Can elevated CO2 improve salt tolerance in olive trees?

ARTICLE IN PRESS Journal of Plant Physiology 165 (2008) 631—640

www.elsevier.de/jplph

Can elevated CO2 improve salt tolerance in olive trees? Juan Carlos Melgara,, James P. Syvertsenb, Francisco Garcı´a-Sa ´nchezc a

Departmento de Agronomı´a, Universidad de Co ´rdoba, Edificio ‘Celestino Mutis’, Campus Universitario de Rabanales, Ctra. Madrid-Ca´diz km. 396, 14071 Co ´rdoba, Spain b UF/IFAS, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850, USA c Centro de Edafologı´a y Biologı´a Aplicada del Segura, CSIC, Campus Universitario de Espinardo, Espinardo 30100, Murcia, Spain Received 17 September 2006; received in revised form 12 December 2006; accepted 3 January 2007

KEYWORDS CO2 enrichment; Olea europaea; Photosynthesis; Salt stress; Salt tolerance

Summary We compared growth, leaf gas exchange characteristics, water relations, chlorophyll fluorescence, and Na+ and Cl concentration of two cultivars (‘Koroneiki’ and ‘Picual’) of olive (Olea europaea L.) trees in response to high salinity (NaCl 100 mM) and elevated CO2 (eCO2) concentration (700 mL L1). The cultivar ‘Koroneiki’ is considered to be more salt sensitive than the relatively salt-tolerant ‘Picual’. After 3 months of treatment, the 9-month-old cuttings of ‘Koroneiki’ had significantly greater shoot growth, and net CO2 assimilation ðACO2 Þ at eCO2 than at ambient CO2, but this difference disappeared under salt stress. Growth and ACO2 of ‘Picual’ did not respond to eCO2 regardless of salinity treatment. Stomatal conductance (gs) and leaf transpiration were decreased at eCO2 such that leaf water use efficiency (WUE) increased in both cultivars regardless of saline treatment. Salt stress increased leaf Na+ and Cl concentration, reduced growth and leaf osmotic potential, but increased leaf turgor compared with non-salinized control plants of both cultivars. Salinity decreased ACO2 , gs, and WUE, but internal CO2 concentrations in the mesophyll were not affected. eCO2 increased the sensitivity of PSII and chlorophyll concentration to salinity. eCO2 did not affect leaf or root Na+ or Cl concentrations in salt-tolerant ‘Picual’, but eCO2 decreased leaf and root Na+ concentration and root Cl concentration in the more salt-sensitive ‘Koroneiki’. Na+ and Cl accumulation was associated with the lower water use in ‘Koroneiki’ but not in ‘Picual’. Although eCO2 increased WUE in salinized leaves and decreased salt ion uptake in the relatively salt-tolerant ‘Koroneiki’, growth of these young olive trees was not affected by eCO2. & 2007 Elsevier GmbH. All rights reserved.

Corresponding author. Tel.: +34 957 218 498; fax: +34 957 218 569.

E-mail address: [email protected] (J.C. Melgar). 0176-1617/$ - see front matter & 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2007.01.015

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Introduction The most important olive-growing areas are located in arid and semiarid regions of the world that have a Mediterranean-like climate. Around the Mediterranean Basin, olive trees have traditionally been cultivated without supplemental irrigation. However, the use of irrigation is increasing in olive orchards because of enhanced yields and profits (Orgaz and Fereres, 2004). Because olive trees are considered moderately tolerant to salinity (Maas and Hoffman, 1977), irrigation water with high salt concentration is often used without consideration for the negative effects of poor water quality on olive tree growth and productivity. Responses of olive trees to salt stress include decreases in growth and net CO2 assimilation (ACO2 ) in leaves, with changes in leaf water relations and leaf morphology (Bongi and Loreto, 1989; Tattini et al., 1995). Responses to salinity stress vary with cultivar (Marı´n et al., 1995), age of plant (Abd El Rahman and Sharkawi, 1968; Bernstein, 1975), and duration of exposure. Ion toxicity symptoms in leaves have been described when leaf Na+ and Cl content exceed 0.2% and 0.5% d.w., respectively (Bernstein, 1975). Thus, Cl uptake from NaCl and its transport to the shoot in olive trees is generally lower and less of a problem than Na+ (Bongi and Loreto, 1989; Tattini et al., 1992). Salt tolerance in olive trees has been related to the their ability to decrease leaf osmotic potential (Gucci et al., 1997) and salinity tolerance in different olive tree cultivars has been linked to Na+ and/or Cl ion exclusion mechanisms or to the retention of salt ions in roots (Tattini et al., 1994), preventing the accumulation of Na+ and/or Cl in shoots (Gucci and Tattini, 1997). Consequently, the high salt tolerance of olive genotypes such as ‘Arbequina’, ‘Picual’, and ‘Lechı´n de Sevilla’ (Marı´n et al., 1995) has been associated with their capacity to restrict Na+ accumulation in leaves. Although there have been studies on the potential use of olive cultivars as rootstocks to improve stress tolerance (Hartmann, 1958; Caballero and Del Rio, 2004), olive trees are generally grown from their own rooted cuttings (Caballero and Del Rio, 2004). Information on salt tolerance in olive cultivars that could be used as potential rootstocks could be put to practical commercial use. Plant growth is usually closely linked to water use, as faster growing larger plants use more water than smaller plants. Salt tolerance can also be related to low water use (Moya et al., 1999) because saltstressed plants that use relatively little water should accumulate less salt than plants that use more water. Plant growth and ACO2 can be increased by

