and 94.7% for ‘Gaidourelia’, ‘Kalamon’, ‘Koroneiki’ and‘Megaritiki’, respectively, in comparison to C.
As a consequence of lower stomatal conductance under waterstress, transpiration (E) and internal CO2 concentration (Ci) werealso affected. Transpiration was significantly lower in FC33 treat-ment in all cultivars, throughout the experimental period (Fig. 1C).However, E values in C and FC66 plants in June were higher thanthose in May, whereas in FC33 plants, June values were lower than
A. Petridis et al. / Plant Physiology and Biochemistry 60 (2012) 1e114
May. In May, the Ci was not affected by the water stress regime, butsignificant cultivar and cultivar � water stress effects were recor-ded, with ‘Megaritiki’ presenting the lowest values in FC33 treat-ment (Fig. 1D). Nevertheless, in June, Ci increased in the FC33 plants,except in ‘Gaidourelia’. The increase in Ci of FC33 plants incomparison to the C plants of ‘Kalamon’, ‘Koroneiki’ and ‘Megaritiki’was 40.62%, 64.35% and 23.85%, respectively.
In both periods of measurements, the minimum chlorophyllfluorescence (Fo) of dark-adapted leaves was significantly increasedin FC33 treatment for all cultivars (Fig. 2A). Values of Fo recorded inJune were slightly higher than those in May. Maximum chlorophyllfluorescence (Fm) decreased in the FC33 for all cultivars and periods(Fig. 2B). As in F0, June Fm values were slightly higher than those ofMay. Concerningmaximumquantumyield efficiency of PSII (Fv/Fm),the olive cultivars showed similar behaviour in both periods(Fig. 2C). Under FC66 the Fv/Fm ratio was at C levels, while FC33 hadlower values, except in ‘Gaidourelia’, which presented similar valuesto FC66 and C. The decrease of Fv/Fm in May was 20.38%, 28.34%,8.61% and 9.76% and in June 1.57%, 16.10%, 11.25% and 11.93% for‘Gaidourelia’, ‘Kalamon’, ‘Koroneiki’ and ‘Megaritiki’, respectively.
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15/5/2010
A
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Fig. 2. Leaf chlorophyll fluorescence parameters of four olive cultivars (G, Gaidourelia; Ka, Kafield capacity 66%; FC33, field capacity 33%). (A) Minimum chlorophyll fluorescence (F0) andquantum efficiency of PSII (Fv/Fm). Vertical bars represent means of 3 replications � S.E (p
2.2. Changes in polyphenol concentration
During May, the total phenol content (TPC) was not differentamong stress treatments, except for ‘Koroneiki’, where TPCincreased (63.36%) in the FC33 treatment compared to C. After 60days of water stress, significant differences were recorded amongcultivars and water regimes. In general, ‘Gaidourelia’ showed thehighest TPC followed by ‘Kalamon’, ‘Megaritiki’ and ‘Koroneiki’.FC33 treatment increased TPC in all cultivars (Fig. 3).
Significant differences in oleuropein concentration were foundamong cultivars and water regimes in both periods of measure-ments (Fig. 4). On average, ‘Kalamon’ had the highest oleuropeinconcentration and ‘Megaritiki’ the lowest one. For all cultivars,June values were higher than those of May. In both periodsa slight increase in the FC66 treatment and a sharper one in theFC33 treatment were detected compared to C plants. Especially inJune, the increase of oleuropein concentration in the FC33 treat-ment, compared to C, was 4.93, 2.81, 7.19 and 4.56-foldshigher for ‘Gaidourelia’, ‘Kalamon’, ‘Koroneiki’ and ‘Megaritiki’,respectively.
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lamon; Ko, Koroneiki; M, Megaritiki) under contrasting water regimes (C, control; FC66,(B) Maximum chlorophyll fluorescence (Fm) in the dark-adapted state; (C) Maximum
� 0.05).
