Environmental and Experimental Botany 79 (2012) 37– 43 Contents lists available at SciVerse ScienceDirect Environmental and Experimental Botany journa l h omepa g e: www.elsev ier .com/ Salinity ds cultiva hip Antonios , Ch a School of Agri b Laboratory of ssalon 57001 Thermi- c School of Agri 4, Gre a r t i c l Article history: Received 11 Ja Accepted 19 Ja Keywords: Antioxidant activity Hydroxytyrosol Olea europaea L. Olive roots Oleuropein Salinity of al ental l phe aline also tested the possible relationship between oleuropein and glucose, since the latter compound is a part of the former molecule and accumulates under saline conditions. The data indicate that salinity stimulated the biosynthesis of phenols and oleuropein, especially in leaves, whereas the hydroxytyrosol concentration was either negatively or not affected by the salt stress. Oleuropein was the main phenolic compound in both tissues regardless of NaCl treatments. In leaves, glucose showed a totally inverse response to salinity than that of oleuropein, while a highly negative correlation existed between these 1. Introdu Olive (O Mediterran ity, particu (Chartzoula tinuously b yields and quality irrig unavoidabl and Misopo salts (Theri among cult One of tal stresses increased l gen species ∗ Correspon E-mail add 0098-8472/$ – doi:10.1016/j. two substances (R = −0.90, −0.80, −0.88 and −0.84 for ‘Zard’, ‘Ascolana’, ‘Koroneiki’ and ‘Arbequina’, respectively). A possible explanation for this relationship is that oleuropein acts as a glucose-reservoir for osmoregulation or high energy-consuming processes required for plant adaptation to salinity. A highly significant correlation was recorded between total phenol content and antioxidant activity in both leaves and roots. Finally, there is no indication pointing to translocation of phenolic compounds between leaves and root owing to exposure to salt stress. © 2012 Elsevier B.V. All rights reserved. ction lea europaea L.), one of the major tree crops in the ean region, is often exposed to high root-zone salin- larly during the long and warm/dry summer season kis, 2005; Remorini et al., 2009). Its cultivation is con- eing extended to irrigated land because of enhanced profits, but in several areas the availability of good- ation water is limited and the use of brackish water is e (Chartzoulakis et al., 2006; Melgar et al., 2009; Therios linos, 1988). Olive trees present medium tolerance to os and Misopolinos, 1988) with marked differences ivars (Gucci et al., 1997). the major consequences of various environmen- , including salinity, is oxidative stress mediated by evels of reactive oxygen species (ROS). Reactive oxy- have the potential to interact with many cellular ding author. Tel.: +30 2310 998625; fax: +30 2310 998674. ress:
[email protected] (A. Petridis). components, causing significant damage to membranes and other cellular structures (Goreta et al., 2007). Plants produce a large num- ber of antioxidants aimed at scavenging or detoxifying ROS (Grace, 2005). Phenolic compounds act as antioxidants that protect plants against the damaging effects of increased ROS levels due to salt stress (Tattini et al., 2006). Oleuropein and hydroxytyrosol (3′,4′-dihydroxyphenylethanol) are two important phenolic compounds for the olive tree. These compounds are responsible for crucial nutritional and organolep- tic properties of olives and olive oil and are, also, involved in plant defense against pathogens and herbivores (Ortega-García and Peragón, 2009). Oleuropein is a heterosidic ester of ˇ-glucosylated elenolic acid and hydroxytyrosol and can be easily transformed by endogenous or exogenous supply of the enzyme ˇ-glucosidase into glucose and oleuropein aglycon (Ranalli et al., 2006). It is, also, the most abundant phenolic compound in olive leaves and fruits and is responsible for the characteristic bitterness of olive fruit and the browning that occurs in green table olives either after impact and