J.C. Melgar et al. elevated CO2 (eCO2) concentrations while at the same time stomatal conductance is usually decreased (Roden and Ball, 1996; Sebastiani et al., 2002). Thus, growing plants at eCO2 almost always lead to higher water use efficiency (WUE); eCO2 offers a tool to uncouple plant growth from water use (Yeo, 1999). Growing olive trees with contrasting salinity tolerance under conditions of salt stress and eCO2 may elucidate mechanisms of salinity tolerance. If salt uptake is indeed coupled with water uptake, leaves of plants grown at eCO2 with higher WUE should also contain lower salt concentrations than those grown at ambient CO2 (aCO2) (Ball and Munns, 1992; Garcı´a-Sa ´nchez and Syvertsen, 2006). Thus, we hypothesized that salt tolerance would be enhanced in olive tree cuttings grown at eCO2. We tested the effects of eCO2 concentration (700 mL L1) and high salinity (100 mM NaCl) on growth and net gas exchange characteristics of two olive cultivars (Olea europaea L.) with different tolerances to salinity. ‘Koroneiki’ is considered to be relatively salt sensitive and ‘Picual’ is considered to be salt tolerant (Marı´n et al., 1995; Gucci and Tattini, 1997). The eCO2 concentration of about twice ambient (700 mL L1) was selected to be sufficient to decrease stomatal conductance by 33–50% and leaf transpiration by 20–27% while at the same time increase plant growth (Kang et al., 1995; Zhang et al., 1999). We used a salinity concentration of 100 mM NaCl because it has been considered to be a critical threshold for reductions in olive tree growth (Loreto and Bongi, 1987; Chartzoulakis et al., 2002). Materials and methods Plant material and growth conditions The study was conduced at the University of Florida Citrus Research and Education Center (Lake Alfred, FL, USA, 28.09 N, 81.73 W; elevation 51 m). Six-month-old rooted cuttings of O. europaea L. (cv. ‘Picual’ and cv. ‘Koroneiki’) were purchased from a commercial nursery, bare rooted and grown in 1.5 L containers filled with previously autoclaved Candler fine soil sand. Plants were well established by watering three times per week with 100 mL of half-strength Hoagland’s solution, sufficient to drain from the bottom of all pots. Twenty-four uniform plants of each cultivar were grown in either of two identical temperature-controlled greenhouses made of clear double-walled polycarbonate. During the experimental period, the two greenhouses had almost identical growth conditions except for CO2 concentration. Maximum PAR (LI-170, LICOR, Inc., Lincoln, NE, USA) measured above the plants was 1500 mmol m2 s1 with natural photoperiods, average day/night temperature was 36/21 1C, and relative humidity varied from 40% to

ARTICLE IN PRESS Elevated CO2 improves salt tolerance 100%. One greenhouse was continuously supplied with supplemental CO2 from a compressed CO2 cylinder; CO2 concentration was monitored with an infrared gas analyzer (S-151, Qubit Systems Inc., Kingston, Ontario, Canada) equipped with an automatic switching solenoid to maintain the eCO2 concentration at about twice ambient (700720 mL L1). Whenever the CO2 concentration dropped to 690 mL L1, CO2 was automatically injected and mixed into the greenhouse until the concentration reached 710 mL L1. The other well-ventilated greenhouse maintained an aCO2 concentration, which was about 360 mL L1. The salt treatment was started at the same time as the CO2 treatment by applying daily increments of 20 mM NaCl in the irrigation water to reach the final NaCl concentration of 100 mM. The NaCl was added to the half-strength Hoagland’s nutrient solution while the non-salinized control plants continued to receive only nutrient solution. eCO2 and salinity treatments were maintained for 12 weeks so the experiment ended when trees were 9 months old. The experimental design was a 2  2  2 factorial of two cultivars (‘Picual’ and ‘Koroneiki’)  two CO2 treatments (360 and 700 mL L1)  two salt treatments (0 and 100 mM NaCl), with six replicate trees in each treatment. Leaf water relations and net gas exchange Measurements of leaf water relations were performed 2 and 11–12 weeks after the initiation of treatments using individual leaves from the middle of the shoot of each tree. Pre-dawn (06:00–08:00 h) leaf water potential (Cw) was measured using a Scholander-type pressure chamber (PMS instrument, Corvallis, OR, USA) equipped with a magnifying lens to observe end points (Scholander et al., 1965). Following Cw measurements, leaves were immediately wrapped in aluminum foil, frozen by immersing in liquid nitrogen and stored at 18 1C. Leaf osmotic potential (Cp) was measured on sap pressed from the thawed tissue at 2571 1C with a vapor pressure osmometer (Model 5100B, Wescor, Logan, UT, USA). Turgor potential (Cp) was calculated as the difference between the Cp and Cw expressed in MPa. Using leaves adjacent to those used for water relation measurements, net CO2 assimilation rate ðACO2 Þ, stomatal conductance (gs), leaf evapotranspiration (Elf), the ratio of intercellular to atmospheric CO2 (Ci/Ca), and leaf WUE ðWUE ¼ ACO2 =E lf Þ were determined with a LICOR portable photosynthesis system (LI-6200, LI-COR Inc., Lincoln, NE, USA) using a 250 cm3 cuvette. The LICOR-6200 was equipped with a high-intensity red LED light source (Model QB1205LI-670, Quantum Devices Inc., Barneveld, WI, USA) to maintain a PAR at 800 mmol m2 s1. This light level was sufficient to support maximum rates of photosynthesis in sun-acclimated leaves (Tattini et al., 1995; Chartzoulakis et al., 2002). All gas exchange measurements were recorded in the morning (08:00–10:00 h) to avoid high afternoon temperatures and low humidity. During all gas exchange measurements, leaf temperatures were 3272 1C and leaf to air vapor pressure difference (D) was 2.470.4 kPa within the cuvette in both greenhouses.

