PHYSIOLOGIA PLANTARUM 106: 184189.

1999
Printed in Ireland all rights reser6ed

Copyright Physiologia Plantarum 1999

ISSN 0031-9317

Intrinsic water use efficiency at the pollination stage as a parameter for

drought tolerance selection in Phaseolus 7ulgaris
C. Pimentela,*, D. Laffrayb and P. Louguetb
a

Depto. de Fitotecnia, I.A., Uni6. Fed. Rural do Rio de Janeiro, Itagua, 23851 -970, RJ, Brazil
Lab. de Physiologie Vegetale, Faculte des Sciences, Uni6. Paris-Val de Marne, A6. du Gen. de Gaulle, F-94 010, Creteil-Cedex, France
*Corresponding author, e-mail: greenman@amcham.com.br

Received 25 November 1998; revised 22 February 1999

Genotype differences in gas exchange during ontogeny and

water stress responses at the vegetative and pollination stages
were evaluated in four lines of Phaseolus 7ulgaris L. In the
cultivar Carioca, net photosynthetic rate (A) and stomatal
conductance (gs) were lower at the vegetative stage (20 days
after sowing [DAS]) and maximum at the pollination stage (39
DAS), followed by a decrease at the flowering stage (46 DAS)
and a dramatic fall at the grain-filling stage (60 DAS).
Among the lines studied, the stomata of A320 closed faster
than those of the other lines when water stress occurred at 20

or 39 DAS. The cultivar SC-90298823 had greater stomatal

conductance at 39 DAS and a higher photosynthetic level than
the other lines. Stomata of Ouro negro remained partially
open during the water stress at the pollination stage (39 DAS)
and supported a positive net photosynthetic rate (A). Differences were also found between lines in intrinsic water use
efficiency (IWUE) at 39 DAS, but not at 20 DAS. The
possibility of using IWUE at the pollination stage is discussed,
in view of its use as one of the parameters for a drought
tolerance breeding program in bean lines.

Introduction
Water availability is one of the most important constraints
for plant productivity, mostly affecting the growth of leaves
and roots, stomatal conductance, photosynthesis and dry
matter accumulation (Blum 1997). Water stress tolerance is
considered a multigenic characteristic, and beans (Phaseolus
6ulgaris L.) have two main mechanisms for adapting to
water stress: stomatal control (Laffray and Louguet 1990)
and root development (Kuruvadi and Aguilera 1990).
Past research on adaptation of common beans has
demonstrated that differences in yield under water stress
were primarily due to variation in the root habit (Norman et
al. 1995) and White et al. (1990) pointed out the correlation
between gas exchange and root density in the water deficit
responses of beans. Stomatal conductance control in a photosynthetically efficient genotype can cause a decrease in leaf
transpiration, maintaining growth and yield (Ehleringer
1990).
Beans have a lower photosynthetic rate than grasses
because of a low CO2 mesophyll conductance from substomatal cavities through the cell wall, membranes and
liquid to fixation sites (von Caemmerer and Evans 1991).

Leaf anatomy and chloroplast distribution probably play a

role in this process (Nobel 1991). Roupsard et al. (1996)
showed that stomatal closure is probably the main factor
reducing CO2 concentration in the chloroplast during
drought in oak, a C3 species with a low photosynthetic rate.
However, Jones (1998) argued that the stomata play a
relatively small role (20%) in total photosynthetic limitation,
but they may play a major role in determining the difference
in assimilation rate within plants.
The production of beans can be decreased by more than
50% when water stress occurs during the pollination or
flowering stages (Norman et al. 1995), and there are differences in the effect of gas exchange on production and
adaptation among bean cultivars evaluated at the pollination stage (Bascur et al. 1985). Early stages of reproduction
are more susceptible to losses from a limited water supply
than any other stage of development in reproductive crops
(Kramer and Boyer 1995). At those times, there was a
positive correlation between net CO2 exchange and final
yield. Although the requirement for photosynthate by flowers, at this critical stage, is relatively low, a threshold level of

Abbre6iations A, net photosynthetic rate; DAS, days after sowing; gs, stomatal conductance; IWUE, intrinsic water use efficiency.