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Fig. 3. Leaf total phenol content of four olive cultivars under contrasting water regimes (C, control; FC66, field capacity 66%; FC33, field capacity 33%). Vertical bars represent meansof 3 replications � S.E. and same colour bars with different letters are significantly different (p � 0.05).
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Fig. 4. Leaf oleuropein concentration of four olive cultivars under contrasting water regimes (C, control; FC66, field capacity 66%; FC33, field capacity 33%). Vertical bars representmeans of 3 replications � S.E. and same colour bars with different letters are significantly different (p � 0.05).
A. Petridis et al. / Plant Physiology and Biochemistry 60 (2012) 1e11 5
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.)
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G Ka Ko M
Fig. 6. Antioxidant activity of four olive cultivars (G, Gaidourelia; Ka, Kalamon; Ko,Koroneiki; M, Megaritiki) under contrasting water regimes (C, control; FC66, fieldcapacity 66%; FC33, field capacity 33%). Vertical bars represent means of 3replications � S.E (p � 0.05).
A. Petridis et al. / Plant Physiology and Biochemistry 60 (2012) 1e116
In May, significant genotypic differences and genotype � waterstress interaction were observed, concerning hydroxytyrosolconcentration (Fig. 5). Overall, ‘Gaidourelia’ showed the highesthydroxytyrosol concentration compared to the other cultivars. In allcultivars no constant trend was observed among water regimes. InJune the trend of hydroxytyrosol concentration changed. In allcultivars hydroxytyrosol levels significantly decreased with waterstress, with the lowest values in FC33 (33.81%, 51.82%, 35.45% and52.54% for ‘Gaidourelia’, ‘Kalamon’, ‘Koroneiki’ and ‘Megaritiki’,respectively). Overall, as in oleuropein concentration, June values ofhydroxytyrosol were higher than those of May.
2.3. Changes in antioxidant activity and lipid peroxidation
The antioxidant activity was measured in June (Fig. 6). Upon thewhole, ‘Gaidourelia’ presented the highest antioxidant activity(13.44 mmol A.A. g�1 d.w.), followed by ‘Kalamon’, ‘Koroneiki’ and‘Megaritiki’ (11.76, 10.90 and 10.55 mmol A.A. g�1 d.w., respec-tively). FC33 plants had higher antioxidant activity than FC66 and Cin all cultivars, while the FC66 treatment did not differ from the Cplants.
Fig. 7 shows the malondialdehyde (MDA) content of the culti-vars under water stress in both periods. In May, MDA content of‘Gaidourelia’ and ‘Megaritiki’ was similar among water regimes,while in ‘Kalamon’ and ‘Koroneiki’ it was higher in FC33 treatment.‘Gaidourelia’ had the highest MDA content. June MDA values werehigher than in May for all cultivars, due to the progression of waterstress. In June, differences were recorded among cultivars, with‘Kalamon’ presenting the greatest lipid peroxidation and ‘Gai-dourelia’ the lowest one.
a aa
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Fig. 5. Leaf hydroxytyrosol concentration of four olive cultivars under contrasting waterrepresent means of 3 replications � S.E. and same colour bars with different letters are sig
3. Discussion
Water stress affected photosynthetic performance of the oliveplants, without any significant differences among cultivars. TheFC33 treatment resulted in a significant decrease in stomatalconductance, due to stomata closure, which in turn restricted netCO2 assimilation rate and water loss through transpiration (Fig. 1).Decrease in A and gs was observed from the first month of the
b ba
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regimes (C, control; FC66, field capacity 66%; FC33, field capacity 33%). Vertical barsnificantly different (p � 0.05).
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.)
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A
B
Fig. 7. MDA content of four olive cultivars (G, Gaidourelia; Ka, Kalamon; Ko, Koroneiki;M, Megaritiki) under contrasting water regimes (C, control; FC66, field capacity 66%;FC33, field capacity 33%). (A) 15/5/2010; (B) 15/6/2010. Vertical bars represent means of3 replications � S.E (p � 0.05).
