Changes in water status and osmolyte contents in leaves and roots of olive plants ( Olea europaea L.) subjected to water deficit

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Two-year-old olive trees (Olea europaea L., cv. Coratina) were subjected to a 15-day period of water deficit, followed by 12 days of rewatering. Water deficit caused decreases in predawn leaf water potential (Ψw), relative water content and osmotic
  ORIGINAL PAPER Changes in water status and osmolyte contents in leaves and rootsof olive plants ( Olea europaea  L.) subjected to water deficit Bartolomeo Dichio   Giovanna Margiotta   Cristos Xiloyannis   Sabino A. Bufo   Adriano Sofo   Tommaso R. I. Cataldi Received: 7 February 2008/Revised: 11 August 2008/Accepted: 2 September 2008/Published online: 23 September 2008   Springer-Verlag 2008 Abstract  Two-year-old olive trees ( Olea europaea  L.,cv. Coratina) were subjected to a 15-day period of waterdeficit, followed by 12 days of rewatering. Water deficitcaused decreases in predawn leaf water potential ( W w ),relative water content and osmotic potential at full turgor( W p 100 ) of leaves and roots, which were normally restoredupon the subsequent rewatering. Extracts of leaves androots of well-watered olive plants revealed that the mostpredominant sugars are mannitol and glucose, whichaccount for more than 80% of non-structural carbohydratesand polyols. A marked increase in mannitol contentoccurred in tissues of water-stressed plants. During waterdeficit, the levels of glucose, sucrose and stachyosedecreased in thin roots (with a diameter \ 1 mm), whereasmedium roots (diameter of 1–5 mm) exhibited no differ-ences. Inorganic cations largely contribute to  W p 100  andremained stable during the period of water deficit, exceptfor the level of Ca 2 ? , which increased of 25% in water-stressed plants. The amount of malate increased in bothleaves and roots during the dry period, whereas citrate andoxalate decreased. Thin roots seem to be more sensitive towater deficit and its consequent effects, while mediumroots present more reactivity and a higher osmotic adjust-ment. The results support the hypothesis that the observeddecreases in  W w  and active osmotic adjustment in leavesand roots of water-stressed olive plants may be physio-logical responses to tolerate water deficit. Keywords  Drought    Water stress    Osmotic adjustment   Mannitol    Organic acids Introduction Olive tree ( Olea europaea  L.) is a woody species typicallycultivated in most Mediterranean countries where plantsare often exposed to long periods of water deficit during thedry season (Connor and Fereres 2005). Among fruit treespecies, olive tree is able to tolerate a broad range of adverse environmental factors, including the low avail-ability of water in soil (Bacelar et al. 2007), salinity (Tattini et al. 1996), chilling and high temperature stress (Bongi and Long 1987) and high irradiance levels (Sofo et al. 2004).The uncommon capability of adaptation of olive treeagainst water deficit is due to a variety of morphologicaland physiological adaptations, such as the regulation of stomata aperture and transpiration (Nogue´s and Baker2000), the regulation of gas exchange (Xiloyannis et al.2004), the appearance of leaf anatomical alterations(Chartzoulakis et al. 1999), the ability of extracting water from the soil due to a deep root system and to a high waterpotential gradient between canopy and roots (Ferna´ndezet al. 1997), and a very developed osmotic adjustment (Chartzoulakis et al. 1999; Dichio et al. 2006). This last contribution describes changes in the osmotic potential of leaves and roots due to accumulation of organic osmolytes Communicated by T. Buckley.B. Dichio ( & )    G. Margiotta    C. Xiloyannis   S. A. Bufo    A. SofoDipartimento di Scienze dei Sistemi Colturali,Forestali e dell’Ambiente, Universita` degli Studi dellaBasilicata, Via dell’Ateneo Lucano, 10, 85100 Potenza, Italye-mail: bartolomeo.dichio@unibas.itT. R. I. CataldiDipartimento di Chimica, Universita` degli Studi della Basilicata,Via N. Sauro 85, 85100 Potenza, Italy  1 3 Trees (2009) 23:247–256DOI 10.1007/s00468-008-0272-1  confined to the cytoplasmic compartment and vacuoles of cells (Munns 1988). Osmotic adjustment reduces the osmotic component of the total water potential and allows plant tissues tomaintain water even at low xylem and soil water poten-tials, so maintaining turgor and metabolic activity, andindirectly growth and productivity during prolonged waterdeficit (Hanson and Hits 1982; Rhodes and Samaras 1994). The recognized metabolic benefits of osmolyteaccumulation may depend on either active augmentationof them within cells (i.e., active osmotic adjustment) orloss of water from the plant tissues and the consequentconcentration of solutes (i.e., passive osmotic adjustment)or both.The contribution of passive osmotic adjustment in olivetree varies on the basis of plant physiological conditionsand genotypes but it represents approximately a half of thetotal osmotic adjustment, whereas the other half is due to ex novo  synthesis of osmotically active compounds (Dichioet al. 2006). The levels of specific sugar compounds andthe extent of their involvement in osmotic adjustmentdepend on the plant species, genotype within the samespecies and environmental stress conditions (McCue andHanson 1990). In particular, studies performed on peach( Prunus persica  [L.] Batsch.) (Lo Bianco et al. 2000) have demonstrated the important role of sorbitol and sucrose inplants subjected to water deficit. Osmotic adjustment of   Rosaceous  fruit trees, such as apple (  Malus domestica  [L.]Borkh.) (Wang and Stutte 1992) and cherry ( Prunus cer-asus  L. and  P. avium x pseudocerasus ) (Ranney et al. 1991) during water deficit is mainly accompanied by the accu-mulation of sorbitol formed by glucose reduction.Mannitol, another alditol, is the most widely distributedhexitol in nature (Williamson et al. 2002) and has beenidentified as the major active solute, which increases itslevel in leaves of   Fraxinus excelsior   L. under water deficitconditions (Guicherd et al. 1997). Other important osmo- lytes in plants are organic acids (such as malic, oxalic andcitric acids), inorganic cations, inorganic anions (mainlysulphate, phosphate and chloride) and some amino acids,such as proline and glycine betaine (Ingram and Bartels1996; Nuccio et al. 1999). Understanding the mechanisms by which olive plantstolerate water deficit is essential for selecting more tolerantplant cultivars. The objective of this work was to study theinfluence of water deficit and a following rewatering onosmolyte contents in olive plants and the contribution of osmolyte accumulation to osmotic adjustment. As there arefew reports concerning the carbohydrate and polyolscomposition and their physiological role in woody planttissues other than leaves, changes in the osmolyte contentsat increasing levels of water deficit were also investigatedin olive roots. Materials and methods Plant material and water-stress conditionsA group of 50 olive trees ( Olea europaea  L. cv. Coratina),two year old and own rooted, was transplanted on March2000 in 18-L pots containing approximately a 2:1 mixtureof soil (83.1% sand, 8.8% silt and 9.0% clay) and peat.Cultivar Coratina is indigenous of southern Italy charac-terized by a high degree of tolerance against water deficit if compared to other olive cultivars and by expanded leaveswith an elliptic–lanceolate shape (Xiloyannis et al. 2004).Until 5 July, when water-stress period was initiated, plantswere grown outside under natural conditions in Metapontoat ‘Pantanello’ Agricultural Experimental Station(40  24 0 N, 16  48 0 E) and were supplied with 3–4 g of slownitrogen release complex fertilizer (Nitroposka Gold 15N - 9P - 16K   ?  2Ca  ?  7Mg; Compo Agricoltura, CesanoMaderno, MI, Italy) every 25–30 days. Plants were sub- jected to 15 days of water deficit followed by 12 days of rewatering conditions. Before the onset of water-stressperiod, each pot was covered with plastic films and alu-minium sheets to minimize evaporation from soil surfaceand to avoid exposure of soil to direct sunlight, respec-tively. During the plant growth, the soil water content wasconstantly maintained at about 85% of field water capacityby adding at the end of daily sunlight the amount of waterlost through transpiration.During the period of water deficit, ten plants were usedas controls (control plants, CP) and irrigated daily tomaintain an optimum soil water content; the remainingplants were stressed (stressed plants, SP) by withholdingwater as to achieve four stress levels at following predawnleaf water potential ( W w ) values:  - 0.45 MPa (CP; day 0from the beginning of water deficit),  - 1.5 MPa (day 7), - 3.9 MPa (day 11) and  - 6.0 MPa (day 15). CP weremaintained at a predawn  W w  of   - 0.45 MPa during thewhole experimental period. The water soil conditions of well-watered plants were restored during the subsequent12 days of rewatering.Plant