184

Physiol. Plant. 106, 1999

carbon accumulation is required to stimulate fertilization

(Wardlaw 1990).
In order to evaluate gas exchange adaptation, Osmond et
al. (1980), Cornic et al. (1983), and Jones (1985) proposed to
select drought-tolerant plants, mostly among C3 plants, by
analyzing the intrinsic water use efficiency (IWUE), i.e., the
rate of photosynthesis obtained for any given stomatal
conductance (A/gs).
The present study was carried out to evaluate A and gs at
the vegetative, pollination, flowering and grain-filling stages
and to analyze the IWUE at the vegetative and pollination
stages under water stress. The trial was conducted on lines
that showed good performance in the field, with a lesser
effect of root growth on gas exchange. Differences in root
development were lower in plants growing in pots because
the root system developed in the same volume of soil. Ismail
et al. (1994) demonstrated, with cowpea plants growing in
three pot sizes, that the differences in the carbon isotope
discrimination and water use efficiency among cultivars were
correlated with the concentration of ABA in the xylem sap,
but not with pot size. Our objective was to evaluate the use
of the IWUE at the pollination stage as one of the parameters for a breeding program to improve water stress
tolerance.

Materials and methods

Four bean lines were grown under controlled conditions of
14 h of light with a photon flux density of 360 mmol m 2
s 1 and maximum and minimum temperatures of 28 and
22C, in 5-l pots containing a 50/50 peat/vermiculite mixture. They were permanently irrigated using a controlled
system and once a week received a diluted commercial
nutrient solution containing 21.5 g l 1 N-NO3 , 63.0 g l 1
K, 17.5 g l 1 P, 9.4 g l 1 S, 8.5 g l 1 Mg, 6.0 g l 1 Ca and
micronutrients.
The selected lines were SC-90298823 (a new line for high
temperature zones developed by CNPAF-EMBRAPA), Carioca (the cultivar most commonly cultivated in Brazil), Ouro
negro (a new black seeded cultivar) and A320 (a line that
maintains high leaf water potential under drought conditions; Pimentel et al. 1991). In a first experiment, the line
Carioca was used to compare the gas exchange at different
stages, and in a second experiment, water stress effects were
studied in all four lines. In the field and in these experiments, the lines have the same growth habit, intermediate
between type II and III (type II is an upright indeterminate
habit, with an erect stem without a guide, and type III is a
bush indeterminate habit, with a prostate stem and variable
ability to climb; Grahan and Ranalli 1997), maturity at 75
days after sowing (DAS), and practically the same shoot
weight and leaf area.
In the first experiment, the line Carioca was sown at
different moments to have plants at different stages. Data
for the ontogenic study were collected on plants at 20 DAS
(vegetative stage), at 39 DAS (pollination stage), at 46 DAS
(flowering stage) and at 60 DAS (grain-filling stage). The
pots were arranged in a completely randomized design (1
genotype 4 age groups), with three replicates. The meaPhysiol. Plant. 106, 1999

