Directory UMM :Data Elmu:jurnal:T:Tree Physiology:vol17.1997:
Tree Physiology 17, 13--21
© 1997 Heron Publishing----Victoria, Canada
Analysis of leaf water relations in leaves of two olive (Olea europaea)
cultivars differing in tolerance to salinity
RICCARDO GUCCI,1 LEONARDO LOMBARDINI1 and MASSIMILIANO TATTINI2
1
Dipartimento di Coltivazione e Difesa delle Specie Legnose, Sezione Coltivazioni Arboree, Università di Pisa, Via del Borghetto 80, Pisa 56124, Italy
2
Istituto sulla Propagazione delle Specie Legnose -- CNR, Via Ponte di Formicola 76, 50018 Scandicci (FI), Italy
Received January 8, 1996
Summary One-year-old rooted cuttings of olive (Olea
europaea L. cvs. Frantoio and Leccino) were grown either
hydroponically or in soil in a greenhouse. Plants were exposed
to NaCl treatments (0, 100, and 200 mM) for 35 days, followed
by 30 to 34 days of relief from salt stress to determine whether
previously demonstrated genotypic differences in tolerance to
salinity were related to water relations parameters. Exposure
to high salt concentrations resulted in reductions in predawn
water potential (Ψw), osmotic potential at full turgor (ΨπFT),
osmotic potential at turgor loss point (ΨπTLP), and relative water
content (RWC) in both cultivars, regardless of the growth
substrate. Leaf Ψw and RWC returned to values similar to those
of controls by the end of the relief period. The effect of salinity
on Ψπ appeared earlier in Leccino than in Frantoio. Values for
ΨπFT were − 2.50, − 2.87, and − 3.16 MPa for the 0, 100, and
200 mM salt-treated Frantoio plants, respectively, and − 2.23,
− 2.87, and − 3.37 MPa for the corresponding Leccino plants.
Recovery of Ψπ was complete for plants in the 100 mM salt
treatment, but not for plants in the 200 mM salt treatment,
which maintained an increased pressure potential (Ψp) compared to control plants. Net solute accumulation was higher in
Leccino, the salt-sensitive cultivar, than in Frantoio. In controls
of both cultivars, cations contributed 39.9 to 42.0% of the total
ΨπFT, mannitol and glucose contributed 27.1 to 30.8%, and
other soluble carbohydrates contributed 3.1 to 3.6%. The osmotic contribution of Na+ increased from 0.1--2.1% for nontreated plants to 8.6--15.5% and 15.6--20.0% for the 100 mM
and 200 mM salt-treated plants, respectively. The mannitol
contribution to ΨπFT reached a maximum of 9.1% at the end of
the salinization period. We conclude that differences between
the two cultivars in leaf water relations reflect differences in
the exclusion capacities for Na+ and Cl− ions.
Keywords: osmotic adjustment, pressure-volume isotherms,
relief from salinity, turgor loss point.
Frantoio and Leccino, the two cultivars used in the present
study, differ in their capacity to exclude Na+ or Cl− ions from
the shoot when NaCl is applied to the rooting medium (Tattini
et al. 1992, Tattini 1994). A previous study showed that increasing the NaCl concentration from 0 to 200 mM significantly decreased leaf water potential (Ψw) in the salt-tolerant
Frantoio cultivar; however, this reduction was accompanied by
a decrease in osmotic potential (Ψπ), so that leaf turgor of the
salinized plants was maintained (Tattini et al. 1995).
Salt stress changes the water relations of most higher plants,
and salt tolerance often depends on drought tolerance (Greenway and Munns 1980, Flowers and Yeo 1986). Changes in Ψw,
Ψπ, leaf succulence, cell wall extensibility, and bulk leaf
modulus of elasticity (ε) have been reported for herbaceous
crops exposed to increased salt concentrations (Longstreth and
Nobel 1980, Neumann et al. 1988, Erdei and Taleisnik 1993).
No similar studies have been reported for olive. Olive trees are
drought tolerant, and leaves can reach extremely low values of
Ψw and relative water content (RWC) (− 3.5 MPa and 75--80%,
respectively) before losing turgor (Hinckley et al. 1980, Lo
Gullo and Salleo 1988, Larsen et al. 1989). Seasonal and
diurnal declines in leaf Ψw are mainly attributed to inelastic
cell walls, osmotic adjustment, and transpirational losses,
which continue even at low Ψw (Lo Gullo and Salleo 1988). In
addition, olive leaves often experience a diurnal osmotic stress
during the dry season because the high hydraulic resistance of
the stem causes the development of water deficits in leaves
(Larsen et al. 1989, Lo Gullo and Salleo 1990).
In the present study, olive plants of two cultivars, growing in
either hydroponic or soil culture, were subjected to salinization
and subsequent relief. The objective was to elucidate the contribution of changes in leaf water relations to the differences in
salt tolerance exhibited by the cultivars.
Materials and methods
Introduction
Plant material and salinity treatments
Olive (Olea europaea L.) is more salt tolerant than other
woody crops and has great potential for cultivation in areas,
such as the Mediterranean region, where salinity is becoming
a major problem. Salt tolerance in olive is cultivar-dependent.
One-year-old rooted cuttings of the olive cultivars Frantoio
and Leccino were grown in a greenhouse from April to September 1994, either hydroponically or in soil. Plants were
trained to two shoots, as previously described (Tattini et al.
14
GUCCI, LOMBARDINI AND TATTINI
1995). About 300 plants of both cultivars were transplanted to
a quartz sand hydroponic culture system at Scandicci
(43°45′ N, 11°11′ E) on March 1, 1994 and acclimated until
April 27, 1994, when salt treatments were initiated. Plants
received half-strength Hoagland solution twice a day (Tattini
et al. 1995). A separate group of 150 plants from the same
batch of rooted cuttings was grown in 1-liter containers at
Scandicci and then transferred to Pisa (43°43′ N, 10°25′ E) on
June 24, 1994. The plants were grown in a substrate of 1/1/1
fertilized peat/perlite/field soil. Plants in containers were irrigated two to four times per week, depending on the evaporative
demand, and received either half-strength Hoagland solution
or a fertilizer solution containing 5 g l −1 of 18/9/18 N,P,K once
or twice a week.
Plants in both hydroponic and soil culture were subjected to
35 days of increased salinity, followed by 30 to 34 days of
relief. In hydroponic cultures, 0, 100, or 200 mM NaCl was
added to the standard nutrient solution (Tattini et al. 1995). In
soil cultures, NaCl concentrations were increased by 50 mM
day −1 until final concentrations of 0, 100, and 200 mM were
reached, containers were covered with plastic film to reduce
evaporation from the soil surface, and plants were watered
with excess solution to prevent the occurrence of salt gradients
in the container profile. The concentration of the soil solution
leaching from the bottom of the container after irrigation was
periodically checked with a Wescor 5500 vapor pressure osmometer (Wescor Inc., Logan, UT) and was usually within
20% of the concentration of the nutrient solution. At the end of
the salinization period, stress was relieved by supplying the
nutrient solution only (Tattini et al. 1995).
A randomized complete block design was adopted with nine
plants per treatment arranged in blocks according to location
in the greenhouse.
Leaf water relations
Relative water content (RWC) was determined on three to four
fully expanded leaves of similar age (15 to 25 days old).
Leaves were excised before dawn, weighed fresh (FW) and
placed in vials to rehydrate in the dark for 20 h. Preliminary
experiments had indicated that full rehydration was complete
after 18 h. The following morning, leaf turgid weight (TW)
was measured and then leaves were dried at 80 °C for 48 h and
dry weight (DW) was determined. The RWC was calculated
from the equation:
RWC = 100[(FW − DW )/(TW − DW )].
(1)
Predawn Ψw of three to six leaves per treatment was measured at approximately 10-day intervals with a pressure chamber (TecnoGas, Pisa, Italy), according to a standard technique
(Ritchie and Hinckley 1975). After the Ψw measurement, the
leaves were immediately frozen in liquid nitrogen and stored
at − 80 °C for determination of Ψπ with a Wescor 5500 vapor
pressure osmometer (Tattini et al. 1995). Leaf turgor potential
(Ψp) was calculated as the difference between Ψw and Ψπ.
Osmotic potential at full turgor (ΨπFT) was determined from
pressure-volume curves or calculated using the equation proposed by Wilson et al. (1979):
Ψ π FT = Ψπ [(RWC − A)/(100 − A)],
(2)
where A is the apoplastic water fraction, estimated at 4.4% in
olive leaves (see analysis of pressure-volume isotherms). Osmotic adjustment resulting from net solute accumulation was
estimated as the difference between total change in Ψπ and the
change resulting from dehydration (D) and non-osmotic volume (V). In this procedure, changes in Ψπ and ΨπFT (∆Ψπ and
∆ΨπFT) induced by salinity were calculated by comparing
pre-dawn Ψπ of plants in NaCl treatments with predawn Ψπ of
control plants, according to the following equations (Girma
and Krieg 1992):
∆Ψ π = Ψ π ,salt − Ψ π,control
(3)
∆Ψ π FT = Ψ π FT,salt − Ψ πFT,contr ol.
(4)
The contribution of dehydration (D) to changes in Ψπ was
calculated as:
D = ∆Ψπ − ∆Ψ π FT .
(5)
Similarly, changes in solute concentration resulting from
changes in the non-osmotic volume at 100% hydration were
estimated from changes in the TW/DW ratio between the
control and the various salt treatments (Girma and Krieg
1992):
(TW/DW)control − (TW /DW)salt
V=
∆Ψ π FT .
(TW/DW)control
(6)
Leaf mass per unit area and succulence index (water per unit
leaf area) were measured at the end of the salinity and relief
periods on four to six fully expanded leaves of plants growing
in hydroponic culture. Leaf area was measured with a leaf area
meter (Delta-T Devices, Burwell, Cambridge, U.K.).