A. Petridis et al. / Plant Physiology and Biochemistry 60 (2012) 1e11 7
experiment. On the other hand, the FC66 treatment had no effect onleaf gas exchange parameters, suggesting that photosyntheticapparatus in olive trees is very resistant to mild water deficits andthat stomata constitute the main limiting factor of carbon uptakeunder drought [12]. The higher E rates of FC66 and C plants in Junecompared to May can be attributed to the higher evaporativedemand due to the higher vapor pressure deficit (VPD) of June.Interestingly, only in FC33 treatment we found an increase in Ciconcentration in spite of lower gs, suggesting that non stomatallimitations also occurred in this treatment, with the exception of‘Gaidourelia’ (Fig. 1D). Since gas exchange parameters weremeasured at midday, the increase of Ci concentration is in accor-dance to Bacelar et al. [30], who also found an increase in Ci frommorning to midday. Increase in Ci (except ‘Gaidourelia’) coincidedwith Fv/Fm values lesser than 0.7 in June, suggesting non stomatallimitations in A.
Chlorophyll fluorescence gives information about the state ofPhotosystem II (PSII), showing the extent to which PSII is using theenergy absorbed by chlorophyll and the extent to which it is beingdamaged by excess light. The flow of electrons through PSII isindicative, under many conditions, of the overall rate of photo-synthesis, thus, giving the potential to estimate photosyntheticperformance. PSII is also accepted to be the most vulnerable part ofthe photosynthetic apparatus to light induced damage and damageto PSII will often be the first manifestation of stress in a leaf.Consequently, chlorophyll fluorescence can give insights into the
ability of a plant to tolerate environmental stresses and into theextent to which those stresses have damaged the photosyntheticapparatus [31]. Fv/Fm ratio have been suggested as quantitativemeasures of the photochemical efficiency of the PSII system and thephoton yield of oxygen evolution under different environmentalstresses [32], with optimal values around 0.83 for most plantspecies [31]. Under severe water stress, limitations of net CO2assimilation may promote an imbalance between photochemicalactivity at PSII, leading to an overexcitation and subsequent pho-toinhibitory damage of PSII reaction centre [33,34]. In our experi-ment the analysis of chlorophyll fluorescence revealed that non-stomatal limitations are also responsible for the photosyntheticrate decrease in the FC33 treatment. The changes of chlorophylla indices F0, Fm and Fv/Fm can be another indication that high lightintensities combined with water stress can cause an inhibition ofnet photosynthetic rates. The reduction of Fv/Fm ratio was mainlydue to a decrease in the variable fluorescence, which resulted froma decrease in Fm and a gradual increase in F0. Physiologically, thedecrease in Fv/Fm ratio and in Fv indicates a reduction in thephotochemical efficiency of the PSII complex, which could be due toinefficient energy transfer from the light-harvesting Chl a/b complex to the reaction center [32]. However, damage to thereaction center may not be ruled out [35]. The decrease in Fm couldbe due to structural alterations in the PSII complex, causinga decrease in the photochemistry of PSII, an increase in the decay ofexcitation energy as fluorescence, an increase in the radiationlessdecay, or the transfer of excitation energy in favor of PSI. Further-more, ROS such as O2
�, H2O2,�OH and 1O2 are involved in the pho-
todamage to PSII [36]. The similar reduction of Fv/Fm in the FC33treatment, after 30 and 60 days from the initiation of the experi-ment, may suggest that optimal photochemical efficiency of PSIIdoes not alter over a long period of time, which might be due to anadaptation response exhibited by olive plants. This adaptationcould be attributed to enhancement of the antioxidant machinery,partly phenolic compounds, which were increased under waterdeficit conditions (see below).