surements were made on different leaves sampled on three

different plants. At all of these stages, gas exchange was
measured on the medium leaflet of the oldest and first
trifoliolate leaf and on the third and fifth trifoliolate leaves,
using an infrared gas analyzer in an open circuit (ADC
model 225-MK3; ADC, UK). The first and oldest leaf was
mature, but was not considered to be parasitic for the rest of
the plant (Wardlaw 1990). The third leaf was the youngest
fully expanded leaf with maximum photosynthate export.
The fifth leaf was at 50% of expansion and beginning to
export photoassimilates (Foyer and Galtier 1996). The
whole leaflet attached to the plant was placed in a temperature-controlled chamber with forced ventilation to obtain a
high boundary layer conductance. The chamber, fitted with
a heat-reflecting glass, was illuminated with a photon flux
density of 780 mmol m 2 s 1, and the ambient air in the
chamber was at 25C, with a vapor pressure deficit (VPD) of
0.010 mol mol 1. The air temperature was maintained by a
water jacket circuit in an aluminium-walled chamber undersurface, and the VPD was controlled by bubbling air first
through water at a temperature well above the required dew
point and then through water at the dew point temperature
for this VPD, before entering the chamber (Long and Hallgren 1993).
The second experiment was conducted to evaluate water
stress and rehydration effects on the four lines at two
growth stages. Therefore, a drying treatment was applied by
withholding water at 20 and 39 DAS and then rehydrating
for 2 days when the pre-dawn leaf water potential (Cl) was
near 1.5 MPa. The four lines were sown together and the
measurements were made first on plants at the youngest age
(20 DAS) and later on others plants at the second age (39
DAS). The pots were also laid out in a completely randomized design (4 lines 2 age groups, at 20 and 39 DAS), with
three replicates on different leaves at the same position on
three different plants. The tension in the xylem was measured with a Scholander pressure chamber in a central leaf
on the same plant in which gas exchange measurements
were made. These measurements were assumed to measure
the Cl. For beans, a Cl of 1.5 MPa is the minimum for
full recovery (Boyer 1978) and was achieved in 5 days
during the vegetative stage and 3 days during the pollination
stage. Gas exchange measurements were performed on the
middle leaflet of the youngest fully expanded leaf, which had
a maximum photoassimilate export.
Net photosynthetic rate (A) and stomatal conductance
(gs) were calculated according to von Caemmerer and Farquhar (1981). The IWUE was derived by a second-order
polynomial relation between A and gs and calculated by
dividing A by gs (Osmond et al. 1980). The values of
transpiration (E) were not shown because they were proportional to gs. Both were calculated from the difference between air humidity at the entrance and exit of the chamber.
The maximum value of E was around 2.5 mol m 2 s 1 at
20 DAS for all lines. At 39 DAS, E was 3.2 mol m 2 s 1
in SC-90298823 and around 2.0 mol m 2 s 1 for the other
lines.
The data were subjected to analysis of variance
(ANOVA), and means were compared and segregated using
the Tukey test.
185

Results
During the ontogeny of Carioca, A was lower at the vegetative stage, maximum at the pollination stage and decreased
at the flowering stage and, more dramatically, during grain
filling (Table 1). The measured values of A agree with those
presented by von Caemmerer and Evans (1991). These
variations in A were not related to equivalent changes in gs,
which were in the same range at the vegetative and pollination stages. Nevertheless, the gs values were greater than at
the flowering and grain-filling stages. Maximum gas exchange was observed at the vegetative stage in the fifth leaf
and at the pollination stage in the third leaf. At the flowering and grain-filling stages, no significant differences were
detected between the third and fifth leaves.
Under water deficit at the vegetative and pollination stages,
the Cl of line A320 was significantly higher (Fig. 1) than the
values for the other lines. Before water stress, SC-9029883 had
a significantly higher A than the other lines, both at 20 DAS
(Fig. 2a) and 39 DAS (Fig. 2b). The gs was not significantly
different from A320 and Carioca at 20 DAS (Fig. 3a), but was
significantly higher than in the other lines at 39 DAS (Fig. 3b).
The cultivar Carioca showed the lowest A before water stress
at 20 DAS. At 39 DAS, the lowest A was found in Ouro negro.
The imposition of water stress caused a decrease in A and
gs for all lines, with a reduced Cl at the two stages. However, both A and gs of line A320 reached zero at a higher Cl
at 20 DAS (Fig. 2a, Fig. 3a) and 39 DAS (Fig. 2b, Fig. 3b)
compared to the other lines. The rapid stomatal closure of
A320 was the cause of its higher Cl during the water stress
compared to the other lines (Fig. 1).
The SC-9029883 and Carioca lines showed the same
behavior with respect to A, gs and Cl at 20 and 39 DAS.
However, the cultivar Ouro negro, in which A and gs
reached zero at the same Cl value as the other lines at 20
DAS, maintained a positive A and displayed only a slight
decrease of gs at 39 DAS.
Table 1. Photosynthetic rate (A) and stomatal conductance (gs) of
the genotype Carioca, at 20 (vegetative stage), 39 (pollination
stage), 46 (flowering stage) and 60 (grain-filling stage) days after
sowing (DAS), in the first oldest leaf (1) and in the third (3) and
fifth trifoliolate leaves (5). In the columns, values followed by
different letters are significant by difference at 5% (for leaf) at each
developmental stage.
A (mmol m2 s1)

gs (mol m2 s1)