633 Chlorophyll fluorescence Using the same leaves used for gas exchange measurements, chlorophyll fluorescence was measured with a pulse-modulated fluorometer (Model OS1-Fl, OptiSciences, Hudson, NH, USA) and used to determine changes in the efficiency of light utilization for electron transport. Measurements were performed on six leaves per treatment for both light-exposed and dark-acclimated leaves. Leaves were acclimated to the dark using light exclusion clips (FL-DC, Opti-Sciences, Hudson, NH, USA) for at least 20 min prior to fluorescence measurements. The parameters of maximum quantum efficiency (Fv/Fm) of photosystem II were calculated as Fv/Fm ¼ (FmF0)/Fm, where Fm and F0 were maximum and minimum fluorescence of dark-acclimated leaves, respectively (Maxwell and Johnson, 2000; Jifon and Syvertsen, 2003). Effective quantum yield (Y) was measured as Y ¼ (F0 M–F0 )/F0 M, where F0 M and F0 were maximum and steady-state fluorescence yield in the light, respectively. Non-photochemical quenching (qN) was measured as qN ¼ (Fm–F0 M)/(Fm–F0 0). Plant transpiration and leaf area After 12 weeks of treatment, whole plant transpiration (Ewp) was measured for each replicate tree during two selected clear days and the daily values were averaged. Each pot was bagged with clear plastic, sealed at the base of stem to inhibit evaporation from the soil, and weighed early in the morning and again in the afternoon. Transpiration rate was calculated as weight lost during daylight hours corrected for total leaf area per plant and expressed in units of mg H2O cm2 h1. Total leaf area was measured after harvesting with an area meter (LI-3000, LI-COR Inc., Lincoln, NE, USA) in combination with a transparent belt conveyor accessory (LI-3050A, LI-COR Inc., Lincoln, NE, USA). Chlorophyll analysis Chlorophyll was extracted from two leaf disks (0.45 cm2 each), removed from the same leaves used for gas exchange measurements (avoiding major veins). Leaf disks were placed into glass screw cap vials with 5 mL of N,N-dimethylformamide, as described by Moran and Porath (1980). After at least 72 h in the dark, solution absorbances were determined at 647 and 664 nm with a UV–vis spectrophotometer (Model UV2401PC, Shimadzu, Riverwood Drive, Columbia, MD, USA). Total leaf chlorophyll concentration was calculated according to the equations of Inskeep and Bloom (1985) and expressed as mg cm2. Growth and leaf nutrient concentration Plants were harvested at the end of 12 weeks, and leaves, stems, and roots were separated. Leaves and roots were briefly rinsed with deionized water, oven-dried at 60 1C for at least 48 h, weighed, and ground to a powder. Tissue Cl

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concentration was measured using a silver ion titration chlorodimeter (HBI Chlorodimeter, Haake Buchler, Sandle Brook, NJ, USA) after the tissue had been extracted with 0.1 N solution of nitric acid and 10% acetic acid (Garcı´aSa ´nchez and Syvertsen, 2006). Leaf tissue Na+ concentration was determined by an analytical laboratory (Waters Agricultural Lab, Camilla, GA, USA) and root Na+ concentration was determined with an inductively coupled plasma atomic emission spectrometer after digestion with nitric/percloric acid (2:1, v:v; Garcı´a-Sa ´nchez and Syvertsen, 2006). Statistical analysis Data were subjected to analysis of variance with two olive cultivars  two CO2 levels  two salinity levels and six replicate plants per treatment. Treatment means were compared by Duncan’s multiple range test using the SPSS statistical package (SPSS, Chicago, IL, USA). Linear regression was used to describe relationships between selected variables, and analysis of covariance was used to compare slopes of relationships.

Results Growth Although there were no cultivar differences in total plant dry weight (TPDW), ‘Koroneiki’ had greater leaf area and leaf dry weight, resulting in a higher shoot/root weight ratio than ‘Picual’ (Table 1). Under non-saline conditions, TPDW was increased about 37% by eCO2 in both cultivars,

although significant differences only occurred in ‘Koroneiki’. This increase was due primarily to the increases in leaf and stem dry weight (data not shown). Thus, shoot/root dry weight ratio was also significantly increased in ‘Koroneiki’ by eCO2, compared with plants grown at aCO2. Since salinity reduced leaf area, TPDW, dry weights of leaves and roots, and shoot/root weight ratio for both ‘Koroneiki’ and ‘Picual’ regardless of CO2 treatment, there was no increase in growth of salt-stressed plants by eCO2. There was a significant CO2  salt interaction in leaf area, leaf dry weight, and TPDW; these parameters were enhanced at eCO2 in nonsalinized plants but were not affected by eCO2 in salinized plants regardless of cultivar.

Gas exchange and transpiration After 2 weeks of treatment, ‘Picual’ had higher overall ACO2 , WUE, and lower Ci/Ca than ‘Koroneiki’ but these cultivar differences disappeared after 12 weeks (Table 2). Under non-saline conditions, the effects of eCO2 on ACO2 were different in ‘Koroneiki’ and ‘Picual’. After 2 weeks, eCO2 increased ACO2 and decreased gs in ‘Picual’ plants, whereas in ‘Koroneiki’ plants, eCO2 decreased gs but ACO2 was not affected. After 12 weeks, ‘Koroneiki’ plants grown at eCO2 had a significantly higher ACO2 but similar gs relative to plants grown at aCO2. In ‘Picual’, however, gs in eCO2 plants was lower

Table 1. Effects of NaCl (0 or 100 mM NaCl) and CO2 concentration (360 or 700 mL L1) during growth on leaf area (cm2), leaf DW (g), root DW (g), total plant DW (g), and shoot/root (dimensionless) of ‘Koroneiki’ and ‘Picual’ leaves following a 12-week experimental period Cultivar