Many kinds of environmental stresses have been shown toincrease the levels of ROS in plant cells, including high light [37],drought [15] and salinity [38]. The role of oxidative stress as anunderlying factor in plant response to environmental stress is nowwidely accepted [39]. It is therefore noteworthy that many envi-ronmental conditions that cause oxidative stress are associatedwith the induction of phenylpropanoid metabolism in plants. Thissuggests that phenolics are involved in protection against oxidativestress under adverse environmental conditions [14]. In our study,a late induction of TPC was observed for all cultivars, in response towater stress, since TPC increased after 60 days from the beginningof the experiment (Fig. 3). The fact that after two months there wasno increase in TPC under mild water stress implies that the anti-oxidant machinery of olive trees functions sufficiently and/or thatolive is able to withstand prolonged periods of mild water stress.Our results are in contrast to those of Bacelar et al. [30], who foundnegligible increase in TPC, as water stress progressed. This could beascribed to the different environmental conditions and age of thetrees, since the eight-years-old olive plants used by these authorsmight need a longer period to show differences in TPC. Changes inolive leaf-TPC have been observed in various environmentalstresses [22,29,40,41] and it was claimed that phenolics acted asantioxidants. Apart from their role as antioxidants, phenoliccompounds have been considered to act as screening agents againstthe damaging effects of UV-B radiation, which usually accompanieswater deficit [14]. Hydroxycinnamic acids and flavonoids exhibithigh UV absorbance [42]. The latter are, also, thought to protect thephotosynthetic apparatus [43] and shield, at least in part, DNAfrom the damaging effects of UV radiation [44]. ‘Gaidourelia’
A. Petridis et al. / Plant Physiology and Biochemistry 60 (2012) 1e118
accumulated higher TPC than the other cultivars under waterdeficit conditions, suggesting that genotypic differences existconcerning phenol accumulation. The fact that ‘Gaidourelia’possessed higher antioxidant activity (Fig. 6) and lower lipid per-oxidation (Fig. 7) than the other cultivars, while presenting higherTPC, clearly shows the protective effect of phenolic compoundsagainst oxidative stress. This is highly important and furtherresearch should be conducted to exploit high phenolic potentialcultivars. It also suggests, along with PSII stability, a higher capacityof ‘Gaidourelia’ to withstand drought conditions.
Water stress increased oleuropein concentration in all cultivarsfrom the first month of the experiment (Fig. 4) and the increasewas slight in the FC66 treatment and sharp in the FC33 treatmentduring both dates of measurements. However, June values werehigher than those of May, as in the case of TPC. The latter could beattributed to greater exposure time to water stress as well as tohigher sunlight irradiance combined with higher temperaturesduring June. The increase in oleuropein concentration is anotherbiochemical response to water stress and it is more pronounced aswater stress becomes greater. The high oleuropein concentration,as a function of water stress, may be related to its antioxidantactivity and therefore may offer protection against the inducedoxidative damage. In total, ‘Kalamon’ had higher oleuropeinconcentration presenting, however, the lowest increase in the FC33compared to C. The highest increase was recorded in ‘Koroneiki’followed by ‘Gaidourelia’ and ‘Megaritiki’. The increase in oleur-opein concentration could be an important trait for areas wherewater availability is a limiting factor, in order to obtain betterquality of olives and olive oil. In addition, oleuropein increasemight offer other benefits, such as protection to ovipositing by‘Olive Fruit Fly’, as suggested by Daane and Johnson [45] whospeculated that susceptibility to ovipositing increases as oleur-opein decreases. Petridis et al. [41] also found an increase inoleuropein concentration under saline conditions, suggestinga common response of olive tree to various stressful environ-mental conditions. Hydroxytyrosol concentration did not showany specific trend among cultivars during May; nevertheless, inJune, FC33 plants showed lower hydroxytyrosol values in allcultivars compared to FC66 and C (Fig. 5). The highest decrease inhydroxytyrosol concentration due to water stress was recorded in‘Gaidourelia’ followed by ‘Kalamon’, ‘Koroneiki’ and ‘Megaritiki’.Since hydroxytyrosol is one of the main phenolic compounds inolive and possesses high antioxidant activity [46], one wouldexpect it to increase under adverse conditions. However, this wasnot confirmed in our experiment. Reduction in hydroxytyrosolconcentration was previously reported by Ortega-García and Per-agón [22] and Petridis et al. [41], under cold and salt stress,respectively. Perhaps hydroxytyrosol reduction is a response ofolive tree to environmental stresses. A possible explanationis that hydroxytyrosol is used in oleuropein synthesis. Whetherthis is true or not requires further research, to uncover the rela-tionship between closely related compounds under water stressconditions.
