Journal of Experimental Botany, Vol. 50, No. 330, pp. 127–138, January 1999
Acclimation to long-term water deficit in the leaves of
two sunflower hybrids: photosynthesis, electron
transport and carbon metabolism
Dejana Panković1,3, Zvonimir Sakač1, Slavko Kevrešan2 and Marijana Plesničar1
1 Oil Crops Department, Institute of Field and Vegetable Crops, Novi Sad, Yugoslavia
2 Faculty of Agriculture, University of Novi Sad, Novi Sad, Yugoslavia
Received 24 March 1998; Accepted 14 August 1998
Abstract
Introduction
The influence of long-term water deficit on photosynthesis, electron transport and carbon metabolism of
sunflower leaves has been examined. Water deficit
was imposed from flower bud formation up to the
stage of full flowering in the field on two sunflower
hybrids with different drought tolerance. CO assim2
ilation and stomatal conductance of the intact leaves,
determined at atmospheric CO and full sunlight
2
(1500–2000 mmol quanta m−2 s−1), decreased with
water deficit. Maximum quantum efficiency of PSII
(F /F ) and relative quantum yield of PSII (W ) deterv m
II
mined under similar experimental conditions, did not
change significantly in severely stressed leaves. The
strong inhibition of the plateau region of the light
response curve, determined at high CO (5%) in
2
water-deficient sunflower leaves, indicates that
photosynthesis is also limited by non-stomatal factors. The decreased slope and the plateau of the
CO response curves show that the capacity of
2
carboxylation and RuBP regeneration decreased in
severely stressed intact leaves. Rubisco specific
activity decreased in severely stressed leaves, but
Rubisco content increased under prolonged drought.
The increase of Rubisco content was significantly
higher in leaves of the drought-tolerant sunflower
hybrid indicating that a higher Rubisco content could
be one factor in conferring better acclimation and
higher drought tolerance.
Despite great progress in understanding the effects of
water stress on photosynthesis, there is still no unified
concept of the events which reduce photosynthetic efficiency (Lawlor, 1995). There are several reports which
underline the stomatal limitation of photosynthesis as a
primal event, which is then followed by adequate changes
of photosynthetic reactions (Dietz and Heber, 1983;
Cornic et al., 1989; Cornic and Briantais, 1991; Brestic
et al., 1995). A decrease of photosynthesis in sunflower
leaves due to water deficit, has been attributed to both
stomatal and non-stomatal limitations (Ben et al., 1987;
Graan and Boyer, 1990; Lauer and Boyer, 1992; Ort
et al., 1994). Non-stomatal limitation of photosynthesis
in sunflower leaves has been attributed to reduced carboxylation efficiency ( Wise et al., 1991), reduced RuBP
regeneration (Gimenez et al., 1992; Tezara and Lawlor,
1995) or to a reduced amount of functional Rubisco
( Kanechy et al., 1995). Recent literature on the functioning of PSII under reversible water deficit is consistent.
PSII is well protected (Cornic et al., 1989; Jefferies, 1994)
by increased non-radiative energy dissipation (Brestic
et al., 1995) and/or by increased photorespiration (Lawlor
and Fock, 1977; Heber and Krause, 1980; Heber et al.,
1996).
So far, the influence of water stress on photosynthesis
has usually been examined in the early developmental
stages of plants grown under controlled conditions.
However, cultivated sunflower plants rarely experience
drought during early development. It has been shown
that drought near anthesis exerts the most negative effect
to sunflower seed yield (Rawson and Turner, 1982). At
Key words: Helianthus annuus, drought tolerance, gas
exchange, chlorophyll fluorescence, Rubisco.
3 To whom correspondence should be addressed. Fax: +381 21 413 833. E-mail: [email protected]
© Oxford University Press 1999
128
Panković et al.
anthesis, leaf area, which determines the photosynthetic
capacity of the plant, and seed yield are positively correlated (Rawson et al., 1980). Another feature of many
experiments has been an examination of the effects of
short-term water stress on photosynthesis. Even when
named ‘long-term water stress’ water was withheld only
for several days (Ben et al., 1987; Fredeen et al., 1991;
Zrenner and Stitt, 1991). Such short-term approaches do
not include the phenomenon of acclimation (Conroy
et al., 1988) which is usually well developed in mature
field-grown plants ( Wise et al., 1991; Ort et al., 1994). In
this study two sunflower hybrids have been chosen which
have different drought tolerance on the basis of seed yield
(Škorić, 1992). The influence of long-term water deficit
on electron transport and carbon assimilation has been
studied in leaves of the two hybrids grown in the field, in
order to distinguish the potential for acclimation of fully
developed and expanding leaves.
Materials and methods
Growth conditions
Two sunflower (Helianthus annuus L.) hybrids with different
drought tolerance were chosen on the basis of seed yield under
drought conditions (Škorić, 1992). The hybrids were selected
at the Institute of Field and Vegetable Crops in Novi Sad, and
under optimal water supply they are both high yielding. Plants
of the more tolerant hybrid, NS-H-43, and the less tolerant
hybrid, NS-H-26 were grown in the field at 4.7 plants m−2 in
chernozem soil (field capacity 200 mm H O). One set of plants
2
was also grown in pots (10 l ) kept in the field.
Mean maximum and minimum temperatures for April, May,
June, and July were 17.2/5.7, 25.2/12.3, 26.2/13.9, and
28.3/13.9 °C, relative air humidity was 67, 58, 63, and 60% and
solar radiation was 15.7, 19.0, 22.0, and 22.4 MJ d−1, respectively. All plants were irrigated regularly to maintain the soil
water content close to field capacity until flower bud formation
(R1, Schneiter and Miller, 1981). Then the irrigation treatment
continued on control plants and the drought treatment on the
other plants was imposed by witholding water and using an
automatic transparent shelter during rain. The y of leaves
W
under the drought treatment dropped from −0.7 MPa to
−2.3 MPa. Over the following 37 d the plants developed
through the following phases of flowering: R2, R5.1–R5.9. In
order to examine leaves with maximum photosynthetic activity,
leaves close to positions 10, 15 and 20 were examined in phases
R1-R2, R5.1–R5.4 and R5.5–R5.9, respectively (English et al.,
1979; Panković et al., 1991) (Fig. 1).
Measurements of photosynthesis
Leaves at their maximum photosynthetic activity were used for
measurements. Stomatal conductance and CO assimilation rate
2
were measured on intact leaves in the field, by LI-6000 (Li-Cor,
USA), under atmospheric CO and full sunlight (1500–
2
2000 mmol quanta m−2 s−1). About 100 cm2 of leaf was clamped
in a 4 l chamber. Leaf temperature varied between 28 °C and
33 °C. CO response curves were also determined by LI-6000
2
on intact leaves in the field at full sunlight. Elevated [CO ] was
2
introduced by breathing into the chamber, prior to clamping it
on a leaf ( Wise et al., 1991). After several min the CO
2
exchange rate stabilized and measurements started at a CO
2
concentration of #600 ppm. As the leaf consumed CO each
2
following measurement was made at lower CO partial pressure.