Pressure-volume isotherms
Pressure-volume (P-V) isotherms were developed for three to
six fully expanded leaves collected at the end of the salinity
and relief periods. Additional leaf material was sampled from
plants growing in hydroponic culture 15 days after the beginning of relief. The P-V isotherms were constructed following
the free transpiration technique of Wilson et al. (1979). In brief,
leaves from plants cultivated hydroponically were collected in
the afternoon and handled as described for RWC determination. Plants grown in soil culture were rehydrated by saturating
the substrate with water and enclosing the plants in black
plastic bags at room temperature for 18 h before leaf excision.
The pressure chamber temperature was maintained at 20 °C
and the flow of nitrogen gas into the chamber was regulated at
0.005 MPa s −1. Pressurization was stopped when sap was
visible on at least 50% of the cut surface of the petiole.
WATER RELATIONS IN OLIVE UNDER SALINITY
Pressure was then slowly reduced by 0.10--0.15 MPa and then
increased until sap flowed again. At this point, the balance
pressure was recorded as the leaf Ψw. Leaves were dried on the
laboratory bench at 23 °C to achieve progressive dehydration
of the tissue, and the Ψw determination was repeated nine to 15
times. Four to 10 data points in the region of zero turgor and
three to seven data points in the positive turgor range were
typically obtained for one P-V isotherm. A period of 8--9 h was
necessary to produce a complete P-V isotherm.
Pressure-volume isotherms were analyzed by type II transformation (Tyree and Richter 1982). The point indicating departure from linearity corresponded to the turgor loss point
(TLP). The TLP was determined iteratively by non-linear least
squares analysis (Schulte and Hinckley 1985). Fully turgid
weight was estimated as the intercept of the linear regression
(R2 > 0.99) of fresh weight versus Ψw between 0 and − 1.4 MPa
balancing pressures (Wilson et al. 1979). The estimated leaf
TW was then used to calculate the RWC corresponding to each
Ψw. Individual P-V isotherms were analyzed by fitting a least
square linear regression (R2 > 0.995) to four to 10 data points
situated in the linear region of each data set, except in three
cases when only three data points were obtained in the linear
region. The intersection of this straight line with the y-axis
corresponded to the osmotic potential at full turgor (ΨπFT),
whereas the intersection with the x-axis (Ψw−1 = 0) gave the
value of the apoplastic water fraction (Schulte and Hinckley
1985). Bulk leaf modulus of elasticity (ε) was calculated by
non-linear least square analysis using a modified exponential
equation (Schulte and Hinckley 1985). A total of 110 P-V
isotherms were analyzed, 18 of which were eliminated because
they did not meet the statistical requirements.
Analysis of cations and soluble carbohydrates, and
calculation of osmotic contribution
Cations (Na+, K+, Ca2+ and Mg2+) and soluble carbohydrates
(glucose, galactose, mannitol, fructose, myo-inositol, sucrose,
raffinose and stachyose) were analyzed in leaves sampled for
Ψπ at the beginning and end of the stress and relief periods.
Cation concentrations were determined with a Perkin-Elmer
M1100 emission-absorption spectrophotometer (Perkin Elmer
Co., Norwalk, CT), as described previously (Tattini et al.
1995). Soluble carbohydrates were determined by HPLC: carbohydrates were separated on an 8 × 300 mm SC1011 column
(Showa Denko, Tokyo, Japan) equipped with an 8 × 50 mm
SC1011 pre-column eluted with water/acetonitrile (95/5, v/v)
at a flow rate of 0.5 ml min −1. The columns were maintained
at 90 °C by a column heater module (Tattini et al. 1996).
Osmotic contribution of solutes was calculated by the Van’t
Hoff equation:
Ψ πι = −0.002479(RDW )C,
(7)
where Ψπι indicates the contribution (in MPa) of individual
solutes to Ψπ, RDW is the relative dry weight at saturation
(kg m −3), C is the molar concentration of solutes (mol kg −1),
and − 0.002479 m3 MPa mol −1 is the RT value at 25 °C. Solutes
15
were assumed to have an ideal osmotic behavior (Alarcon et
al. 1993).
Results
Effects of salt exposure on leaf water relations
Salinity markedly decreased leaf predawn Ψw of both Frantoio
and Leccino olive plants grown in hydroponic culture. Differences in Ψw were significant from 12 days after the beginning
of salinization and increased with increasing NaCl concentration of the external solution (Figures 1a and 1b). The effect of
salinity on leaf Ψπ appeared earlier in Leccino than in Frantoio.
Leaf Ψπ of Leccino was reduced from 22 days after the beginning of salinization, whereas Ψπ of Frantoio was affected only
after 35 days (Figures 1c and 1d). As a result, Leccino plants
showed a higher bulk leaf Ψp than controls after 22 and 35 days
of treatment, whereas Frantoio plants had a lower Ψp than
controls after 12 days (Figures 1e and 1f). Predawn Ψw of
salt-treated plants returned to control values at the end of the
relief period in both cultivars. At that time, recovery of Ψπ was
complete for plants in the 100 mM NaCl treatment, but not for
plants in the 200 mM treatment, which had lower Ψπ values
than the control plants. Because Ψπ of plants in the 200 mM
treatment did not return to control values during relief, Ψp was
higher for 200 mM salt-treated plants than for plants in the
other treatments (Figures 1e and 1f). Similar trends in Ψw, Ψπ
and Ψp were obtained for both cultivars grown in soil culture,
and both recovered their initial Ψw eight days after the beginning of relief (data not shown). Recovery of Ψπ for the 100 mM
salt-treated plants was complete eight days after the beginning
of relief in both cultivars (data not shown).
There were no differences in predawn RWC between Frantoio and Leccino plants during the salt treatment or relief
period (Figure 2). Twenty days of salinization were sufficient
to decrease the RWC of both cultivars, and this decrease was
more evident in plants in the 200 mM than in plants in the
100 mM NaCl treatment. Recovery of RWC to control values
was complete 27 days after the beginning of the relief period
(Figure 2). The leaf DW/FW ratio increased more in Frantoio
than in Leccino when both cultivars were exposed to increased
salt concentrations, but differences were significant only between the 0 and 200 mM NaCl treatments in Frantoio plants.
There were no significant differences in dry weight to leaf area
ratio (DW/LA) or succulence index between salt treatments or
cultivars (Table 1).
Because there were no major differences between P-V isotherms derived from plants grown in hydroponic culture and
plants grown in soil culture, the data are discussed together
(Figures 3 and 4). Salinity had a similar effect on leaf RWCTLP
of both cultivars. By the end of the stress period, leaf RWCTLP
had decreased from 85.5--88.3% for Leccino control plants to
81.8--76.5% for Leccino plants in the 200 mM NaCl treatment,
and from 85.2--90.6% to 77.7--85.4% for the corresponding
treatments in Frantoio. Both Ψ π TLP and ΨπFT were decreased
by exposure to increased salt concentration. In general, Ψ π TLP
and ΨπFT followed parallel patterns during the salt stress and,
during the relief period, their values remained lower than the
16
GUCCI, LOMBARDINI AND TATTINI
Figure 1. Changes in predawn leaf
water potential (Ψw), osmotic potential (Ψπ), and turgor potential (Ψp) of
olive cultivars Frantoio (a, c, e) and
Leccino (b, d, f ) exposed to different
NaCl concentrations in hydroponic
culture. Vertical dotted lines indicate
the beginning of the relief period.
Data points are means ± SE of three
to six replicate leaves, except for Ψp
which was calculated as the difference between mean values of Ψw and
Ψπ.
corresponding values in control plants (Figures 3 and 4). For
plants salinized hydroponically, the decrease of both Ψ πTLP
and ΨπFT was more marked in Leccino than in Frantoio (Figures 3 and 4). Salinity had no apparent effect on ε of either
cultivar (data not shown).
Factors contributing to salt-induced changes in leaf osmotic
potential
The differences between Ψπ of control and treated plants (∆Ψπ)
increased either when salt concentration was increased or
stress duration was extended (Table 2). The value of ∆Ψπ was
higher in Leccino than in Frantoio (maxima of 1.54 and
0.96 MPa, respectively, Table 2). The contribution of non-os-
motic volume changes induced by salt stress accounted for less
than 8.7% of the observed ∆Ψπ in both cultivars. Compared to
controls, the contribution of dehydration (MPa) increased by
56--58% in the 100 mM NaCl treatment and by 20--21% in the
200 mM NaCl treatment during the 23--35-day period of
stress. During the 23--35-day period, as compared with the
0--22-day period, the difference in ∆Ψπ between the 0 and
100 mM treatments increased by 118--122%, and the difference in ∆Ψπ between the 0 and 200 mM treatments increased
by 54--81%. Therefore, dehydration contributed less to total
∆Ψπ in the 23--35-day period of salt stress than in the 0--22-day
period (Table 2). Net solute accumulation was 6.2--7.3%
higher in Leccino than in Frantoio during the first 22 days of
Figure 2. Changes in predawn relative
water content (RWC) of olive cultivars
Frantoio (a) and Leccino (b) exposed
to different NaCl concentrations in hydroponic culture. Vertical dotted lines
indicate the beginning of the relief period. Data points are means ± SE of
three to six replicate leaves.
WATER RELATIONS IN OLIVE UNDER SALINITY
Table 1. Changes in dry weight/fresh weight ratio (DW/FW), leaf
mass/leaf area ratio (DW/LA) and succulence index (SI) of olive
leaves after 35 days of salinization of hydroponically grown olive
plants. Data were analyzed by a two-factor analysis of variance. The
LSD refers to means (n = 4--6) significantly different at P < 0.05
within each column.
