Annals of Botany

AOBPreview originally published online on August 13, 2008
Annals of Botany 2008 102(4):609-622; doi:10.1093/aob/mcn134

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Responses to Changes in Ca²⁺ Supply in Two Mediterranean Evergreens, Phillyrea latifolia and Pistacia lentiscus, During Salinity Stress and Subsequent Relief

Massimiliano Tattini^* and Maria Laura Traversi

Istituto per la Valorizzazione del Legno e delle Specie Arboree, IVALSA, Consiglio Nazionale delle Ricerche, Via Madonna del Piano 10, I-50019, Sesto F.no, Firenze, Italy

^* For correspondence. E-mail m.tattini{at}ivalsa.cnr.it

Received: 6 May 2008    Returned for revision: 6 June 2008    Accepted: 25 June 2008    Published electronically: 14 August 2008

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS APPENDIX 1 APPENDIX 2 LITERATURE CITED

 

Background and Aims: Changes in root-zone Ca²⁺ concentration affect a plant's performance under high salinity, an issue poorly investigated for Mediterranean xerophytes, which may suffer from transient root-zone salinity stress in calcareous soils. It was hypothesized that high-Ca²⁺ supply may affect differentially the response to salinity stress of species differing in their strategy of Na⁺ allocation at organ level. Phillyrea latifolia and Pistacia lentiscus, which have been reported to greatly differ for Na⁺ uptake and transport rates to the leaves, were studied.

Methods: In plants exposed to 0 mM or 200 mM NaCl and supplied with 2·0 mM or 8·0 mM Ca²⁺, under 100 % solar irradiance, measurements were conducted of (a) gas exchange, PSII photochemistry and plant growth; (b) water and ionic relations; (c) the activity of superoxide dismutase and the lipid peroxidation; and (d) the concentration of individual polyphenols. Gas exchange and plant growth were also estimated during a period of relief from salinity stress.

Key Results: The performance of Pistacia lentiscus decreased to a significantly smaller degree than that of Phillyrea latifolia because of high salinity. Ameliorative effects of high-Ca²⁺ supply were more evident in Phillyrea latifolia than in Pistacia lentiscus. High-Ca²⁺ reduced steeply the Na⁺ transport to the leaves in salt-treated Phillyrea latifolia, and allowed a faster recovery of gas exchange and growth rates as compared with low-Ca²⁺ plants, during the period of relief from salinity. Salt-induced biochemical adjustments, mostly devoted to counter salt-induced oxidative damage, were greater in Phillyrea latifolia than in Pistacia lentiscus.

Conclusions: An increased Ca²⁺ : Na⁺ ratio may be of greater benefit for Phillyrea latifolia than for Pistacia lentiscus, as in the former, adaptive mechanisms to high root-zone salinity are primarily devoted to restrict the accumulation of potentially toxic ions in sensitive shoot organs.

Key words: Calcium–sodium interactions, gas exchange, Na allocation, Na uptake and transport, oxidative damage, Phillyrea latifolia, Pistacia lentiscus, polyphenols, PSII photochemistry, relief from salinity, water relations

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS APPENDIX 1 APPENDIX 2 LITERATURE CITED

 
Pistacia lentiscus (Anacardiaceae) and Phillyrea latifolia (Oleaceae) are evergreen sclerophylls, largely distributed in dry/warm areas of the Mediterranean basin, well-adapted to severe conditions of drought in very unfertile soils (Filella et al., 1998; Tattini et al., 2006). Both species are currently being investigated for their potential to restore vegetation in dry-land areas of the Mediterranean basin, which may suffer from excess soil salinity during the warm summer-season (Margaris, 1981; Gucci et al., 1997a).

Phillyrea latifolia is effective in limiting the transport of potentially toxic ions and to maintain favourable K⁺:Na⁺ ratios in actively growing shoot organs, mostly due to a reduced transport of water (Tattini and Gucci, 1999; Tattini et al., 2002). Salt-induced osmotic stress is largely countered by an increase in the concentration of soluble carbohydrates, particularly mannitol (Tattini et al., 2002). This ‘low-sodium strategy’ (Flowers and Yeo, 1995; Munns, 2002) exposed Phillyrea latifolia plants to (transient) severe water stress in response to an increase in root-zone NaCl, but allowed a prompt recovery of gas exchange performance once salinity stress was relieved (Tattini et al., 1997; Tattini et al., 2002). This strategy is ecologically relevant, as leaching of salts from the soil by late-summer/early-autumn rainfalls allow Mediterranean evergreens to assimilate carbon and produce new growth at considerable rates during periods of relief from high salinity, because of still-optimal sunlight irradiance and air temperature (Cerling et al., 1993; Tattini and Traversi, 2008).

Pistacia lentiscus, a species largely distributed in salty wetlands of the Mediterranean coasts (Pignatti, 1997), has been poorly investigated for adaptive strategies to high root-zone salinity (Tattini et al., 2006). Transport rates of Na⁺ to the leaves accounted for most of the Na⁺ uptake rates, and leaf Na⁺ concentrations as high as 600 mM on a bulk-tissue-water basis (irrespective of leaf age) were detected in Pistacia lentiscus supplied with 200 mM NaCl and exposed to full sunlight over a 2-month period (Tattini et al., 2006). It is conceivable that a very efficient mechanism of vacuolar salt-sequestration operates in Pistacia lentiscus leaves (Niu et al., 1995; Munns, 2002), which are not equipped with specialized organs to excrete ions from the leaf (Pignatti, 1997).

Dry-lands of the Mediterranean basin are very rich in Ca²⁺ (White and Broadley, 2003), and most fruit-tree crops cannot be cultivated in such areas, with the notable exceptions of Olea europaea and Pistacia vera, two species closely related to Phillyrea latifolia and Pistacia lentiscus (Melgar et al., 2006; Tattini and Traversi, 2008). Consequently, how changes in root Ca²⁺ may affect response mechanisms and performances of these species when suffering from excess soil salinity is of great eco-physiological and horticultural value. In simple terms, an increase in Ca²⁺ supply under high root-zone NaCl concentration enhances the selectivity for the uptake and the transport of K⁺ over Na⁺, by inhibiting the unidirectional influx of Na⁺ into the roots and decreasing the Na⁺-stimulated efflux of K⁺ (Maathuis and Amtmann, 1999; Zhu, 2002). An increase in cytoplasmic Ca²⁺ may activate both Ca²⁺-dependent protein kinases and a Ca²⁺-calmodulin dependent PP-2B protein phosphatase capable of modifying the Na⁺ and K⁺ uptake systems to have a high affinity for K⁺ (Knight et al., 1997; Zhu, 2002). These Ca²⁺-activated mechanisms are thought to be mostly responsible for the increased salt tolerance in plants supplied with high Ca²⁺ (Pardo et al., 1998; White and Broadley, 2003). Changes in Ca²⁺ supply may also affect other key mechanisms of acclimation of plants to elevated root-zone NaCl concentrations, such as those linked to the salt-induced increase in ABA biosynthesis, which is responsible for stomatal closure (Bowler and Fluhr, 2000; Wilkinson and Davies, 2002; Scrase-Field and Knight, 2003). Nevertheless, contradictory results have been reported for the effect of Ca²⁺ on stomatal conductance in attached leaves (Atkinson, 1991; Sohan et al., 1999; Yang et al., 2003; Shabala et al., 2005), which may help explain the sometimes inconsistent effects of high Ca²⁺ supply on whole-plant performance under root-zone salinity stress (Ashraf and Naqvi, 1991; Schmidt et al., 1993; Cramer, 2002; Hernández et al., 2003; Tester and Davenport, 2003).