2
Each CO response curve took about 15 min. To avoid the
2
condensation of water vapour in the system two sacks with dry
silica gel were added to absorb moisture. This prevented an
increase in air humidity and did not allow the calculation of
stomatal conductance as well as C . Thus changes of assimilation
i
rates were estimated as a function of external CO .
2
Light response curves of photosynthetic O evolution were
2
determined on leaf discs (10 cm2) in a closed chamber (LD2,
Hansatech, UK ) at 25 °C and 5% CO (Delieu and Walker,
2
1981; Walker, 1990). Control leaf discs were put on a moistened
pad and droughted leaf discs on a dry pad, to avoid recovery
during the measurement. The effect of CO concentration on
2
the rate of photosynthesis was tested prior to experimentation
(see results, Table 4). Different CO concentrations (5, 10 and
2
15%) in the chamber were achieved by the application of 400 ml
of carbonate-bicarbonate buffers (Table 1).
The following procedure was used for the light response
curve of photosynthetic O evolution. After calibration of the
2
oxygen electrode, a leaf disc was illuminated in the chamber at
800 mmol quanta m−2 s−1 for 5 min, followed by 5 min of
darkness. After measurement of the dark respiration rate, the
light response curve over a range of 14 photon flux densities
(PFDs) was determined starting from the lowest light intensity.
The leaf chamber was illuminated from the top by LH36U
(Light-emitting diodes, peak emission at 650 nm, Hansatech,
UK ) up to 800 mmol quanta m−2 s−1 and with a Björkman
lamp for the highest light intensity (1800 mmol quanta m−2 s−1;
LS2, Hansatech, UK ). Leaf-disc electrode and the associated
apparatus was connected to a personal computer, and light
response curve measurements were conducted by the ‘Leaf Disc’
computer program ( Walker, 1990). This program facilitates
calibration and automatically changes photon flux density.
Analysis and storage of data is automated, and the determination of the quantum yield (QY ) and maximum rate of
photosynthesis is speeded up. Maximum rates of photosynthesis
were used for the determination of relative mesophyll limitation
(RML) of photosynthesis as
–A
)/A
)×100,
maxC maxWS maxC
where A
and A
are the maximum rates of photosynmaxC
maxWS
thesis in control and water-stressed leaves, respectively.
RML=((A
Determination of leaf water status
After the measurements of photosynthesis, nearby leaves were
used for the determination of water status. Leaf water potential
was measured with a pressure chamber (Soil Moisture
Equipment Corporation, USA). Relative water content (RWC )
was determined gravimetrically. After measurement of fresh
weight, 10 leaf discs of 2 cm2 were kept in the dark, floating on
distilled water in Petri dishes. After attaining constant turgid
weight (4 h), discs were dried. Relative water content (%) was
Table 1. Carbonate/bicarbonate buffers used for obtaining different CO concentrations in leaf-disc oxygen electrode chamber
2
(Rabinovič, 1951)
Buffer no.
pH
[ K CO ]
2 3
(mol l−1)
[ KHCO ]
3
(mol l−1)
[CO ]
2
(%)
9
11
11
9.4
8.5
8.5
0.3
0.06
0.09
1.7
1.11
1.66
5
10
15
Drought and sunflower photosynthesis 129
Fig. 1. The effect of drought on leaf area of examined leaves. Time 0 corresponds to 60 d after sowing, when plants were in the phase of flower
bud formation, R1. Results are means of at least three replicates, vertical bars indicate standard errors of the means; (Ω) control plants;
(#) drought treatment. Drought treatment was imposed over the following 37 d while plants developed throught R2 (I ), R5.1 to R5.4 (II ) and
from R5.5 to R5.9 (III ), i.e. flower bud development and flowering.
calculated from the ratio of differences between fresh weight
and dry weight, and turgid weight and dry weight.
Chlorophyll fluorescence
Measurements of modulated chlorophyll fluorescence emission
(MFM System Hansatech, UK ) from the upper surface of the
intact leaf, were made after bringing the plant in the pot to the
laboratory. After the measurement, water status of the same
leaf was determined as already described. Laboratory-built
high-intensity light source (quartz-iodide lamp with reflector:
24 V, 250 W ) ( Walker, 1990), equipped with a stabilized power
supply unit (Farnell Instruments Ltd., UK ), was adapted at
the Faculty of Technical Sciences in Novi Sad, to be simultaneously used as a source of actinic light and saturation pulses.
A dark-adapted leaf (20 min) was initially exposed to the weak
modulated measuring beam (LED, Ealing 35–5404 short pass
filter with cut off at 620 nm, 1 mmol quanta m−2 s−1), photodiode fluorescence detector was protected with long pass filter
(Schott RG665, cut off at 665 nm). This was followed by
simultaneous exposure to continuous white light and a saturating light pulse (c. 5000 mmol quanta m−2 s−1, 990 ms) to
determine maximum quantum efficiency of PSII (F /F ). At
v m
steady-state photosynthesis, pulses of saturating light were given
every 30 s to reduce fully the primary electron acceptor and
remove photochemical quenching. At atmospheric CO and
2
two light intensities (100 or 800 mmol quanta m−2 s−1),
chlorophyll fluorescence quenching parameters q , (photochemP
ical ) and q (non-photochemical ) (Schreiber et al., 1986),
N
quantum efficiency of PSII (W ) and excitation efficiency of
II
open PSII centres (W ) (Genty et al., 1989) were measured at
exc
steady-state photosynthesis. After that the same leaf was used
for the measurement of y .
W
Soluble proteins, Rubisco content and activity
For the determination of ribulose-1,5-bisphosphate carboxylase/
oxygenase (Rubisco) activity, Rubisco content and content of
soluble proteins, immediately after the measurement of CO
2
exchange rate in the field, leaf discs (5 cm2) were quickly
homogenized in 1 ml of extraction medium: 50 mmol l−1
HEPES, pH 7.4; 20 mmol l−1 MgCl ; 1 mmol l−1 EDTA; 2%
2
glycerol (w/v); 0.05% Triton X-100 (w/v); with freshly added
20 mmol l−1 dithiothreitol; 2 mmol l−1 benzamidine; 2 mmol l−1
e-amino-caproic acid; 0.1% BSA; 0.5 mmol l−1 PMSF (phenylmethylsulphonyl fluoride); and some polyclar AT, in a glass
homogenizer on ice (Lauerer et al., 1993). After extraction and
centrifugation (1 min, 13 000 rpm) the supernatant was divided
into aliquots and frozen in liquid nitrogen.
To measure the initial activity of Rubisco, aliquots including
0.5 mmol l−1 Ru1,5bisP, 100 mmol l−1 TRIS-HCl pH 8.1,
10 mmol l−1 MgCl , 10 mmol l−1 NaHCO , 1 mmol l−1 EDTA,
2
3
2.5 mmol l−1 DTT, and 5 mmol l−1 NaH14CO (0.4 Ci mol−1),
3
were assayed by 14CO incorporation immediately after extrac2
tion (Quick et al., 1991). Aliquots for determination of
fully activated Rubisco, were thawed on ice and assayed by
14CO incorporation after preincubation for 4 min at 30 °C
2
in 100 mmol l−1 TRIS-HCl pH 8.1, 20 mmol l−1 MgCl ,
2
10 mmol l−1 NaHCO , 1 mmol l−1 EDTA, and 5 mmol l−1
3
DTT. The percentage of activation was calculated as the ratio
of these two activities.