wounding during harvesting, or during subsequent technological treatments (Malik and Bradford, 2006; Soler-Rivas et al., 2000; Therios, 2009). Hydroxytyrosol is a phenolic alcohol see front matter © 2012 Elsevier B.V. All rights reserved. envexpbot.2012.01.007 -induced changes in phenolic compoun rs (Olea europaea L.) and their relations Petridisa,∗, Ioannis Theriosa, Georgios Samourisb culture, Laboratory of Pomology, Aristotle University of Thessaloniki, 541 24, Greece Hygiene and Technology of Food of Animal Origin, Veterinary Research Institute of The Thessaloniki, Greece culture, Laboratory of Apiculture-Sericulture, Aristotle University of Thessaloniki, 541 2 e i n f o nuary 2012 nuary 2012 a b s t r a c t Phenolic compounds are constituents when plants are exposed to environm to determine the phenolic status (tota activity of four olive cultivars under s locate /envexpbot in leaves and roots of four olive to antioxidant activity risoula Tananakic iki, National Agricultural Research Foundation (N.AG.RE.F), ece l higher plants. However, their biosynthesis is often induced stresses, such as salinity. The aim of the present research was nol content, oleuropein and hydroxytyrosol) and antioxidant conditions in two different plant parts (leaves and root). We 38 A. Petridis et al. / Environmental and Experimental Botany 79 (2012) 37– 43 present in the O. europaea, but, unlike oleuropein, it is not confined to this botanical family (Soler-Rivas et al., 2000). Health benefits of these compounds have been extensively investigated. It has been reported that oleuropein and other related phenolic com- pounds, suc and demeth lower risk et al., 1998 (Owen et a pounds pre et al., 1999; Althoug ing fruit de reported (A 2008; Ryan in environm has attract stress, oleu cold stress, Peragón, 20 effect of sa sunlight con tions increa to full sunli of the olive The aim of two phe tyrosol, to concomitan and root). the correla that, due to metabolism nal of defe compartme more, since an active r hypothesis molecules. phenolic sta possible me awareness response to caused by t 2. Materia 2.1. Plant m Four oliv Arbequina, rooted plan bags contai to the green thetic activ at midday, days with 2 (Hoagland The sand–p salinity leve and equal t plants were accumulate The experim treatments 2.2. Sample preparation and extraction of phenolic compounds At the end of the experiment, roots and fully expanded leaves from the mid-section of current year shoots were sampled. All s w lized and . An a met tered o ex to po naly PTFE rboh bohy xtrac d wa r to d tal p tota t 760 act w dist cuba room troph allic igh p ic com C an eries or (D ed-ph m pa ined at a fl ising nd a 0% at llow ions rd so etec ing re atogr antit poin 99, tivel rd su ytyr rd so trati volu high oluti h as hydroxytyrosol, tyrosol, verbascoside, ligustroside yloleuropein, act as antioxidants that contribute to a of coronary diseases (Beauchamp et al., 2005; Visioli ; Wiseman et al., 1996) and several types of cancers l., 2000; Tripoli et al., 2005). Furthermore, these com- sent antimicrobial and antiviral activity (Bisingnano Uccella, 2001). h changes in oleuropein and hydroxytyrosol levels dur- velopment and maturation of olive trees have been miot et al., 1986; Bouaziz et al., 2004; Damak et al., et al., 2003), the involvement of these compounds ental stresses and particularly in root-zone salinity ed limited attention. In one study, concerning cold ropein concentration increased at moderate and heavy while hydroxytyrosol decreased (Ortega-García and 09). Remorini et al. (2009), studying the interactive linity and solar irradiance, showed that under normal ditions the total phenol and the secoiridoid concentra- sed in the salt-treated plants when they were exposed ght, but not significantly influenced in the shaded side trees. of the present work was to determine the contribution nolic compounds, namely oleuropein and hydroxy- the response of O. europaea L. to salinity