water statusThroughout the periods of water deficit and rewatering,plant water status was determined by concomitant mea-surements of relative water content (RWC) and W w , as theyare two significant indicators of the degree of water deficit.The values of  W w  were measured at predawn (04:30 h) onexpanded leaves taken from each plant along the mediansegment of new-growth shoots. Each excised leaf wasimmediately put inside a polyethylene bag for  W w  mea-surement with a Sholander pressure chamber (Model 600,PMS Instruments Co. Corvallis, OR, USA). In order to 248 Trees (2009) 23:247–256  1 3  obtain the RWC values of olive leaves and roots, afterharvest, the plant tissues were weighed for fresh mass (FM)determination and then placed in beakers sealed withparafilm and hydrated in the dark for 12–24 h. Aftermeasurement of saturated mass (SM), the plant sampleswere dried at 80  C for 48 h to determine dry mass (DM).The RWC values were evaluated as:RWC  ¼  100 FM    DM ð Þ =  SM    DM ð Þ½  ð 1 Þ Throughout the drought and rewatering treatments,tissues were hydrated for 12–24 h before determinationof osmotic potential at full turgor ( W p 100 ). Cell contentswere extracted using plastic syringes to squeezehomogeneously the tissue and to extrude 100  l L cell-content samples. Each sample was used to determine theosmolarity with a vapour pressure osmometer (Wescormodel 2000, Logan, UT, USA) calibrated against a saltsolution. The values of   W p 100  of expressed sap werecalculated from the Van’t Hoff relation as given by Nobel(1983): W p 100 ð MPa Þ ¼ ð 0 : 02437m 3 MPamol  1 Þ ½ osmolarity ð molm  3 Þ ð 2 Þ Active osmotic adjustment (  A DW p 100 ), as a result of thenet accumulation of osmolytes in the symplast, was definedas the difference between the value of   W p 100  in stressedplants (SP) and the respective value in control plants (CP).Plant material preparationDuring the periods of water deficit and rewatering, leavesandrootsweresampledbeforedawntominimizevariationinsolute accumulation during light exposure. For root sam-pling, the selected plants were removed from pots, gentlyseparated from the attached soil and carefully washed withdistilled water. After sampling, roots were divided in twogroups:mediumroots(maturationzone;withadiameter( [ )of 1–5 mm)and thin roots (elongation zone; [ \ 1 mm).Allplant tissues were immediately stored at  - 80  C untillyophilization. Samples were collected from three plantshaving the same W w , with each sample containing five fullyexpandedleavesfromthemid-sectionofcurrent-yearshootsand 10–15 g FM of roots. The lyophilized tissues wereground with an IKA analytical mill Model A10 (Aldrich,Milan, Italy) and stored in airtight vials at room temperatureuntil analysis. All the osmolyte concentrations wereexpressed as mmol per kg of dry mass (DM).Low molecular weight non-structural carbohydratesand polyolsLow molecular weight non-structural carbohydrates andpolyols (NSC–P) were extracted as described by Cataldiet al. (2000). A weighed amount (approximately 50 mg) of  lyophilized tissue, added of internal standard 3- O -meth-ylglucopyranose at the concentration of 40  l M, wassuspended in 16 mL of deionized water (Milli Q grade,Millipore, Bedford, MA, USA). The suspension was thenshaken for 15 min and centrifuged at 3,000 rpm for 10 min(Model 4225, ALC s.r.l., Italy). Prior to analysis, theaqueous extract was filtered through single use 0.22  l m-pore-size nylon filter (Aldrich, St Louis, MO) and passedon a cartridge On Guard A (Dionex, Sunnyvale, CA) toremove anion contaminants. Excellent recoveries wereobtained for each sugar constituent in both analyzed olivetissues, which on average ranged from 97  ±  1% forstachiose to 104  ±  3% for glucose.Carbohydrates and polyols were analyzed by high-per-formance anion-exchange chromatography (HPAEC) inconjunction with pulsed amperometric detection (PAD)using a DX-500 liquid chromatograph (Dionex). Solublesugars were separated on a Dionex CarboPac PA1 col-umn (250  9  2 mm id) equipped with a guard column(50  9  2 mmid)andelutedwithNaOH12 mM  ? Ba(AcO) 2 1 mM at a flow rate of 0.4 mL min - 1 . Column and pre-column were maintained at a temperature of 22  ±  1  C by awater jacket coupled with a circulating water bath modelWK4DS (Colora, Messtechnik GmbH, Germany). Thefollowing triple-potential waveform was used to detectcarbohydrates and polyols as described previously(Cataldi et al. 2000): detection potential,  E  DET  = ? 0.25 V( t  DEL  =  240 ms,  t  INT  =  200 ms), oxidation potential E  OX  = ? 