Lipid peroxidation has been defined as oxidative degradation ofpolyunsaturated lipids that contain more than two carbonecarbondouble covalent bonds [32]. MDA, a decomposition product ofpolyunsaturated fatty acids hydroperoxides, has been utilised veryoften as a suitable biomarker for lipid peroxidation, which is aneffect of oxidative damage [17]. An increase in lipid peroxidationwasobserved at both FC66 and FC33 treatments, as the duration of waterstress progressed. It is also evident that the peroxidation processwas regulated by the intensity of water stress treatment as the rateof MDA formation increased with the intensity of water deficit. Sofoet al. [18] also found an increase in MDA content in water stressedolive plants, whichwas accompanied bya corresponding increase of
lipoxygenase activity. MDA content has also been reported torespond to salt stress and sunlight irradiance [29].
Water stress has been shown to increase the levels of ROS inplant cells [15]. In our experiment, antioxidant activity was affectedby both water stress treatment and cultivar. ‘Gaidourelia’ hadhigher antioxidant activity than the other tested cultivars, sug-gesting that the ability of olive plants to scavenge ROS is cultivardependent. Antioxidant activity was triggered only in the FC33treatment in all cultivars, suggesting that olive can toleratemoderate water stress levels. Plants produce a wide range of anti-oxidants whose major function is to scavenge or, otherwise,detoxify ROS [14]. Phenolics are involved in protection againstwater stress, which is confirmed by the high positive correlationrecorded between TPC and antioxidant activity. Indeed, R2 valueswere 0.95, 0.92, 0.95 and 0.75 for ‘Gaidourelia’, ‘Kalamon’, ‘Kor-oneiki’ and ‘Megaritiki’, respectively. High correlation was alsorecorded between oleuropein and antioxidant activity (R2 was 0.84,0.81, 0.83 and 0.84 for ‘Gaidourelia’, ‘Kalamon’, ‘Koroneiki’ and‘Megaritiki’, respectively) confirming its high antioxidant capacity[46] and its participation in the antioxidant scavengingmechanism.
In conclusion, our results demonstrated that water stressaffected the physiological and biochemical parameters examined inthis work. The extent to which those parameters were affected,depended on the degree and duration of water stress and ongenotype. The decrease in photosynthesis was mainly due tostomatal closure. However, non-stomatal limitations were alsoobserved in the highest stress levels related to the imbalance inphotochemical activity at PSII, leading to an overexcitation andsubsequent photoinhibitory damage of PSII reaction centre.Therefore, chlorophyll fluorescence measurement, along with Ciconcentration determination, could be used as early screeningmethods in an olive cultivar water stress improvement program.The increase in the content of phenolic compounds is in agreementwith data in the literature indicating that this is a response to thegeneration of ROS. Oleuropein, the main phenolic compound ofolive, increased in response to water stress, highlighting itsimportance in the olive antioxidant defense mechanism. In fact, theantioxidant role of phenolic compounds and oleuropein in partic-ular, was confirmed by their high correlation with antioxidantactivity. However, the severity as well as the duration of waterstress might exceed olive antioxidant activity, since MDA levels andsubsequent oxidative damage increased after two months of waterdeficit. Gaidourelia could be considered as the most tolerant towater stress, showing higher phenolic concentration and antioxi-dant activity and lower lipid peroxidation and photochemicaldamage. Finally, mild water stress conditions can be successfullyused in olive to enhance oleuropein concentration, without dis-turbing the tree physiology. This is highly important both in termsof water saving and enhancement of health promoting compounds.Nevertheless, these results should be validated under field condi-tions on bearing olive trees to confirm the data produced in thisexperiment.