NaCl
(mM)
Cultivar
DW/FW
DW/LA
(mg cm −2)
SI
(mg cm −2)
0
100
200
Frantoio
Frantoio
Frantoio
0.347
0.383
0.399
13.0
13.7
14.1
24.5
22.1
21.3
0
100
200
Leccino
Leccino
Leccino
0.366
0.368
0.377
12.8
12.9
12.4
22.2
22.1
20.1
0.049
2.3
5.1
LSD
salt exposure, but was similar for the two cultivars in the
23--35-day period. Osmotic adjustment resulting from net solute accumulation reached a maximum of 70.9 and 73.6% of
total ∆Ψπ in Frantoio and Leccino, respectively, at the end of
the salinization period. During the first three weeks of salinization, net solute accumulation (percent) was higher for plants
17
in the 100 mM treatment than for plants in the 200 mM
treatment (Table 2).
Cations contributed 39.9 and 42.1% to leaf ΨπFT in Frantoio
and Leccino control plants, respectively (Table 3). Glucose and
mannitol, the two most abundant soluble carbohydrates in
olive leaves (Tattini et al. 1996), contributed 27.1--30.8%.
Salinity increased the osmotic contribution by Na+ in both
cultivars. Leccino plants had a lower exclusion capacity for
Na+ than Frantoio plants, and Na+ contribution to total ΨπFT
was lower in Frantoio than in Leccino plants. Contribution to
ΨπFT by other cations (K+, Ca2+, Mg2+) was diminished in
salt-exposed plants. Mannitol concentration increased by 37-39% in both cultivars, but its relative contribution was less than
9.2% in both cultivars. Glucose contributed 17.0--22.7% of
total ΨπFT. The osmotic contribution by other soluble carbohydrates (fructose, myo-inositol, sucrose, raffinose and
stachyose) was less than 3.7% of total ΨπFT in control plants
and was reduced to 1.9--2.1% of the total ΨπFT in the 200 mM
salt-treated plants. The difference in calculated ΣΨπι between
control and salt-treated plants (65--68% and 71--73% of ΨπFT,
respectively) was caused by the increase in Cl− in salt-treated
leaf tissue (Tattini et al. 1995), which was not accounted for in
the calculations of Table 3. Differences between the contribution to osmotic pressure of cations and of soluble carbohy-
Figure 3. Changes in relative water
content at turgor loss point
(RWCTLP), leaf osmotic potential at
turgor loss point (ΨπTLP) and at full
turgor (ΨπFT) in leaves of salt-treated
olive cultivars Frantoio (a, c, e) and
Leccino (b, d, f) grown in hydroponic
culture. Histograms represent means
of three to six replicates, except for
200 mM Leccino (relief 34 days),
with only two replicates. Bars indicate SE.
18
GUCCI, LOMBARDINI AND TATTINI
Figure 4. Changes in relative water
content at turgor loss point
(RWCTLP), leaf osmotic potential at
turgor loss point (ΨπTLP) and at full
turgor (ΨπFT) in leaves of salt-treated
cultivars Frantoio (a, c, e) and Leccino (b, d, f ) grown in soil culture.
Histograms represent means of three
or four replicates, except for Leccino
plants in the 200 mM NaCl treatment
(relief ), with only one replicate. Bars
indicate SE.
drates increased for Leccino, but not for Frantoio, over the
0--100 mM range of NaCl concentration (Figure 5). Both
cultivars behaved similarly over the 100--200 mM range, although values of differential osmotic pressure were still lower
for Frantoio than for Leccino. Although both carbohydrate and
cation concentrations increased, the overall response was a
result primarily of the increase in cation concentrations (Figure 5).
Discussion
Through the analysis of the water relations characteristics of
olive leaves, we observed a common response to salinity,
regardless of the cultivar or growth substrate. Early responses
to salinity included decreases in Ψw and RWC (Figures 1
and 2), similar to those reported for other species (Bañuls and
Primo-Millo 1992, Alarcon et al. 1993, Walker et al. 1993).
Table 2. Components of changes in leaf osmotic potential (∆Ψπ) induced by salinity in leaves of olive plants grown in hydroponic culture. Period
of stress refers to days of exposure to high salt concentration. Values were calculated from means of three to six data points per treatment.
Period of
stress (days)
NaCl
(mM)
Cultivar
∆Ψπ
(MPa)
Dehydration
Non-osmotic
volume
Net solute
accumulation
MPa
%
MPa
%
MPa
%
0--22
23--35
100
100
Frantoio
Frantoio
--0.23
--0.51
− 0.09
− 0.14
39.1
27.4
− 0.003
− 0.009
1.2
1.7
−0.14
−0.36
59.7
70.9
0--22
23--35
200
200
Frantoio
Frantoio
--0.53
--0.96
− 0.25
− 0.30
47.2
31.2
− 0.004
− 0.054
0.7
5.6
−0.28
−0.61
52.1
63.2
0--22
23--35
100
100
Leccino
Leccino
--0.44
--0.96
− 0.12
− 0.19
27.3
19.8
− 0.016
− 0.063
3.7
6.6
−0.30
−0.71
67.0
73.6
0--22
23--35
200
200
Leccino
Leccino
--1.00
--1.54
− 0.33
− 0.40
33.0
26.0
− 0.087
− 0.133
8.7
8.6
−0.58
−1.01
58.3
65.4
WATER RELATIONS IN OLIVE UNDER SALINITY
19
Table 3. Contribution of solutes to leaf osmotic potential at full turgor (ΨπFT) for Frantoio and Leccino olive plants after 35 days of exposure to 0,
100 or 200 mM NaCl in hydroponic culture. ‘‘Other sugars’’ includes fructose + myo-inositol, galactose, sucrose and raffinose + stachyose. ΣΨπι
indicates the sum of calculated osmotic components of individual solutes. Other solutes were calculated as the difference between ΨπFT and ΣΨπι.
Percent values indicate percentage of the ΨπFT, as determined from pressure-volume isotherms. Each value is the mean of three or four
measurements.
Solute
Frantoio
Leccino
0 mM
MPa
+
Na
+
K
2+
Ca
100 mM
%
MPa
200 mM
%
0 mM
MPa
%
MPa
100 mM
%
200 mM
MPa
%
MPa
%
−0.002
0.1
− 0.25
8.6
−0.49
15.6
−0.05
2.1
− 0.47
15.5
−0.67
20.0
−0.65
26.1
− 0.54
19.0
−0.51
16.1
−0.53
24.0
− 0.44
14.6
−0.47
13.9
−0.27
10.8
− 0.20
6.9
−0.17
5.3
−0.27
12.0
− 0.21
7.1
−0.19
5.7
2+
Mg
−0.07
2.9
− 0.05
1.9
−0.05
1.5
−0.09
4.0
− 0.08
2.5
−0.06
1.8
Glucose
−0.49
19.5
− 0.57
19.8
−0.54
17.0
−0.51
22.7
− 0.56
18.7
−0.58
17.2
Mannitol
−0.19
7.6
− 0.26
9.1
−0.26
8.1
−0.18
8.1
− 0.25
8.3
−0.25
7.4
Other sugars −0.09
3.6
− 0.08
2.8
−0.06
1.9
−0.07
3.1
− 0.07
2.3
−0.07
2.1
ΣΨπι
−1.77
70.8
− 1.95
67.9
−2.07
65.5
−1.69
75.8
− 2.07
69.0
−2.29
68.2
ΨπFT
−2.50
100.0
− 2.87
100.0
−3.16
100.0
−2.23
100.0
− 3.00
100.0
−3.37
100.0
Other
−0.73
29.2
− 0.92
32.1
−1.09
34.5
−0.54
24.2
− 0.93
31.0
−1.07
31.8
The decrease in RWC was a result of the high salt concentration of the external solution, which caused osmotic stress and
dehydration at the cellular level (Greenway and Munns 1980).
The high ε typical of the olive leaf (Lo Gullo and Salleo 1988),
combined with leaf dehydration, probably led to the substantial drop in Ψw during salt stress and the rapid recovery of Ψw
on relief of stress. The salt-induced decrease in Ψw was accompanied by a decrease in Ψπ, thus maintaining the Ψp of
salinized plants at values similar to, or higher than, Ψp values
of control plants (Figure 1). A rapid recovery in growth parameters of 100 mM salt-treated plants occurred at the end of
the 30-day relief period, even though Ψp in these plants was
lower than that of the 200 mM salt-treated plants, which
resumed growth more slowly (Tattini et al. 1995). These data
indicate that reductions in the expansion rate of growing cells
that occurred during salt stress was not related to changes in
turgor of mature leaves. Higher Ψp and lower ΨπFT, ΨπTLP and
RWCTLP values in leaves of 200 mM salt-treated plants at the
end of the 30-day relief period, compared to the beginning
(Figures 1, 3, and 4) were indicative of hardening processes
and may represent fundamental mechanisms of adaptation to
salinity or drought (Richardson and McCree 1985). There was
no evidence of changes in ε of olive leaves during the salinity
or relief period in our study, but the intensity of the salt
treatment (duration × concentration) may not have been sufficient to affect ε. This possibility is supported by the observation that changes in leaf morphological characteristics were
less pronounced than those reported in a previous study (Bongi
and Loreto 1989) in which higher NaCl concentration
(250 mM), longer duration of stress (90 days), and older leaf
material (6-month-old) were used.
Figure 5. Differences between the contributions to total osmotic potential (MPa) by cations and soluble carbohydrates (calculated from the
respective values in Table 3) in leaves of Frantoio and Leccino plants
after 35 days of exposure to 0, 100, or 200 mM NaCl in hydroponic
culture.
Non-osmotic volume changes resulting from polymer accumulation were minor in both cultivars because only a small
amount of starch is present in olive leaves exposed to increased
salinity (Tattini et al. 1996). Dehydration components were
higher in the 200 mM NaCl treatment than in the 100 mM
20
GUCCI, LOMBARDINI AND TATTINI
NaCl treatment because higher NaCl concentrations increased
cellular water loss (Table 2). Net solute accumulation was
greater in Leccino plants than in Frantoio plants, suggesting
that Ψπ values reflected the respective ability of the cultivars to
exclude Na+ and Cl− (Table 3; Tattini et al. 1992, Tattini 1994).