Total soluble proteins were determined from aliquots by a
modified method of Lowry (Peterson, 1977). Each sample
(50 mg protein) was separated by SDS-PAGE (Laemmli, 1970).
Rubisco standard (Sigma) was applied at three increasing
concentrations on each gel plate. After separation, Rubisco
protein was quantified by densitometric analysis of gels at
580 nm (ISCO scanner) with an integrator (Hewlet-Packard
3396 II ).
Statistical analysis
Values shown in the tables and figures are means of 4–6
replicates, as indicated. Comparisons between means were
evaluated by ANOVA 2 or t-test, as indicated, at P=0.01 and
0.05 level of error (SigmaStat 2.0, Jandel Sci.)
Results
Leaf characteristics and water status
The area of leaves used for measurements in the control
and the water deficit treatment is presented in Fig. 1. All
plants were irrigated until the flower bud formation stage
(R1; Schneiter and Miller, 1981). After that only control
130
Panković et al.
plants were irrigated. The effects of drought were examined over the following 30 d as the plants developed
through the following phases: R2 (I ), R5.1–R5.4 ( II )
and R5.5–R5.9 ( III ) ( Fig. 1). In order to do the experiments on leaves with maximum photosynthetic activity,
leaves near to positions 10, 15 and 20 were examined in
phases I, II and III, respectively ( English et al., 1979;
Panković et al., 1991). When plants were exposed to
drought, the area of leaves examined developed to a lesser
extent. In NS-H-43 the decrease was about 25% and in
NS-H-26 it was about 37%. The total number of leaves
and the timing of leaf appearance were slightly decreased
under drought in both hybrids ( Table 2). Water deficit
was imposed about 60 d after sowing, when all leaves
were initiated ( Table 2). At this point leaves at positions
10–15 were nearly fully developed, leaves at positions
15–20 were about 50% and leaves at positions 20–25 were
about 25% of their maximum area (Panković et al., 1991).
Leaf water deficit gradually increased from about
−0.8 MPa at time 0 to about −2.3 MPa at the end of
experiment (Fig. 2). So leaves 10–15 experienced mild
water deficit (−1.0 to −1.5 MPa) when they were almost
fully developed. Leaves 15–20 and 20–25 were still developing and spent 50% and 75% of their growth cycle under
increasing drought and at the moment of measurement
they were severely stressed (−1.5 to −2.3 MPa).
Therefore, results on mild water deficit effects refer to
data obtained on leaves 10–15, and those on severe water
deficit effects refer to data obtained on leaves 15–20
and 20–25.
Leaf characteristics changed after a prolonged drought.
Beside the smaller area of water-deficient leaves their
specific leaf weight (SLW ) also changed. SLW of control
leaves was 4.9±0.5 mg cm−2 for the hybrid NS-H-43 and
5.2±0.4 mg cm−2 for the hybrid NS-H-26. Drought
induced an increase in SLW to 5.8±0.2 mg cm−2 and
5.9±0.5 mg cm−2, respectively.
Leaves of field-grown sunflower plants under regular
irrigation had a relative water content (RWC ) of approximately 80% and a leaf water potential of about
−0.8 MPa. In leaves subjected to gradually increasing
drought over 1 month, both parameters decreased, RWC
to approximately 60% and y to −2.3 MPa. According
W
to the regression lines, changes in RWC with y seemed
W
to be similar in leaves of both hybrids ( Fig. 2).
Nevertheless, in the region of the lowest water potential,
leaves of NS-H-43 tend to have higher RWC values than
leaves of NS-H-26.
Photosynthesis and electron transport in ambient conditions
CO exchange rates and stomatal conductance were
2
investigated in field conditions, i.e. at atmospheric CO
2
and sunlight (1500–2000 mmol quanta m−2 s−1). Leaves
of NS-H-26 when well supplied with water had higher
stomatal conductance than leaves of NS-H-43. With the
decrease of y stomatal conductance decreased to the
W
same low level of less than 5 mmol H O m−2 s−1 ( Fig. 3).
2
Rates of photosynthesis in control conditions were similar
in leaves of both hybrids #25 mmol CO m−2 s−1. With
2
the decrease of y photosynthetic rate dropped to
W
#15 mmol CO m−2 s−1 in leaves of NS-H-43, and to less
2
than 10 mmol CO m−2 s−1 in leaves of NS-H-26.
2
Although CO exchange rates and stomatal conductivit2
ies decreased with increasing water deficit, C , calculated
i
Fig. 2. Relative water content and leaf water potential measured in
leaves of drought-tolerant (Ω) NS-H-43 and drought-susceptible (#)
NS-H-26 sunflower hybrids under increasing long-term water deficit in
the field. Lines were obtained by regression analysis of data (Sigma
Plot V, Jandel Sci.).
Table 2. The effect of drought on the leaf appearance at positions 10–15, 15–20 and 20–25, determined in days after sowing when leaf
reached 5 cm2, and on the total number of leaves per plant
The results are means of 10 plants, standard errors of the mean were less than ±1 day and ±1 leaf, respectively.
Hybrid
NS-H-43
NS-H-26
Treatment
Control
Water deficit
Control
Water deficit
Leaf area (cm2)
Total number of leaves
Position 10–15
Position 15–20
Position 20–25
41
44
40
42
46
49
43
47
51
55
48
51
30
28
31
28
Drought and sunflower photosynthesis 131
Fig. 3. CO exchange rate (mmol CO m−2 s−1) (#) and stomatal conductance (mol H O m−2 s−1) ($) measured in leaves of drought-tolerant
2
2
2
(NS-H-43) and drought-susceptible (NS-H-26) sunflower hybrids, under increasing water deficit. y of control leaves was #−0.8 MPa, and of the
W
leaves under severe water deficit was <−2.0 MPa. Measurements were performed by LI-6000 under atmospheric [CO ] and natural light (1500–
2
2000 mmol quanta m−2 s−1) between 9 a.m. and 12 a.m.
as in von Caemmerer and Farquhar (1981), ranged from
150–350 mmol mol−1 in both control and water-stressed
leaves.
Maximum quantum efficiency of PSII did not change
significantly with water deficit (Table 3). Quenching
coefficients of fluorescence and the efficiency of excitation
of open PSII centres (W ) were also determined under
exc
atmospheric CO and two light intensities. At low light
2
intensity (100 mmol quanta m−2 s−1) neither of these
parameters changed significantly with water deficit (results
not shown). At 800 mmol quanta m−2 s−1 there was a
slight decrease of q and W , and as a result W decreased
P
exc
II
by less than 20% in severely water-deficient leaves
( Table 3).
CO and light response curves
2
CO response curves were measured on intact leaves in
2
the field, starting from the highest CO and at high light
2
intensity (#2000 mmol quanta m−2 s−1). Curves are the
result of three separate measurements shown with different symbols (Fig. 4). In control leaves of both hybrids
an increase in CO from 300 ppm to 600 ppm stimulated
2
the CO exchange rate by 30–40% ( Fig. 4A, C ). Water
2
deficit induced the decrease of both the slope and the
plateau of the CO response curve in leaves of both
2
hybrids ( Fig. 4B, D). Inhibition of the plateau region was
higher in leaves of NS-H-26. Since stomatal conductance
was similarly inhibited under drought in leaves of both
hybrids ( Fig. 3) it appears that both carboxylation efficiency (slope) and RuBP regeneration (plateau) were
inhibited under water deficit, and that RuBP regeneration
was more inhibited in leaves of NS-H-26.