CO2

Salt

Leaf area

Leaf DW

Root DW

TPDW

Shoot/root

Koroneiki

360

0 100 0 100

338b 83c 455a 80c

4.74b 1.47c 6.88a 1.67c

2.82a 1.28b 3.06a 1.01b

17.08b 5.24c 23.42a 4.75c

4.94b 3.18cd 6.54a 3.70bcd

0 100 0 100

274b 60c 333b 46c

3.63b 0.96c 4.87b 0.83c

2.58a 1.66b 3.06a 1.65b

13.81b 5.21c 18.95ab 5.20c

4.30bc 2.32d 5.14b 2.19d

13.1** 7.9** 152.6*** n.s. n.s. 7.2* n.s.

n.s. n.s. 58.9*** n.s. n.s. n.s. n.s.

n.s. 4.5* 104.8*** n.s. n.s. 5.3* n.s.

12.7** 5.9* 51.8*** n.s. n.s. n.s. n.s.

700 Picual

360 700

ANOVA, F-values Cultivar CO2 Salt Cultivar  CO2 Cultivar  salt CO2  salt Cultivar  CO2  salt

8.3** n.s. 180*** n.s. n.s. 5.3* n.s.

Values are mean of six replicates. Within each column, means followed by the same letters are not significantly different at 5%. n.s., *, **, *** indicate non-significant differences or significant differences at Po0.05, 0.01, or 0.001, respectively.

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Table 2. Effects of NaCl (0 or 100 mM NaCl) and CO2 concentration (360 or 700 mL L1) during growth on CO2 assimilation rate ( ACO2 , mmol CO2 m2 s1), stomatal conductance (gs, mol H2O m2 s1), leaf water use efficiency (WUE, mmol CO2 mol1 H2O) and intercellular atmospheric CO2 concentration (Ci/Ca, dimensionless) of ‘Koroneiki’ and ‘Picual’ leaves, measured 2 and 12 weeks after initiating the treatments Cultivar

CO2 Salt ACO2

gs

WUE

Ci/Ca

2 weeks 12 weeks 2 weeks 12 weeks 2 weeks 12 weeks 2 weeks 12 weeks Koroneiki

360 700

Picual

360 700

0 100 0 100

13.59bc 12.32bc 14.10bc 6.38d

17.84cd 11.84de 27.04a 14.06de

0.56a 0.40bc 0.31c 0.22d

0.65b 0.43cd 0.60b 0.31e

1.13bc 1.13bc 1.71a 0.89c

2.23b 1.56bc 3.10a 2.20b

0.83bc 0.80de 0.84b 0.88a

0.83a 0.84a 0.85a 0.86a

0 100 0 100

14.91bc 11.36c 19.24a 16.04ab

21.08bc 8.36e 26.06ab 12.85de

0.51a 0.34bc 0.37bc 0.25d

0.82a 0.37de 0.55bc 0.26e

1.33b 1.14bc 2.03a 2.00a

2.19b 1.11c 3.04a 1.98b

0.81cde 0.79de 0.82bcd 0.79e

0.83a 0.87a 0.85a 0.85a

n.s. 128.9*** 83.5*** 10.2** n.s. 4.1* n.s.

n.s. 21.1*** 111.5*** n.s. n.s. n.s. n.s.

21.8*** 29.4*** 8.6** 12.1** n.s. n.s. 7.9**

n.s. 25.7*** 34.1*** n.s. n.s. n.s. n.s.

30.8*** 12.28** n.s. 12.7** 5.0* 5.1* 11.37**

n.s. n.s. n.s. n.s. n.s. n.s. n.s.

ANOVA, F-values Cultivar CO2 Salt Cultivar  CO2 Cultivar  salt CO2  salt Cultivar  CO2  salt

20.6*** n.s. 22.2*** 18.6*** n.s. n.s. 4.1*

n.s. 14*** 64.5*** n.s. n.s. n.s. n.s.

Values are mean of six replicates. Within each column, means followed by the same letters are not significantly different at 5%. n.s., *, **, *** indicate non-significant differences or significant differences at Po0.05, 0.01, or 0.001, respectively.

25 C-aCO2

20

Ewp (mg cm-2 h-1)

than in those grown at aCO2, but ACO2 was not significantly increased by eCO2. In both nonsalinized cultivars, WUE was higher at eCO2 than at aCO2. Salinity consistently decreased gs for both cultivars at both CO2 levels, and also decreased ACO2 and WUE of ‘Koroneiki’ plants at eCO2. Salinity also decreased ACO2 and WUE in ‘Picual’ plants, but only after 12 weeks. Nonetheless, at this time, the WUE of salt-stressed ‘Picual’ plants at eCO2 remained higher than at aCO2. After 12 weeks of treatment, the calculated Ci/Ca ratio was unchanged by salinity or CO2 treatment. eCO2 decreased Ewp of ‘Koroneiki’ and ‘Picual’ below that at aCO2 regardless of salinity (Figure 1). Salinity also consistently decreased Ewp in all plants compared with the non-salinized treatment except in ‘Koroneiki’ at aCO2, where the decrease was not significant. There was a positive correlation (Po0.05) between Ewp (measured by weight loss) and Elf (by gas exchange) in both ‘Koroneiki’ (r ¼ 0.49*) and ‘Picual’ (r ¼ 0.59*).