This exclusion capacity tended to become saturated when salt
exposure was prolonged or when NaCl concentration was
increased, consistent with current models of ion exclusion in
salt-tolerant non-halophytic species (Greenway and Munns
1980, Gorham et al. 1985, Marcum and Murdoch 1994). Saturation occurred sooner in Leccino than in Frantoio. Leccino
plants initially had a higher net solute accumulation than
Frantoio plants, but the two cultivars had similar values of net
solute accumulation at the end of the salinization period (Table 2). Differences in the lipid composition of root cell membranes may partially explain the different ion-exclusion
capacities of the two cultivars during salt stress (Heimler et al.
1995).
Osmotic adjustment in olive leaves under salt stress was
accomplished primarily by accumulation of inorganic ions,
despite the osmotic contribution by soluble carbohydrates.
Although our measurements did not account for some classes
of osmotically important compounds, their relative osmotic
contribution can be estimated from data reported in the literature (Tattini et al. 1993, Heimler et al. 1995, Tattini et al. 1995).
Leaf Cl− concentration was close to that of Na+ (Bongi and
Loreto 1989, Tattini et al. 1992, Heimler et al. 1995), with
presumably comparable osmotic effects. Other anions (nitrate,
sulfate, and phosphate) and amino compounds contribute little
to ΨπFT, and their leaf concentrations are unaffected by salinity
(Tattini et al. 1993, Tattini et al. 1995).
The two cultivars did not differ in their potential to accumulate soluble carbohydrates as an adaptive response to salinity.
The maximum osmotic contribution of carbohydrates was observed at an external concentration of 100 mM NaCl, indicating that the potential to accumulate sugars was fully reached
in the 0--100 mM range (Table 3). Both mannitol and glucose
concentrations increase in response to small increases in leaf
Na+ content (Tattini et al. 1996), but glucose was more abundant than mannitol and consequently contributed more to ΨπFT.
The accumulation of mannitol is considered to be an important
contributor to the maintenance of growth and metabolism in
herbaceous species exposed to increased salinity (Tarczynski
et al. 1993, Everard et al. 1994), and a similar role has been
proposed in olive leaf tissue (Tattini et al. 1996). Transport
from the vacuole and redistribution between cellular compartments must occur in order for compatible solutes and osmotic
agents to counteract the accumulation of toxic ions during salt
stress (Marcum and Murdoch 1994, Colmer et al. 1995). Assuming that 20% of total cell volume is occupied by the
cytoplasm in mesophyll cells of olive, mannitol concentration
appears to exceed the critical threshold (300--400 mOsmol
kg −1) of basal cytoplasmic osmotic potential indicated for
compatible solutes to be effective (Gorham et al. 1985). However, it is not known what conditions are necessary or what
form is needed for sugar-alcohols to exert their protective role
in the cytoplasm.
In conclusion, there were no major differences in the leaf
water relations or in the capacity of the olive cultivars Leccino
and Frantoio to accumulate mannitol, soluble carbohydrates,
or other osmolytes in response to salt stress. Changes in osmotic contribution reflected the different exclusion capacities
of the cultivars for Na+ and Cl−. Like other glycophytic species
with medium tolerance to salinity, osmotic adjustment in olive
was mainly achieved by accumulation of inorganic ions with
relatively little energy expenditure.
Acknowledgments
We thank Maria Assunta Coradeschi, Piero Puntoni, and Laura Traversi for skillful technical assistance. Research was supported by the
National Research Council of Italy, Special Project RAISA, Subproject No. 2, Paper No. 2814. R. Gucci was partially supported by a
contribution Fondo di Ateneo of University of Pisa.
References
Alarcon, J.J., M.J. Sanchez-Blanco, M.C. Bolarin and A. Torrecillas.
1993. Water relations and osmotic adjustment in Lycopersicon
esculentum and L. pennellii during short-term salt exposure and
recovery. Physiol. Plant. 89:441--447.
Bañuls, J. and E. Primo-Millo. 1992. Effects of chloride and sodium
ion on gas exchange properties and water relations of Citrus plants.
Physiol. Plant. 86:115--123.
Bongi, G. and F. Loreto. 1989. Gas exchange properties of saltstressed olive (Olea europaea L.) leaves. Plant Physiol. 90:1408-1416.
Colmer, T.D., E. Epstein and J. Dvorak. 1995. Differential solute
regulation in leaf blades of various ages in salt-sensitive wheat and
a salt-tolerant wheat × Lophopyrum elongatum (Host) A. Löve
amphiploid. Plant Physiol. 108:1715--1724.
Erdei, L. and E. Taleisnik. 1993. Changes in water relation parameters
under osmotic and salt stresses in maize and sorghum. Physiol.
Plant. 89:381--387.
Everard, J.D., R. Gucci, S.C. Kann, J.A. Flore and W.H. Loescher.
1994. Gas exchange and carbon partitioning in the leaves of celery
(Apium graveolens L.) at various levels of root zone salinity. Plant
Physiol. 106:281--292.
Flowers, T.J. and A.R. Yeo. 1986. Ion relations of plants under drought
and salinity. Aust. J. Plant Physiol. 13:75--91.
Girma, F.S. and D.R. Krieg. 1992. Osmotic adjustment in sorghum. I.
Mechanisms of diurnal osmotic potential changes. Plant Physiol.
99:577--582.
Gorham, J., R.G. Wyn Jones and G. McDonnell. 1985. Some mechanisms of salt tolerance in crop plants. Plant Soil 89:15--40.
Greenway, H. and R. Munns. 1980. Mechanisms of salt tolerance in
nonhalophytes. Annu. Rev. Plant Physiol. 31:149--190.
Heimler, D., M. Tattini, S. Ticci, M.A. Coradeschi and M.L. Traversi.
1995. Growth, ion accumulation and lipid composition of two olive
genotypes under salinity. J. Plant Nutr. 18:1723--1734.
Hinckley, T.M., F. Duhme, A.R. Hinckley and H. Richter. 1980. Water
relations of drought hardy shrubs: osmotic potential and stomatal
reactivity. Plant, Cell Environ. 3:131--140.
Larsen, F.E., S.S. Higgins and A. Al Wir. 1989. Diurnal water relations
of apple, apricot, grape, olive and peach in an arid environment
(Jordan). Sci. Hortic. 39:211--222.
Lo Gullo, M.A. and S. Salleo. 1988. Different strategies of drought
resistance in three Mediterranean sclerophyllous trees growing in
the same environmental conditions. New Phytol. 108:267--276.
WATER RELATIONS IN OLIVE UNDER SALINITY
Lo Gullo, M.A. and S. Salleo. 1990. Wood anatomy of some trees with
diffuse- and ring-porous wood: some functional and ecological
interpretations. Giorn. Bot. Ital. 124:601--613.
Longstreth, D.J. and P.S. Nobel. 1979. Salinity effects on leaf anatomy. Consequences for photosynthesis. Plant Physiol. 63:700--703.
Marcum, K.B. and C.L. Murdoch. 1994. Salinity tolerance mechanisms of six C4 turfgrasses. J. Am. Soc. Hortic. Sci. 119:779--784.
Neumann, P.M., E. Van Volkenburgh and R.E. Cleland. 1988. Salinity
stress inhibits bean leaf expansion by reducing turgor, not cell wall
extensibility. Plant Physiol. 88:233--237.
Richardson, S.G. and K.J. McCree. 1985. Carbon balance and water
relations of sorghum exposed to salt and water stress. Plant Physiol.
79:1015--1020.
Ritchie, G.A. and T.M. Hinckley. 1975. The pressure chamber as an
instrument for ecological research. Adv. Ecol. Res. 9:165--254.
Schulte, P.J. and T.M. Hinckley. 1985. A comparison of pressure-volume curve data analysis techniques. J. Exp. Bot. 36:1590--1602.
Tarczynski, M.C., R.G. Jensen and H.J. Bohnert. 1993. Stress protection of transgenic tobacco plants by production of the osmolyte
mannitol. Science 259:508--510.
Tattini, M. 1994. Ionic relations of aeroponically-grown olive genotypes during salt stress. Plant Soil 161:251--256.
Tattini, M., P. Bertoni and S. Caselli. 1992. Genotypic responses of
olive plants to sodium chloride. J. Plant Nutr. 15:1467--1485.
21
Tattini, M., D. Heimler, M.L. Traversi and A. Pieroni. 1993.
Polyamine analysis in salt stressed plants of olive (Olea
europaea L.). J. Hortic. Sci. 68:613-617.
Tattini, M., R. Gucci, M.A. Coradeschi, C. Ponzio and J.D. Everard.
1995. Growth, gas exchange and ion content in Olea europaea
plants during salinity stress and subsequent relief. Physiol. Plant.
95:203--210.
Tattini, M., R. Gucci, A. Romani, A. Baldi and J.D. Everard. 1996.
Changes in non-structural carbohydrates in olive (Olea europaea)
leaves during root zone salinity stress. Physiol. Plant. 98:117--124.
Tyree, M.T. and H. Richter. 1982. Alternate methods of analysing
water potential isotherms: some cautions and clarifications. II.
Curvilinearity in water potential isotherms. Can. J. Bot. 60:911-916.
Walker, R.R., D.H. Blackmore and Sun Qing. 1993. Carbon dioxide
accumulation and foliar ion concentrations in leaves of lemon
(Citrus limon L.) trees irrigated with NaCl or Na2SO4. Aust. J. Plant
Physiol. 20:173--185.
Wilson, J.R., M.J. Fisher, E.D. Schulze, G.R. Dolby and M.M. Ludlow. 1979. Comparison between pressure-volume and dewpointhygrometry techniques for determining the water relations
characteristics of grass and legume leaves. Oecologia 41:77--88.