and the t oxidative stress, focusing on two plant organs (leaves Furthermore, we tested the antioxidant activity and tion that might exist with phenols. We hypothesized high salinity, an up-regulation of the phenylpropanoid may alter and equip leaves with an extraordinary arse- nse compounds, operating in different cells and cell nts, capable of protecting leaves and roots. Further- glucose is a part of oleuropein molecule and plays ole in the process of osmoregulation, we tested the that a close relationship might exist between these two To our knowledge, this is the first report examining the tus of the roots under saline conditions and testing the tabolic relationship between the two plant organs. The of the role of phenols and their behaviour in olive tree salinity, may aid the prevention of any imminent injury his abiotic stress factor. l and methods aterial, growth conditions and experimental design e (O. europaea L.) cultivars (Zard, Iran; Ascolana, Italy; Spain; Koroneiki, Greece) were studied. Uniform self- ts, 40 cm in height, were transplanted to 3 L black plastic ning a sand–perlite mixture (1:1, v/v) and transferred house in a mean temperature of 25 ◦C and a photosyn- e radiation (PAR) of approximately 500 �mol m−2 s−1 on a clear day. Olive plants were irrigated every 2 00 mL of a half-strength Hoagland’s nutrient solution and Arnon, 1950), containing 0, 75 and 125 mM NaCl. erlite medium does not adsorb nutrients; thus, its actual l did not increase over time, but it was almost constant o that of each treatment solution. Every 15 days the irrigated with 300 mL distilled water to leach out any d salts in the substrate. The experiment lasted 5 months. ental design was a completely randomized one with 3 and 6 replicates per treatment. sample lyophi verized −20 ◦C of 80% was fil The tw ferred HPLC a a 20 � 2.3. Ca Car After e distille in orde ysis. 2.4. To The cally a of extr 375 �L was in ing at a spec UK). G 2.5. H phenol HPL tem (S detect revers i.d., 5 � mainta eluted compr acid) a from 1 and fo condit standa were d match chrom For qu a five R2 = 0.9 respec standa hydrox Standa concen priate of the dard s ere immediately transferred to the laboratory and . After lyophilization, both leaves and roots were pul- stored in 50 mL polyethylene plastic screw cap tubes at liquot of 250 mg for each tissue was extracted in 10 mL hanol on a shaker at 200 rpm for 30 min. The mixture through a filter paper and the procedure was repeated. tracts were combined and an aliquot of 2 mL was trans- lypropylene screw cap vials and stored at −80 ◦C until sis. Prior to analyses, the extracts were filtered through syringe filter. ydrate extraction drates were extracted as described by Vemmos (1999). tion the samples were reduced to dryness and 2 mL of ter were added to the residue. The mixture was shaken issolve the carbohydrates and then used for HPLC anal- henol content (TPC) l phenol content was determined spectrophotometri- nm, using the Folin–Ciocalteu reagent. A 125 �L aliquot as combined with 2.5 mL of Folin–Ciocalteu reagent, illed water and 2 mL sodium carbonate. The mixture ted in a water-bath at 50 ◦C for 5 min and after cool- temperature the optical density was measured with otometer (Camspec M106 spectrophotometer, Leeds, acid was used to develop standard curves. erformance liquid chromatography (HPLC) analysis of pounds alyses were performed using a Perkin Elmer HPLC sys- 200) equipped with a Perkin Elmer LC-200 diode array AD). Oleuropein and hydroxytyrosol were separated by ase HPLC using a Spherisorb ODS-2 (250 mm × 4.6 mm rticle size, MZ-Analysentechnik GMBH, Mainz) column, at 35 ◦C during chromatographic runs. The column was ow rate of 1 mL min−1 with a gradient of solvent system water (solvent A, pH adjusted to 3.1 with phosphoric cetonitrile (solvent B). Solvent B was increased linearly zero time to 30% at 10 min, held isocratically for 4 min ed by further linear ramping to 40% at 19 