conditions refer to the classical approach of Maas and Hoffman 1977, relating final yield to salinity
of the soil water extract for example, Francois, 1996. The works that explained the causes of
reduced growth and yield through investigations on leaf area expansion and gas-exchange refer to
water stress, but not to salt stress under field conditions. Rawson and Munns 1984 showed
the high sensitivity of leaf growth to salinity con- ditions for potted sunflower plants. They observed
a leaf area reduction of about 50 with 5 dS m
− 1
electrical conductivity in the nutrient solution. Giorio et al. 1996 also noticed the high sensitiv-
ity of leaf area expansion to salinity in open field experiments, in response to small reductions in
total leaf water potential. Such ‘phenotypic plas- ticity’ in modulating leaf area in response to small
reductions in leaf water potential represents one of the most relevant adaptive properties of
sunflower Connor and Sadras, 1992.
In addition to leaf area expansion the ‘sink- size’ for carbon assimilation, productivity de-
pends also on the carboxylation capacity per unit leaf area the ‘sink-intensity’ for carbon assimila-
tion, compounded by stomatal and non-stomatal contributions to photosynthesis. Most of the stud-
ies on stomatal and non-stomatal components in sunflower refer to water stress conditions but
report conflicting results Lawlor, 1995. While some authors underline stomatal limitation as the
main cause of carbon assimilation reduction Plesnicar et al., 1995, others attribute the de-
crease in photosynthetic rate to non-stomatal lim- itation Gimenez et al., 1992; Tezara and Lawlor,
1995.
Under salinity conditions, the majority of the works dealing with stomatal and non-stomatal
limitation to photosynthesis concern various crops but sunflower. In bean, a salt-sensitive spe-
cies, the reduction in assimilation was found to be mostly due to stomatal limitation Brugnoli and
Lauteri, 1991, and to both stomatal and non- stomatal limitations Seemann and Critchley,
1985. Among other salt-sensitive species, Bethke and Drew 1992 on pepper and Chartzoulakis et
al. 1995 on kiwi ascribed the observed reduction in photosynthesis to non-stomatal limitation.
These conflicting results are also found for more salt-tolerant species. For instance, Brugnoli and
Lauteri 1991 indicated that stomata played the major role in limiting photosynthesis of cotton,
whereas Dunn and Neales 1993 suggested that non-stomatal components limited the photosyn-
thesis of barley. All these works were conducted in a controlled environment with a variable range
of boundary conditions and thus resulting in a low degree of comparability. Moreover, no infer-
ences could be drawn for open-field conditions, where the degree of stress development, intensity
and duration is such that plants can activate adaptation mechanisms for adjusting to the
changing environment Lawlor, 1995.
Concerning sunflower grown in the field, no information is available on the stomatal and non-
stomatal limiting components of photosynthesis under salinity, with few studies confined to inves-
tigation on stomatal response. Katerji et al. 1994 showed a decrease in stomatal conductance with
increasing salt concentration of irrigation water of potted sunflower plants, while Giorio et al. 1996
found no response of stomatal conductance with salinity of plants grown in the field.
From that already reported, our hypothesis is that if leaf area modulation remains the most
important stress avoidance mechanism under salinity and if stomatal closure is of less signifi-
cance, non-stomatal limitations to photosynthesis should be the least or not at all induced by
salinity. This hypothesis would be consistent with the
optimization theory
of plant
processes Cowan, 1982; Givnish, 1986.
Thus, in order to verify it, the present study investigated the effect of gradually developing
salinity stress on leaf expansion, leaf gas-exchange parameters, and stomatal and non-stomatal limi-
tations to carbon assimilation.
2. Materials and methods
The main experiment was carried out in the field, where the high buffering capacity of the soil
exposed the crop to a relatively mild salinity stress. A second experiment was carried out in
pots, although always in the open to increase the level of salinity obtained in the field.
2
.
1
. The field experiment The field trial took place in 1996 at the experi-
mental farm of C.N.R.-ISPAIM located in Vitu- lazio Caserta, Southern Italy 40°07 N, 14°50
E, 25 m above sea level, having a typical Med- iterranean climate. The soil is an alluvial mont-
morillonite clay loam type, defined as Mollic Haplaquept according to the USDA soil classifi-
cation, with physical and chemical characteristics through the upper 1.2 m depth as reported in
Table 1.