S-aCO2

ab

a

C-eCO2

bc

S-eCO2

bc 15

c

c d

d

10

5

0

Koroneiki

Picual Rootstocks

Figure 1. Effects of NaCl (control, C ¼ 0 mM, or salinized, S ¼ 100 mM) and CO2 concentration (aCO2 ¼ 360 mL L1 or eCO2 ¼ 700 mL L1) on whole plant transpiration rate (Ewp) of ‘Koroneiki’ and ‘Picual’ olive cultivars. Each value is the mean of six samples (7S.E.). Different letters within each figure indicate significant differences at Po0.05 (Duncan’s test). Data are from 12 weeks after the experiment started.

Water relations ‘Picual’ generally had lower leaf (more negative) water potential (Cw), higher osmotic potential (Cp),

and lower turgor potential (Cp) than ‘Koroneiki’ (Table 3). These leaf water relation parameters were not affected by eCO2 in non-salinized plants of

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Table 3. Effects of NaCl (0 or 100 mM NaCl) and CO2 concentration (360 or 700 mL L1) during growth on mean water potential (Cw, MPa), osmotic potential (Cp, MPa), and turgor pressure (Cp, MPa) of ‘Koroneiki’ and ‘Picual’ leaves following a 12-week experimental period Cultivar

CO2

Salt

Cw

Cp

Cp

Koroneiki

360

0 100 0 100

0.26a 0.43b 0.33ab 0.58cd

2.13a 3.51c 2.13a 3.68c

1.87c 3.08a 1.80c 3.11a

0 100 0 100

0.38ab 0.38ab 0.44bc 0.66c

1.89a 3.08b 1.87a 3.42c

1.51d 2.69b 1.42d 2.76b

16.0*** n.s. 369.6*** n.s. n.s. n.s. n.s.

34.0*** n.s. 398.7*** n.s. n.s. n.s. n.s.

700 Picual

360 700

ANOVA, F-values Cultivar CO2 Salt Cultivar  CO2 Cultivar  salt CO2  salt Cultivar  CO2  salt

4.1* 17.6*** 22.0*** n.s. n.s. 4.5* n.s.

Values are mean of six replicates. Within each column, means followed by the same letters are not significantly different at 5%. n.s., *, **, *** indicate non-significant differences or significant differences at Po0.05, 0.01, or 0.001, respectively.

either cultivar. However, the significant CO2  salt interaction for Cw was due to the lower Cw in salinized plants at eCO2 than at aCO2 in both cultivars. Salinity decreased Cw in ‘Koroneiki’ and Cp in both cultivars at both CO2 levels, such that Cp was increased by salinity.

Chlorophyll fluorescence and leaf chlorophyll concentration There were no cultivar effects on chlorophyll fluorescence characteristics except that effective quantum yield of light-acclimated ‘Koroneiki’ leaves (Y) was higher and non-photochemical quenching (qN) was generally lower than in ‘Picual’ (Table 4). In non-salinized plants, eCO2 decreased F0 and Fm for ‘Koroneiki’ and ‘Picual’ (about 20% and 30%, respectively), decreased Y, and increased qN in leaves of ‘Koroneiki’ plants. All salinized plants at eCO2 had significantly lower Fv/Fm and Y, and higher qN than salinized plants at aCO2. F0 was lower at eCO2 than at aCO2 in salinized plants but differences were only significant for ‘Picual’. Leaf chlorophyll concentration was not affected by eCO2 in non-salinized plants. However, the significant chlorophyll CO2  salt interaction was due to the salinity-induced 25% decrease in chlorophyll at eCO2 that did not occur at aCO2 in either cultivar.

Leaf Na+ and Cl concentration Although Na+ and Cl concentrations in leaves and roots of both cultivars were increased by salinity, root Na+ and Cl concentrations were higher in ‘Koroneiki’ than in ‘Picual’ (Figure 2), reflecting their differences in salinity tolerance. In ‘Koroneiki’, root and leaf Na+ and root Cl concentrations were significantly decreased by eCO2, but leaf Cl concentration was not affected in saline plants grown at eCO2. In ‘Picual’, root and leaf Na+ tended to increase at eCO2, but Na+ and Cl concentrations in leaves and roots were not significantly affected by eCO2.

Discussion eCO2 significantly increased shoot growth parameters (leaf area, leaf and stem dry weight) in ‘Koroneiki’ but not in ‘Picual’. This growth response difference could have been due to the effect of eCO2 increasing leaf ACO2 in ‘Koroneiki’ but not in ‘Picual’. Increased plant growth almost always occurs at eCO2 along with increased photosynthesis (Syvertsen et al., 2000; Jifon et al., 2002). Root dry weight was not affected by eCO2, which might suggest that CO2 enrichment enhanced shoot growth more than root growth. It is possible,

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Table 4. Effects of NaCl (0 or 100 mM NaCl) and CO2 concentration (360 or 700 ppm) during growth on minimum (F0) and maximum (Fm) fluorescence of dark-acclimated leaves, maximum quantum yield of dark-acclimated leaves (Fv/Fm), effective quantum yield of light-acclimated leaves (Y), non-photochemical quenching (qN), and total chlorophyll concentration (mg cm2) of ‘Koroneiki’ and ‘Picual’ leaves following a 12-week experimental period Cultivar

CO2

Salt

Koroneiki

360

0 100 0 100

97.9a 95.5ab 79.9cd 85.5bc

0 100 0 100

101.75a 98.25a 80.00cd 72.20d

700 Picual

360 700

F0

Fm

Fv/Fm

Y

qN

Total chlorophyll

576a 518b 416c 355d

0.83a 0.81a 0.81ab 0.75c

0.69a 0.59b 0.60b 0.47c

0.55d 0.66bcd 0.69bc 0.91a

6.32a 5.95ab 5.52ab 4.42c

570a 555ab 417c 339d

0.82a 0.82a 0.81ab 0.78b

0.64ab 0.57b 0.58b 0.38d

0.59cd 0.73b 0.67bc 0.91a

6.10a 6.45a 6.17a 4.92bc

n.s. 248.6*** 23.1*** n.s. n.s. n.s. n.s.

n.s. 26.1*** 11.9** n.s. n.s. 5.8* n.s.