To overcome stomatal limitation, light response curves
under 5% CO were also analysed. Some authors have
2
even used a range of CO concentrations from 5–15%,
2
depending on the degree of dehydration of leaves (Cornic
et al., 1989). According to Terashima et al. (1988),
Walker (1990) and Graan and Boyer (1990), 5% CO
2
should be high enough to overcome tight closure of
stomata and should ensure that CO is not limiting for
2
photosynthesis. On the other hand measurement of photosynthesis in the region of CO saturation much above 5%
2
Table 3. Modulated chlorophyll fluorescence emission measured on intact leaves of drought-tolerant (NS-H-43) and drought-susceptible
hybrid (NS-H-26)
Control leaves had leaf water potential between −0.8 and −1.0 MPa. In leaves exposed to mild water deficit y ranged between −1.0 and
W
−1.5 MPa. Severely stressed leaves had y from −1.5 to −2.3 MPa. Maximum quantum yield of PSII (F /F ) was determined after at least
W
v M
20 min of dark relaxation. W was measured in steady-state photosynthesis under atmospheric CO and at 800 mmol quanta m−2 s−1. Results are
II
2
means of 4–8 measurements. Standard errors of the means are also shown.
Parameter
F /F
v m
W
II
NS-H-43
NS-H-26
Control
Mild water
deficit
Severe water
deficit
Control
Mild water
deficit
Severe water
deficit
0.85±0.00
0.23±0.01
0.85±0.00
0.23±0.01
0.84±0.00
0.18±0.04
0.85±0.00
0.21±0.01
0.85±0.00
0.23±0.05
0.84±0.00
0.19±0.01
132
Panković et al.
Fig. 4. Response of CO exchange rate to decreasing external [CO ]. Graphs (A) and (B) refer to data measured in leaves of the drought-tolerant
2
2
hybrid (NS-H-43), and (C ) and (D) to data measured in leaves of the drought-susceptible hybrid (NS-H-26). (A, C ) Control conditions, y
W
between −0.8 and −1.0 MPa; (B, D) severe water deficit, y from −1.5 to −2.3 MPa. The results of three measurements on separate plants are
W
presented by different symbols. CO response curves were obtained by LI-6000. Initially, CO was increased in the chamber and after several min
2
2
the dependence of CO exchange rate on external [CO ] was measured starting from the highest [CO ] (#600 ppm) and at full sunlight (1500–
2
2
2
2000 mmol quanta m−2 s−1)].
CO could induce a decrease of photosynthesis in leaves
2
with optimal water status and it could induce metabolic
recovery in water-stressed leaves, as discussed by
Terashima (1992). This is why it was decided to measure
photosynthesis of both control and stressed leaves under
the same CO concentration and to make sure that 5%
2
CO under these experimental conditions was overcoming
2
stomatal limitation and was not limiting for photosynthesis ( Table 4). The steady-state rate of photosynthesis
was measured after approximately 20 min of illumination,
at 800 and 1700 mmol quanta m−2 s−1. At both light
intensities in control and water-deficient leaves of the
hybrid NS-H-43, the rate of photosynthethic O evolution
2
was saturated at 5% CO , but rates were lower in water2
stressed leaves (similar data obtained for leaves of the
other examined hybrid, NS-H-26, are not presented ). To
confirm that this reduction is due to water stress and not
due to undersaturating CO , another experiment was
2
performed. The petioles of detached control leaves were
recut in degassed destilled water and leaves were illuminated to maintain the transpiration stream. Leaf diffusion
resistance was measured (LI 60, Li-Cor, USA) before
and after the addition of ABA (50 mM ) (Graan and
Boyer, 1990; Lauer and Boyer, 1992). When leaf diffusion
resistance increased to values corresponding to those of
stomatal resistance of water-stressed leaves (Mathews and
Boyer, 1984), the dependence of photosynthetic O evolu2
tion on the same range of the CO concentrations was
2
examined. The rate of photosynthesis in ABA-treated
leaves was 85% of the control leaves at 5% CO , and
2
was not stimulated by the further increase of CO
2
concentration.
The QY of photosynthesis, determined from the linear
part of the light response curve (up to 150 mmol
quanta m−2 s−1) and the maximum rate of photosynthesis
(at 1800 mmol quanta m−2 s−1), in leaves under controlled
conditions and under the influence of mild and severe
water deficit are presented in Fig. 5. QY of photosynthesis
Drought and sunflower photosynthesis 133
Table 4. Dependence of the rate of photosynthetic O evolution on CO concentration in a leaf disc O electrode, at two light intensities
2
2
2
and 25 °C, for control and water-deficient leaves of hybrid NS-H-43
Leaf water potential of control leaves was about −0.8 MPa and of water-deficient leaves was about −2.0 MPa. To obtain different CO
2
concentrations in the gas phase, 400 ml of carbonate/bicarbonate buffers (Rabinovič, 1951) were applied on filter pads in the chamber. Results are
means of three to five measurements. SE of the means are also presented.
Treatment
Light intensity
(mmol quanta m−2 s−1)
Photosynthesis (mmol O m−2 s−1)
2
5% CO
Control
Water deficit
800
1700
800
1700
2
37.69±0.13
66.39±7.26
15.11±0.70
17.90±1.23
10% CO
2
38.71±1.98
64.00±5.20
16.46±0.44
17.94±1.57
15% CO
2
37.69±0.29
62.64±5.51
15.17±1.61
18.30±2.30
stress), our data (Panković et al., 1997) show that under
short-term water stress the recovery of these parameters
is better in leaves of the same hybrid.
Rubisco activity and content
Fig. 5. Quantum yield of photosynthesis (A) and maximum rate of
photosynthesis (mmol O m−2 s−1) (B) were determined from light
2
response curves on leaf discs of drought-tolerant (NS-H-43: empty
bars) and drought-susceptible (NS-H-26: filled bars) sunflower hybrids
exposed to long-term water deficit in the field. Measurements were
performed under 5% CO starting from the lowest light intensity up to
2
the 1800 mmol quanta m−2 s−1. I, II and III refer to control leaves (y ,
W
−0.8 to −1.0 MPa), leaves exposed to mild (y , −1.0 to −1.5 MPa)
W
and severe water deficit (y , −1.5 to −2.3 MPa), respectively. Data
W
are presented as means of 4–8 measurements. S.E. of the means are
presented (vertical bars). Statistical significance of water stress effects
was tested by ANOVA-2, at **P=0.01 and *P=0.05.