Sunflower Helianthus annuus, hybrid Turbo- sol, was sown on 14 June 1996, in rows 0.75 m
apart in three nearby 24 × 15 m
2
plots, with final plant density of 5 plants m
− 2
. The crop received a presowing fertilization, with 96 kg ha
− 1
ureic nitrogen and 95 kg ha
− 1
P
2
O
5
, and was grown under
healthy conditions.
Furrow irrigation
started at 36 days after planting DAP when plants had eight fully expanded leaves, and was
scheduled according to the soil water balance method applied over 1.35 m depth. The control
treatment I
was irrigated with fresh water of 0.9 dS m
− 1
electrical conductivity EC
w
, dS m
− 1
and the salt treatments, I
1
and I
2
, with saline solutions having EC
w
equal to 7.8 and 15.6 dS m
− 1
, respectively, obtained by adding commercial sea-salt NaCl to irrigation water. Soil salinity
was monitored measuring the electrical conductiv- ity in 1:2.5 soil extract, in the 0 – 0.6 m soil layer,
and at different stages of the crop cycle, by a laboratory conductimeter model Micro CM
2201; Crison Instruments SA, Barcelona, Spain. An empirical factor of 3.7 was used to calculate
the equivalent saturation extract EC
e
, dS m
− 1
.
2
.
2
. The pot experiment The trial took place in 1997 at the experimental
field of the Mediterranean Agronomic Institute, located in Valenzano Bari, Southern Italy
41°03 N, 16°52 E, 72 m above sea level, about 200 km from the field experimental site of
Vitulazio.
The same hybrid Turbosol as in the previous experiment was sown on 12 May 1997 in pots of
35 dm
3
, filled with coarse river sand and clay- loam soil in a 3:1 ratio on weight basis, mixed
with 200 g per pot of a ternary fertilizer 3030 30. The resulting pH of the saturated extract was
7.6 and the soil cation exchange capacity CEC was 55 9 7 mmol kg
− 1
. Five treatments with five pots per treatment
one plant per pot were established by adding, like in the field trial, different amounts of com-
mercial sea-salt NaCl to the irrigation water. During the season, the control treatment I
was irrigated with fresh water EC
w
= 0.9 dS m
− 1
and the salt treatments I
1
, I
2
, I
3
, and I
4
with a saline solution having EC
w
of 3.9, 7.8, 11.7 and 15.6 dS m
− 1
, respectively. Irrigation started at 37 DAP, when the plants
had eight fully expanded leaves, and continued during the whole life cycle applying one to two
waterings per day, allowing abundant leaching in order to maintain a stable salt content in the root
zone. Electrical conductivity was measured on the drainage water using the same type of conduc-
timeter used for the field experiment. In the same irrigation water, a nutrient solution was added to
keep
the plants
in healthy
and vigorous
conditions.
2
.
3
. Leaf area de6elopment and water status In the field experiment, green leaf area LA,
cm
2
was determined along the crop cycle by non-destructive measurements of the leaf lamina
length Le, cm and maximal width Wi, cm, using an empirical equation Giorio et al., 1996.
Measurements of Le and Wi were taken on all leaves of five plants per plot randomly selected
and repeated for the same plants every five days. In the pot experiment, measurements were carried
out only at the flowering stage on the third leaf from the top for all plants to have an adequate
magnitude of the surface area of the leaves em- ployed for gas-exchange determinations.
In the field experiment, midday leaf water po- tential
C
l
, MPa was measured on four fully-ex- panded and well-exposed leaves per plot, during
most of the crop cycle, by a Scholander pressure chamber Model 3000; Soil Moisture Equipment
Corp., Santa Barbara, CA, USA.
P .
Steduto et
al .