13.8*** 49.8*** 53.1*** n.s. n.s. 7.6** 4.4*

7.6* 39.4*** 42.5*** n.s. n.s. 4.8* n.s.

n.s. 14.9*** 5.8* n.s. n.s. 5.6* n.s.

ANOVA Cultivar CO2 Salt Cultivar  CO2 Cultivar  salt CO2  salt Cultivar  CO2  salt

n.s. 47.5*** n.s. n.s. n.s. n.s. n.s.

Values are mean of six replicates. Within each column, means followed by the same letters are not significantly different at 5%. n.s., *, **, *** indicate non-significant differences or significant differences at Po0.05, 0.01, or 0.001, respectively.

however, that the pot size of 1.5 L may have constrained root growth. Other plants, such as oak–palmetto or soybean, increased root development when grown at eCO2 (Del Castillo et al., 1989; Day et al., 1996), but the response of the shoot/ root weight ratio was variable (Hunt et al., 1998). Although salt stress reduced growth in both cultivars, shoot growth was decreased more than root growth such that salinity consistently decreased the shoot/root weight ratio (Table 1) as reported for other olive cultivars under salt stress (Tattini et al., 1995; Chartzoulakis et al., 2002). In our experiment, based on growth and leaf WUE, ‘Koroneiki’ and ‘Picual’ had a similar tolerance to salinity. However, the lower Na+ and Cl accumulation in roots of ‘Picual’ supported its greater salt tolerance than ‘Koroneiki’ (Marı´n et al., 1995; Chartzoulakis et al., 2002). Since salinized plants grown under eCO2 had similar growth parameters and gas exchange characteristics as salinized plants grown under aCO2 at the end of the experiment, salt stress negated the effects of eCO2 in both cultivars. Thus, the measured growth parameters and ACO2 were more affected by 100 mM salt treatment than by eCO2. However, when pepino was salinized with only 25 mM NaCl (Chen et al., 1999) or melon with 50 mM NaCl (Mavrogianopoulos et al., 1999), growth was higher at eCO2 concen-

tration than at aCO2. It would be interesting to repeat these studies using a range of salinities to determine whether eCO2 could improve salinity tolerance of olive plants at more moderate levels of salinity. In leaves of olive trees, salinity-induced reductions in the rate of photosynthesis have been attributed in part to stomatal closure (Bongi and Loreto, 1989), but also to the inactivation of metabolic processes leading to a decrease of Rubisco and to the inhibition of photochemical reactions (Loreto et al., 2003). In addition, low photosynthesis under moderate salinity stress appeared to be attributable primarily to stomatal limitations and also to biochemical limitations on photosynthesis when stress became severe (Flexas and Medrano, 2002). Recent studies using severe salt stress (200 mM NaCl) in olive trees, however, revealed decreases in stomatal conductances but not in the biochemical capacity to assimilate CO2 (Centritto et al., 2003). In our experiment, although ACO2 and gs were both reduced by salinity, there were no changes in Ci/Ca (Table 2); so, stomatal conductance was not responsible for the reduction in ACO2 by salt treatment (Farquhar and Sharkey, 1982). Thus, the reduction in ACO2 was mainly due to direct biochemical effects in the mesophyll, and not to stomatal limitations. Similar

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Figure 2. Effects of NaCl (control, C ¼ 0 mM, or salinized, S ¼ 100 mM) and CO2 concentration (aCO2 ¼ 360 mL L1 or eCO2 ¼ 700 mL L1) on Na+ and Cl concentrations (% DW) in leaf and root tissue of ‘Koroneiki’ and ‘Picual’ olive cultivars. Each value is the mean of six samples (7S.E.). Different letters within each figure indicate significant differences at Po0.05 (Duncan’s test). Data are from 12 weeks after the experiment started.

responses have been reported for salinized citrus (Garcı´a-Sa ´nchez and Syvertsen, 2006). Although ACO2 was increased at eCO2 in salinized olive plants, this increase was not significant. eCO2 also increased the sensitivity of leaf chlorophyll and related fluorescence parameters to salinity stress such that salinized plants grown at eCO2 had lower values of Fv/Fm and Y than salinized plants at aCO2 (Table 4). This reduction in Fv/Fm (photoinhibition) could be a consequence of damage to PSII or due to a photoprotective response of the light reaction of photosynthesis (Demmig-Adams and Adams, 1992). Damage to PSII can lead to a lower Fm and higher F0 (Franklin et al., 1992), but in our experiment, Fm decreased while F0 remained constant. Salt treatment at aCO2 did not affect Fv/Fm in either cultivar, although a decrease in Fv/Fm has been reported for other cultivars at high salt concentration (200 mM; Flexas and Medrano, 2002). In addition, salt treatment reduced Y in all plants, regardless of CO2 level. Reduction in effective quantum yield in salt-stressed plants is thought to be related to the increase in quenching excess energy through

non-photochemical mechanisms (Demmig-Adams and Adams, 1992). Although salinity altered leaf water relations of both olive cultivars, turgor potential in saltstressed plants was higher than in non-salinized plants. Thus, leaf water relations were not responsible for reductions in ACO2 and gs by the salt treatment. High leaf Cl and Na+ concentrations can act as osmotic solutes in the osmoregulation process (Tattini et al., 1997). In addition, the lower Cp observed in salinized Picual at eCO2 than at aCO2 could have been partly related to the effect of eCO2 in reducing Na+ accumulation in salinized ‘Koroneiki’ plants. Salt tolerance in glycophytes is associated with the ability to limit the uptake and/or the transport of Na+ and Cl– from the root zone to aerial parts (Greenway and Munns, 1980). Most of the olive cultivars, at low and moderate salinity, show an exclusion capacity for Na+ such that accumulation of potentially toxic ions in the aerial parts is prevented (Loreto et al., 2003). Thus, the concentration of Na+ and Cl can be higher in the root than in the leaf