min. Initial were reached in 10 min. A 20 �L aliquot of extract or lution was injected for each run and elution profiles ted at 280 nm. Phenolic compounds were identified by tention time and the UV spectra of a peak in the extract am with the peak of a known standard compound. ative measurements of hydroxytyrosol and oleuropein, t calibration curve (R2 = 0.999, y = 19364.34x − 27.31; y = 4470.32x − 2.92, for hydroxytyrosol and oleuropein, y) was developed on the basis of the corresponding bstances. Stock standard solutions of oleuropein and osol were prepared in methanol at the 1 mg mL−1 level. lutions of oleuropein/hydroxytyrosol were prepared at ons of 2.5, 5, 10, 25 and 50 �g mL−1 by diluting appro- mes of the stock standard solution in water. Because oleuropein concentration in several samples, stan- ons were, also, prepared at concentrations of 50, 100, A. Petridis et al. / Environmental and Experimental Botany 79 (2012) 37– 43 39 150 and 300 �g mL−1. The calibration curve was linear (R2 = 0.997, y = 4057.61x + 12530.48). 2.6. HPLC analysis of glucose HPLC an 1200 HPLC refractive in on a 305 m (Hamilton C The column 10 �L aliqu run. Glucos in the extra compound. calibration on the basi solutions of 500, 700 an 2.7. FRAP a The ferr (Benzie and idant activi TPTZ (2,4,6- with an int FRAP assay reflect the t found in th lyzed spectr expressed a 2.8. Statisti The dat (ANOVA). C differences ple range te Pearson’s co of 95% (P ≤ SPSS 17.0 fo 3. Results 3.1. Total p plant tissue Phenolic plants. How when plant (Dixon and including s oxidative st also, associa (Grace, 200 dants in var low temper given to sa this experim dant activit relationship The leaf salinity, ex slight at 75 of 125 mM NaCl, TPC was more than double compared with that of control plants in all cultivars tested. ‘Ascolana’ recorded the great- est increase (129%) followed by the cvs ‘Koroneiki’ (127%), ‘Zard’ (112%) and ‘Arbequina’ (110%). Remorini et al. (2009), studying the tive l sun crea sunli olive full l ccor ts ha ut no f sali mM l (Zar . Ion tes io of p (Mun NaC ion c ess, orted oms ine c oots ibly d l valu ts cau gy d rath tive r oxi plain rent hen gh at e in t and t-TP trol in le ntly phe lic co ach o r to f n (20 euro leve f and erat rmin ytyr of 5.1 of red t pein pou s in as fa urop d by alyses were performed using an Agilent Technologies system equipped with an Agilent Technologies 1200 dex detector (RID). Separation of glucose was achieved m × 7.8 mm i.d., 5 �m particle size Hamilton column o.), maintained at 30 ◦C during chromatographic runs. was eluted at a flow rate of 1 mL min−1 with water. A ot of extract or standard solution was injected for each e was identified by matching retention time of the peak ct chromatogram with the peak of a known standard For quantitative measurements of glucose, a five point curve (R2 = 0.999, y = 89.57x + 1438.74) was developed s of the corresponding standard substance. Standard glucose were prepared at concentrations of 100, 200, d 1000 �g mL−1. ssay ic reducing antioxidant ability of plasma (FRAP) assay Strain, 1996) was used, in order to evaluate the antiox- ty of the samples. FRAP method rely on the reduction of tri-pyridyl-s-triazine)-Fe3+ complex to TPTZ-Fe2+ form, ense blue colour and absorption maximum at 593 nm. is a non-specific method and the absorption alterations otal reducing power of all the antioxidant substances e test solution. A triplicate of each treatment was ana- ophotometrically. The FRAP values of the samples were s �mol l-ascorbic acid equivalents (A.A.E.) g−1 d.w. cal analysis a were subjected to one-way analysis of variance omparison between means to determine significant (P ≤ 0.05) was performed using the Duncan’s multi- st. Correlation between variables was determined with rrelation coefficient test, considering a confidence level 0.05). All statistical analyses were performed using the r Windows statistical package (SPSS, Chicago, IL, USA). and discussion henol