En 6
ironmental and
Experimental
Botany
44 2000
243 –
255
246
Table 1 Soil physical and chemical characteristics of the experimental site
Field capacity Wilting point
Bulk density pH
CEC Total CaCO
3
Texture Organic matter
Total nitrogen t m
− 3
mmol kg
− 1
‰ Clay-loam
39.4 9 2.9 21.7 9 1.7o
1.28 9 0.1 7.6 9 0.2
230 9 19 2.51 9 0.3
1.31 9 0.1 0.18 9 0.02
Osmotic potential c
s
, MPa determinations were carried out for both the field and pot
experiments on
the same
leaves where
gas-exchanges had been measured. Ten leaf diskettes of 2 cm
2
each were frozen in liquid nitrogen and squeezed at fixed pressure to extract
the cellular sap. The osmolality of the sap was measured
using a
micro-osmometer 1313
DR-Autocal-Hermann; Roebling
Messtechnik, Berlin, Germany and was then converted into
osmotic potential using Morse’s equation Morse, 1914.
2
.
4
. Leaf gas-exchange measurements Gas-exchange parameters and the assimilation
A, mmol m
− 2
s
− 1
versus substomatal CO
2
con- centration c
i
, mmol mol
− 1
response curve Ac
i
of leaves were measured in both field and pot experiments, with two portable photosynthesis
open systems model Li-6400; LiCor Inc., Lin- coln, Nebraska, USA.
The Ac
i
response curve was generated after setting, inside the leaf chamber, the vapour pres-
sure deficit VPD, kPa and leaf temperature con- stant at about the same values as those measured
in the surrounding atmosphere of the leaf. Light intensity at 2000 mmol
photon
m
− 2
s
− 1
was pro- vided by a red light diode source emitting at 670
nm. An external CO
2
tank provided different CO
2
concentrations in the range 0 – 2000 mmol mol
− 1
. The Ac
i
measurements started at CO
2
concen- tration of 2000 mmol mol
− 1
and continued down to 40 mmol mol
− 1
in a step-wise fashion with shorter and shorter intervals 200, 150, 100 and
50. At each step, gas exchange variables were recorded after 15 – 20 min to achieve steady-state
conditions, and about 2 h were needed to obtain each Ac
i
curve. Calculations of gas-exchange parameters were performed according to von
Caemmerer and Farquhar 1981. Water use effi- ciency WUE
N
, mmol mmol
− 1
kPa, normalized for VPD, was derived as the ratio of assimilation
rate to transpiration rate. Ac
i
curves were taken around noon from 11:00 to 15:30, on exposed
uppermost leaves usually the third from the top over a clipped leaf surface of 6.0 cm
2
. In the field experiment, measurements started
at 46 DAP, when plants had ten fully expanded leaves, and continued every 4 – 5 days until 76
DAP. In the pot experiment, Aci curves were all taken at the flowering stage 80 DAP, after
exposure to salinity treatments from 37 DAP.
2
.
5
. Stomatal and non-stomatal limitation estimates
In the present work, the analysis of the Ac
i
curve follows
the model
of Farquhar
and Sharkey 1982. The data of each Ac
i
curve were interpolated by non-linear regression to fit a non-
rectangular hyperbola equation Jones, 1983. The fitted equation A = fc
i
was used to derive the carboxylation efficiency a
, mol m
− 2
s
− 1
, the maximum photosynthetic rate at saturated
light and CO
2
A
max
, mmol m
− 2
s
− 1
, the sensi- tivity of A to c
i
variation g, mol m
− 2
s
− 1
, the CO
2
compensation point G, mmol mol
− 1
, and the light respiration R
l
, mmol m
− 2
s
− 1
. The a and g parameters were calculated from the first
derivative of the equation Ac
i
at c
i
= 0 and
c
i
= operational, respectively. The R
l
and A
max
parameters were obtained from A = fc
i
when c
i
= 0 and c
i
= , respectively. The CO
2
compen- sation point was derived as the value of c
i
when A = 0.
The relative contributions of stomatal l
s
, and non-stomatal l
m
, limitations to photosyn- thesis were calculated according to the differential
method Jones, 1985 as
l
s
= g
g + g
sc
1 and
l
m
= 1 − l
s
2 where g
sc
mol m
− 2
s
− 1
is the stomatal conduc- tance for CO
2
, corresponding to the reciprocal of the slope of the line joining the point where c
i
equals ambient CO
2
concentration to the assimi- lation value at operational c
i
. The Student’s t-test was applied to compare
statistically the results between treatments.
3. Results