ARTICLE IN PRESS Elevated CO2 improves salt tolerance (Chartzoulakis et al., 2002). In our experiment, however, Na+ and Cl concentrations in the leaves generally were higher than in roots. Consequently, we did not observe any exclusion of Na+ and/or Cl in the roots of either cultivar. At 100 mM NaCl, the exclusion capacity of roots may have been exceeded even in relatively salt-tolerant ‘Picual’ such that these ions were rapidly accumulated in the aerial parts as has been reported for citrus rootstock seedlings (Ca ´mara-Zapata et al., 2004). Salinized ‘Koroneiki’ plants grown at eCO2 had lower concentrations of Na+ in leaf and root, and lower Cl in root than plants grown at aCO2. Since salinized ‘Koroneiki’ plants grown at both aCO2 and eCO2 had similar growth, any dilution effect by increased growth at eCO2 was not responsible for these differences. Water and salt uptake can be closely coupled (Moya et al., 1999) and the lower Ewp (Figure 1) and higher WUE (Table 2) at eCO2 than at aCO2 may be related to the decreased ion concentration in leaves and roots for salt-sensitive ‘Koroneiki’ but not in ‘Picual’. Reductions in leaf transpiration in tomatoes (Maggio et al., 2002) and in citrus (Moya et al., 1999) were related to decreases in leaf Cl concentration supporting the idea that water and salt uptake can be linked, but there are exceptions (Garcı´a-Sa ´nchez and Syvertsen, 2006). In conclusion, based on growth parameters, tolerance to 100 mM NaCl of ‘Koroneiki’ and ‘Picual’ olive trees was not affected by eCO2. Leaf WUE of salt-stressed olive trees was increased at eCO2, but despite the decreased leaf and root Na+ and root Cl concentrations at eCO2 in ‘Koroneiki’, eCO2 did not significantly improve salt-induced reductions in growth or net gas exchange. This may have been related to the increased sensitivity of PSII and chlorophyll concentration to salinity. In addition, salt uptake in salt-sensitive ‘Koroneiki’ appeared to be linked to reduced water uptake because Ewp was also lower at eCO2 than at aCO2. This did not occur in the relatively salt-tolerant ‘Picual’.

Acknowledgments J.C. Melgar was a visiting Ph.D. student in J. Syvertsen’s lab, supported by UF/IFAS and the Comisio ´n Interministerial de Ciencia y Tecnologı´a, Spain, Project No. AGL2001-2447. Dr. F. Garcı´aSa ´nchez was funded by a postdoctoral fellowship from the Ministerio de Educacio ´n, Cultura y Deportes of Spain (AGL2003-O8502-CO4-02/AGR). Grateful acknowledgement is made to Eva Ros and Jill Dunlop for their skilled technical assistance.

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References Abd El Rahman AA, Sharkawi MH. Effect of salinity and water supply on olive. Plant Soil 1968;28:280–90. Ball MC, Munns R. Plant responses to salinity under elevated atmospheric concentrations of CO2. Aust J Bot 1992;40:515–25. Bernstein L. Effect of salinity and sodicity on plant growth. Annu Rev Phytopathol 1975;13:295–312. Bongi G, Loreto F. Gas-exchange properties of saltstressed olive (Olea europaea L.) leaves. Plant Physiol 1989;90:1408–16. Caballero J, Del Rio C. Me´todos de multiplicacio ´n. In: Barranco D, Ferna ´ndez-Escobar R, Rallo L, editors. El Cultivo del Olivo. Madrid: Mundi-Prensa; 2004. p. 93–123. Ca ´mara-Zapata JM, Garcı´a-Sa ´nchez F, Martı´nez V, Nieves M, Cerda ´ A. Effect of NaCl on citrus cultivars. Agronomie 2004;24:155–60. Centritto M, Loreto F, Chartzoulakis K. The use of low [CO2] to estimate diffusional and non-diffusional limitations of photosynthetic capacity of salt-stressed olive samplings. Plant Cell Environ 2003;26: 585–94. Chartzoulakis K, Loupassaki M, Bertaki M, Androulakis I. Effects of NaCl salinity on growth, ion content and CO2 assimilation rate of six olive cultivars. Sci Hortic 2002;96:235–47. Chen K, Hu G, Keutgen N, Janssens MJJ, Lenz F. Effects of NaCl salinity and CO2 enrichment on pepino (Solanum muricatum Ait.) I. Growth and yield. Sci Hortic 1999;81:25–41. Day FP, Weber EP, Hinkle CR, Drake BG. Effects of elevated CO2 on fine root length and distribution in an oak–palmetto ecosystem in central Florida. Global Change Biol 1996;2:143–8. Del Castillo D, Acock B, Reddy VR, Acock MC. Elongation and branching of roots on soybean plants in a carbon dioxide-enriched aerial environment. Agron J 1989; 81:692–5. Demmig-Adams B, Adams WW. Carotenoid composition in sun and shade leaves of plants with different life forms. Plant Cell Environ 1992;15:411–9. Farquhar GD, Sharkey TD. Stomatal conductance and photosynthesis. Annu Rev Plant Physiol 1982;33: 317–45. Flexas J, Medrano H. Drought-inhibition of photosynthesis in C3 plants: stomatal and non-stomatal limitations revisited. Ann Bot 2002;89:183–9. Franklin LA, Levavasseur G, Osmond CB, Henley JH, Ramus J. Two components of onset and recovery during photoinhibition of Ulva rotundata. Planta 1992;186:399–408. Garcı´a-Sa ´nchez F, Syvertsen JP. Salinity tolerance of Cleopatra mandarin and Carrizo citrange citrus rootstock seedlings is affected by CO2 enrichment during growth. J Am Soc Hortic Sci 2006;131:24–31. Greenway H, Munns R. Mechanisms of salt tolerance in nonhalophytes. Annu Rev Plant Physiol 1980;31: 149–90.