Here the hypothesis was tested that high-Ca²⁺ supply will differentially affect the performance of species, which either avoid large fluxes of Na⁺ and Cl^– to the leaves (Phillyrea latifolia) or use these potentially toxic ions to decrease leaf osmotic potential (Pistacia lentiscus) in response to high salinity. In this experiment, Phillyrea latifolia and Pistacia lentiscus were exposed to 0 mM or 200 mM NaCl and supplied with Ca²⁺ at 2·0 mM (referred to as low-Ca²⁺ supply and low-Ca²⁺ plants throughout the paper) or 8·0 mM (high-Ca²⁺ supply and high-Ca²⁺ plants) under 100 % solar radiation, as soil salinity concentration rises during clear days in arid or semi-arid zones of the Mediterranean basin (Margaris, 1981; Tattini et al., 2006). Measurements were conducted of (a) gas exchange, net carbon gain and plant growth; (b) net fluxes of Na⁺, Cl^–, and K⁺, and the allocation of Na⁺ at whole-plant and organ levels; (c) water relations and the contributions of individual solutes to salt-induced osmotic adjustment; (d) photosynthetic performances and photosystem II (PSII) photochemistry; (e) the activity of superoxide dismutase (SOD) and the degree of lipid peroxidation [estimated in terms of malondialdehyde (MDA) concentration]; and (f) leaf polyphenol concentration and composition, together with calculations of ‘newly assimilated carbon’ allocated to polyphenols. Gas exchange, growth rates and Na⁺ allocation were also measured during a period of relief from salinity stress, as most evergreens inhabiting Mediterranean dry-lands are exposed to fluctuating root-zone salinity over either the whole-growth season or the long period of a leaf's life span.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS APPENDIX 1 APPENDIX 2 LITERATURE CITED

 
Plant material and growth condition
One-year-old Phillyrea latifolia and Pistacia lentiscus plants were grown at Follonica (42°46'N, 10°53'E) in 4·5-L pots with artificial substrate (consisting of sphagnum peat: pumice: sand, 40 : 40 : 20, v/v/v) from 5 July to 20 September 2005. On average, plants received a daily irradiance of 12·1 MJ m^–2 over the PAR (400–700 nm) waveband, as measured with a Li-1800 spectroradiometer (equipped with a remote cosine receptor; Li-Cor Inc., Lincoln, NE, USA), on a total of 25 d, both clear and cloudy, over the whole experimental period. The low-Ca²⁺ standard nutrient solution had the following composition (mM): Ca(NO₃)₂, 2·0; KNO₃, 2·0; KH₂PO₄, 0·5; Mg(SO₄)₂, 0·4. The high-Ca²⁺ standard nutrient solution was obtained by adding 6·0 mM CaCl₂ to the low-Ca²⁺ standard nutrient solution. In both Ca²⁺ treatments, micronutrients were as in a 1/3 Hoagland solution (Hoagland and Arnon, 1950). Plants were supplied with 0 mM or 200 mM NaCl, three times a week (final salinity concentration was reached by the end of a 4-d period by daily increments of 50 mM NaCl), over a 5-week period followed by a 5-week period of relief from salinity stress, during which plants were supplied with the low-Ca²⁺ standard nutrient solution. The experiment was a completely randomized design, each pot (plant) representing a replicate.

Gas exchange, PSII photochemistry and plant growth
Gas exchange characteristics at saturating light (i.e. at 900 µmol m^–2 s^–1 over the PAR waveband) were measured on medial leaves under laboratory conditions, using a portable Li-Cor 6400 (Li-Cor Inc.) infrared gas analyser operating at 34 ± 0·5 Pa ambient CO₂. The response of CO₂ assimilation rate (A_sat) to changes in intercellular CO₂ concentration (c_i) (A/c_i curves at saturating light) were analysed by keeping the leaf chamber at an air flux of 0·5 Pa CO₂, over a 40-min period, to stimulate stomatal opening. A/c_i curves were then constructed by varying the external CO₂ concentration using a Li 6400-01 CO₂ mixer, and the ‘apparent’ maximum rate of carboxylation by Rubisco (referred to as carboxylation efficiency throughout this paper) was calculated as in Guidi et al. (2008). Note that the analysis actually underestimated V_c,max, as in leaves in sunshine the intercellular CO₂ partial pressure may largely exceed CO₂ inside the chloroplasts, due to strong mesophyll limitations to CO₂ diffusion (Evans and von Caemmerer, 1996; Centritto et al., 2003). However, mesophyll anatomy did not vary in response to Ca²⁺ supply (data not shown), which allowed comparison of V_c,max of low- vs. high-Ca²⁺ leaves. Also note that both LMA (leaf mass per unit leaf area; 14·7 ± 1·5 in Phillyrea latifolia or 15·6 ± 1·1 mg d. wt cm^–2 in Pistacia lentiscus, mean ± s.d., n = 16) and whole-leaf thickness (325·7 ± 18·7 or 343·4 ± 17·8 µm in Phillyrea latifolia or Pistacia lentiscus, respectively) did not significantly vary in the species examined. Daily assimilated CO₂ (CO₂^daily) was calculated by in situ measurements of net photosynthesis, which were conducted at 3-h intervals during the day, from 0600 h to 2000 h, and night-time respiration rates, which were determined at midnight and 0400 h. Net daily carbon gain was then calculated by the integration procedures reported in Valladares and Pearcy (1997) and in Tattini et al. (2004).

Steady-state modulated chlorophyll fluorescence was performed using a portable PAM-2000 fluorometer (Walz, Effeltrich, Germany) connected to a Walz 1030-B leaf-clip holder through a Walz 2010-F trifurcated fibre optic as reported in Tattini et al. (2005). In detail, the maximum efficiency of PSII photochemistry of both dark-adapted (F_v/F_m) leaves and leaves exposed to saturating light (_PSII) was estimated using the protocols of Schreiber et al. (1986) and of Genty et al. (1989). Briefly, after measuring the minimal fluorescence (F₀), using a modulated light pulse <1 µmol m^–2 s^–1 (to avoid appreciable variable fluorescence), and the maximum fluorescence yield (F_m), through a 0·8-s saturating pulse (at 20 kHz) of white light at 8000 µmol m^–2 s^–1, the calculation F_v/F_m = (F_m – F₀)/F_m of dark-adapted (over a 40-min period) leaves was made. The actual efficiency of PSII photochemistry was calculated as _PSII = (F'_m – F_s)/F'_m. F'_m, i.e. the maximal fluorescence in light conditions,, was determined at 900 µmol m^–2 s^–1 of photons over the PAR waveband together with the steady-state fluorescence level (F_s).

