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 Garcı́a-Sánchezc

a
Departmento de Agronomı́a, Universidad de Córdoba, Edificio ‘Celestino Mutis’,
Campus Universitario de Rabanales, Ctra. Madrid-Cádiz km. 396, 14071 Córdoba, Spain
b
UF/IFAS, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850, USA
c
Centro de Edafologı́a y Biologı́a 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 Summary
CO2 enrichment;
We compared growth, leaf gas exchange characteristics, water relations, chlorophyll
Olea europaea;
fluorescence, and Na+ and Cl concentration of two cultivars (‘Koroneiki’ and
Photosynthesis;
‘Picual’) of olive (Olea europaea L.) trees in response to high salinity (NaCl 100 mM)
Salt stress;
and elevated CO2 (eCO2) concentration (700 mL L1). The cultivar ‘Koroneiki’ is
Salt tolerance
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: pa2mejij@uco.es (J.C. Melgar).

0176-1617/$ - see front matter & 2007 Elsevier GmbH. All rights reserved.
doi:10.1016/j.jplph.2007.01.015
 ARTICLE IN PRESS
632
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 (Marı́n 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 ‘Lechı́n de Sevilla’ (Marı́n
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 salt-
stressed 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 de-
creased (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 toler-
ance. 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; Garcı́a-Sánchez 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 (Marı́n 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 experi-
mental period, the two greenhouses had almost identical
growth conditions except for CO2 concentration. Max-
imum 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 concentra-
tion dropped to 690 mL L1, CO2 was automatically
injected and mixed into the greenhouse until the
concentration reached 710 mL L1. The other well-venti-
lated 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 ex-
change 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 measure-
ments, chlorophyll fluorescence was measured with
a pulse-modulated fluorometer (Model OS1-Fl, Opti-
Sciences, 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-accli-
mated 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 measure-
ments. The parameters of maximum quantum effi-
ciency (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 chlor-
ophyll 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
 ARTICLE IN PRESS
634 J.C. Melgar et al.

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 (Garcı́a-
Sánchez and Syvertsen, 2006). Leaf tissue Na+ concentration
was determined by an analytical laboratory (Waters Agri-
cultural 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; Garcı́a-Sánchez 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 ‘Kor-
oneiki’ 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 non-
salinized 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 ‘Koronei-
ki’ 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 338b 4.74b 2.82a 17.08b 4.94b

100 83c 1.47c 1.28b 5.24c 3.18cd
700 0 455a 6.88a 3.06a 23.42a 6.54a
100 80c 1.67c 1.01b 4.75c 3.70bcd
Picual 360 0 274b 3.63b 2.58a 13.81b 4.30bc
100 60c 0.96c 1.66b 5.21c 2.32d
700 0 333b 4.87b 3.06a 18.95ab 5.14b
100 46c 0.83c 1.65b 5.20c 2.19d
ANOVA, F-values

Cultivar 8.3** 13.1** n.s. n.s. 12.7**

CO2 n.s. 7.9** n.s. 4.5* 5.9*
Salt 180*** 152.6*** 58.9*** 104.8*** 51.8***
Cultivar  CO2 n.s. n.s. n.s. n.s. n.s.
Cultivar  salt n.s. n.s. n.s. n.s. n.s.
CO2  salt 5.3* 7.2* n.s. 5.3* n.s.
Cultivar  CO2  salt 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.
 ARTICLE IN PRESS
Elevated CO2 improves salt tolerance 635

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 0 13.59bc 17.84cd 0.56a 0.65b 1.13bc 2.23b 0.83bc 0.83a
100 12.32bc 11.84de 0.40bc 0.43cd 1.13bc 1.56bc 0.80de 0.84a
700 0 14.10bc 27.04a 0.31c 0.60b 1.71a 3.10a 0.84b 0.85a
100 6.38d 14.06de 0.22d 0.31e 0.89c 2.20b 0.88a 0.86a
Picual 360 0 14.91bc 21.08bc 0.51a 0.82a 1.33b 2.19b 0.81cde 0.83a
100 11.36c 8.36e 0.34bc 0.37de 1.14bc 1.11c 0.79de 0.87a
700 0 19.24a 26.06ab 0.37bc 0.55bc 2.03a 3.04a 0.82bcd 0.85a
100 16.04ab 12.85de 0.25d 0.26e 2.00a 1.98b 0.79e 0.85a
ANOVA, F-values

Cultivar 20.6*** n.s. n.s. n.s. 21.8*** n.s. 30.8*** n.s.

CO2 n.s. 14*** 128.9*** 21.1*** 29.4*** 25.7*** 12.28** n.s.
Salt 22.2*** 64.5*** 83.5*** 111.5*** 8.6** 34.1*** n.s. n.s.
Cultivar  CO2 18.6*** n.s. 10.2** n.s. 12.1** n.s. 12.7** n.s.
Cultivar  salt n.s. n.s. n.s. n.s. n.s. n.s. 5.0* n.s.
CO2  salt n.s. n.s. 4.1* n.s. n.s. n.s. 5.1* n.s.
Cultivar  CO2  salt 4.1* n.s. n.s. n.s. 7.9** n.s. 11.37** 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.