ARTICLE IN PRESS 640 Gucci R, Tattini M. Salinity tolerance in olive. Hortic Rev 1997;21:177–214. Gucci R, Lombardini L, Tattini M. Analysis of leaf water relations of two olive cultivars (Olea europaea L.) differing in tolerance to salinity. Tree Physiol 1997; 17:13–21. Hartmann HT. Rootstock effects in the olive. Proc Am Soc Hortic Sci 1958;72:242–51. Hunt HW, Morgan JA, Read JJ. Simulating growth and root–shoot partitioning in prairie grasses under elevated atmospheric CO2 and water stress. Ann Bot 1998;81:489–501. Inskeep WP, Bloom PR. Extinction coefficients of chlorophyll a and b in N,N-dimethylformamide and 80% acetone. Plant Physiol 1985;77:483–5. Jifon JL, Syvertsen JP. Moderate shade can increase net gas exchange and reduce photoinhibition in citrus leaves. Tree Physiol 2003;23:119–27. Jifon J, Graham JH, Drouillard DL, Syvertsen JP. Growth depression of mycorrhizal citrus seedlings grown at high phosphorus supply is mitigated by elevated CO2. New Phytol 2002;153:133–42. Kang SZ, Cai HJ, Liu XM. Effects of the atmospheric CO2 concentration increase on the water use efficiency and evapotranspiration of spring wheat. Acta Univ Agric Boreali-occidentalis 1995;23:1–5. Loreto F, Bongi G. Control of photosynthesis under salt stress in the olive. In: Prodi F, Rossi F, Cristoferi G, editors. Proceedings of the international conference on agrometeorology. Cesena: Fondazione Cesena Agricoltura; 1987. p. 411–20. Loreto F, Centritto M, Chartzoulakis K. Photosynthetic limitations in olive cultivars with different sensitivity to salt stress. Plant Cell Environ 2003;26:595–601. Maas EV, Hoffman GJ. Crop salt tolerance – current assessment. J Irrig Drain Div 1977;103:115–34. Maggio A, Dalton FN, Piccinni G. The effects of elevated carbon dioxide on static and dynamic indices for tomato salt tolerance. Eur J Agron 2002;16: 197–206. Marı´n L, Benlloch M, Ferna ´ndez-Escobar R. Screening of olive cultivars for salt tolerance. Sci Hortic 1995;64: 113–6. Mavrogianopoulos GN, Spanakis J, Tsikalas P. Effect of carbon dioxide enrichment and salinity on photosynthesis and yield in melon. Sci Hortic 1999;79:51–63. Maxwell K, Johnson GN. Chlorophyll fluorescence – a practical guide. J Exp Bot 2000;51:659–68.

J.C. Melgar et al. Moran R, Porath D. Chlorophyll determination of chlorophyllous pigments extracted with N,N-dimethylformamide. Plant Physiol 1980;65:478–9. Moya JL, Primo-Millo E, Talo ´n M. Morphological factors determining salt tolerance in citrus seedlings: the shoot to root ratio modulates passive root uptake of chloride ions and their accumulation in leaves. Plant Cell Environ 1999;22:1425–33. Orgaz F, Fereres E. Riego. In: Barranco D, Ferna ´ndezEscobar R, Rallo L, editors. El cultivo del olivo. Madrid: Mundi-Prensa; 2004. p. 269–88. Roden JS, Ball MC. Growth and photosynthesis of two eucalypt species during high temperature stress under ambient and elevated [CO2]. Global Change Biol 1996;2:115–28. Scholander P, Hammel H, Bradstreet E, Hemmingsen E. Sap pressure in vascular plants. Negative hydrostatic pressure can be measured in plants. Science 1965;148: 339–46. Sebastiani L, Minnocci A, Tognetti R. Genotypic differences in the response to elevated CO2 concentration of one-year-old olive cuttings (Olea europaea L. cv Frantoio and Moraiolo). Plant Biosyst 2002;136: 199–207. Syvertsen JP, Lee LS, Grosser JW. Limitations on growth and net gas exchange of diploid and tetraploid citrus rootstock cultivars grown at elevated CO2. J Am Soc Hortic Sci 2000;125:228–34. Tattini M, Bertoni P, Caselli S. Genotypic responses of olive plants to sodium chloride. J Plant Nutr 1992; 15:1462–85. Tattini M, Ponzio C, Coradeschi MA, Tafani R, Traversi ML. Mechanisms of salt tolerance in olive plants. Acta Hortic 1994;356:181–4. Tattini M, Gucci R, Coradeschi MA, Ponzio C, Everard JD. Growth, gas exchange and ion content in Olea europaea plants during salinity stress and subsequent relief. Physiol Plant 1995;95:203–10. Tattini M, Lombardini L, Gucci R. The effect of NaCl stress and relief on gas exchange properties of two olive cultivars differing in tolerance to salinity. Plant Soil 1997;197:87–93. Yeo A. Predicting the interaction between the effects of salinity and climate change on crop plants. Sci Hortic 1999;78:159–74. Zhang FC, Kang SZ, Ma QL. The effects of the atmospheric CO2 concentration increase on physiological characters and growth cotton. J Basic Sci Eng 1999;7:267–73.