Relative growth rate was calculated by the increase in shoot dry weight (W), over the period t₁ – t₀ on plants harvested at the end of salinity stress and the relief periods, using the equation, RGR = (ln W₁ – ln W₀)/t₁ – t₀. The shoot dry weight did not differ markedly in the examined species at the beginning of the experiment, i.e. 7·2 ± 0·9 or 8·7 ± 1·3 g (mean ± s.d., n = 8) for Phillyrea latifolia and Pistacia lentiscus, respectively.

Ions and soluble carbohydrates
Ground dried-tissues were extracted with ultrapure water for 2 h in a boiling water bath for chloride quantification, which was performed with a Quanta 4000E ion capillary electrophoresis unit (Waters, Milford, MA, USA), based on the protocols previously reported in Tattini and Gucci (1999). Cation analysis was performed with a Perkin-Elmer 1100 emission-absorption spectrophotometer (Perkin-Elmer, Norwalk, CT, USA) on tissue previously digested in a microwave digestion unit (Mls 1200 Mega, Milestone Italia srl, Bergamo, Italy). The amount of Na⁺ in different plant organs (moles organ^–1) were calculated by multiplying the tissue elemental concentration by the dry weight of the relative organ at the end of both salinization and relief periods. Net Na⁺, Cl^– and K⁺ fluxes were calculated using the equation (Tattini and Gucci, 1999):

(1)

X is the elemental content (Na⁺, Cl^– or K⁺) of the whole plant (J_X,plant); WR is the root dry weight; t₁ – t₀ is the time interval. Rates of Na⁺, Cl^– or K⁺ transport to the shoot (J_X,shoot) or to the leaves (J_X,leaf) were calculated using the elemental contents in corresponding organs.

Soluble carbohydrates were identified and quantified via HPLC-RI as reported previously (Tattini et al., 1996). In Phillyrea latifolia >90 % of the soluble carbohydrate pool (on a tissue-water molar basis) was composed of glucose and mannitol (Tattini et al., 2002). In Pistacia lentiscus, other than sucrose, glucose and fructose, significant amounts (approx. 40 % of total soluble carbohydrates on a peak area basis) of a sugar were detected with an M-H⁺ of 225 m/z. The identity of this compound is still undetermined, although a retention time very close to that of the internal standard, sorbitol, allows us to hypothesize that it is a sugar-alcohol structure. Quantification of this compound was performed using the calibration curve of mannitol.

Leaf water relations and osmotic adjustment
Water potential (_w), osmotic potential () and relative water content (RWC) were measured on leaves sampled at predawn using a standard methodology (Tattini et al., 2002). Leaf osmotic potential was measured on expressed sap of frozen and thawed leaves using a freezing-point Osmomat 030 osmometer (Gonotec, Berlin, Germany) equipped with a 15-µL measuring cell.

Leaf osmotic potential at full turgor _FT was then calculated as:

(2)

AWF is the apoplastic water fraction, which was estimated from the analysis of pressure/volume isotherms (Gucci et al., 1997b) at 4 % and 5 % in Phillyrea latifolia and P. lentiscus, respectively. The osmotic contributions of individual solutes (ⁱ) to _FT were calculated by the Van't Hoff equation:

(3)

where ⁱ indicates the contribution (–MPa) of individual solutes, RDW is the relative dry weight at saturation (kg m^–3), C is the molal concentration of solutes (mol kg^–1), and 0·002479 m³ MPa mol^–1 is the RT value at 25 °C.

SOD activity and MDA content
SOD (EC 1.15.1.1) was measured in fresh leaf material, as reported in Guidi et al. (2008), based on the protocol of Beyer and Fridovich (1987). Briefly, leaf tissue was homogenized in four volumes (w/v) of an ice-cold buffer containing 0·1 M Tris–HCl, 0·1 mM EDTA and 0·05 % Triton X-100. The homogenates were filtered thorough four layers of cheesecloth and centrifuged at 4 °C at 12 000 g for 20 min. The supernatants were used for the SOD assay. The reaction mixture contained 66 mM phosphate buffer (pH 7·8), 75 µM nitroblue tetrazolium, 13 mM methionine, 2·0 µM riboflavin and an appropriate aliquot of enzyme extract. The reaction was started by placing the reaction tube at 200 µmol m^–2 s^–1 for 10 min. The development of the purple coloration was then determined by measurement of the absorbance at 560 nm (A₅₆₀) in a spectrophotometer blanked with SOD assay buffer. An inhibition curve of A₅₆₀ was constructed with an increasing volume of sample. One unit of SOD was defined as that contained in the volume of extract that caused 50 % of the fraction of the nitroblue tetrazolium reduction inhibited by SOD (Beyer and Fridovich, 1987). Each extract was assayed twice.

Leaf lipid peroxidation was estimated by measuring the concentration of MDA, using the protocol of Hodges et al. (1999), which takes into account the possible influence of interfering compounds in the assay for TBA (2-thiobarbituric acid)-reactive substances.

Quantification of individual polyphenols
Polyphenols were identified and quantified following the protocols previously reported in Tattini et al. (2005, 2006). Briefly, 60–80 mg freeze-dried tissue was extracted with 3x 15 mL EtOH/H₂O solution (75/25; v/v) adjusted to pH 3·2 with formic acid. The supernatant was then partitioned with 3x 15 mL n-hexane, reduced to dryness and finally rinsed with 1 mL H₂O/MeOH/CH₃CN (20/60/20; v/v/v). Aliquots (15–30 µL) were injected into an HP1100 liquid chromatograph equipped with a diode array detector, and managed by a HP workstation (all from Hewlett & Packard, Palo Alto, CA, USA). The column was an 8 x 250 mm LiChrosorb RP₁₈ (Merck, Darmstadt, Germany), maintained at 26 °C, and equipped with an 8 x 10 mm LiChrosorb RP₁₈ pre-column. The eluent was H₂O (pH 3·2 by H₃PO₄)/CH₃CN, and polyphenols separated using the seven-step linear gradient solvent system with a flow rate of 1 mL min^–1 during a 106-min run developed by Romani et al. (1996). In Pistacia lentiscus, galloyl quinic derivatives, which were quantified at 280 nm using a calibration curve of gallic acid, and flavonoids (i.e. myricetin 3-O-rhamnoside, myricetin 3-O-galactoside, and myricetin 3-O-glucuronide, which were quantified at 360 nm using a calibration curve of myricetin 3-O-rhamnoside, were identified (Romani et al., 2002). Trace amounts (<4 % of the total flavonoid concentration) of quercetin 3-O-glycosides were also detected in Pistacia lentiscus leaves: since their contribution to the flavonoid pool was unaffected by salt or Ca²⁺ treatment, they have not been reported here. Polyphenols in Phillyrea latifolia leaves mostly consisted of hydroxyl-tyrosol and caffeic acid derivatives (verbascoside and echinacoside), oleuropein and flavonoid glycosides (Tattini et al., 2000). Flavonoids (namely quercetin 3-O-rutinoside, luteolin 7-O-glucoside, luteolin 4'-O-glucoside and two apigenin 7-O-glycosides) were quantified at 350 nm, using calibration curves of individual compounds (Tattini et al., 2005). Verbascoside and echinacoside were quantified at 330 nm, and oleuropein at 280 nm as reported in Tattini et al. (2004).