Stage

Leaf

Vegetative
(20 DAS)

0.18a

0.041a

3
5
1

4.44b
6.59c
5.27a

0.066ab
0.131b
0.019a

3
5
1

13.29c
11.99b
1.13a

0.150c
0.095b
0.030a

3
5
1

5.65b
5.01b
0.59a

0.070b
0.056b
0.019a

3
5

2.57b
2.96b

0.050b
0.049b

Pollination
(39 DAS)

Flowering
(46 DAS)

Grain filling
(60 DAS)

186

Fig. 1. Leaf water potential (Cl) on the youngest most developed

leaf of the four lines SC-9029883 ( ), Carioca (), Ouro negro
(2) and A320 ( ), during water stress and rehydration, at 20 DAS
(a) and at 39 DAS (b). (a) LSD5% =0.12 MPa. (b) LSD5% = 0.09
MPa.

The relation between A and gs was best fitted with a

second-order polynomial, and the slope described the IWUE
in the vegetative (20 DAS) and pollination (39 DAS) stages
(Fig. 4). Differences appeared among lines at the pollination, but not at the vegetative stage. At 20 DAS, A320,
Carioca and Ouro negro showed the greatest A, with a
higher gs, while SC-9029883 showed the highest A value
among lines, with a lower gs. At 39 DAS, SC-9029883 had
the highest A, but also the highest gs. A320 and Carioca
showed the best IWUE at the pollination stage, having a
high A with low gs (Fig. 4b) and, consequently, E.
The values of IWUE during water stress were the same
for all lines at 20 DAS (Table 2), except on the fourth day
of water stress, when Ouro negro showed the highest value.
However, for plants at pollination stage (39 DAS), there
were significant differences among lines, and the highest
IWUE values during water stress were obtained for Carioca
and Ouro negro (Table 2).

Discussion
Carbohydrate accumulation during the pollination stage,
when the plants show their maximum A, will be of primary
Physiol. Plant. 106, 1999

importance for the next stages in the growth of pods and

seeds (Table 1). If a climatic constraint occurs at this
stage, bean production will be affected dramatically (Bascur et al. 1985). Because most (90%) of the pods that
reach maturity were formed from early flowers, the reduction of photosynthesis induced by drought at this stage
will cause the abscission of these flowers and thus a lower
productivity (Norman et al. 1995).
When a water deficit was applied, the gas exchange
reached zero faster in plants at 39 DAS (3 days) than in
plants at 20 DAS (5 days), despite the same Cl values
(Fig. 1). This was caused by the greater total leaf area at
39 DAS compared to 20 DAS.
Under water stress, line A320 closed stomata more and
maintained Cl higher than the other lines in the two
stages at 20 and 39 DAS (Fig. 3), as also shown by
Pimentel et al. (1991). This is a desirable drought avoidance mechanism in beans (Subbarao et al. 1995), but the
reduction of A caused by stomatal closure probably made
the plant use its own reserves. If the water deficit was not
prolonged, there would be little yield loss.
There can be non-uniform stomatal closure under water
stress, called patchiness, which causes variations of A and
CO2 concentration in the chloroplasts, but Cheeseman
(1991) showed that stomatal patchiness cannot account for
changes in the relation between A and intercellular CO2

Fig. 3. Stomatal conductance (gs) and leaf water potential (Cl)

relations on the youngest fully expanded leaf of the four lines
SC-9029883 ( ), Carioca (), Ouro negro (2) and A320 ( ),
during water stress at 20 DAS (a) and at 39 DAS (b). (a) LSD5% =
0.04 mol m 2 s 1. (b) LSD5% =0.1 mol m 2 s 1.