0.80 V ( t  OX  =  180 ms), reduction potential E  RED  = - 0.25 V ( t  RED  =  360 ms). Low molecular weightnon-structural carbohydrates and polyols were identified bycomparison with retention times of sugar standards injectedunder the same experimental conditions. As mannitol andglucose were present at relatively high levels in olive tissueextracts, a 16-fold dilution was usually accomplished beforeinjection.Inorganic and organic anionsInorganic and organic anions were extracted with wateraccording to Russo and Karmarkar (1998) and analyzed byion chromatography with conductivity detection (IC-CD)using a DX-500 liquid chromatograph (Dionex). Inorganicanions were separated on an IonPac AS12A column(Dionex), whereas both inorganic and organic anions wereseparated on a microbore IonPac AS11-HC column (Dio-nex). The AS12A column was eluted with Na 2 CO 3 2.7 mM  ?  NaHCO 3  0.3 mM at 22  ±  1  C at a flow rate of 1.0 mL min - 1 . The AS11-HC column was eluted with 10–30 mM NaOH gradient in order to reduce the base linedistortions at 30  ±  1  C at a flow rate of 0.38 mL min - 1 .The NaOH eluent was prepared daily starting from a 50% Trees (2009) 23:247–256 249  1 3  (w/w) NaOH solution under helium pressure by using aDionex EO1 eluent/solvent organizer. The quantification of anions was performed using the calibration curves obtainedfor each anion by plotting the peak area ratios of eachcompound to an internal standard (IS) against the con-centrations of each analyte identified. The internal standardused was nitrite, whose chromatographic peak was mark-edly separated by other peaks and not present in the plantsamples examined.Inorganic cationsInorganic cations were extracted by an acid digestion,according to Walinga et al. (1995). A weighed amount(approximately 400 mg) of lyophilized tissue was added to10 mL of 95% H 2 SO 4  and 4 mL of HNO 3  concentrated.After mineralization for 2 h at 370  C, the material wassuspended in pure water and then filtered through paper(Whatman International Ltd, Maidstone, England). Prior toanalysis, the aqueous extract was 50 9 diluted to be includedin the linearity ranges defined for each cation. The levels of Na ? , K  ? , Ca 2 ? and Mg 2 ? were determined by flame atomicabsorption spectrophotometry using a Spectra AA-30 flamespectrophotometer (Varian, Mulgrave, Victoria, Australia)with cathode lamps (Photron Pty. Ltd, Australia). The lampamperagewas5.0 mAforNa ? andK  ? analysis,and3.5 mAfor Ca 2 ? and Mg 2 ? analysis. A mixture of acetylene and air(flametemperatureof2,400–2,700  K)wasusedtovolatilizeand atomize samples. For Na ? , K  ? , Ca 2 ? and Mg 2 ? anal-ysis, the wave lengths selected were 589.0, 766.5, 422.7 and285.2 nm, respectively, whereas the concentration rangesused were 0.15–0.60, 0.5–2.0, 1–4 and 0.1–0.4  l g mL - 1 ,respectively. Results Effects of water deficit on plant water statusThe values of   W w  and RWC of olive tissues were signifi-cantly affected by water availability (Fig. 1). The waterpotential decreased rapidly after the 7 days of water deficitand then increased after 15 days, when plants were rewa-tered (Fig. 1a). At the end of the rewatering period, SPreached the same  W w  value of CP (Fig. 1a). In parallel,RWC in SP decreased, reaching the value of 59  ±  3% inleaves after 15 days of water deficit (Fig. 1b), whereasslight differences of RWC were observed in medium andthin roots (Fig. 1c, d). After 12 days from the beginning of rewatering, RWC in all the tissues reached the values of CP(Fig. 1). During the whole experimental period, CP showedno major changes of   W w  and RWC (Fig. 1b, d).Thevaluesof  W p 100 inleavesandmediumrootsdecreasedwith decreasing  W w , and they were lower in leaves than inroots during the whole experimental period (Fig. 2a). At thebeginning of the experiment, the mean of   W p 100  was - 2.61  ±  0.02 MPa in leaves,  - 1.26  ±  0.11 MPa in med-ium roots and  - 0.57  ±  0.02 MPa in thin roots (Fig. 2a).Waterdeficitdeterminedadeclineinthevaluesof   A DW p 100 ,particularlymarkedinleavesandmediumroots( - 0.68 MPa Fig. 1  Changes in leaf waterpotential ( W w ) and relativewater content (RWC) in leaves,medium roots and thin roots of water-stressed ( closed symbols )and control ( open symbols )plants.  Vertical dotted lines indicate the beginning of therewatering period. Values arethe means ( ± SE) of threemeasurements from threeselected plants.  