The effects of mild and severe water deficit on the content
of total soluble proteins and Rubisco content and activity
are shown in Table 5. Rubisco activity per mg of Rubisco
protein decreased with drought, 20% in leaves of NS-H-43
and 12% in leaves of NS-H-26. However, an increase in
Rubisco protein relative to total soluble proteins was
detected under the influence of prolonged drought. In leaves
of NS-H-43 Rubisco protein increased by 80%, while in
leaves of NS-H-26 it increased by 43%. As a consequence,
Rubisco activity expressed per unit leaf area increased with
drought from #30 mmol CO m−2 s−1 (Tezara and Lawlor,
2
1995; Woodrow, 1994), by 2.9 times in NS-H-43, and by
1.9 times in NS-H-26. The content of total soluble proteins
per unit leaf area also increased, by 95% and 43% under
severe water deficit in leaves of NS-H-43 and NS-H-26,
respectively. In leaves of NS-H-43 most changes were statistically significant already under mild water deficit, while
in leaves of NS-H-26 significant differences appeared under
severe water deficit. The differences in Rubisco activity and
content and total soluble protein content between control
and severely stressed leaves of NS-H-43 and NS-H-26 were
checked by t-test, and are significant at both, P=0.05 and
P=0.01 (Table 6).
The decrease in Rubisco activation with water deficit
was less than 10%. In control and severely stressed leaves
it changed from 81±4% to 74±2% in hybrid NS-H-43
and from 89±3% to 83±2% in hybrid NS-H-26.
Discussion
was inhibited under severe water deficit in leaves of
NS-H-43 and NS-H-26, by 26% and 31%, respectively
( Fig. 5A). The maximum rate of photosynthesis was
inhibited by more than 60% in severely stressed leaves of
both hybrids (Fig. 5B). Although statistical analysis of
the data indicates that changes in QY and A
in leaves
max
of NS-H-43 are expressed earlier (Fig. 5) (mild water
Stomatal and non-stomatal limitation of photosynthesis
under long-term drought
These results indicate that long-term water deficit leads
to both stomatal and non-stomatal limitation of photosynthesis in leaves of sunflower plants grown in the field.
This confirms the results of other authors, who studied
134
Panković et al.
Table 5. Rubisco activity, content of total soluble proteins, Rubisco protein and Rubisco specific activity in leaves of drought-tolerant
(NS-H-43) and drought-susceptible (NS-H-26) sunflower hybrids exposed to long-term drought
y of control leaves ranged from −0.8 to −1.0 MPa. Leaves under mild water deficit had y from −1.0 to −1.5 MPa. Under severe water deficit
W
W
y ranged from −1.5 to −2.3 MPa. Results are means of six measurements. Statistically significant differences between treatments are denoted by
W
*P=0.05, and **P=0.01 (ANOVA-2).
NS-H-43
Control
Mild water deficit
Severe water deficit
LSD (P=0.01)
(P=0.05)
NS-H-26
Control
Mild water deficit
Severe water deficit
LSD (P=0.01)
(P=0.05)
Rubisco activity
(mmol CO m−2 s−1)
2
Total soluble proteins
(g m−2)
Rubisco protein
(mg mg−1 protein)
Rubisco specific activity
(mmol CO mg−1 Rubisco min−1)
2
32.03
53.09**
92.13**
6.77
10.34**
13.24**
0.30
0.37
0.54**
0.97
0.89
0.78*
0.11
0.08
0.13
0.09
0.30
0.33
0.43**
0.83
0.83
0.73
0.10
0.07
0.17
0.12
12.99
9.13
37.45
44.81
70.91**
15.04
10.57
2.80
1.97
9.20
9.68
13.18**
1.44
1.01
Table 6. The comparison of differences in Rubisco activity and content, and total soluble proteins between control and severely stressed
leaves in two hybrids
Means of six replicates and standard deviations are presented. The significance of differences beween genotypes was tested by t-test, with * for
P=0.05 and ** for P=0.01.
Rubisco activity (mmol CO m−2 s−1)
2
Total soluble proteins (g m−2)
Rubisco protein (mg mg−1 protein)
Rubisco specific activity (mmol CO mg−1 Rubisco min−1)
2
the effect of water deficit on sunflower under different
experimental conditions (Ben et al., 1987; Graan and
Boyer, 1990; Lauer and Boyer, 1992; Ort et al., 1994).
Lawlor (1995) has suggested that contradictory results
about the importance of limitation to CO diffusion by
2
stomata (Cornic and Briantais, 1991; Brestic et al., 1995;
Lal and Edwards, 1996) versus metabolic regulation (Graan
and Boyer, 1990; Lauer and Boyer, 1992; Tezara and
Lawlor; 1995, Kanechy et al., 1995, 1996) in water-stressed
leaves, could be caused by different plant species being
investigated under different environmental conditions.
Indeed, Quick et al. (1992) have examined four plant species
and they found a difference between them in stomatal
compared to non-stomatal control of photosynthesis in
water-stressed leaves. Among examined species, they have
investigated the effect of short-term water stress on young
sunflower plants and concluded that the inhibition of photosynthesis was due to closure of stomata. In our results,
which are similar to those of Ort et al. (1994), both stomatal
and non-stomatal limitation of photosynthesis occurred in
drought-stressed field-grown sunflower plants. Our conclusion is based on the fact that CO exchange rate and
2
stomatal conductance decreased under atmospheric CO ,
2
and that substantial inhibition of photosynthesis under 5%
CO was also detected.
2
NS-H-43
NS-H-26
60.10±6.58
6.47±1.34
0.24±0.06
0.19±0.04
33.46±4.39
3.98±0.47
0.13±0.05
0.10±0.02
**
**
**
**
In leaves of both hybrids stomatal conductance
decreased more sharply than photosynthetic CO
2
exchange rate (Fig. 3). This could mean a higher inhibition of photosynthesis by stomatal closure than by biochemical activity. On the contrary, the co-ordinate
decrease of C was not observed. The neglected cuticular
i
transpiration in C calculation probably contributes to
i
the overestimation of C and also enhances the noise at
i
low leaf water potentials (Boyer et al., 1997). Others have
also shown that the decrease of photosynthetic and
stomatal conductance in sunflower leaves is not followed
by changes in C (Guidi and Soldatini, 1997). This could
i
reflect the same relative stomatal limitation of photosynthesis in control and water stress conditions. Mathews
and Boyer (1984) have also shown that drought did not
induce the increase of relative stomatal limitation of
photosynthesis in sunflower leaves.
Our results confirm that photosynthetic capacity of
sunflower leaves decreased when RWC of leaves declined
bellow 70% ( Kaiser, 1987; Cornic et al., 1989). Relative
mesophyll limitation of photosynthesis, determined from
maximum rates of photosynthesis under 5% CO ,
2
increased from 14.6% (NS-H-43) and 5.0% (NS-H-26)
under mild water stress, to 64.1% (NS-H-43) and 66.5%
(NS-H-26) under severe water stress.
Drought and sunflower photosynthesis 135
It has been shown that several processes of photosynthesis were affected. According to the degree of inhibition
of slopes and plateau regions of light-response curves Pi
regeneration was inhibited the most, but RuBP regeneration was also affected. Although it was not possible to
determine A/C curves, the changes of assimilation rate
i
to external CO (Fig. 4) indicate that RuBP regeneration
2
(plateau) and carboxylation efficiency (slope) were also
decreased in water-deficient leaves. Von Caemmerer and
Farquhar (1984) observed that RuBP carboxylation and
regeneration capacities change in parallel in response to
defoliation and changes in light intensity. They found
that under short-term water stress, RuBP regeneration
capacity was more affected with the tendency that CO
2
assimilation rates, both at high and low p(CO ), were
2
affected as stress progressed. Specific activity of Rubisco
decreased for only up to 20%, which means that this was
not a primary cause for the inhibition of photosynthesis.