content according to salinity level and kind of compounds are constitutive compounds of all higher ever, phenylpropanoid metabolism is often induced s are exposed to a wide range of environmental stresses Paiva, 1995). Under certain environmental conditions, alinity (Mittova et al., 2003), plants may experience ress as a result of increased production of ROS and are, ted with the induction of phenylpropanoid metabolism 5). Although phenolics have been studied as antioxi- ious stresses such as, high light intensity, UV radiation, ature, pathogens and ozone, limited attention has been line conditions, especially for the olive tree. Thus, in ent, we determined the phenolic status and antioxi- y of four olive cultivars under saline conditions and the between the two different constitutive plant parts. -TPC increased in all cultivars (Fig. 1) with increasing cept in ‘Arbequina’ at 75 mM NaCl. The increase was mM and abrupt at 125 mM NaCl. Especially, at the level interac norma tions in to full of the under are in a Roo vars, b level o at 125 contro quina) regula lation leaves 50 mM exclus salt str transp sympt ate sal in the r is poss contro ponen of ener leaves, cally ac greate also ex Infe ently w Althou increas other h the roo the con similar differe tion of pheno thus, e in orde Peragó 3.2. Ol salinity Lea to mod to dete Hydrox times fication compa oleuro lic com extract rosol w Ole affecte effect of salinity and solar irradiance, showed that under light conditions the TPC and the secoiridoid concentra- sed in the salt-treated plants when they were exposed ght, but not significantly influenced in the shaded side trees. In our experiment the olive plants were grown ight conditions in the greenhouse and, thus, the results dance with those of Remorini et al. (2009). d a different behaviour than leaves (Fig. 1). In all culti- t in ‘Arbequina’, TPC was nearly doubled at the middle nity in comparison to the non-salinized control, while NaCl it decreased again to the levels measured in the d, Ascolana) or to even lower values (Koroneiki, Arbe- exclusion and compartmentalization at the root level n concentration in the xylem sap preventing accumu- otentially toxic ions in the photosynthetically active ns and Tester, 2008). At low or moderate salinity (up to l) most olive tree cultivars exhibit considerable sodium apacity and this mechanism works effectively against but at high salinities in salt sensitive cultivars, Na+ is and deposited to the aerial parts, resulting in toxicity (Chartzoulakis, 2005). Our results indicate that moder- onditions induce the formation of phenolic compounds . Under high salinity, root phenylpropanoid metabolism epressed, since TPC was equal or lower than that of the es. This could be ascribed to the injury of the cell com- sed by the toxic ions at root level, and/or to a priority isposal for the synthesis of phenolic compounds in the er than the roots, in order to protect the photosyntheti- leaves of the olive tree. The accumulation of Na+ and the dative stress at high salinities in the aerial parts could the abrupt increase of TPC in the leaves. ially, the roots and leaves of olive tree behaved differ- the root system was exposed to high NaCl salinity. 75 mM NaCl the TPC increased in both plant parts, the he leaves was small, while in the roots was great. On the , the leaf-TPC was over-doubled at 125 mM NaCl, while C was either not altered or reduced in comparison with . Although in control plants the TPC concentration was aves and roots, while in the two NaCl treatments it was influenced, there is no indication pointing to transloca- nolic compounds between leaves and roots. Probably, mpounds are not transported within the olive tree and, rgan has the capacity to synthesize the essential amount unction properly as suggested also by Ortega-García and 10). pein and hydroxytyrosol concentration according to l and kind of plant tissue root phenolic extracts of four olive cultivars