4. Experimental
4.1. Plant material, growth conditions and experimental design
The trials were conducted at the Agricultural Farm of AristotleUniversity of Thessaloniki (northern Greece, 40�340N, 22�580E) onfour Greek olive cultivars (O. europaea L. cvs ‘Gaidourelia’, ‘Kala-mon’, ‘Koroneiki’ and ‘Megaritiki’), between 15 April and 15 June,2010. One-year-old self-rooted plants were grown in a glasshousein 7 L pots filled with sandy-loam soil from the experimental site(55.2% sand, 29.6% silt, 15.2% clay; 26% field capacity). Meantemperature, relative humidity and photosynthetic photon flux
A. Petridis et al. / Plant Physiology and Biochemistry 60 (2012) 1e11 9
density, inside the glasshouse, were 25�e32 �C, 50e65% and about1200 mmol m�2 s�1 at midday, on a clear day, respectively. The potswere covered with plastic film and aluminum foil to restrictevaporation from the soil surface and to minimize the radiantheating of the root system. The experimental containers wererandomly arranged and periodically rotated to minimize the effectsof environmental heterogeneity. Plants were irrigated every twodays with 200 mL of half-strength Hoagland’s nutrient solution for2 months before the experiment. In mid-April, uniform plants(based on height, leaf number and total leaf area) of each cultivarwere selected for the experiment. At the initiation of the experi-ment, plants were separated into three groups of six, and threewater treatments were developed on the basis of field capacity. Inthe control (C), thewater addedwas equal to the amount transpired(100% of field capacity). In the other two treatments, drought stresswas imposed until the end of the experiment by restoring 66%(Field Capacity 66%, FC66) and 33% (Field Capacity 33%, FC33) of thetotal water transpired.
The application of water stress conditions during the experi-mental period was as follows: at the initiation of the experimentfield capacity was determined. The value of field capacity (%)multiplied by the standard soil weight of each pot represented theamount of water that should be added in order to reach fieldcapacity. After determining the amount of water and prior trans-planting, the weight of each plant was estimated so that the finalweight of the containers included the weight of soil, plant, pot andwater. The olive plants were weighed every two days and theamount of the water transpired was determined and restored,depending on the irrigation level. In order to monitor plant waterstatus for each treatment, midday leaf water potential (Jw) wasmeasured 30 and 60 days after the initiation of the experiment. Leafwater potential measurements were conducted on two fullyexpanded leaves from the mid-section of current year’s shoots onthree plants per treatment, using a Scholander pressure chamber.
4.2. Gas exchange and chlorophyll a fluorescence measurements
The measurements of net CO2 assimilation rate (A), stomatalconductance (gs), transpiration rate (E) and internal CO2 concen-tration (Ci) were performed twice (30 and 60 days from thebeginning of the experiment) at midday, on a clear day, witha portable gas-exchange meter (ADC Bioscientific LCpro þ System,Hoddesdon, UK) on two fully expanded leaves from themid-sectionof current year’s shoots, on three plants per treatment. The leaveswere marked at the beginning of the experiment. Measurementswere conducted at midday because the water stress effects on oliveplants are greater at this time than in the morning [47]. One hourbefore the beginning of the measurements, the plants were trans-ferred outdoors for adaptation to ambient light intensity andtemperature, where the measurements were conducted (meantemperature 21.3 �C and 28.4 �C for 15 May and 15 June, respec-tively). Vapor pressure deficit (VPD) was calculated from airtemperature and relative humidity data, recorded by an automatedweather station placed inside the agricultural farm of the AristotleUniversity of Thessaloniki. The values of VPD duringmeasurementswere 7.83 mbar and 14.24 mbar for May and June, respectively.