The CO₂-based accumulation of phenolics (Phenolic^CO2) was calculated as in Guidi et al. (2008):

(4)

Total assimilated CO₂ was calculated by integrating measurements of daily net assimilated CO₂, which were estimated on days 15, 25 and 35 during salinity stress and on days 15 and 30 after the onset of the relief treatment, on leaves newly developed under different treatments (Tattini et al., 2004). ‘Phenolics’ is the concentration of polyphenols in leaves at the end of the whole experimental period, i.e. in plants exposed to 5 weeks of salinity stress followed by 5 weeks of relief from salinity.

Experimental design and statistics
The experiment was a completely randomized design with at least 40 plants per treatment. Stomatal conductance (g_s) and net CO₂ assimilation rate (A_sat) were measured on fully developed medial leaves of four replicate plants at eight sampling dates during the whole experimental period. Daily assimilated CO₂, i.e. diurnal net assimilation – night-time respiration rates, apparent maximum carboxylation efficiency (V_C,max) and maximal (F_v/F_m) and actual (_PSII) efficiency of PSII photochemistry, were estimated for five replicate apical (approx. 1 week old), medial (2–3 weeks old) and basal (4–5 weeks old) leaves at the end of the salinity period. On the same leaves Na⁺ concentration was determined, on a tissue dry-weight molar basis. Shoot dry-weight increments were estimated on eight replicate plants harvested at the end of both the salinity stress and the relief periods for calculation of RGR. The Na⁺ and K⁺ contents were estimated in different plant parts of these eight replicate plants to both calculate Na⁺, Cl^– and K⁺ fluxes during salinity stress, as well as Na⁺ allocation in different plant parts at the end of the salinity stress and relief periods. Leaf water (_w) and osmotic () potentials, and RWC were measured, and the contributions of individual solutes (ⁱ) to osmotic potential at full turgor (_FT) were calculated on five replicate medial leaves, taken from five replicate plants, sampled at 10-d intervals during the salinity period. The activity of SOD and the MDA concentration were measured for five replicate leaf samples (each consisting of apical, medial and basal leaves) sampled from five replicate plants, after 15 d and 35 d of salinity stress. The concentrations of individual polyphenols were measured on five replicate samples (each consisting of apical, medial and basal leaves) at the end of the whole experimental period.

All the data were subjected to ANOVA, except those concerning water relations and ion fluxes. Data for gas exchange and growth rates were subjected to a four-way ANOVA with species, salt, Ca²⁺ and treatment period as fixed factors, with their interaction factors (see Appendix 1). CO₂^daily, V_C,max and chlorophyll fluorescence-derived parameters of different-aged leaves in salt-treated plants were subjected to a three-way ANOVA with species, leaf age and Ca²⁺ as fixed factors with their interaction factors. Data for Na⁺ allocation in the whole plant and in different plant organs of salt-treated plants were subjected to a three-way ANOVA with species, Ca²⁺ and treatment period as fixed factors with their interaction factors (Appendix 2). Data for the SOD activity, the MDA and polyphenol concentrations were subjected to a three-way ANOVA with species, salt and Ca²⁺ as fixed factors with their interaction factors (data of time-course experiments for SOD and MDA have been pooled together prior to statistical analysis).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS APPENDIX 1 APPENDIX 2 LITERATURE CITED

 
Gas exchange and growth rates
Salinity stress significantly decreased stomatal conductance (g_s) and net CO₂ assimilation rate (A_sat) irrespective of species (Fig. 1 and Appendix 1). There were significant inter-specific differences in both A_sat and g_s of control plants, since A_sat declined much less in Pistacia lentiscus (–27 %) than in Phillyrea latifolia (–52 %) during the salinity period (F_NaClxspecies = 14·5, P < 0·01, d.f. = 127; ANOVA performed pooling together measurements during salinity stress only). Overall, Ca²⁺-supply did not significantly affect gas exchange performance in the species examined, over the whole period of salinity stress. Indeed, high-Ca²⁺ slightly decreased both A_sat and g_s during the first 2 weeks of treatment, whereas g_s (+15 %) and A_sat (+21 %) were greater in salt-treated high-Ca²⁺ than in corresponding low-Ca²⁺ plants, especially in Phillyrea latifolia (+28 % or +32 % for g_s and A_sat, respectively), during the last 3 weeks of treatment (Fig. 1A, C). Interestingly, gas exchange performance in previously salt-treated plants supplied with high-Ca²⁺ recovered to significantly greater rates (particularly in Phillyrea latifolia) than the corresponding low-Ca²⁺ counterparts during the period of relief from salinity (F_Ca2+xperiod = 4·9, P < 0·05, d.f. = 255).

View larger version (22K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 1. (A, B) Stomatal conductance (gs) and (C, D) net CO2 assimilation rate at saturating light (Asat) in Phillyrea latifolia (A, C) and Pistacia lentiscus (B, D) supplied with 0 mM or 200 mM NaCl under low- or high-Ca2+ supply over a 5-week period of salinity treatment followed by 5 weeks of relief from salinity stress. Measurements were conducted on medial (2–3 weeks old) leaves. Data points are means ± s.d., n = 4. The ANOVA table is given in Appendix 1.

 
Similarly, RGR_shoot in the species examined was greatly affected by changes in root-zone NaCl, especially in Phillyrea latifolia, but did not vary appreciably in response to changes in root-zone Ca²⁺ during the salinity period (Fig. 2 and Appendix 1). By contrast, the recovery of RGR_shoot in high-Ca²⁺ plants previously treated with 200 mM NaCl (on average +52 % as compared with RGR_shoot during salinity stress) was greater than that in low-Ca²⁺ counterparts (on average +33 %) during the period of relief from salinity stress, particularly in Phillyrea latifolia (Fig. 2).

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 2. Relative growth rate of Phillyrea latifolia and Pistacia lentiscus plants supplied with 0 mM or 200 mM NaCl under low- (2 mM) or high- (8 mM) Ca2+ supply at the end of salinity or relief periods. RGR was calculated on the basis of shoot dry weight increments. Bars are means ± s.d., n = 8. The ANOVA table is given in Appendix 1.