The accumulation of soluble carbohydrates has been
reported both in the case of osmotically induced water
deficit on leaf discs (Quick et al., 1989) and in that of
water stress experienced by plant as soil dries out ( Zrenner
and Stitt, 1991; Quick et al., 1992). An increased level of
soluble carbohydrates under water deficit was also
detected in leaves of sunflower plants grown in field
( Fredeen et al., 1991; Sakač et al., 1995). A marked drop
in translocation of 14C-labelled photosynthates was measured from water-stressed sunflower leaves ( Watson and
Wardlaw, 1981). According to these and our results, it
seems that in water-deficient leaves the export of soluble
carbohydrates was first affected, which could lead to the
feedback inhibition of photosynthesis, decreased RuBP
regeneration and Rubisco specific activity. Increased content of Rubisco and as a consequence increased in vitro
activity per unit area represents an acclimation to longterm water deficit, as will be discussed later.
Electron transport under long-term drought
As shown by others (Cornic et al., 1989; Jefferies, 1994)
maximum quantum yield of PSII photochemistry was not
significantly decreased under drought conditions and,
although CO assimilation was decreased by approxi2
mately 50%, photoinhibition did not occur ( Table 3).
Water deficit induced a decrease of q and W in leaves
P
II
of Phaseolus vulgaris L. (Cornic et al., 1989; Cornic and
Briantais, 1991; Brestic et al., 1995). In leaves of Digitalis
lanata (Stuhlfauth et al., 1988) and Solanum tuberosum
L. (Jefferies, 1994) there were no changes of q , q , and
P N
W under drought. Similarly, in our experimental condiII
tions q and q did not change significantly over the
P
N
range of measured leaf water potentials and W only
II
slightly decreased in severely stressed leaves. This indicates
that electron transport was not limiting under severe
water stress, and that the flux of electrons through PSII
was maintained similarly as in control leaves. Obviously,
the capacity of alternative electron acceptors in waterdeficient sunflower leaves is high. The increase of photorespiration in sunflower leaves has been reported (Lawlor
and Fock, 1977; Scheuermann et al., 1991). Scheuermann
et al. (1991) have found the difference in balance of
photochemical and non-photochemical energy dissipation
between water-stressed sunflower and bean leaves. In
water-stressed sunflower leaves q was more involved in
P
energy dissipation, which reflected the internal refixation
of photorespired CO . In water-stressed bean leaves this
2
process was less expressed, and q increased markedly.
N
Our results on QY of photosynthesis and W indicate that
II
the inhibition of QY in drought-stressed leaves under 5%
CO (non-photorespiratory conditions) was caused by O
2
2
reduction in the Mehler-peroxidase reaction. Biehler and
Fock (1996) have shown that in drought-stressed wheat
leaves approximately 30% of the photosynthetic electrons
are consumed in the Mehler-peroxidase reaction.
Acclimation to long-term water deficit
It has been shown that under severe short-term water
stress ‘patchy’ stomatal closure can lead to the underestimation of A and overestimation of C ( Terashima
i
et al., 1988). This could be pronounced in heterobaric
leaves, such as sunflower leaves. But if plants are exposed
to long-term drought in field conditions, as in our experiment, heterogenous stomatal closure is not to be expected
(Gunasekera and Berkowitz, 1992; Wise et al., 1992;
Hirasawa et al., 1995).
Short-term water deficit investigated on sunflower
plants at flowering, also led to the decrease of stomatal
conductance, photosynthesis, RuBP and Pi regeneration
and the carboxylation, but its inhibitory effect was much
higher (Panković, 1996). The main difference between
short-term and long-term water deficit, investigated here,
was the significant increase of Rubisco protein in sunflower leaves under prolonged drought. In our experiments, leaf water deficit gradually increased with time.
Leaves 10–15 experienced mild water deficit (−1.0 to
−1.5 MPa) when they were almost fully developed.
Leaves 15–20 and 20–25 still developed and spent 50%
and 75% of their growth cycle under increasing drought.
At the moment of measurement they were severely
stressed (−1.5 to −2.3 MPa). The changes in the content
of total soluble proteins and Rubisco content and activity
( Table 5) with drought indicate that even almost fully
developed leaves are able to adjust to water deficit to
some extent. They tend to increase the ratio of Rubisco
protein to total soluble proteins and Rubisco activity per
unit leaf area, but these changes are not always statistically significant. On the other hand leaves that developed
mostly under the drought can acclimate by increased
Rubisco partitioning to total soluble proteins and higher
Rubisco activity per unit leaf area ( Table 6).
136
Panković et al.
It has been shown that the turnover (synthesis and
degradation) of Rubisco protein can change even in fully
expanded leaves under the influence of different environmental conditions (Blenkinsop and Dale, 1974; Simpson,
1978). However, at the time of writing, an increase of
carbon allocation towards Rubisco protein under water
stress has not been reported. Either there have been no
changes ( Tezara and Lawlor, 1995) or in one instance a
degradation of Rubisco was detected ( Kanechi et al.,
1995). Those results were obtained under short-term
water stress and with plants in earlier stages of development. It has already been shown that experimental conditions, such as growth conditions and source–sink status
of the plant, are very important for plant response to, for
example, increased CO (Sage, 1994). Results obtained
2
with Rubisco transgenic plants suggest that Rubisco
control over photosynthesis is high at high light intensity,
high temperature and low C (Lauerer et al., 1993; Krapp
i
et al., 1994). In addition, when nitrogen is in excess to
demands, it is allocated to Rubisco (Chapin et al., 1990;
Quick et al., 1991). These results suggest that sunflower
plants when flowering acclimate to long-term water deficit
by investing more carbon into Rubisco.
Intraspecific variations for transpiration efficiency of
dry matter production ( Virgona et al., 1990), osmotic
adjustment (Chimenti and Hall, 1993) and leaf water use
efficiency (Plesničar et al., 1993) in sunflower have been
observed. Our results show that ambient photosynthesis
rates at 300 ppm CO ( Fig. 3) and 600 ppm CO ( Fig. 4)
2
2
were less inhibited in severely stressed leaves of droughttolerant hybrid NS-H-43. Although specific activity of
Rubisco decreased, Rubisco partitioning to total soluble
proteins increased under water deficit, so Rubisco activity
per unit leaf area in severely stressed leaves was higher in
the drought-tolerant hybrid. Stitt and Schulze (1994)
concluded that Rubisco as a ‘reserve’ protein could be
advantageous in providing greater efficiency of photosynthesis in response to fluctuating conditions within a day.
With higher stomatal conductance higher Rubisco content
would provide higher photosynthesis.
Acknowledgements
We are grateful to Professor Ulrich Heber ( University of
Würzburg, Germany) for useful suggestions during the preparation of the manuscript. We also wish to thank Dr Elspeth
MacRae (DSIR FATS, Auckland, New Zealand) for critical
reading of this manuscript and correcting the English text. This
work was supported in part by the Ministry of Science and
Technology of the Republic of Serbia.