Chlorophyll a fluorescence was measured with a portable fluo-rometer (type 05-30, Opti-Sciences, Tynssboro, MA) on two fullyexpanded leaves (the same ones used for photosynthesis) on threereplications per treatment. Chlorophyll fluorescence from PSII wasrecorded at 692 nm. Prior to measurements, the attached leaveswere dark-adapted for 30 min in leaf-clips. The sensor unit wasconnected to the main control box by a cable, housed an opticalassembly, which provided powerful illumination (actinic lightbeam) to the leaf and detected the consequent fluorescence signals.
The sensor was placed over the leaf clip, so that daylight wasexcluded. Values for maximum fluorescence yield (Fm) andminimum fluorescence yield (F0) from the fluorescence inductioncurve were used for calculation of the Fv/Fm ratio (optimal photo-chemical efficiency of PSII). The fluorescence indices F0, Fm, Fv andFv/Fm were automatically calculated and displayed. F0 is the initialfluorescence (all PSII reaction centers are oxidized) and Fm themaximum fluorescence (complete reduction of PSII reactioncenters).
4.3. Sample preparation and extraction of phenolic compounds
Fully expanded leaves from the mid-section of current year’sshoots were sampled. All samples were immediately transferred tothe laboratory and lyophilized. After lyophilization, leaves werepulverized and stored in 50 mL polyethylene plastic screw captubes at �20 �C. An aliquot of 250 mg from each tissue wasextracted in 10 mL of 80% methanol on a shaker at 200 rpm for30 min. The mixture was filtered and the procedure was repeated.The two extracts were combined and an aliquot of 2 mL wastransferred to polypropylene screw cap vials and stored at �80 �Cuntil HPLC analysis. Prior to analyses, the extracts were filteredthrough a 20m PTFE syringe filter.
4.4. Total phenol content (TPC)
The total phenol content was determined spectrophotometri-cally at 760 nm, using the Folin-Ciocalteu reagent [48]. A portion of125 mL extract was combined with 2.5 mL of Folin-Ciocalteureagent, 375 mL distilled water and 2 mL sodium carbonate(75 g L�1). The mixture was incubated in a water-bath for 5 min at50 �C and after cooling at room temperature the optical density wasmeasured with a spectrophotometer (Camspec M106 spectropho-tometer, Leeds, UK). Gallic acid at concentrations of 12, 28, 40 and56 mg mL�1 was used to develop standard curves.
4.5. High performance liquid chromatography (HPLC) analysis ofphenolic compounds
HPLC analyses were performed with a Perkin Elmer HPLCsystem (Series 200) equipped with a Perkin Elmer LC-200 diodearray detector (DAD). Oleuropein and hydroxytyrosol were sepa-rated by reversed-phase HPLC using a Spherisorb ODS-2(250 mm � 4.6 mm i.d., 5 mm particle size, MZ-AnalysentechnikGMBH, Mainz) column, maintained at 35 �C during chromato-graphic runs. The column was eluted at a flow rate of 1 mL min�1
with a gradient of solvent system comprising water (solvent A, pHadjusted to 3.1 with phosphoric acid) and acetonitrile (solvent B).Solvent B was increased linearly from 10% at zero time to 30% at10 min, held isocratically for 4 min and followed by further linearramping to 40% at 19min. Initial conditions were reached in 10min.A 20 mL aliquot of extract or standard solutionwas injected for eachrun and elution profiles were detected at 280 nm. Phenoliccompounds were identified bymatching retention time and the UVspectra of a peak in the extract chromatogram with the peak ofa known standard compound. For quantitative measurements ofoleuropein and hydroxytyrosol, a five point calibration curve(R2¼ 0.999, y¼ 19364.34x� 27.31; R2¼ 0.999, y¼ 4470.32x� 2.92,for hydroxytyrosol and oleuropein, respectively) was developed onthe basis of the corresponding standard substances. Stock standardsolutions of oleuropein and hydroxytyrosol were prepared inmethanol at the 1 mg mL�1 level. Standard solutions of oleuropein/hydroxytyrosol were prepared at concentrations of 2.5, 5,10, 25 and50 mg mL�1 by diluting appropriate volumes of the stock standardsolution in water.