subjected e and heavy salt stress were analyzed by HPLC in order e the concentrations of oleuropein and hydroxytyrosol. osol and oleuropein were identified with retention 7 and 14.32 min, respectively. To confirm the identi- the above compounds, except of retention times, we heir UV (280 nm) spectra with standard solutions of and hydroxytyrosol. Oleuropein was the main pheno- nd found in the chromatographic profile of methanolic both leaves and roots; the concentration of hydroxyty- r lower than that of oleuropein (Figs. 2 and 3). ein concentration in the leaves of all cultivars was NaCl concentration in the nutrient solution (Fig. 2). 40 A. Petridis et al. / Environmental and Experimental Botany 79 (2012) 37– 43 aA 60 .w .) Leaves Root s ZAR D a 60 70 .w .) Leaves Roo ts ASCOLANA Fig. 1. Total p the same colou Hence, oleu exception o est salinity occurred in 5.5, 2.5 and ‘Ascolana’, other wood by excludin accumulati tionally, the by a steep i particularly et al., 2010) oleuropein response in high oleuro capacity an oxidative d molecule an nous enzym glucose pla salinity in o may act as consuming above hypo cose concen between ol terized by a c b B B 0 10 20 30 40 50 125750 T o ta l P h en o ls ( m g g -1 d NaCl (mM) 0 10 20 30 40 50 T o ta l P h en o ls ( m g g -1 d c b a B A C 0 10 20 30 40 50 60 70 125750 T o ta l P h en o ls ( m g g -1 d .w .) Leaves Roo ts KORONE IKI 0 10 20 30 40 50 60 70 T o ta l P h en o ls ( m g g -1 d .w .) NaCl ( mM) henol concentrations in leaves and roots of four olive cultivars treated with 0, 75 or 125 r followed by different letters are significantly different (P ≤ 0.05). ropein concentration declined at 75 mM NaCl, with the f ‘Zard’, which remained at control’s level. At the high- treatment an abrupt and great increase of oleuropein all cultivars, since oleuropein concentration was 18.5, 3.8 folds greater than the control plants for ‘Zard’, ‘Koroneiki’ and ‘Arbequina’, respectively. As occurs in y evergreens, olive adapts to root-zone salinity mostly g Na+ and Cl− at the root level and, thus, preventing Na+ on in the leaves (Tattini, 1994; Tattini et al., 1996). Addi- salt induced osmotic imbalance is partially countered ncrease in the concentration of soluble carbohydrates, the “osmoprotectant” sugar–alcohol mannitol (Cimato . In this experiment we have shown that the increase in concentration is another biochemical salinity induced the olive leaves, but only at high levels of salinity. The pein concentration may be related to its antioxidant d, therefore, oleuropein may offer protection against amage due to salinity. Glucose is a part of oleuropein d can be easily released by the endogenous or exoge- e ˇ-glucosidase (Ranalli et al., 2006). On the other hand, ys an active role in the process of osmotic adaptation to live tree (Tattini et al., 1996). Consequently, oleuropein a glucose-reservoir for osmoregulation or high energy- processes required for plant adaptation to salinity. The thesis was strengthened by measurements of the glu- tration in the leaves. We observed a close relationship europein concentration and glucose, which is charac- significant negative correlation (Pearson’s correlation coefficients ‘Ascolana’, site respons This close r the prefere and provide further rese Ortega-Gar concentrati along with common re from oxida An oppo recorded in an increase the same ex was great, tion decline all cultivars The notion (e.g. TPC, o relationship not shown restricted a for the esse sidering th TPC in both it is highl c b B A B 125750 NaCl (mM) b b a A A B 125750 Leaves Roo ts ARBE QUINA NaCl (mM) mM NaCl. Results are expressed as means ± standard errors. Bars of R (P < 0.01) = −0.90, −0.80, −0.88 and −0.84 for ‘Zard’, ‘Koroneiki’ and ‘Arbequina’, respectively) and an oppo- e of both substances as salinity increased (Figs. 2 and 4). elationship is highly interesting since it could explain nce of olive tree to oleuropein as a salt-tolerance agent s a novel insight