 
Ion relations and osmotic adjustment
The significantly greater effects of high-Ca²⁺ supply on gas exchange features and growth rates detected in Phillyrea latifolia than in Pistacia lentiscus were consistent with the species-specific Ca²⁺-effects on Na⁺ fluxes and allocation at both whole-plant and leaf level (Figs 3 and 4). Net Na⁺ uptake rates (J_plant) decreased by 10 % or 25 % in salt-treated Pistacia lentiscus and Phillyrea latifolia, respectively, in response to high-Ca²⁺ supply (Fig. 3A). Major effects of high-Ca²⁺ supply were observed for the rates of net Na⁺ transport to the leaves, which declined by 25 % in Pistacia lentiscus or by 48 % in Phillyrea latifolia passing from low- to high-Ca²⁺ salt-treated plants. Ca²⁺-induced reductions in Cl^– transport to the leaves (Fig. 3B) were also greater for Phillyrea latifolia (–28 %) than for Pistacia lentiscus (–15 %). Both net uptake and net transport rates of K⁺ were affected to a smaller degree than net Na⁺ fluxes by changes in Ca²⁺ supply (Fig. 3C), so that the selectivity ratio of K⁺ to Na⁺ fluxes (S_K,Na = J_K+/J_Na+ x [Na⁺]_ext/[K⁺]_ext) actually increased in response to high-Ca²⁺ supply, particularly in Phillyrea latifolia (e.g. S_K/Na,leaf ranged from 52·4 in low- to 64·6 in high-Ca²⁺ plants). Similarly whole-plant Na⁺, which was 81 % greater in Pistacia lentiscus than in Phillyrea latifolia, decreased most in the latter (–28 %) than in the former (–11 %) in response to high-Ca²⁺ during the salinity period (Fig. 4 and Appendix 2). Furthermore, the amount of Na⁺ in the leaf declined to a greater extent in Phillyrea latifolia (–39 %) than in Pistacia lentiscus (–14 %) in response to high-Ca²⁺ supply, irrespective of the treatment period (Fig. 4).

View larger version (15K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 3. Sodium (A), chloride (B) and potassium (C) fluxes (J, µmol g–1 d. wt d–1) at the level of whole-plant (Jplant), shoot (Jshoot) and leaves (Jleaf) in Phillyrea latifolia and Pistacia lentiscus supplied with 200 mM NaCl under low- (2 mM) or high- (8 mM) Ca2+ supply. Ion quantification and calculations of net ion fluxes were performed on eight replicate plants at the end of the salinity period: Data are means ± s.d.

 

View larger version (26K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 4. The amount of Na+ in different organs of salt-treated Phillyrea latifolia and Pistacia lentiscus plants at the end of both 5 weeks of treatment with low- (2 mM) or high- (8 mM) Ca2+ supply and 5 weeks of relief from salinity stress. Measurements and calculations were performed on eight replicate plants. The ANOVA table is given in Appendix 2. Leaf Na+ concentrations are given in Fig. 5.

 
The species examined differed considerably in the relative contribution of dehydration (D = – _FT) to salt-induced changes in leaf bulk osmotic potential, as D contributed 48 % or 29 % to in Phillyrea latifolia or Pistacia lentiscus, respectively, over the whole-period of salinity imposition (Table 1). Estimated leaf turgor potential (_p) of salt-treated plants was 0·08 MPa lower in Phillyrea latifolia and 0·09 MPa higher in Pistacia lentiscus than _p of controls. High-Ca²⁺ leaves of salt-treated plants had a smaller _p and a greater D than the low-Ca²⁺ counterparts, especially in Phillyrea latifolia. Nevertheless, in high-Ca²⁺ leaves, Cl^– and above all Na⁺ contributed less to salt-induced changes in _FT than in high-Ca²⁺ leaves, particularly in Phillyrea latifolia (Table 1). The contribution of K⁺ to _FT, which increased in response to high salinity, was minimally affected by changes in root-zone Ca²⁺ concentrations. In contrast, soluble carbohydrates contributed to _FT more in high-Ca²⁺ (on average 44 %) than in low-Ca²⁺ (37 %) plants, especially in Phillyrea latifolia (62·5 % or 50 % of _FT in high- or low-Ca²⁺ leaves, respectively).

View this table: [in this window] [in a new window]   TABLE 1. Salt-induced changes in turgor potential (p), bulk osmotic potential (), osmotic potential at full turgor (FT), the relative contributions of individual solutes (i) to FT in leaves of P. latifolia and P. lentiscus supplied with low (2 mM) or high (8 mM) root zone Ca2+ concentrations

 
Photosynthetic performances and PSII photochemistry
Daily assimilated CO₂ (CO₂^daily) differed markedly between different-aged leaves of salt-treated plants (F_leaf = 49·5, P < 0·01, Fig. 5A). In Phillyrea latifolia CO₂^daily was 50 % less in basal than in apical leaves, whereas CO₂^daily differed slightly (15 %) between different-aged leaves in Pistacia lentiscus. CO₂^daily increased by 35 % in response to high-Ca²⁺ supply in basal leaves of Phillyrea latifolia, whereas CO₂^daily differed for <10 % in apical leaves of low- or high-Ca²⁺ plants (Fig. 5A). A significant inverse correlation emerged (CO₂^daily = 139·8 – 0·24 Na⁺; R² = 0·861, P < 0·001) between CO₂^daily and leaf Na⁺ concentration, which increased considerably passing from apical (143·6 µmol g^–1 d. wt) to basal leaves (338·4 µmol g^–1 d. wt) and from high-Ca²⁺ (208·4 µmol g^–1 d. wt ) to low-Ca²⁺ leaves (275·2 µmol g^–1 d. wt), in salt-treated Phillyrea latifolia. Leaf Na⁺ concentration and maximum carboxylation efficiency were similarly correlated in salt-treated Phillyrea latifolia, as V_C,max decreased sharply (Fig. 5B) passing from apical (V_C,max = 72·8 ± 3·2 µmol m^–2 s^–1, mean ± s.d., n = 10) to basal leaves (V_C,max = 45·9 ± 4·5 µmol m^–2 s^–1). In contrast, V_C,max varied little with leaf age in salt-treated Pistacia lentiscus (V_C,max = 71·2 ± 4·3, mean ± s.d., n = 30). It is likely that mesophyll anatomy differed between apical and basal (thicker) leaves of both species (data not available); thus V_c,max in the basal leaves was probably under-estimated when compared with apical ones (Loreto et al., 2003; Flexas et al., 2006; Guidi et al., 2008). Nevertheless, leaf anatomy did not vary in response to high Ca²⁺, irrespective of leaf age and species (data not shown, but see Tattini and Traversi, 2008).

View larger version (12K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 5. (A) Daily net CO2 assimilation rate (CO2daily), (B) apparent maximum carboxylation efficiency (VC,max), (C) maximal (Fv/Fm) and (D) actual efficiency of PSII photochemistry (PSII) as related with Na+ concentration (tissue dry-weight molar basis) in apical, medial and basal leaves of salt-treated Phillyrea latifolia and Pistacia lentiscus supplied with low- (2 mM) or high- (8 mM) Ca2+ root-zone concentration at the end of 5 weeks of treatment. Measurements were conducted on five replicate leaves. **, P < 0·001; *, P < 0·05; n.s., not significant.

 
PSII photochemistry varied to a markedly greater degree in response to salinity stress than in response to high-Ca²⁺ supply, particularly in Phillyrea latifolia (F_species = 715·6, P < 0·001, d.f. = 119; Fig. 5C, D). Both F_v/F_m and _PSII were significantly smaller in basal than in apical leaves (F_leaf = 270·8, P < 0·01), especially in Phillyrea latifolia. High-Ca²⁺ leaves had a greater maximal (0·804 ± 0·016 vs. 0·787 ± 0·013, mean ± s.d., n = 30; Fig. 5C) and actual efficiency of PSII photochemistry (0·345 ± 0·027 vs. 0·328 ± 0·025) than low-Ca²⁺ leaves (Fig. 5D).