A. Petridis et al. / Plant Physiology and Biochemistry 60 (2012) 1e1110
4.6. FRAP assay
The ferric reducing antioxidant ability of plasma (FRAP) assay[49] was used to evaluate the antioxidant activity of the samples.FRAP method relies on the reduction of TPTZ (2,4,6-tri-pyridyl-s-triazine)-Fe3þ complex to TPTZ-Fe2þ form, with an intense bluecolour and absorption maximum at 593 nm. FRAP assay is a non-specific method and the absorption alterations reflect the totalreducing power of all the antioxidant substances found in the testsolution. A triplicate of each treatment was analysed spectropho-tometrically. Briefly, a weighted amount of 1 g of sample wasextracted twicewith 10mL of a solution consisting of 50%methanolin 1.2 M HCl (1:10 w/v) at 4 �C for 24-h intervals. For the FRAP assayan FRAP reagent was prepared by mixing acetate buffer (0.3 M, pH3.6), 10 mM TPTZ in 40 mM HCl and 20 mM FeCl3 at 10:1:1 (v/v/v)ratio. The FRAP reagent (3 mL) and sample extract (10 mL) wereadded in a test tube, vortexed and incubated immediately in awater-bath at 37 �C for 4 min. After cooling at room temperature theabsorbance was recorded at 593 nm. All solutions were prepared atthe day of measurements. An aqueous solution of 100 mM ascorbicacid was used for calibration of the instrument. Total antioxidantactivity is expressed as mmol ascorbic acid equivalent (AAE) g�1 d.w.
4.7. Lipid peroxidation
Malondialdehyde, the compound used as an index of lipid per-oxidation, was determined by a selective third-order derivativespectrophotometric method [50]. A 300 mg lyophilized sample wasthoroughly homogenized (Ultra Turrax homogenizer, Model T25basic, IKA Labortechnik, Germany) in the presence of 8 mL aqueoustrichloroacetic acid (5%) and 5 mL of butylated hydroxytoluene inhexane (0.8%), and the mixture was centrifuged at 3000 g for 5 min.The upper layer was discarded and a 2.5 mL aliquot from the bottomlayerwasmixedwith1.5mLof aqueous2-thiobarbituric acid (0.8%) tobe further incubated at 70 �C for 30min. After incubation themixturewas cooled to room temperature and submitted to conventionalspectrophotometry (Shimadzu, Model UV-1601, Tokyo, Japan) in therange of 400e650 nm. Third-order spectra were produced by digitaldifferentiation of the normal spectra using a derivative wavelengthdifference setting of 21 nm. The concentration of malondialdehyde(ng g�1 d.w.) in analysed extracts was calculated on the basis of theheight of the third-order peak at 521.5 nm by referring to slope andintercept data of the computed least-squares fit of the standardcalibration curve prepared using 1,1,3,3-tetraethoxypropane.
4.8. Statistical analysis
The data were subjected to one-way (for water treatmentdifferences) or two-way (for cultivar and water availability effects)analysis of variance (ANOVA) using the SPSS 17.0 for Windowsstatistical package (SPSS, Chicago, IL, USA). Comparison among themeans to determine significant differences (P � 0.05) were per-formed using the Duncan’s multiple range test.
Acknowledgements
The authors are most grateful to the Veterinary Research Insti-tute of Thessaloniki (NAGREF), which provided us with the HPLCunit and had been accommodating us during our experimentation.
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