© 1997 Heron Publishing----Victoria, Canada
Analysis of leaf water relations in leaves of two olive (Olea europaea)
cultivars differing in tolerance to salinity
RICCARDO GUCCI,1 LEONARDO LOMBARDINI1 and MASSIMILIANO TATTINI2
1
Dipartimento di Coltivazione e Difesa delle Specie Legnose, Sezione Coltivazioni Arboree, Università di Pisa, Via del Borghetto 80, Pisa 56124, Italy
2
Istituto sulla Propagazione delle Specie Legnose -- CNR, Via Ponte di Formicola 76, 50018 Scandicci (FI), Italy
Received January 8, 1996
Summary One-year-old rooted cuttings of olive (Olea
europaea L. cvs. Frantoio and Leccino) were grown either
hydroponically or in soil in a greenhouse. Plants were exposed
to NaCl treatments (0, 100, and 200 mM) for 35 days, followed
by 30 to 34 days of relief from salt stress to determine whether
previously demonstrated genotypic differences in tolerance to
salinity were related to water relations parameters. Exposure
to high salt concentrations resulted in reductions in predawn
water potential (Ψw), osmotic potential at full turgor (ΨπFT),
osmotic potential at turgor loss point (ΨπTLP), and relative water
content (RWC) in both cultivars, regardless of the growth
substrate. Leaf Ψw and RWC returned to values similar to those
of controls by the end of the relief period. The effect of salinity
on Ψπ appeared earlier in Leccino than in Frantoio. Values for
ΨπFT were − 2.50, − 2.87, and − 3.16 MPa for the 0, 100, and
200 mM salt-treated Frantoio plants, respectively, and − 2.23,
− 2.87, and − 3.37 MPa for the corresponding Leccino plants.
Recovery of Ψπ was complete for plants in the 100 mM salt
treatment, but not for plants in the 200 mM salt treatment,
which maintained an increased pressure potential (Ψp) compared to control plants. Net solute accumulation was higher in
Leccino, the salt-sensitive cultivar, than in Frantoio. In controls
of both cultivars, cations contributed 39.9 to 42.0% of the total
ΨπFT, mannitol and glucose contributed 27.1 to 30.8%, and
other soluble carbohydrates contributed 3.1 to 3.6%. The osmotic contribution of Na+ increased from 0.1--2.1% for nontreated plants to 8.6--15.5% and 15.6--20.0% for the 100 mM
and 200 mM salt-treated plants, respectively. The mannitol
contribution to ΨπFT reached a maximum of 9.1% at the end of
the salinization period. We conclude that differences between
the two cultivars in leaf water relations reflect differences in
the exclusion capacities for Na+ and Cl− ions.
Keywords: osmotic adjustment, pressure-volume isotherms,
relief from salinity, turgor loss point.
Frantoio and Leccino, the two cultivars used in the present
study, differ in their capacity to exclude Na+ or Cl− ions from
the shoot when NaCl is applied to the rooting medium (Tattini
et al. 1992, Tattini 1994). A previous study showed that increasing the NaCl concentration from 0 to 200 mM significantly decreased leaf water potential (Ψw) in the salt-tolerant
Frantoio cultivar; however, this reduction was accompanied by
a decrease in osmotic potential (Ψπ), so that leaf turgor of the
salinized plants was maintained (Tattini et al. 1995).
Salt stress changes the water relations of most higher plants,
and salt tolerance often depends on drought tolerance (Greenway and Munns 1980, Flowers and Yeo 1986). Changes in Ψw,
Ψπ, leaf succulence, cell wall extensibility, and bulk leaf
modulus of elasticity (ε) have been reported for herbaceous
crops exposed to increased salt concentrations (Longstreth and
Nobel 1980, Neumann et al. 1988, Erdei and Taleisnik 1993).
No similar studies have been reported for olive. Olive trees are
drought tolerant, and leaves can reach extremely low values of
Ψw and relative water content (RWC) (− 3.5 MPa and 75--80%,
respectively) before losing turgor (Hinckley et al. 1980, Lo
Gullo and Salleo 1988, Larsen et al. 1989). Seasonal and
diurnal declines in leaf Ψw are mainly attributed to inelastic
cell walls, osmotic adjustment, and transpirational losses,
which continue even at low Ψw (Lo Gullo and Salleo 1988). In
addition, olive leaves often experience a diurnal osmotic stress
during the dry season because the high hydraulic resistance of
the stem causes the development of water deficits in leaves
(Larsen et al. 1989, Lo Gullo and Salleo 1990).
In the present study, olive plants of two cultivars, growing in
either hydroponic or soil culture, were subjected to salinization
and subsequent relief. The objective was to elucidate the contribution of changes in leaf water relations to the differences in
salt tolerance exhibited by the cultivars.
Materials and methods
Introduction
Plant material and salinity treatments
Olive (Olea europaea L.) is more salt tolerant than other
woody crops and has great potential for cultivation in areas,
such as the Mediterranean region, where salinity is becoming
a major problem. Salt tolerance in olive is cultivar-dependent.
One-year-old rooted cuttings of the olive cultivars Frantoio
and Leccino were grown in a greenhouse from April to September 1994, either hydroponically or in soil. Plants were
trained to two shoots, as previously described (Tattini et al.
14
GUCCI, LOMBARDINI AND TATTINI
1995). About 300 plants of both cultivars were transplanted to
a quartz sand hydroponic culture system at Scandicci
(43°45′ N, 11°11′ E) on March 1, 1994 and acclimated until
April 27, 1994, when salt treatments were initiated. Plants
received half-strength Hoagland solution twice a day (Tattini
et al. 1995). A separate group of 150 plants from the same
batch of rooted cuttings was grown in 1-liter containers at
Scandicci and then transferred to Pisa (43°43′ N, 10°25′ E) on
June 24, 1994. The plants were grown in a substrate of 1/1/1
fertilized peat/perlite/field soil. Plants in containers were irrigated two to four times per week, depending on the evaporative
demand, and received either half-strength Hoagland solution
or a fertilizer solution containing 5 g l −1 of 18/9/18 N,P,K once
or twice a week.
Plants in both hydroponic and soil culture were subjected to
35 days of increased salinity, followed by 30 to 34 days of
relief. In hydroponic cultures, 0, 100, or 200 mM NaCl was
added to the standard nutrient solution (Tattini et al. 1995). In
soil cultures, NaCl concentrations were increased by 50 mM
day −1 until final concentrations of 0, 100, and 200 mM were
reached, containers were covered with plastic film to reduce
evaporation from the soil surface, and plants were watered
with excess solution to prevent the occurrence of salt gradients
in the container profile. The concentration of the soil solution
leaching from the bottom of the container after irrigation was
periodically checked with a Wescor 5500 vapor pressure osmometer (Wescor Inc., Logan, UT) and was usually within
20% of the concentration of the nutrient solution. At the end of
the salinization period, stress was relieved by supplying the
nutrient solution only (Tattini et al. 1995).
A randomized complete block design was adopted with nine
plants per treatment arranged in blocks according to location
in the greenhouse.
Leaf water relations
Relative water content (RWC) was determined on three to four
fully expanded leaves of similar age (15 to 25 days old).
Leaves were excised before dawn, weighed fresh (FW) and
placed in vials to rehydrate in the dark for 20 h. Preliminary
experiments had indicated that full rehydration was complete
after 18 h. The following morning, leaf turgid weight (TW)
was measured and then leaves were dried at 80 °C for 48 h and
dry weight (DW) was determined. The RWC was calculated
from the equation:
RWC = 100[(FW − DW )/(TW − DW )].
(1)
Predawn Ψw of three to six leaves per treatment was measured at approximately 10-day intervals with a pressure chamber (TecnoGas, Pisa, Italy), according to a standard technique
(Ritchie and Hinckley 1975). After the Ψw measurement, the
leaves were immediately frozen in liquid nitrogen and stored
at − 80 °C for determination of Ψπ with a Wescor 5500 vapor
pressure osmometer (Tattini et al. 1995). Leaf turgor potential
(Ψp) was calculated as the difference between Ψw and Ψπ.
Osmotic potential at full turgor (ΨπFT) was determined from
pressure-volume curves or calculated using the equation proposed by Wilson et al. (1979):
Ψ π FT = Ψπ [(RWC − A)/(100 − A)],
(2)
where A is the apoplastic water fraction, estimated at 4.4% in
olive leaves (see analysis of pressure-volume isotherms). Osmotic adjustment resulting from net solute accumulation was
estimated as the difference between total change in Ψπ and the
change resulting from dehydration (D) and non-osmotic volume (V). In this procedure, changes in Ψπ and ΨπFT (∆Ψπ and
∆ΨπFT) induced by salinity were calculated by comparing
pre-dawn Ψπ of plants in NaCl treatments with predawn Ψπ of
control plants, according to the following equations (Girma
and Krieg 1992):
∆Ψ π = Ψ π ,salt − Ψ π,control
(3)
∆Ψ π FT = Ψ π FT,salt − Ψ πFT,contr ol.
(4)
The contribution of dehydration (D) to changes in Ψπ was
calculated as:
D = ∆Ψπ − ∆Ψ π FT .
(5)
Similarly, changes in solute concentration resulting from
changes in the non-osmotic volume at 100% hydration were
estimated from changes in the TW/DW ratio between the
control and the various salt treatments (Girma and Krieg
1992):
(TW/DW)control − (TW /DW)salt
V=
∆Ψ π FT .
(TW/DW)control
(6)
Leaf mass per unit area and succulence index (water per unit
leaf area) were measured at the end of the salinity and relief
periods on four to six fully expanded leaves of plants growing
in hydroponic culture. Leaf area was measured with a leaf area
meter (Delta-T Devices, Burwell, Cambridge, U.K.).
