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

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PHYSIOLOGIA PLANTARUM 106: 184–189. 1999 Copyright © Physiologia Plantarum 1999 ISSN 0031-9317 Printed in Ireland — all rights resered

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

drought tolerance selection in Phaseolus ulgaris

C. Pimentela,*, D. Laffrayb and P. Louguetb

aDepto. de Fitotecnia, I .A., Uni . Fed . Rural do Rio de Janeiro, Itaguaı́ , 23851-970 , RJ , Brazil bLab. de Physiologie Végétale, Faculté des Sciences , Uni . Paris-Val de Marne, A. du Gén. de Gaulle, F -94 010 , Créteil -Cedex, France

*Corresponding author, e-mail : [email protected]

Received 25 November 1998; revised 22 February 1999

or 39 DAS. The cultivar SC-90298823 had greater stomatalGenotype differences in gas exchange during ontogeny and

conductance at 39 DAS and a higher photosynthetic level thanwater stress responses at the vegetative and pollination stages

the other lines. Stomata of Ouro negro remained partiallywere evaluated in four lines of Phaseolus ulgaris L. In the

open during the water stress at the pollination stage (39 DAS)cultivar Carioca, net photosynthetic rate (A) and stomatal

and supported a positive net photosynthetic rate (A). Differ-conductance ( g s) were lower at the vegetative stage (20 daysafter sowing [DAS]) and maximum at the pollination stage (39 ences were also found between lines in intrinsic water use

efficiency (IWUE) at 39 DAS, but not at 20 DAS. TheDAS), followed by a decrease at the flowering stage (46 DAS)

and a dramatic fall at the grain-filling stage (60 DAS). possibility of using IWUE at the pollination stage is discussed,

in view of its use as one of the parameters for a droughtAmong the lines studied, the stomata of A320 closed faster

than those of the other lines when water stress occurred at 20 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

ulgaris 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 pho-

tosynthetically 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 sub-

stomatal 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 differ-

ences in the effect of gas exchange on production and

adaptation among bean cultivars evaluated at the pollina-

tion 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 flow-

ers, at this critical stage, is relatively low, a threshold level of

Abbreiations – A, net photosynthetic rate; DAS, days after sowing; g s, stomatal conductance; IWUE, intrinsic water use efficiency.

Physiol. Plant. 106, 1999184


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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/ g s).

The present study was carried out to evaluate A and g s 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 linesthat 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 parame-

ters 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 mol m−2

s−1 and maximum and minimum temperatures of 28 and

22°C, in 5-l pots containing a 50/50 peat/vermiculite mix-

ture. 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), Car-ioca (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 condi-

tions; 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 experi-

ments, 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 mea-

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 tempera-ture-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 mol m−2 s−1, and the ambient air in the

chamber was at 25°C, 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 under-

surface, 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 Häll-

gren 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 ( l) 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 random-

ized 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 mea-

sured 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 measurethe l. For beans, a l 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

( g s) were calculated according to von Caemmerer and Far-

quhar (1981). The IWUE was derived by a second-order

polynomial relation between A and g s and calculated by

dividing A by g s (Osmond et al. 1980). The values of

transpiration (E ) were not shown because they were propor-

tional to g s. Both were calculated from the difference be-

tween 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.

Physiol. Plant. 106, 1999 185


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Results

During the ontogeny of Carioca, A was lower at the vegeta-

tive 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 g s,

which were in the same range at the vegetative and pollina-

tion stages. Nevertheless, the g s values were greater than at

the flowering and grain-filling stages. Maximum gas ex-

change was observed at the vegetative stage in the fifth leaf and at the pollination stage in the third leaf. At the flower-

ing 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 l 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 g s 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

at20 DAS.At 39 DAS, the lowest A was found in Ouro negro.

The imposition of water stress caused a decrease in A and

g s for all lines, with a reduced l at the two stages. How-ever, both A and g s of line A320 reached zero at a higher lat 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 l 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, g s and l at 20 and 39 DAS.

However, the cultivar Ouro negro, in which A and g sreached zero at the same l value as the other lines at 20

DAS, maintained a positive A and displayed only a slight

decrease of g s at 39 DAS.