Values with theasterisk   are significantlydifferent between control andwater-stressed plants ( P  B  0.05,according to Student’s  t  -test)250 Trees (2009) 23:247–256  1 3  and - 1.69 MPaafter15 days,respectively)(Fig. 2b).Attheend of the rewatering period,  A DW p 100  values in leaves andmedium roots were significantly lower than those observedat the beginning of the experiment, whereas no significantdifferences were found in thin roots (Fig. 2b).Changes in low molecular weight non-structuralcarbohydrates and polyols during water deficitAnalyses of leaf extracts from CP revealed that mannitoland glucose were the predominant sugar compounds with avery similar content, representing 43.2 and 45.0%,respectively, of the total amount of NSC–P (Table 1). Inleaf tissues, the levels of raffinose series oligosaccharides,namely raffinose and stachyose, accounted for less than2.0% of total NSC–P, and the levels of the raffinose oli-gosaccharides precursors, namely galactose, sucrose and myo -inositol, were about fourfold higher than those of theraffinose series (Table 1). Fructose was present in leaves ata concentration of 9.0  ±  3.0 mmol kg - 1 DM, which isapproximately 24-fold lower than that of glucose (Table 1).In the roots of CP, the same amount of sugar compounds of leaves samples was found, even though the amounts of each carbohydrate and sugar alcohol changed according tothe specific root size (Table 1). Likewise leaves, the mostabundant sugar compounds in olive roots were mannitoland glucose, which represented together 82 and 70% of thetotal NSC–P in medium and thin roots, respectively(Table 1). In leaves of CP, the total amount of otheridentified carbohydrates and polyols, other than mannitoland glucose, was about 12% (Table 1).Water deficit caused significant increases in mannitolcontent in all the olive tissue examined (Table 1). After15 days of water deficit, the mannitol levels in leaves,medium and thin roots were 294  ±  28, 229  ±  8 and219  ±  19 mmol kg - 1 DM, respectively (Table 1). After12 days of rewatering, the mannitol content of root tissuesdeclined to control levels, whereas leaves exhibited aslightly lower value if compared to CP (Table 1). Glucose,the second most abundant sugar in olive tissues, increasedsharply throughout the drought period (Table 1). No sig-nificant variation of glucose was revealed in medium rootsof SP if compared to CP, whereas thin roots exhibited areduction in all the levels of water deficit (Table 1). At theend of the rewatering period, glucose concentration inleaves of SP reached the same values of CP, whereas itscontent in medium and thin roots was slightly lower thanthat found in CP (Table 1). Sucrose levels in olive tissueswere much lower than those of mannitol and glucose(Table 1). In SP, sucrose exhibited a significant quantita-tive decrease both in leaves and thin roots after 11 days,whereas no significant differences were found in mediumroots (Table 1).The differences in sugar content of other identifiedcompounds in SP were minor, with two exceptions: fruc-tose in leaves and stachyose in thin roots (Table 1).Although fructose content in olive tissues was much lowerthan that of glucose, leaves of SP exhibited a markedincrease in this sugar up to a  W w  of   - 3.9 MPa (after11 days of water deficit) and then a rapid fall at a  W w  of  - 6.0 MPa (after 15 days) (Table 1). The level of stachyoseat the maximum level of water deficit showed a significantdecrease in thin roots of SP with decreasing water poten-tial, reaching a value of about one-tenth of that found at thebeginning of the experiment (Table 1).The contribution of NSC–P to  W p 100  in leaves, mediumroots and thin roots was  - 0.97,  - 0.55 and  - 0.19 MPa inCP, and - 0.96, - 0.98 and - 0.31 MPa in SP, respectively(Table 2). The osmotic contribution of mannitol in all thetissues increased in water-stressed plants, whereas glucose Fig. 2 a  Osmotic potential at full turgor ( W p 100 ) in leaves ( graycolumns ), medium roots ( black columns ) and thin roots ( whitecolumns ) of water-stressed plants during the experimental period.  b Active osmotic adjustment (  A DW p 100 ) in leaves, medium roots andthin roots of water-stressed plants. Values are the means ( ? SE) of three measurements from three plants having the same  W w . Valuesfollowed by  different letters  ( uppercase letters  between tissue typesand  lowercase  between sampling dates) are significantly different( P  B  0.05, according to Student’s  t   test)Trees (2009) 23:247–256 251  1 3
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