Pressure-volume isotherms
Pressure-volume (P-V) isotherms were developed for three to
six fully expanded leaves collected at the end of the salinity
and relief periods. Additional leaf material was sampled from
plants growing in hydroponic culture 15 days after the beginning of relief. The P-V isotherms were constructed following
the free transpiration technique of Wilson et al. (1979). In brief,
leaves from plants cultivated hydroponically were collected in
the afternoon and handled as described for RWC determination. Plants grown in soil culture were rehydrated by saturating
the substrate with water and enclosing the plants in black
plastic bags at room temperature for 18 h before leaf excision.
The pressure chamber temperature was maintained at 20 °C
and the flow of nitrogen gas into the chamber was regulated at
0.005 MPa s −1. Pressurization was stopped when sap was
visible on at least 50% of the cut surface of the petiole.
WATER RELATIONS IN OLIVE UNDER SALINITY
Pressure was then slowly reduced by 0.10--0.15 MPa and then
increased until sap flowed again. At this point, the balance
pressure was recorded as the leaf Ψw. Leaves were dried on the
laboratory bench at 23 °C to achieve progressive dehydration
of the tissue, and the Ψw determination was repeated nine to 15
times. Four to 10 data points in the region of zero turgor and
three to seven data points in the positive turgor range were
typically obtained for one P-V isotherm. A period of 8--9 h was
necessary to produce a complete P-V isotherm.
Pressure-volume isotherms were analyzed by type II transformation (Tyree and Richter 1982). The point indicating departure from linearity corresponded to the turgor loss point
(TLP). The TLP was determined iteratively by non-linear least
squares analysis (Schulte and Hinckley 1985). Fully turgid
weight was estimated as the intercept of the linear regression
(R2 > 0.99) of fresh weight versus Ψw between 0 and − 1.4 MPa
balancing pressures (Wilson et al. 1979). The estimated leaf
TW was then used to calculate the RWC corresponding to each
Ψw. Individual P-V isotherms were analyzed by fitting a least
square linear regression (R2 > 0.995) to four to 10 data points
situated in the linear region of each data set, except in three
cases when only three data points were obtained in the linear
region. The intersection of this straight line with the y-axis
corresponded to the osmotic potential at full turgor (ΨπFT),
whereas the intersection with the x-axis (Ψw−1 = 0) gave the
value of the apoplastic water fraction (Schulte and Hinckley
1985). Bulk leaf modulus of elasticity (ε) was calculated by
non-linear least square analysis using a modified exponential
equation (Schulte and Hinckley 1985). A total of 110 P-V
isotherms were analyzed, 18 of which were eliminated because
they did not meet the statistical requirements.
Analysis of cations and soluble carbohydrates, and
calculation of osmotic contribution
Cations (Na+, K+, Ca2+ and Mg2+) and soluble carbohydrates
(glucose, galactose, mannitol, fructose, myo-inositol, sucrose,
raffinose and stachyose) were analyzed in leaves sampled for
Ψπ at the beginning and end of the stress and relief periods.
Cation concentrations were determined with a Perkin-Elmer
M1100 emission-absorption spectrophotometer (Perkin Elmer
Co., Norwalk, CT), as described previously (Tattini et al.
1995). Soluble carbohydrates were determined by HPLC: carbohydrates were separated on an 8 × 300 mm SC1011 column
(Showa Denko, Tokyo, Japan) equipped with an 8 × 50 mm
SC1011 pre-column eluted with water/acetonitrile (95/5, v/v)
at a flow rate of 0.5 ml min −1. The columns were maintained
at 90 °C by a column heater module (Tattini et al. 1996).
Osmotic contribution of solutes was calculated by the Van’t
Hoff equation:
Ψ πι = −0.002479(RDW )C,
(7)
where Ψπι indicates the contribution (in MPa) of individual
solutes to Ψπ, RDW is the relative dry weight at saturation
(kg m −3), C is the molar concentration of solutes (mol kg −1),
and − 0.002479 m3 MPa mol −1 is the RT value at 25 °C. Solutes
15
were assumed to have an ideal osmotic behavior (Alarcon et
al. 1993).
Results
Effects of salt exposure on leaf water relations
Salinity markedly decreased leaf predawn Ψw of both Frantoio
and Leccino olive plants grown in hydroponic culture. Differences in Ψw were significant from 12 days after the beginning
of salinization and increased with increasing NaCl concentration of the external solution (Figures 1a and 1b). The effect of
salinity on leaf Ψπ appeared earlier in Leccino than in Frantoio.
Leaf Ψπ of Leccino was reduced from 22 days after the beginning of salinization, whereas Ψπ of Frantoio was affected only
after 35 days (Figures 1c and 1d). As a result, Leccino plants
showed a higher bulk leaf Ψp than controls after 22 and 35 days
of treatment, whereas Frantoio plants had a lower Ψp than
controls after 12 days (Figures 1e and 1f). Predawn Ψw of
salt-treated plants returned to control values at the end of the
relief period in both cultivars. At that time, recovery of Ψπ was
complete for plants in the 100 mM NaCl treatment, but not for
plants in the 200 mM treatment, which had lower Ψπ values
than the control plants. Because Ψπ of plants in the 200 mM
treatment did not return to control values during relief, Ψp was
higher for 200 mM salt-treated plants than for plants in the
other treatments (Figures 1e and 1f). Similar trends in Ψw, Ψπ
and Ψp were obtained for both cultivars grown in soil culture,
and both recovered their initial Ψw eight days after the beginning of relief (data not shown). Recovery of Ψπ for the 100 mM
salt-treated plants was complete eight days after the beginning
of relief in both cultivars (data not shown).
There were no differences in predawn RWC between Frantoio and Leccino plants during the salt treatment or relief
period (Figure 2). Twenty days of salinization were sufficient
to decrease the RWC of both cultivars, and this decrease was
more evident in plants in the 200 mM than in plants in the
100 mM NaCl treatment. Recovery of RWC to control values
was complete 27 days after the beginning of the relief period
(Figure 2). The leaf DW/FW ratio increased more in Frantoio
than in Leccino when both cultivars were exposed to increased
salt concentrations, but differences were significant only between the 0 and 200 mM NaCl treatments in Frantoio plants.
There were no significant differences in dry weight to leaf area
ratio (DW/LA) or succulence index between salt treatments or
cultivars (Table 1).
Because there were no major differences between P-V isotherms derived from plants grown in hydroponic culture and
plants grown in soil culture, the data are discussed together
(Figures 3 and 4). Salinity had a similar effect on leaf RWCTLP
of both cultivars. By the end of the stress period, leaf RWCTLP
had decreased from 85.5--88.3% for Leccino control plants to
81.8--76.5% for Leccino plants in the 200 mM NaCl treatment,
and from 85.2--90.6% to 77.7--85.4% for the corresponding
treatments in Frantoio. Both Ψ π TLP and ΨπFT were decreased
by exposure to increased salt concentration. In general, Ψ π TLP
and ΨπFT followed parallel patterns during the salt stress and,
during the relief period, their values remained lower than the
16
GUCCI, LOMBARDINI AND TATTINI
Figure 1. Changes in predawn leaf
water potential (Ψw), osmotic potential (Ψπ), and turgor potential (Ψp) of
olive cultivars Frantoio (a, c, e) and
Leccino (b, d, f ) exposed to different
NaCl concentrations in hydroponic
culture. Vertical dotted lines indicate
the beginning of the relief period.
Data points are means ± SE of three
to six replicate leaves, except for Ψp
which was calculated as the difference between mean values of Ψw and
Ψπ.
corresponding values in control plants (Figures 3 and 4). For
plants salinized hydroponically, the decrease of both Ψ πTLP
and ΨπFT was more marked in Leccino than in Frantoio (Figures 3 and 4). Salinity had no apparent effect on ε of either
cultivar (data not shown).
Factors contributing to salt-induced changes in leaf osmotic
potential
The differences between Ψπ of control and treated plants (∆Ψπ)
increased either when salt concentration was increased or
stress duration was extended (Table 2). The value of ∆Ψπ was
higher in Leccino than in Frantoio (maxima of 1.54 and
0.96 MPa, respectively, Table 2). The contribution of non-os-
motic volume changes induced by salt stress accounted for less
than 8.7% of the observed ∆Ψπ in both cultivars. Compared to
controls, the contribution of dehydration (MPa) increased by
56--58% in the 100 mM NaCl treatment and by 20--21% in the
200 mM NaCl treatment during the 23--35-day period of
stress. During the 23--35-day period, as compared with the
0--22-day period, the difference in ∆Ψπ between the 0 and
100 mM treatments increased by 118--122%, and the difference in ∆Ψπ between the 0 and 200 mM treatments increased
by 54--81%. Therefore, dehydration contributed less to total
∆Ψπ in the 23--35-day period of salt stress than in the 0--22-day
period (Table 2). Net solute accumulation was 6.2--7.3%
higher in Leccino than in Frantoio during the first 22 days of
Figure 2. Changes in predawn relative
water content (RWC) of olive cultivars
Frantoio (a) and Leccino (b) exposed
to different NaCl concentrations in hydroponic culture. Vertical dotted lines
indicate the beginning of the relief period. Data points are means ± SE of
three to six replicate leaves.
WATER RELATIONS IN OLIVE UNDER SALINITY
Table 1. Changes in dry weight/fresh weight ratio (DW/FW), leaf
mass/leaf area ratio (DW/LA) and succulence index (SI) of olive
leaves after 35 days of salinization of hydroponically grown olive
plants. Data were analyzed by a two-factor analysis of variance. The
LSD refers to means (n = 4--6) significantly different at P < 0.05
within each column.