Fig. 1. Leaf water potential ( l) on the youngest most developedleaf of the four lines SC-9029883 (), Carioca (), Ouro negro() and A320 (), during water stress and rehydration, at 20 DAS(a) and at 39 DAS (b). (a) LSD5%=0.12 MPa. (b) LSD5%=0.09MPa.

The relation between A and g s 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 pollina-

tion, but not at the vegetative stage. At 20 DAS, A320,

Carioca and Ouro negro showed the greatest A, with a

higher g s, while SC-9029883 showed the highest A value

among lines, with a lower g s. At 39 DAS, SC-9029883 had

the highest A, but also the highest g s. A320 and Carioca

showed the best IWUE at the pollination stage, having a

high A with low g s (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 highestIWUE 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

Table 1. Photosynthetic rate (A) and stomatal conductance ( g s) of the genotype Carioca, at 20 (vegetative stage), 39 (pollinationstage), 46 (flowering stage) and 60 (grain-filling stage) days aftersowing (DAS), in the first oldest leaf (1) and in the third (3) andfifth trifoliolate leaves (5). In the columns, values followed bydifferent letters are significant by difference at 5% (for leaf) at eachdevelopmental stage.

Stage Leaf A (mol m−2 s−1) g s (mol m−2 s−1)

Vegetative 1 0.041a0.18a(20 DAS)

0.066ab3 4.44b5 0.131b6.59c

Pollination 1 5.27a 0.019a(39 DAS)

3 13.29c 0.150c

5 11.99b 0.095bFlowering 0.030a1 1.13a

(46 DAS)3 5.65b 0.070b

0.056b5.01b5

Grain filling 0.019a1 0.59a(60 DAS)

0.050b2.57b32.96b5 0.049b



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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 (Bas-

cur et al. 1985). Because most (90%) of the pods that

reach maturity were formed from early flowers, the reduc-

tion 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 l values

(Fig. 1). This was caused by the greater total leaf area at39 DAS compared to 20 DAS.

Under water stress, line A320 closed stomata more and

maintained l 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 avoid-

ance 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 ( g s) and leaf water potential ( l)relations on the youngest fully expanded leaf of the four linesSC-9029883 (), Carioca (), Ouro negro () 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 1 relationson the youngest fully expanded leaf of the four lines, SC-9029883(), Carioca (), Ouro negro () and A320 (), during waterstress at 20 DAS (a) and at 39 DAS (b). (a) LSD5%=1.2 mol m

−2

s−1. (b) LSD5%=2.5 mol m−2 s−1.

concentration. One factor reducing CO2 availability in the

chloroplast during drought might be stomatal closure

(Roupsard et al. 1996), but loss of photosynthetic bio-

chemical activity also seems to be involved (Lauer and

Boyer 1992). In our study, besides a rapid stomatal clo-

sure (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 irriga-

tion, where it can keep a high g s 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 show-

ing 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 de-

crease in l (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



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the field for their use in a drought tolerance breeding

program.

Drought tolerance is a multigenic mechanism, and char-

acterization 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 toler-

ance in beans has been root growth, but several other

physiological, morphological and phenological characteris-

tics 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 con-trol 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.

Table 2. Intrinsic water use efficiency (IWUE), in mol CO2 mol−1

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

A320Days SC-9029883 Carioca Ouro negro

At 20 DAS45a1 55a 39a 37a39a51a45a2 67a

92a3 43a71a 47a113bc 195c 0a4 90ab

0a5 0a0a 0a

At 39 DAS1 60a 77b43a 142b

83c 77c 15a2 41b0a3 74b0a0a

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Fig. 4. Intrinsic water use efficiency (IWUE), i.e., the relationbetween the photosynthetic rate (A) and stomatal conductance ( g s)on the youngest fully expanded leaf of the four lines, during waterstress at 20 DAS (a) and at 39 DAS (b). (a) SC-9029883 (),0.59+77.56x−122.89x2, r2=0.71; Carioca (), −0.19+100.92x−253.45x2, r2=0.87; Ouro negro (), 0.30+96.66x−263.59x2, r2=0.85; A320 (), −0.32+91.75−192.17x2,r2=0.97. (b) SC-9029883, −0.22+54.25x−34.99x2, r2=0.99;Carioca, −0.44+151.54x−416.05x2, r2=0.89; Ouro negro,0.90+99.86x−353.29x2, r 2=0.99; A320, 0.13+88.10x+75.79x2,r2=0.99.



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

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