Fig. 2. Photosynthetic rate (A) and leaf water potential C1 relations

on the youngest fully expanded leaf of the four lines, SC-9029883
( ), Carioca (), Ouro negro (2) and A320 ( ), during water
stress at 20 DAS (a) and at 39 DAS (b). (a) LSD5% = 1.2 mmol m 2
s 1. (b) LSD5% = 2.5 mmol m 2 s 1.
Physiol. Plant. 106, 1999

concentration. One factor reducing CO2 availability in the

chloroplast during drought might be stomatal closure
(Roupsard et al. 1996), but loss of photosynthetic biochemical activity also seems to be involved (Lauer and
Boyer 1992). In our study, besides a rapid stomatal closure (Fig. 3) and a consequent reduction in water loss,
line A320 showed a lower stomatal limitation of A, as
pointed out by Jones (1998), than SC-90298883 and Ouro
negro under water deficit at the pollination stage (Fig.
4b).
Line SC-9029883 can be selected for cropping with irrigation, where it can keep a high gs and A, in order to obtain
the highest yield. The response of Carioca, with values of A
close to those of SC-9029883 when well hydrated and showing a high IWUE curve slope (Fig. 4b) and calculated values
(Table 2), explained the wide adaptation of this cultivar
planted in contrasting environments. Line A320 showed the
same IWUE slope as Carioca, but its stomata closed earlier.
Ouro negro, in which the stomata remained slightly open
under water stress at the pollination stage, causing a decrease in Cl (Fig. 1b), might present a better protoplasmic
tolerance than the other lines. However, more studies are
needed to confirm the response of A320 and Ouro negro in
187

Table 2. Intrinsic water use efficiency (IWUE), in mmol CO2 mol1

H2O, of the four lines during 5 days of water stress at 20 days after
sowing (DAS) and during 3 days of water stress at 39 DAS. In the
lines, values followed by different letters are significant by difference at 5% (for genotype) at each day of water stress.

the field for their use in a drought tolerance breeding

program.
Drought tolerance is a multigenic mechanism, and characterization of a drought-tolerant ideotype has not yet
been achieved. Therefore, numerous constitutive traits
must be considered for adaptation to drought (Blum
1997). The most used trait for breeding for drought tolerance in beans has been root growth, but several other
physiological, morphological and phenological characteristics are required for desiccation tolerance. The IWUE at
the pollination stage could be one of the shoot parameters
to be used by plant breeders. Nevertheless, stomatal control is not independent of root activity, and genotypes
with increased root growth under drought could maintain
large stomatal conductance, decreasing the IWUE (White
et al. 1990). Thus, this parameter should be evaluated in
the field together with other traits for drought adaptation
to confirm our results.

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Fig. 4. Intrinsic water use efficiency (IWUE), i.e., the relation

between the photosynthetic rate (A) and stomatal conductance (gs)
on the youngest fully expanded leaf of the four lines, during water
stress at 20 DAS (a) and at 39 DAS (b). (a) SC-9029883 ( ),
0.59 + 77.56x 122.89x 2, r 2 = 0.71; Carioca (), 0.19 +
100.92x253.45x 2, r 2 = 0.87; Ouro negro (2), 0.30 +96.66x
263.59x 2, r 2 = 0.85; A320 ( ), 0.32+ 91.75 192.17x 2,
r 2 =0.97. (b) SC-9029883, 0.22+ 54.25x 34.99x 2, r 2 =0.99;
Carioca, 0.44+ 151.54x416.05x 2, r 2 = 0.89; Ouro negro,
0.90+ 99.86x 353.29x 2, r 2 = 0.99; A320, 0.13+88.10x+75.79x 2,
r 2 =0.99.

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Days
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1
2
3
4
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At 39 DAS
1
2
3

SC-9029883

Carioca

Ouro negro

A320

55a
45a
71a
90ab
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39a
67a
47a
113bc
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37a
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92a
195c
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45a
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43a
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77b
15a
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Edited by I. Terashima
Physiol. Plant. 106, 1999

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