to the role of this molecule. However, arch should be conducted in order to confirm this idea. cía and Peragón (2009) found an increase in oleuropein on in olive trees subjected to cold stress. The latter, our findings, may suggest oleuropein accumulation as a sponse of olive tree to various stresses, protecting them tive damage. site behaviour, regarding oleuropein concentration, was roots (Fig. 2). All cultivars, except ‘Arbequina’, showed in oleuropein concentration at 75 mM NaCl, but not to tent. The increase in the cvs ‘Ascolana’ and ‘Koroneiki’ while in ‘Zard’ was quite small. Oleuropein concentra- d sharply below the control’s values at 125 mM NaCl in and only in ‘Ascolana’ it remained at control’s level. of the different behaviour between leaves and roots leuropein) was also evident in the glucose–oleuropein , since no correlation was found in the roots (data ). Possibly, at high salinity oleuropein biosynthesis is nd other compounds, such as mannitol, are synthesized ntial process of osmoregulation in the root zone. Con- e analogue response of oleuropein concentration and leaves and roots and the obtained chromatograms, y possible that oleuropein is the main phenolic A. Petridis et al. / Environmental and Experimental Botany 79 (2012) 37– 43 41 b b a A A B 0 20 40 60 80 100 120 125750 O le u ro p ei n ( m g g -1 d .w .) NaCl (mM) Leaves Root s ZAR D b c a B A B 0 20 40 60 80 100 120 125750 O le u ro p ei n ( m g g -1 d .w .) NaCl (mM) Leaves Roo ts ASCO LAN A b c a B A C 0 20 40 60 80 100 120 140 125750 O le u ro p ei n ( m g g -1 d .w .) NaCl (mM) Leaves Root s KORONE IKI b c a A A B 0 20 40 60 80 100 120 140 160 125750 O le u ro p ei n ( m g g -1 d .w .) NaCl (mM) Leaves Roo ts ARBEQUINA Fig. 2. Oleuropein concentrations in leaves and roots of four olive cultivars treated with 0, 75 or 125 mM NaCl. Results are expressed as means ± standard errors. Bars of the same colour followed by different letters indicate significantly different values (P ≤ 0.05). a a b A B A 0 50 100 150 200 250 300 350 400 0 75 12 5 H y d ro x y ty ro so l (μ g g -1 d .w .) NaCl (mM) Leaves Root s ZARD b a c B C A 0 100 200 300 400 500 600 700 800 0 75 125 H y d ro x y ty ro so l (μ g g - 1 d .w .) NaCl (mM) Leaves Roo ts ASCOLAN A b a cA A A 0 100 200 300 400 500 600 0 75 12 5 H y d ro x y ty ro so l (μ g g -1 d .w .) NaCl (mM) Leaves Root s KORON EIKI b a cA A A 0 50 100 150 200 250 300 350 400 450 0 75 125 H y d ro x y ty ro so l (μ g g -1 d .w .) NaCl (mM) Leaves Roo ts ARBEQUINA Fig. 3. Hydroxytyrosol concentrations in leaves and roots of four olive cultivars treated with 0, 75 or 125 mM NaCl. Results are expressed as means ± standard errors. Bars of the same colour followed by different letters indicate significantly different values (P ≤ 0.05). 42 A. Petridis et al. / Environmental and Experimental Botany 79 (2012) 37– 43 b a c 0 20 40 60 80 100 120 0 75 12 5 G lu co se ( m g g -1 d .w .) NaCl (mM) ZARD b a c 0 20 40 60 80 100 120 0 75 12 5 G lu co se ( m g g -1 d .w .) NaCl (m M) ASCOLANA ARBE QUI NA Fig. 4. Glucos esult letters indicat compound (Figs. 1 and The tren than that of NaCl, leaf h ‘Zard’. The g the cvs ‘Ko rosol decrea cultivars. Pe at moderat was, also, ob and Peragó Root hyd salinity leve decrease w rosol conce the highest Table 1 Antioxidant ac and cultivar fo NaCl (mM) Antioxidant Control 75 125 Antioxidant Control 75 125 b a c 0 20 40 60 80 100 120 0 75 12 5 G lu co se ( m g g -1 d .w .) NaCl (m M) KORONEIKI 0 20 40 60 80 100 120 G lu co se ( m g g -1 d .w .) e concentrations in leaves of four olive cultivars treated with 0, 75 or 125 mM NaCl. R e significantly different values (P ≤ 0.05). involved in olive tree protection against salinity stress 2). d of leaf hydroxytyrosol concentration was different oleuropein, while salinity increased (Fig. 3). At 75 mM ydroxytyrosol levels increased in all cultivars, except reatest increase was recorded in ‘Ascolana’ followed by roneiki’ and ‘Arbequina’. At 