SOD activity, lipid peroxidation and polyphenol concentration
The activity of SOD was constitutively greater in Pistacia lentiscus (F_species = 89·4, P < 0·001, d.f. = 79), but increased to a significantly greater degree in Phillyrea latifolia (+38 % vs. +15 % in P. lentiscus) because of salinity stress (Fig. 6A). In contrast, salt-induced oxidative damage was similar in both species (MDA increased by 25 % or 23 % in response to high salinity in Phillyrea latifolia or Pistacia lentiscus, respectively), irrespective of root-zone Ca²⁺ concentrations (Fig. 6B). It was observed that both the SOD activity and the MDA concentration significantly varied with the duration of salt treatment (F_time = 5·9, P < 0·05 and = 22·6, P < 0·01 for SOD and MDA, respectively, d.f. = 79). Indeed, SOD activity declined from 157 U g^–1 f. wt at day 15 to 126 U g^–1 f. wt at day 35, particularly in salt-treated Phillyrea latifolia (from 160 to 115 U g^–1 f. wt), whereas it substantially increased, from 98 U g^–1 f. wt at day 15 to 125 U g^–1 f. wt at day 35, in control plants (data not shown). In contrast, the MDA concentration increased, on average by 15 %, from day 15 to day 35, irrespective of salt treatment (data not shown).

View larger version (25K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 6. The activity of superoxide dismutase (SOD, A) and the concentration of malon-dialdehyde (MDA, B) in leaves of Phillyrea latifolia and Pistacia lentiscus supplied with 0 mM or 200 mM NaCl under low- (2 mM) or high- (8 mM) Ca2+ supply. Measurements were conducted on five replicate leaf samples (each consisting of an apical, medial and basal leaf) sampled from five replicate plants at days 15 and 35 during the period of high salinity. Bars are means ± s.d., n = 10 (data have been pooled together prior to statistical analysis). ***, P < 0·0001; n.s., not significant.

 
On the whole, the polyphenol concentration did not vary in response to high salinity (Fig. 7A), due to salt-induced decreases in Phillyrea latifolia (from 88 to 73 mg g^–1 d. wt) or slight increases in Pistacia lentiscus (from 261 to 272 mg g^–1 d. wt). The lack of significant effects of salinity stress on the leaf polyphenol concentration mostly originated from large salt-induced reductions in the amount of carbon actually available for their synthesis (Fig. 5A). In fact, the ‘newly assimilated carbon’ recovered in polyphenols (Phenolic^CO2; Tattini et al., 2004, 2006) increased sharply from 50·0 in controls to 76·0 mg g^–1 of assimilated CO₂ in salt-stressed plants (Fig. 7A). Also note that Phenolic^CO2 was on average 14 % greater in low- than in high-Ca²⁺ leaves (particularly in Phillyrea latifolia, +21 %) despite the fact that the polyphenol concentration did not differ because of the Ca²⁺ supply. Finally, salt stress substantially affected the flavonoid composition in Phillyrea latifolia leaves, as the ratio of flavonoids to other phenolics and that of di-hydroxy (i.e. quercetin 3-O-rutinoide and luteolin 7-O-glucoside) to mono-hydroxy B-ring-substituted flavonoid glycosides (i.e. luteolin 4'-O-glucoside and the two apigenin 7-O-glycosides) substantially increased in salt-treated plants (Fig. 7B). In contrast, neither the ratio of myricetin glycosides to gallotannins nor the composition of the flavonoid pool varied appreciably in response to root-zone NaCl or Ca²⁺ concentrations in Pistacia lentiscus.

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 7. (A)The concentration of polyphenols and (B) the ratio of flavonoids to other polyphenols in leaves of Phillyrea latifolia and Pistacia lentiscus supplied with 0 mM or 200 mM NaCl under low- (2 mM) or high- (8 mM) Ca2+ supply at the end of the whole-experimental period (5 weeks of stress + 5 weeks of relief from stress). Numbers in columns in (A) refer to the increase in leaf polyphenol concentration normalized to total assimilated CO2 (phenolicCO2 = mg g–1 CO2), whereas those in (B) refer to the ratio of di-hydroxy (que 3-O-rut and lut 7-O-glc) to mono-hydroxy B-ring-substituted flavonoid glycosides (lut 4'-O-glc and api 7-O-gly) in P. latifolia. Measurements were conducted on five replicate leaf samples (each consisting of an apical, medial and basal leaf) sampled from five replicate plants. Data are means ± s.d. **, P < 0·001; *, P < 0·05; n.s., not significant.

 

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS APPENDIX 1 APPENDIX 2 LITERATURE CITED

 
Data from these experiments show species-specific effects of high-Ca²⁺ supply on plant performance under salinity stress. The ameliorative effects of an increased root-zone Ca²⁺ : Na⁺ ratio were much greater in Phillyrea latifolia, whose ability to cope with high salinity depends on mechanisms primarily aimed at restricting the entry of potentially toxic ions in the leaf, than in Pistacia lentiscus, which largely uses Na⁺ and Cl^– for salt-induced osmotic adjustment.

Firstly, it is shown here that the greater extent to which gas exchange performance and growth rates increased in salt-treated Phillyrea latifolia (A_sat +15 %, RGR_shoot +30 %) than in Pistacia lentiscus (A_sat +2 %, RGR_shoot +4 %) because of high-Ca²⁺ supply, exclusively depended upon greater recoveries during the period of relief from salinity, not from superior rates during the stress period (Figs 1 and 2). Actually, g_s and A_sat underwent transiently greater reductions in high-Ca²⁺ than low-Ca²⁺ plants, irrespective of NaCl supply and species (Hawkins and Lewis, 1993; De Silva et al., 1994), during the first 2 weeks of treatment (Fig. 1). These findings agree with those of Yang et al. (2003) and seem to support previous suggestions of the pivotal role of Ca²⁺ as signal transduction molecule (Essah et al., 2003; Tester and Davenport, 2003) in ABA-mediated stomatal closure (Bowler and Fluhr, 2000; Wilkinson and Davies, 2002). Intensity of salt stress and plant age may have been responsible for the negligible effect of Ca²⁺ treatments on g_s during the last 3 weeks (Sohan et al., 1999; Yang et al., 2003; Husain et al., 2004).