NaCl
(mM)
Cultivar
DW/FW
DW/LA
(mg cm −2)
SI
(mg cm −2)
0
100
200
Frantoio
Frantoio
Frantoio
0.347
0.383
0.399
13.0
13.7
14.1
24.5
22.1
21.3
0
100
200
Leccino
Leccino
Leccino
0.366
0.368
0.377
12.8
12.9
12.4
22.2
22.1
20.1
0.049
2.3
5.1
LSD
salt exposure, but was similar for the two cultivars in the
23--35-day period. Osmotic adjustment resulting from net solute accumulation reached a maximum of 70.9 and 73.6% of
total ∆Ψπ in Frantoio and Leccino, respectively, at the end of
the salinization period. During the first three weeks of salinization, net solute accumulation (percent) was higher for plants
17
in the 100 mM treatment than for plants in the 200 mM
treatment (Table 2).
Cations contributed 39.9 and 42.1% to leaf ΨπFT in Frantoio
and Leccino control plants, respectively (Table 3). Glucose and
mannitol, the two most abundant soluble carbohydrates in
olive leaves (Tattini et al. 1996), contributed 27.1--30.8%.
Salinity increased the osmotic contribution by Na+ in both
cultivars. Leccino plants had a lower exclusion capacity for
Na+ than Frantoio plants, and Na+ contribution to total ΨπFT
was lower in Frantoio than in Leccino plants. Contribution to
ΨπFT by other cations (K+, Ca2+, Mg2+) was diminished in
salt-exposed plants. Mannitol concentration increased by 37-39% in both cultivars, but its relative contribution was less than
9.2% in both cultivars. Glucose contributed 17.0--22.7% of
total ΨπFT. The osmotic contribution by other soluble carbohydrates (fructose, myo-inositol, sucrose, raffinose and
stachyose) was less than 3.7% of total ΨπFT in control plants
and was reduced to 1.9--2.1% of the total ΨπFT in the 200 mM
salt-treated plants. The difference in calculated ΣΨπι between
control and salt-treated plants (65--68% and 71--73% of ΨπFT,
respectively) was caused by the increase in Cl− in salt-treated
leaf tissue (Tattini et al. 1995), which was not accounted for in
the calculations of Table 3. Differences between the contribution to osmotic pressure of cations and of soluble carbohy-
Figure 3. Changes in relative water
content at turgor loss point
(RWCTLP), leaf osmotic potential at
turgor loss point (ΨπTLP) and at full
turgor (ΨπFT) in leaves of salt-treated
olive cultivars Frantoio (a, c, e) and
Leccino (b, d, f) grown in hydroponic
culture. Histograms represent means
of three to six replicates, except for
200 mM Leccino (relief 34 days),
with only two replicates. Bars indicate SE.
18
GUCCI, LOMBARDINI AND TATTINI
Figure 4. Changes in relative water
content at turgor loss point
(RWCTLP), leaf osmotic potential at
turgor loss point (ΨπTLP) and at full
turgor (ΨπFT) in leaves of salt-treated
cultivars Frantoio (a, c, e) and Leccino (b, d, f ) grown in soil culture.
Histograms represent means of three
or four replicates, except for Leccino
plants in the 200 mM NaCl treatment
(relief ), with only one replicate. Bars
indicate SE.
drates increased for Leccino, but not for Frantoio, over the
0--100 mM range of NaCl concentration (Figure 5). Both
cultivars behaved similarly over the 100--200 mM range, although values of differential osmotic pressure were still lower
for Frantoio than for Leccino. Although both carbohydrate and
cation concentrations increased, the overall response was a
result primarily of the increase in cation concentrations (Figure 5).
Discussion
Through the analysis of the water relations characteristics of
olive leaves, we observed a common response to salinity,
regardless of the cultivar or growth substrate. Early responses
to salinity included decreases in Ψw and RWC (Figures 1
and 2), similar to those reported for other species (Bañuls and
Primo-Millo 1992, Alarcon et al. 1993, Walker et al. 1993).
Table 2. Components of changes in leaf osmotic potential (∆Ψπ) induced by salinity in leaves of olive plants grown in hydroponic culture. Period
of stress refers to days of exposure to high salt concentration. Values were calculated from means of three to six data points per treatment.
Period of
stress (days)
NaCl
(mM)
Cultivar
∆Ψπ
(MPa)
Dehydration
Non-osmotic
volume
Net solute
accumulation
MPa
%
MPa
%
MPa
%
0--22
23--35
100
100
Frantoio
Frantoio
--0.23
--0.51
− 0.09
− 0.14
39.1
27.4
− 0.003
− 0.009
1.2
1.7
−0.14
−0.36
59.7
70.9
0--22
23--35
200
200
Frantoio
Frantoio
--0.53
--0.96
− 0.25
− 0.30
47.2
31.2
− 0.004
− 0.054
0.7
5.6
−0.28
−0.61
52.1
63.2
0--22
23--35
100
100
Leccino
Leccino
--0.44
--0.96
− 0.12
− 0.19
27.3
19.8
− 0.016
− 0.063
3.7
6.6
−0.30
−0.71
67.0
73.6
0--22
23--35
200
200
Leccino
Leccino
--1.00
--1.54
− 0.33
− 0.40
33.0
26.0
− 0.087
− 0.133
8.7
8.6
−0.58
−1.01
58.3
65.4
WATER RELATIONS IN OLIVE UNDER SALINITY
19
Table 3. Contribution of solutes to leaf osmotic potential at full turgor (ΨπFT) for Frantoio and Leccino olive plants after 35 days of exposure to 0,
100 or 200 mM NaCl in hydroponic culture. ‘‘Other sugars’’ includes fructose + myo-inositol, galactose, sucrose and raffinose + stachyose. ΣΨπι
indicates the sum of calculated osmotic components of individual solutes. Other solutes were calculated as the difference between ΨπFT and ΣΨπι.
Percent values indicate percentage of the ΨπFT, as determined from pressure-volume isotherms. Each value is the mean of three or four
measurements.
Solute
Frantoio
Leccino
0 mM
MPa
+
Na
+
K
2+
Ca
100 mM
%
MPa
200 mM
%
0 mM
MPa
%
MPa
100 mM
%
200 mM
MPa
%
MPa
%
−0.002
0.1
− 0.25
8.6
−0.49
15.6
−0.05
2.1
− 0.47
15.5
−0.67
20.0
−0.65
26.1
− 0.54
19.0
−0.51
16.1
−0.53
24.0
− 0.44
14.6
−0.47
13.9
−0.27
10.8
− 0.20
6.9
−0.17
5.3
−0.27
12.0
− 0.21
7.1
−0.19
5.7
2+
Mg
−0.07
2.9
− 0.05
1.9
−0.05
1.5
−0.09
4.0
− 0.08
2.5
−0.06
1.8
Glucose
−0.49
19.5
− 0.57
19.8
−0.54
17.0
−0.51
22.7
− 0.56
18.7
−0.58
17.2
Mannitol
−0.19
7.6
− 0.26
9.1
−0.26
8.1
−0.18
8.1
− 0.25
8.3
−0.25
7.4
Other sugars −0.09
3.6
− 0.08
2.8
−0.06
1.9
−0.07
3.1
− 0.07
2.3
−0.07
2.1
ΣΨπι
−1.77
70.8
− 1.95
67.9
−2.07
65.5
−1.69
75.8
− 2.07
69.0
−2.29
68.2
ΨπFT
−2.50
100.0
− 2.87
100.0
−3.16
100.0
−2.23
100.0
− 3.00
100.0
−3.37
100.0
Other
−0.73
29.2
− 0.92
32.1
−1.09
34.5
−0.54
24.2
− 0.93
31.0
−1.07
31.8
The decrease in RWC was a result of the high salt concentration of the external solution, which caused osmotic stress and
dehydration at the cellular level (Greenway and Munns 1980).
The high ε typical of the olive leaf (Lo Gullo and Salleo 1988),
combined with leaf dehydration, probably led to the substantial drop in Ψw during salt stress and the rapid recovery of Ψw
on relief of stress. The salt-induced decrease in Ψw was accompanied by a decrease in Ψπ, thus maintaining the Ψp of
salinized plants at values similar to, or higher than, Ψp values
of control plants (Figure 1). A rapid recovery in growth parameters of 100 mM salt-treated plants occurred at the end of
the 30-day relief period, even though Ψp in these plants was
lower than that of the 200 mM salt-treated plants, which
resumed growth more slowly (Tattini et al. 1995). These data
indicate that reductions in the expansion rate of growing cells
that occurred during salt stress was not related to changes in
turgor of mature leaves. Higher Ψp and lower ΨπFT, ΨπTLP and
RWCTLP values in leaves of 200 mM salt-treated plants at the
end of the 30-day relief period, compared to the beginning
(Figures 1, 3, and 4) were indicative of hardening processes
and may represent fundamental mechanisms of adaptation to
salinity or drought (Richardson and McCree 1985). There was
no evidence of changes in ε of olive leaves during the salinity
or relief period in our study, but the intensity of the salt
treatment (duration × concentration) may not have been sufficient to affect ε. This possibility is supported by the observation that changes in leaf morphological characteristics were
less pronounced than those reported in a previous study (Bongi
and Loreto 1989) in which higher NaCl concentration
(250 mM), longer duration of stress (90 days), and older leaf
material (6-month-old) were used.
Figure 5. Differences between the contributions to total osmotic potential (MPa) by cations and soluble carbohydrates (calculated from the
respective values in Table 3) in leaves of Frantoio and Leccino plants
after 35 days of exposure to 0, 100, or 200 mM NaCl in hydroponic
culture.
Non-osmotic volume changes resulting from polymer accumulation were minor in both cultivars because only a small
amount of starch is present in olive leaves exposed to increased
salinity (Tattini et al. 1996). Dehydration components were
higher in the 200 mM NaCl treatment than in the 100 mM
20
GUCCI, LOMBARDINI AND TATTINI
NaCl treatment because higher NaCl concentrations increased
cellular water loss (Table 2). Net solute accumulation was
greater in Leccino plants than in Frantoio plants, suggesting
that Ψπ values reflected the respective ability of the cultivars to
exclude Na+ and Cl− (Table 3; Tattini et al. 1992, Tattini 1994).