125 mM NaCl, hydroxyty- sed abruptly below the values of the control plants in all rhaps, hydroxytyrosol biosynthesis is augmented only e salinities. Decrease in hydroxytyrosol concentration served in the cv. ‘Picual’ after cold stress (Ortega-García n, 2009). roxytyrosol concentration (Fig. 3) did not differ at any l in ‘Koroneiki’ and ‘Arbequina’, while in ‘Zard’ a slight as recorded at 75 mM NaCl. In ‘Ascolana’ hydroxyty- ntration decreased at the moderate and increased at salinity level compared with the control. According to tivity in leaves and roots of four olive cultivars treated with 0, 75 or 125 mM NaCl. Resu llowed by different letters are statistically different (P ≤ 0.05). Zard Ascolana activity (�mol A.A.E. g−1 d.w.) – leaves 191.2 ± 12.4 c 228.4 ± 13.4 c 220.0 ± 32.7 b 254.6 ± 6.58 b 479.8 ± 13.9 a 494.6 ± 16.3 a activity (�mol A.A.E. g−1 d.w.) – roots 277.0 ± 7.16 b 248.7 ± 19.4 b 432.0 ± 25.7 a 531.6 ± 5.93 a 278.0 ± 5.33 b 172.2 ± 6.75 c our results, in the root 3.3. Antioxi tissue As salini leaves of a total pheno at 125 mM total pheno NaCl. Root an vars at 75 was over d other two at 125 mM b a c 0 75 12 5 NaCl (mM) s are expressed as means ± standard errors. Bars followed by different lts are expressed as means ± standard error. Columns of each tissue Koroneiki Arbequina 222.9 ± 7.22 c 260.7 ± 1.44 b 288.1 ± 1.44 b 252.1 ± 18.4 b 532.1 ± 21.5 a 496.8 ± 7.39 a 205.0 ± 15.0 b 170.7 ± 8.08 b 491.5 ± 6.33 a 298.0 ± 21.9 a 143.8 ± 1.00 c 146.2 ± 1.08 b it could be assumed that hydroxytyrosol is not involved antioxidative mechanism. dant activity according to salinity level and plant ty progressed the antioxidant activity increased in the ll cultivars (Table 1) and was in proportion with the ls. Hence, at 75 mM NaCl the increase was slight, but it was twice as high as in the control. As in the case of ls, ‘Arbequina’ did not differ from the control at 75 mM tioxidant activity (Table 1) increased in all culti- mM NaCl. Therefore, in ‘Ascolana’ and ‘Koroneiki’ it oubled compared with control plants, while in the cultivars it was almost double. On the other hand, NaCl the antioxidant activity reached that of the A. Petridis et al. / Environmental and Experimental Botany 79 (2012) 37– 43 43 Table 2 Pearson’s correlation coefficient (R) between TPC and antioxidant activity in leaves and roots of four olive cultivars treated with 0, 75 or 125 mM NaCl. Cultivars Correlation coefficient (R) Arbequina Ascolana Koroneiki Zard * Significant ** Significant control plan ‘Koroneiki’) The obta ipate in the from the hi ity in both a down-reg leaves and the decreas to improve showed an oxidase (AP could comp idant defen 4. Conclus Overall, ropein in th moderate s involved in leaves and translocatio each organ amount tha oleuropein serve as glu additional c tree. Acknowled The auth tute of Thes unit and be References Amiot, M.J., Fl compound 823–826. Beauchamp, G Breslin, P.A oil. Nature Ben Ahmed, C Saline wat mulation 11484–11 Benzie, I., Stra of ‘antioxi Bisingnano, G. the in vitr Pharmaco Bouaziz, M., Ch and antiox Tunisia. J. Chartzoulakis, K.S., 2005. 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Salinity-induced changes in phenolic compounds in leaves and roots of four olive cultivars (Olea europaea L.) and their re... 1 Introduction 2 Material and methods 2.1 Plant material, growth conditions and experimental design 2.2 Sample preparation and extraction of phenolic compounds 2.3 Carbohydrate extraction 2.4 Total phenol content (TPC) 2.5 High performance liquid chromatography (HPLC) analysis of phenolic compounds 2.6 HPLC analysis of glucose 2.7 FRAP assay 2.8 Statistical analysis 3 Results and discussion 3.1 Total phenol content according to salinity level and kind of plant tissue 3.2 Oleuropein and hydroxytyrosol concentration according to salinity level and kind of plant tissue 3.3 Antioxidant activity according to salinity level and plant tissue 4 Conclusions Acknowledgements References