The species-specific effect of Ca²⁺ treatments on gas exchange and photosynthetic performances closely depended upon strikingly different species-specific strategies of salt allocation at the organ level (Cheeseman, 1988; Tattini and Gucci, 1999; Munns, 2005; Tattini et al., 2006). Net transport rates of Na⁺ to the leaves decreased much more in the ‘salt-excluder’ Phillyrea latifolia (J_Na,shoot and J_Na,leaf accounted for 57 % and 20 %, respectively, of net Na⁺ uptake rate) than in the ‘salt-includer’ Pistacia lentiscus (J_Na,shoot and J_Na,leaf accounted for 57 % and 39 %, of J_Na,plant) because of high-Ca²⁺ supply (Fig. 3A). It is also noted that the stem contributed little in Pistacia lentiscus (32 %) as compared with Phillyrea latifolia (53 %), to the partitioning of Na⁺ in the whole plant (Fig. 4). This finding, which depended upon strikingly different Na⁺ concentrations (287·5 in Phillyrea latifolia vs. 126·7 µmol g^–1 d. wt in Pistacia lentiscus) and not upon different growth rates of the stem (Fig. 2), suggests a key role of the stem in the partitioning of Na⁺ in salt-tolerant glycophytes (Wolf et al., 1991; Tattini and Gucci, 1999). At the same time, high-Ca²⁺- induced improvement in K⁺/Na⁺ selectivity, which has been reported to be responsible for Ca²⁺-induced enhancements in the salt-tolerance of most species (Maathuis and Amtmann, 1999; Tester and Davenport, 2003), was greater in Phillyrea latifolia than in Pistacia lentiscus (Fig. 3). These issues, although poorly investigated with sometimes contrasting results (Davenport et al., 1997; Hernández et al., 2003), may have ecological relevance. The effect of changes in Ca²⁺ supply on the Cl^– transport rate to the leaves (Fig. 3B) and the leaf Cl^– concentration (which declined from 197 to 168 µmol g^–1 d. wt passing from low- to high-Ca²⁺ leaves; data not shown) was less important than that observed on Na⁺ fluxes and concentrations (Figs 3A and 5). These results conform to a Ca²⁺-specific effect on Na⁺ uptake and transport systems (Maathuis and Amtmann, 1999; Zhu, 2002), and agree with data recently reported for hydroponically grown Olea europea supplied with similar NaCl and Ca²⁺ root-zone concentrations (Tattini and Traversi, 2008).

The greater extent to which Phillyrea latifolia underwent salt-induced osmotic deficit, which was slightly enhanced by high-Ca²⁺ supply (Table 1; Reid and Smith, 2000; Jiang and Huang, 2001; Tattini and Traversi, 2008), may help explain the greater reductions in gas exchange performance as compared with Pistacia lentiscus due to high salinity (Tattini et al., 1997, 2002). However, a transient salt-induced osmotic deficit [estimated _p did not actually differ between control and salt-treated leaves by the end of 5 weeks of treatment (data not shown, but see Tattini et al., 2002)] may have little significance for species growing under natural conditions. Phillyrea latifolia, as for other drought-tolerant evergreen Oleaceae species, suffers from fluctuating root-zone salinity over the whole growing season (Margaris, 1981; Moriana et al., 2002). Species invading hypersaline shores of the Dead Sea avoid transport of soil water until rainfall water is available to the roots (Yakir and Yechiely, 1995). The ability to survive under severe conditions of water-stress (Phillyrea latifolia has been shown to respond better than Pistacia lentiscus to severe drought-stress in the field, Filella et al, 1998) is a key mechanism through which Phillyrea latifolia may adapt to high salinity (Tattini et al., 2002).

On the other hand, Ca²⁺-induced improvements in solute regulation at cellular level (a key determinant of salt tolerance; Colmer et al., 1995; Niu et al., 1995; Hasegawa et al., 2000), which were more important in Phillyrea latifolia than in Pistacia lentiscus (Table 1), were likely to have allowed the former to preserve the photosynthetic apparatus (Fig. 2B, D) from the deleterious effects of high [NaCl]_ext (Allakhverdiev et al., 2000; Shabala et al., 2005; Tattini and Traversi, 2008). It is noted that K⁺ concentration decreased from 340 in low- to 300 µmol g^–1 d. wt in high-Ca²⁺ salt-treated leaves of Phillyrea latifolia, but the leaf K⁺: Na⁺ ratio of 1·72 was greater in the latter (also note a K⁺: Na⁺ ratio of 0·36 in high-Ca²⁺ Pistacia lentiscus leaves) than in the former which was 1·46 (K⁺: Na⁺ ratio was 0·34 in low-Ca²⁺ Pistacia lentiscus leaves, data not shown).

The ameliorative effects of high Ca²⁺ on the photosynthetic performance of both newly developed and old-basal leaves in Phillyrea latifolia may be of particular value under field conditions, as such ‘protected-high-Ca²⁺’ leaves may assimilate carbon to a significantly greater degree than the low-Ca²⁺ counterparts when good-quality water is available to the roots. In contrast, salinity stress and Ca²⁺ supply poorly affected PSII photochemistry in Pistacia lentiscus (Fig. 2C, D), despite a massive accumulation of potentially toxic ions in the leaves (Lu et al., 2003), confirming that an effective compartmentation of potentially toxic ions out of sensitive cell compartments operated in this species (Tattini et al., 2006).

The greater salt-induced physiological and biochemical changes in Phillyrea latifolia (for example, the use of mannitol requires much more energy than using the cheap osmolytes Na⁺ and Cl^– for salt-induced osmotic adjustment; Raven, 1995) than in Pistacia lentiscus, were probably responsible for the strikingly different effects of NaCl and Ca²⁺ treatments on net assimilation rate and shoot growth (Figs 1 and 2). Indeed, the decrease in RGR (–71 %) was markedly greater than that in A_sat (–52 %) in Phillyrea latifolia, whereas these traits decreased similarly in Pistacia lentiscus (RGR by 28 % and A_sat by 24 %) during salinity stress. Similarly, the recovery in RGR (54 % of controls) was substantially lower than that in A_sat (81 % of controls) in salt-treated Phillyrea latifolia during the relief period (Figs 1 and 2). Highly energy-consuming processes aimed to sequester potentially toxic ions, at both tissue and intracellular level, were likely to be still active during the relief period, following the flux of toxic ions to the shoot via the restored water mass flow (Flowers and Yeo, 1989; Tattini et al., 1995; Tattini and Gucci, 1999). Further, the decrease in whole-plant Na⁺ at the end of the relief period (–40 % or –27 % in Phillyrea latifolia or Pistacia lentiscus, respectively; Fig. 4), was mostly due to reductions in root and old-stem Na⁺, suggesting that phloem Na⁺ re-translocation occurred: this highly energetic process may have also contributed to divert energy otherwise available to growth (Gouia et al., 1994; Tattini and Traversi, 2008).

The relationship between antioxidant enzyme activity and salt tolerance has been investigated previously but with contrasting results (Benavides et al., 2000; Broetto et al., 2002). The ability to counter salt-induced oxidative damage, a key determinant of salt-tolerance, has been generally associated with a constitutively greater activity of antioxidant enzymes (Benavides et al., 2000; Chinnusamy and Zhu, 2003; Moradi and Ismail, 2007), as was also observed in the present experiment with regard to the activity of SOD (Fig. 6A). The greater salt-induced enhancement of SOD activity in Phillyrea latifolia than in Pistacia lentiscus leaves agrees with the previous findings of Hernández et al. (2003), such that the scavenger activity against superoxide anions did not differ in the species examined during the whole period of salinity stress (Hernández et al., 1999; Benavides et al., 2000; Hernández and Almansa, 2002). The decrease in SOD activity in P. latifolia leaves as salt-stress progressed (from 160 to 115 U g^–1 f. wt) may have depended on H₂O₂-induced enzyme inactivation, as the concomitant action of high solar radiation and salinity stress has been reported to modulate the SOD activity (Broetto et al., 2002).