References
Ben GY, Osmond CB, Sharkey TD. 1987. Comparisons of
photosynthetic responses of Xanthium strumarium and
Helianthus annuus to chronic and acute water stress in sun
and shade. Plant Physiology 84, 476–482.
Biehler K, Fock H. 1996. Evidence for the contribution of the
Mehler-peroxidase reaction in dissipating excess electrons in
drought-stressed wheat. Plant Physiology 112, 265–272.
Blenkinsop PG, Dale JE. 1974. The effects of shade treatment
and light intensity on ribulose-1,5-diphosphate carboxylase
activity and fraction I protein level in the first leaf of barley.
Journal of Experimental Botany 25, 899–912.
Boyer JS, Wong SC, Farquhar GD. 1997. CO and water vapor
2
exchange across leaf cuticle (epidermis) at various water
potentials. Plant Physiology 114, 185–191.
Brestic M, Cornic G, Fryer MJ, Baker NR. 1995. Does
photorespiration protect the photosynthetic apparatus in
French bean leaves from photoinhibition during drought
stress? Planta 196, 450–457.
Chapin FS, Schulze ED, Mooney HA. 1990. The ecology and
economics of storage in plants. Annual Review of Ecology
and Systematics 21, 423–447.
Chimenti CA, Hall AJ. 1993. Genetic variation and changes
with ontogeny of osmotic adjustment in sunflower (Helianthus
annuus L.). Euphytica 71, 201–210.
Conroy JP, Virgona JM, Smillie RM, Barlow EW. 1988.
Influence of drought acclimation and CO enrichment on
2
osmotic adjustment and chlorophyll a fluorescence of sunflower during drought. Plant Physiology 86, 1108–1115.
Cornic G, Briantais JM. 1991. Partitioning of photosynthetic
electron flow between CO and O reduction in a C leaf
2
2
3
(Phaseolus vulgaris L.) at different CO concentrations and
2
during drought stress. Planta 183, 178–184.
Cornic G, Le Gouallec JL, Briantais JM, Hodges M. 1989.
Effect of dehydration and high light on photosynthesis of
two C plants (Phaseolus vulgaris L. and Elatostema repens
3
(Lour.) Hall f.). Planta 177, 84–90.
Delieu T, Walker DA. 1981. Polarographic measurement
of photosynthetic oxygen evolution by leaf discs. New
Phytologist 89, 165–175.
Dietz KJ, Heber U. 1983. Carbon dioxide gas exchange and the
energy status of the leaves of Primula palinuri under water
stress. Planta 158, 349–356.
English SD, McWilliam JR, Smith RCG, Davidson JL. 1979.
Photosynthesis and partitioning of dry matter in sunflower.
Australian Journal of Plant Physiology 6, 149–164.
Fredeen AL, Gamon JA, Field CB. 1991. Response of photosynthesis and carbohydrate partitionig to limitations in nitrogen
and water availability in field-grown sunflower. Plant, Cell
and Environment 14, 963–970.
Graan T, Boyer JS. 1990. Very high CO partially restores
2
photosynthesis in sunflower at low leaf water potentials.
Planta 181, 378–384.
Genty B, Briantais JM, Baker NR. 1989. The relationship
between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochimica
et Biophysica Acta 990, 87–92.
Gimenez K, Mitchell V, Lawlor D. 1992. Regulation of
photosynthetic rate of two sunflower hybrids under water
stress. Plant Physiology 98, 516–524.
Gunasekera D, Berkowitz GA. 1992. Heterogenous stomatal
closure in response to leaf water deficits is not a universal
phenomenon. Plant Physiology 98, 660–665.
Guidi L, Soldatini GF. 1997. Chlorophyll fluorescence and gas
exchanges in flooded soybean and sunflower plants. Plant
Physiology and Biochemistry 35, 713–717.
Heber U, Bligny R, Streb P, Douce R. 1996. Photorespiration
is essential for the protection of the photosynthetic apparatus
Drought and sunflower photosynthesis 137
of C plants against photoinactivation under sunlight.
3
Botanica Acta 109, 307–315.
Heber U, Krause GH. 1980. What is the physiological role of
photorespiration. Trends in Biochemical Science 2, 32–34.
Hirasawa T, Wakabayashi K, Touya S, Ishihara K. 1995.
Stomatal responses to water deficits and abscisic acid in
leaves of sunflower plants (Helianthus annuus L.) grown under
different conditions. Plant Cell Physiology 36, 955–964.
Jefferies RA. 1994. Drought and chlorophyll fluorescence
in field-grown potato (Solanum tuberosum). Physiologia
Plantarum 90, 93–97.
Kaiser WM. 1987. Effects of water deficit on photosynthetic
capacity. Physiologia Plantarum 71, 142–149.
Kanechi M, Kunitomo E, Inagaki N, Maekawa S. 1995. Water
stress effects on ribulose-1,5-bisphosphate carboxylase and its
relationship to photosynthesis in sunflower leaves. In:
Mathis P, ed. Photosynthesis: from light to biosphere. Vol. IV.
The Hague: Kluwer Academic Publishers, 597–600.
Kanechi M, Uchida N, Yasuda T, Yamaguchi T. 1996. Nonstomatal inhibition associated with inactivation of Rubisco
in dehydrated coffee leaves under unshaded and shaded
conditions. Plant Cell Physiology 37, 455–460.
Krapp A, Chaves MM, David MM, Rodrigues ML, Pereira JS,
Stitt M. 1994. Decreased ribulose-1,5-bisphosphate carboxylase/oxygenase in transgenic tobacco transformed with
‘antisense’ rbcS. VIII. Impact on photosynthesis and growth
in tobacco growing under extreme high irradiance and high
temperature. Plant, Cell and Environment 17, 945–953.
Laemmli UK. 1970. Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227,
680–685.
Lal A, Edwards GE. 1996. Analysis of inhibition of photosynthesis under water stress in the C species Amaranthus
4
cruentus and Zea mays: electron transport, CO fixation and
2
carboxylation capacity. Australian Journal of Plant Physiology
23, 403–412.
Lauer MJ, Boyer JS. 1992. Internal CO measured directly in
2
leaves. Abscisic acid and low leaf water potential cause
opposing effects. Plant Physiology 98, 1310–1316.
Lauerer M, Saftiæ D, Quick WP, Labate C, Fichtner K, Schulze
ED, Wendler R, Leegood RC, Mooney HA, Rodermel SR,
Bogorad L, Stitt M. 1993. Decreased ribulose-1,5bisphosphate carboxylase in transgenic tobacco transformed
with ‘antisense’ rbcS. VI. Effect on plants grown in different
irradiance. Planta 190, 332–345.
Lawlor DW. 1995. The effects of water deficit on photosynthesis.
In: Smirnoff N, ed. Environment and plant metabolism
flexibility and acclimation. Oxford: BIOS, 129–160.
Lawlor DW, Fock H. 1977. Photosynthetic assimilation of
14CO by water-stressed sunflower leaves at low O concentra2
2
tions and the specific activity of products. Journal of
Experimental Botany 28, 320–328.