than in those grown at aCO2, but ACO2 was not 25

C-aCO2
significantly increased by eCO2. In both non- S-aCO2 a
salinized cultivars, WUE was higher at eCO2 than 20 ab C-eCO2

at aCO2. Salinity consistently decreased gs for both bc S-eCO2

Ewp (mg cm-2 h-1)

bc
cultivars at both CO2 levels, and also decreased 15 c c
ACO2 and WUE of ‘Koroneiki’ plants at eCO2. Salinity
d
also decreased ACO2 and WUE in ‘Picual’ plants, but d
10
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 5
treatment, the calculated Ci/Ca ratio was un-
changed by salinity or CO2 treatment. 0
eCO2 decreased Ewp of ‘Koroneiki’ and ‘Picual’ Koroneiki Picual
below that at aCO2 regardless of salinity (Figure 1). Rootstocks
Salinity also consistently decreased Ewp in all plants Figure 1. Effects of NaCl (control, C ¼ 0 mM, or sali-
compared with the non-salinized treatment except nized, S ¼ 100 mM) and CO2 concentration (aCO2 ¼
in ‘Koroneiki’ at aCO2, where the decrease was not 360 mL L1 or eCO2 ¼ 700 mL L1) on whole plant tran-
significant. There was a positive correlation spiration rate (Ewp) of ‘Koroneiki’ and ‘Picual’ olive
(Po0.05) between Ewp (measured by weight loss) cultivars. Each value is the mean of six samples (7S.E.).
and Elf (by gas exchange) in both ‘Koroneiki’ Different letters within each figure indicate significant
(r ¼ 0.49*) and ‘Picual’ (r ¼ 0.59*). differences at Po0.05 (Duncan’s test). Data are from 12
weeks after the experiment started.

Water relations
and lower turgor potential (Cp) than ‘Koroneiki’
‘Picual’ generally had lower leaf (more negative) (Table 3). These leaf water relation parameters
water potential (Cw), higher osmotic potential (Cp), were not affected by eCO2 in non-salinized plants of
 ARTICLE IN PRESS
636 J.C. Melgar et al.

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 0.26a 2.13a 1.87c

100 0.43b 3.51c 3.08a
700 0 0.33ab 2.13a 1.80c
100 0.58cd 3.68c 3.11a
Picual 360 0 0.38ab 1.89a 1.51d
100 0.38ab 3.08b 2.69b
700 0 0.44bc 1.87a 1.42d
100 0.66c 3.42c 2.76b
ANOVA, F-values

Cultivar 4.1* 16.0*** 34.0***

CO2 17.6*** n.s. n.s.
Salt 22.0*** 369.6*** 398.7***
Cultivar  CO2 n.s. n.s. n.s.
Cultivar  salt n.s. n.s. n.s.
CO2  salt 4.5* n.s. n.s.
Cultivar  CO2  salt 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.

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 para-
meters (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 photo-
synthesis (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,
 ARTICLE IN PRESS
Elevated CO2 improves salt tolerance 637

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 F0 Fm Fv/Fm Y qN Total chlorophyll

Koroneiki 360 0 97.9a 576a 0.83a 0.69a 0.55d 6.32a

100 95.5ab 518b 0.81a 0.59b 0.66bcd 5.95ab
700 0 79.9cd 416c 0.81ab 0.60b 0.69bc 5.52ab
100 85.5bc 355d 0.75c 0.47c 0.91a 4.42c
Picual 360 0 101.75a 570a 0.82a 0.64ab 0.59cd 6.10a
100 98.25a 555ab 0.82a 0.57b 0.73b 6.45a
700 0 80.00cd 417c 0.81ab 0.58b 0.67bc 6.17a
100 72.20d 339d 0.78b 0.38d 0.91a 4.92bc
ANOVA

Cultivar n.s. n.s. n.s. 13.8*** 7.6* n.s.

CO2 47.5*** 248.6*** 26.1*** 49.8*** 39.4*** 14.9***
Salt n.s. 23.1*** 11.9** 53.1*** 42.5*** 5.8*
Cultivar  CO2 n.s. n.s. n.s. n.s. n.s. n.s.
Cultivar  salt n.s. n.s. n.s. n.s. n.s. n.s.
CO2  salt n.s. n.s. 5.8* 7.6** 4.8* 5.6*
Cultivar  CO2  salt n.s. n.s. n.s. 4.4* 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 develop-
ment 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 de-
creased 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 accumula-
tion in roots of ‘Picual’ supported its greater salt
tolerance than ‘Koroneiki’ (Marı́n 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 reduc-
tions 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 ap-
peared 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
 ARTICLE IN PRESS
638 J.C. Melgar et al.

5 5
Leaf Na+ concentration (% DW)

Leaf Cl- concentration (% DW)

C-aCO2
4 a a S-aCO2 4
C-eCO2
ab S-eCO2 a
3 a 3
b a a

2 2

1 1
b b
c c c c b b
0 0
Root Na+ concentration (% DW)

Root Cl- concentration (% DW)

2.5 2.5
a
2.0 b 2.0
bc a
c
1.5 b 1.5
c
c
1.0 1.0

0.5 0.5
d d d d
d d d d
0.0 0.0
Koroneiki Picual Koroneiki Picual

Cultivars Cultivars

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
(Garcı́a-Sánchez and Syvertsen, 2006). Although
ACO2 was increased at eCO2 in salinized olive plants,
this increase was not significant. eCO2 also in-
creased 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 treat-
ment 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 salt-
stressed plants was higher than in non-salinized
plants. Thus, leaf water relations were not respon-
sible 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 (Cámara-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 (Garcı́a-Sánchez 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
Comisión Interministerial de Ciencia y Tecnologı́a,
Spain, Project No. AGL2001-2447. Dr. F. Garcı́a-
Sánchez was funded by a postdoctoral fellowship
from the Ministerio de Educación, 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.
639
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