Lipid peroxidation increased only slightly in response to high salinity, indicating that both species were effective in countering salt-induced oxidative damage (Fig. 6B). It is likely that light-induced biochemical adjustments equipped leaves with an effective system capable of countering salt-induced reactive oxygen species (ROS) generation, broadly ‘cross-tolerance’ (Karpinski et al., 1999; Pastori and Foyer, 2002; Guidi et al., 2008), as plants experienced several weeks of full solar irradiance in the present experiment. These light-induced biochemical adjustments (Grace and Logan, 1996) may have been also partially responsible for the lack of significant effects of Ca²⁺ on both the SOD activity (Jiang and Huang, 2001) and the oxidative damage in the species examined (Fig. 6A, B). Furthermore, the marked increases in mannitol concentration in response to salinity stress should have allowed Phillyrea latifolia leaves (which are likely to have experienced a greater salt-induced ROS generation than Pistacia lentiscus; see relative F_v/F_m and _PSII; Fig. 5C, D) to effectively scavenge the highly reactive hydroxyl radical (Shen et al., 1997; Conde et al., 2007) and, hence to reduce damage to membrane lipids (Fig. 6B).

Salinity stress markedly altered the secondary metabolism, as assimilated carbon allocated to carbon-based secondary compounds was greater in salt-treated than in control leaves, irrespective of species (Fig. 7A; Tattini et al., 2004, 2006). It is hypothesized that salt-induced increase in the ROS content may have up-regulated the expression of genes involved in phenylpropanoid biosynthetic pathways (Jabs et al., 1997; Mackerness et al., 2001; Babu et al., 2003), although the increase in soluble carbohydrates may have also shifted cell metabolism to defence compound biosynthesis (Koch, 1996; Arnold et al., 2004). This idea is corroborated by the greater enhancement in Phenolic^CO2 coupled with the sharp increase in the ratio of effective antioxidant to poor antioxidant flavonoids observed in Phillyrea latifolia than in Pistacia lentiscus (Fig. 7B; Tattini et al., 2005; Agati et al., 2007). A superior up-regulation of flavonoid metabolism coupled with a greater enhancement in the activity of SOD, as previously reported to occur in a salt-sensitive rice genotype (Walia et al., 2005), may have equipped P. latifolia with an efficient system capable of inhibiting the generation of (Rice-Evans et al., 1996; Brown et al., 1998) as well as to scavenge (Tattini et al., 2005; Agati et al., 2007) free radicals, and hence, to preserve the leaves from severe oxidative damage.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS APPENDIX 1 APPENDIX 2 LITERATURE CITED

 
Based on reductions in carbon acquisition and growth rates, Pistacia lentiscus was ‘more tolerant’ than Phillyrea latifolia to salinity stress, which conforms to the relative species distribution in the Mediterranean basin. However, survival more than growth maintenance is the key target of the adaptive strategies of Phillyrea latifolia, as well as of other evergreen sclerophylls inhabiting Mediterranean dry-lands, to adverse environmental conditions. High-Ca²⁺ supply ameliorated the performance of Phillyrea latifolia more than that of Pistacia lentiscus under high salinity. The enhanced control of Na⁺ uptake to sensitive shoot organs of Phillyrea latifolia when with high-Ca²⁺ supply, however, exposed it to a transiently more severe water deficit during the salt stress, but enabled a faster recovery in gas exchange performance when salt-induced osmotic stress was removed by supplying plants with good quality water. Salt tolerance in long-lived species is difficult to estimate during a short period of intense salinity, as in this study, as drought-tolerant species such as Phillyrea latifolia seem to decrease transpiration in order to avoid a massive flux of unwanted ions following the water mass flow. Thus, mechanisms operate to counter the ionic rather than the osmotic component of an increase in root-zone NaCl. Furthermore, salinity stress and high-Ca²⁺ enhanced the arsenal of antioxidant compounds in Phillyrea latifolia leaves, probably at the expense of growth, which may effectively protect long-lived leaves from adverse environmental conditions.

	APPENDIX 1

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS APPENDIX 1 APPENDIX 2 LITERATURE CITED

 

Summary of four-way ANOVA of effects of species, NaCl, calcium supply (Ca²⁺) and treatment period (salinity stress or relief from salinity) as fixed factors, with their interaction factors, on net CO₂ assimilation rate at saturating light (A_sat), stomatal conductance (g_s) and RGR of the shoot in Phillyrea latifolia and Pistacia lentiscus.

Treatment effect (F-ratio) Asat (µmol m–2 s–1) gs (mmol m–2 s–1) RGRshoot (d–1 x 103) Species 1·5 n.s. 1·2 n.s. 403·6*** NaCl 194·9*** 212·6*** 493·7*** Ca2+ 0·5 n.s. 0·7n.s. 1·2 n.s. Period 46·9*** 38·9*** 7·6* Species x NaCl 3·2 n.s. 2·6 n.s. 72·4*** Species x Ca2+ 0·9 n.s. 1·7 n.s. 4·5* Species x period 1·6 n.s. 2·1 n.s. 9·4*

Data of time-course experiment for gas exchange features have been pooled together prior to statistical analysis (four replicate leaves at eight sampling dates over the whole experimental period; d.f. = 255). RGR was calculated on eight replicate plants at the end of the salinity and relief periods (d.f. = 127).

	APPENDIX 2

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS APPENDIX 1 APPENDIX 2 LITERATURE CITED

 

Summary of three-way ANOVA of effects of species (sp), calcium supply (Ca), and treatment period (per, salinity stress or relief from salinity) as fixed factors, with their interaction factors, on the allocation of Na⁺ at whole plant and organ levels in salt-treated Phillyrea latifolia and Pistacia lentiscus.

Parameter d.f. Fsp FCa Fper FspxCa Fspxper FCaxper FspxCax per Whole-plant Na+ (mol plant–1) 63 1284·5*** 142·6*** 347·4*** 1·6 n.s. 21·1*** 0·2 n.s. 4·1* Root Na+ (mol organ–1) 63 259·0*** 55·4*** 216·3*** 0·2 n.s. 20·9*** 0·4 n.s. 4·6* Old-stem Na+ (mol organ–1) 63 124·2*** 47·5*** 188·6*** 2·2 n.s. 4·1* 0·1 n.s. 4·4* New-stem Na+ (mol organ–1) 63 379·8*** 88·4*** 92·7*** 16·5** 4·2* 6·6* 6·9* Leaf Na+ (mol organ–1) 63 1254·6*** 34·6*** 25·1*** 1·4 n.s. 5·3* 0·1 n.s. 0·3 n.s.

	LITERATURE CITED

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