Mathews MA, Boyer JS. 1984. Acclimation of photosynthesis
to low leaf water potentials. Plant Physiology 74, 161–166.
Ort DR, Oxborough K, Wise RR. 1994. Depressions of
photosynthesis in crops with water deficits. In: Baker NR,
Bowyer JR, eds. Photoinhibition of photosynthesis from
molecular mechanisms to the field. Oxford: Bios Scientific
Publishers, 315–329.
Panković D. 1996. Photosynthesis in sunflower leaves (Helianthus
annuus L.) under water deficit. PhD thesis, University of
Belgrade.
Panković D, Kevrešan S, Plesničar M, Sakač Z, Ćupina T. 1997.
The effect of water deficit on ribulose-1,5,-bisphosphate
carboxylase/oxygenase and photosynthesis in sunflower
leaves. In: Jevtić S, Pekić S, eds. Drought and plant production.
Belgrade: Agricultural Research Institute ‘Serbia’, 365–371.
Panković D, Sakač Z, Plesničar M, Ćupina T, Škorić D. 1991.
Leaf expansion and photosynthesis during growth and
development of NS sunflower hybrids and inbred lines. Helia
14, 55–62.
Peterson GL. 1977. A simplification of the protein assay method
of Lowry et al. which is more generally applicable. Analytical
Biochemistry 83, 346–356.
Plesničar M, Sakač Z, Panković D, Ćupina T, Škorić D. 1993.
Efficiency of carbon assimilation and water utilization in
several NS sunflower lines and hybrids. Helia 18, 21–30.
Quick WP, Siegl G, Neuhaus E, Feil R, Stitt M. 1989. Shortterm water stress leads to a stimulation of sucrose synthesis
by activating sucrose-phosphate synthase. Planta 177,
535–546.
Quick WP, Schurr U, Scheibe R, Schulze E-D, Rodermel
SR, Bogorad L, Stitt M. 1991. Decreased ribulose-1,5bisphosphate carboxylase-oxygenase in transgenic tobacco
transformed with ‘antisense’ rbcS. I. Impact on photosynthesis
in ambient growth conditions. Planta 183, 542–554.
Quick WP, Chaves MM, Wendler R, David M, Rodrigues ML,
Passaharinho JA, Pereira JS, Adcock MD, Leegood RC,
Stitt M. 1992. The effect of water stress on photosynthetic
carbon metabolism in four species grown under field conditions. Plant, Cell and Environment 15, 25–35.
Rabinovič E. 1951. In: Fotosintez, Vol 1, Moskva: Izdatelstvo
inostrannoj literaturi, 179–186.
Rawson HM, Constable GA, Howe GN. 1980. Carbon production of sunflower cultivars in field and controlled environments. II. Leaf growth. Australian Journal of Plant Physiology
7, 575–586.
Rawson HM, Turner NC. 1982. Recovery from water stress in
five sunflower (Helianthus annuus L.) cultivars. I Effects of
the timing of water application on leaf area and seed
production. Australian Journal of Plant Physiology 9, 437–448.
Sage RF. 1994. Acclimation of photosynthesis to increasing
atmospheric CO : the gas exchange perspective.
2
Photosynthesis Research 39, 351–368.
Sakač Z, Panković D, Plesničar M, Ćupina T. 1995.
Photosynthesis and carbohydrate accumulation in the
leaves of two sunflower hybrids subjected to drought.
INTERDROUGHT 95, Montpellier, France, 31.08–3.09.
Schneiter AA, Miller JF. 1981. Description of sunflower growth
stages. Crop Science 21, 901–903.
Scheuermann R, Biehler K, Stuhlfauth T, Fock HP. 1991.
Simultaneous gas exchange and fluorescence measurements
indicate differences in response of sunflower, bean and maize
to water stress. Photosynthesis Research 27, 189–197.
Schreiber U, Schliwa U, Bilger W. 1986. Continuous recording
of photochemical and non-photochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer. Photosynthesis Research 10, 51–62.
Simpson E. 1978. Biochemical and genetic studies of the
synthesis and degradation of RuP carboxylase. In:
2
Siegelmann HW, Hind G, eds. Photosynthetic carbon assimilation. New York: Plenum Press, 113–125.
Stitt M, Schulze D. 1994. Does Rubisco control the rate of
photosynthesis and plant growth? An exercise in molecular
ecophysiology. Plant, Cell and Environment 17, 465–487.
Stuhlfauth T, Sultmeyer DF, Weinz S, Fock HP. 1988.
Fluorescence quenching and gas exchange in water-stressed
C plant, Digitalis lanata. Plant Physiology 86, 246–250.
3
Škorić D. 1992. Achievements and future directions of sunflower
breeding. Field Crops Research 30, 231–270.
138
Panković et al.
Terashima I. 1992. Anatomy of non-uniform leaf photosynthesis.
Photosynthesis Research 31, 195–212.
Terashima I, Wong S-C, Osmond CB, Farquhar GD. 1988.
Characterisation of non-uniform photosynthesis induced by
abscisic acid in leaves having different mesophyll anatomies.
Plant Cell Physiology 29, 385–394.
Tezara W, Lawlor DW. 1995. Effects of water stress on the
biochemistry and physiology of photosynthesis in sunflower.
In: Mathis P, ed. Photosynthesis: from light to biosphere.
Vol. IV. The Hague: Kluwer Academic Publishers, 625–628.
Virgona JM, Hubick KT, Rawson HM, Farquhar GD, Downes
RW. 1990. Genotypic variation in transpiration efficiency,
carbone-isotope discrimination and carbon allocation during
early growth in sunflower. Australian Journal of Plant
Physiology 17, 207–214.
von Caemmerer S, Farquhar GD. 1984. Effects of partial
defoliation, changes of irradiance during growth, short-term
water stress and growth at enhanced p(CO ) on the
2
photosynthetic capacity of leaves of Phaseolus vulgaris L.
Planta 160, 320–329.
von Caemmerer S, Farquhar GD. 1981. Some relationships
between the biochemistry of photosynthesis and the gas
exchange of leaves. Planta 153, 376–387.
Walker DA. 1990. The use of oxygen electrode and fluorescence
probes in simple measurements of photosynthesis. Sheffield:
Oxygraphics Ltd.
Watson BT, Wardlaw IF. 1981. Metabolism and export of 14Clabelled photosynthate from water-stressed leaves. Australian
Journal of Plant Physiology 8, 143–153.
Wise RR, Ortiz-Lopez A, Ort DR. 1992. Spatial distribution of
photosynthesis during drought in field-grown and acclimated
and nonacclimated growth chamber-grown cotton. Plant
Physiology 100, 26–32.
Wise RR, Sparrow DH, Ortiz-Lopez A, Ort DR. 1991.
Biochemical regulation during the mid-day decline of photosynthesis in field-grown sunflower. Plant Science 74, 45–52.
Woodrow IE. 1994. Control of steady-state photosynthesis in
sunflowers growing in enhanced CO . Plant, Cell and
2
Environment 17, 277–286.
Zrenner R, Stitt M. 1991. Comparison of the effect of rapidly
and gradually developing water-stress on carbohydrate metabolism in spinach leaves. Plant, Cell and Environment
14, 939–946.