This exclusion capacity tended to become saturated when salt
exposure was prolonged or when NaCl concentration was
increased, consistent with current models of ion exclusion in
salt-tolerant non-halophytic species (Greenway and Munns
1980, Gorham et al. 1985, Marcum and Murdoch 1994). Saturation occurred sooner in Leccino than in Frantoio. Leccino
plants initially had a higher net solute accumulation than
Frantoio plants, but the two cultivars had similar values of net
solute accumulation at the end of the salinization period (Table 2). Differences in the lipid composition of root cell membranes may partially explain the different ion-exclusion
capacities of the two cultivars during salt stress (Heimler et al.
1995).
Osmotic adjustment in olive leaves under salt stress was
accomplished primarily by accumulation of inorganic ions,
despite the osmotic contribution by soluble carbohydrates.
Although our measurements did not account for some classes
of osmotically important compounds, their relative osmotic
contribution can be estimated from data reported in the literature (Tattini et al. 1993, Heimler et al. 1995, Tattini et al. 1995).
Leaf Cl− concentration was close to that of Na+ (Bongi and
Loreto 1989, Tattini et al. 1992, Heimler et al. 1995), with
presumably comparable osmotic effects. Other anions (nitrate,
sulfate, and phosphate) and amino compounds contribute little
to ΨπFT, and their leaf concentrations are unaffected by salinity
(Tattini et al. 1993, Tattini et al. 1995).
The two cultivars did not differ in their potential to accumulate soluble carbohydrates as an adaptive response to salinity.
The maximum osmotic contribution of carbohydrates was observed at an external concentration of 100 mM NaCl, indicating that the potential to accumulate sugars was fully reached
in the 0--100 mM range (Table 3). Both mannitol and glucose
concentrations increase in response to small increases in leaf
Na+ content (Tattini et al. 1996), but glucose was more abundant than mannitol and consequently contributed more to ΨπFT.
The accumulation of mannitol is considered to be an important
contributor to the maintenance of growth and metabolism in
herbaceous species exposed to increased salinity (Tarczynski
et al. 1993, Everard et al. 1994), and a similar role has been
proposed in olive leaf tissue (Tattini et al. 1996). Transport
from the vacuole and redistribution between cellular compartments must occur in order for compatible solutes and osmotic
agents to counteract the accumulation of toxic ions during salt
stress (Marcum and Murdoch 1994, Colmer et al. 1995). Assuming that 20% of total cell volume is occupied by the
cytoplasm in mesophyll cells of olive, mannitol concentration
appears to exceed the critical threshold (300--400 mOsmol
kg −1) of basal cytoplasmic osmotic potential indicated for
compatible solutes to be effective (Gorham et al. 1985). However, it is not known what conditions are necessary or what
form is needed for sugar-alcohols to exert their protective role
in the cytoplasm.
In conclusion, there were no major differences in the leaf
water relations or in the capacity of the olive cultivars Leccino
and Frantoio to accumulate mannitol, soluble carbohydrates,
or other osmolytes in response to salt stress. Changes in osmotic contribution reflected the different exclusion capacities
of the cultivars for Na+ and Cl−. Like other glycophytic species
with medium tolerance to salinity, osmotic adjustment in olive
was mainly achieved by accumulation of inorganic ions with
relatively little energy expenditure.
Acknowledgments
We thank Maria Assunta Coradeschi, Piero Puntoni, and Laura Traversi for skillful technical assistance. Research was supported by the
National Research Council of Italy, Special Project RAISA, Subproject No. 2, Paper No. 2814. R. Gucci was partially supported by a
contribution Fondo di Ateneo of University of Pisa.
References
Alarcon, J.J., M.J. Sanchez-Blanco, M.C. Bolarin and A. Torrecillas.
1993. Water relations and osmotic adjustment in Lycopersicon
esculentum and L. pennellii during short-term salt exposure and
recovery. Physiol. Plant. 89:441--447.
Bañuls, J. and E. Primo-Millo. 1992. Effects of chloride and sodium
ion on gas exchange properties and water relations of Citrus plants.
Physiol. Plant. 86:115--123.
Bongi, G. and F. Loreto. 1989. Gas exchange properties of saltstressed olive (Olea europaea L.) leaves. Plant Physiol. 90:1408-1416.
Colmer, T.D., E. Epstein and J. Dvorak. 1995. Differential solute
regulation in leaf blades of various ages in salt-sensitive wheat and
a salt-tolerant wheat × Lophopyrum elongatum (Host) A. Löve
amphiploid. Plant Physiol. 108:1715--1724.
Erdei, L. and E. Taleisnik. 1993. Changes in water relation parameters
under osmotic and salt stresses in maize and sorghum. Physiol.
Plant. 89:381--387.
Everard, J.D., R. Gucci, S.C. Kann, J.A. Flore and W.H. Loescher.
1994. Gas exchange and carbon partitioning in the leaves of celery
(Apium graveolens L.) at various levels of root zone salinity. Plant
Physiol. 106:281--292.
Flowers, T.J. and A.R. Yeo. 1986. Ion relations of plants under drought
and salinity. Aust. J. Plant Physiol. 13:75--91.
Girma, F.S. and D.R. Krieg. 1992. Osmotic adjustment in sorghum. I.
Mechanisms of diurnal osmotic potential changes. Plant Physiol.
99:577--582.
Gorham, J., R.G. Wyn Jones and G. McDonnell. 1985. Some mechanisms of salt tolerance in crop plants. Plant Soil 89:15--40.
Greenway, H. and R. Munns. 1980. Mechanisms of salt tolerance in
nonhalophytes. Annu. Rev. Plant Physiol. 31:149--190.
Heimler, D., M. Tattini, S. Ticci, M.A. Coradeschi and M.L. Traversi.
1995. Growth, ion accumulation and lipid composition of two olive
genotypes under salinity. J. Plant Nutr. 18:1723--1734.
Hinckley, T.M., F. Duhme, A.R. Hinckley and H. Richter. 1980. Water
relations of drought hardy shrubs: osmotic potential and stomatal
reactivity. Plant, Cell Environ. 3:131--140.
Larsen, F.E., S.S. Higgins and A. Al Wir. 1989. Diurnal water relations
of apple, apricot, grape, olive and peach in an arid environment
(Jordan). Sci. Hortic. 39:211--222.
Lo Gullo, M.A. and S. Salleo. 1988. Different strategies of drought
resistance in three Mediterranean sclerophyllous trees growing in
the same environmental conditions. New Phytol. 108:267--276.
WATER RELATIONS IN OLIVE UNDER SALINITY
Lo Gullo, M.A. and S. Salleo. 1990. Wood anatomy of some trees with
diffuse- and ring-porous wood: some functional and ecological
interpretations. Giorn. Bot. Ital. 124:601--613.
Longstreth, D.J. and P.S. Nobel. 1979. Salinity effects on leaf anatomy. Consequences for photosynthesis. Plant Physiol. 63:700--703.
Marcum, K.B. and C.L. Murdoch. 1994. Salinity tolerance mechanisms of six C4 turfgrasses. J. Am. Soc. Hortic. Sci. 119:779--784.
Neumann, P.M., E. Van Volkenburgh and R.E. Cleland. 1988. Salinity
stress inhibits bean leaf expansion by reducing turgor, not cell wall
extensibility. Plant Physiol. 88:233--237.
Richardson, S.G. and K.J. McCree. 1985. Carbon balance and water
relations of sorghum exposed to salt and water stress. Plant Physiol.
79:1015--1020.
Ritchie, G.A. and T.M. Hinckley. 1975. The pressure chamber as an
instrument for ecological research. Adv. Ecol. Res. 9:165--254.
Schulte, P.J. and T.M. Hinckley. 1985. A comparison of pressure-volume curve data analysis techniques. J. Exp. Bot. 36:1590--1602.
Tarczynski, M.C., R.G. Jensen and H.J. Bohnert. 1993. Stress protection of transgenic tobacco plants by production of the osmolyte
mannitol. Science 259:508--510.
Tattini, M. 1994. Ionic relations of aeroponically-grown olive genotypes during salt stress. Plant Soil 161:251--256.
Tattini, M., P. Bertoni and S. Caselli. 1992. Genotypic responses of
olive plants to sodium chloride. J. Plant Nutr. 15:1467--1485.
21
Tattini, M., D. Heimler, M.L. Traversi and A. Pieroni. 1993.
Polyamine analysis in salt stressed plants of olive (Olea
europaea L.). J. Hortic. Sci. 68:613-617.
Tattini, M., R. Gucci, M.A. Coradeschi, C. Ponzio and J.D. Everard.
1995. Growth, gas exchange and ion content in Olea europaea
plants during salinity stress and subsequent relief. Physiol. Plant.
95:203--210.
Tattini, M., R. Gucci, A. Romani, A. Baldi and J.D. Everard. 1996.
Changes in non-structural carbohydrates in olive (Olea europaea)
leaves during root zone salinity stress. Physiol. Plant. 98:117--124.
Tyree, M.T. and H. Richter. 1982. Alternate methods of analysing
water potential isotherms: some cautions and clarifications. II.
Curvilinearity in water potential isotherms. Can. J. Bot. 60:911-916.
Walker, R.R., D.H. Blackmore and Sun Qing. 1993. Carbon dioxide
accumulation and foliar ion concentrations in leaves of lemon
(Citrus limon L.) trees irrigated with NaCl or Na2SO4. Aust. J. Plant
Physiol. 20:173--185.
Wilson, J.R., M.J. Fisher, E.D. Schulze, G.R. Dolby and M.M. Ludlow. 1979. Comparison between pressure-volume and dewpointhygrometry techniques for determining the water relations
characteristics of grass and legume leaves. Oecologia 41:77--88.