Environmental and Experimental Botany 51 (2004) 45–56

Photosynthetic gas exchange, chlorophyll fluorescence and some

associated metabolic changes in cowpea (Vigna unguiculata)
during water stress and recovery
R.P. Souza a , E.C. Machado a,∗ , J.A.B. Silva b , A.M.M.A. Lagôa a , J.A.G. Silveira c
a Centro de Ecofisiologia e Biofı́sica, Instituto Agronômico de Campinas, CP 28, 13001-970 Campinas, SP, Brazil
bDepartamento de Fisiologia Vegetal, Universidade Estadual de Campinas, CP 6109, 13083-970 Campinas, SP, Brazil
c Departamento de Bioquı́mica e Biologia Molecular, Universidade Federal do Ceará, CP 6020, 60451-970 Fortaleza, CE, Brazil

Received 22 October 2002; received in revised form 15 May 2003; accepted 19 May 2003

Abstract

The responses of photosynthetic gas exchange and chlorophyll fluorescence along with changes in carbohydrate and proline
levels were studied in cowpea (Vigna unguiculata) during water stress and recovery. Three experiments were conducted under
greenhouse and laboratory conditions. Decreased CO2 assimilation rates during water stress were largely dependent on stomatal
closure, which reduced available internal CO2 and restricted water loss through transpiration. During the initial phase of stress,
photochemical activity was not affected, as revealed by lack of alterations in fluorescence parameters associated with photosystem
II (PSII) activity. Development of non-radiative energy dissipation mechanisms was evidenced during stress by increases in
non-photochemical quenching and decreases in efficiency of excitation capture by open centers. At an advanced phase of stress,
a down-regulation of PSII activity was observed along with some impairment of photochemical activity, as revealed by decreases
in the maximum quantum yield of PSII (Fv/Fm). However, this impairment did not limit the overall photosynthetic process, since
assimilation rates recovered, upon rewatering, independent of the still present decreased Fv/Fm values. Complete recovery of all
gas exchange and fluorescence parameters occurred 3 days after rewatering. However, on the first day after water stress relief,
assimilation rates only partially recovered in spite of the availability of internal CO2 , suggesting some non-stomatal limitation
of photosynthesis. Accordingly, the downregulation of PSII activity observed during stress persisted at this time. Our results
on carbohydrate metabolic changes revealed an accumulation of soluble sugars in water-stressed leaves, which also persisted
for 1 day after rewatering. This finding suggest a transient end-product inhibition of photosynthesis, contributing to a minor
non-stomatal limitation during stress and initial phase of recovery. Increases in proline level were small and their onset was
delayed after stress imposition, so that it may rather be a consequence and not a stress-induced beneficial response.
© 2003 Elsevier B.V. All rights reserved.

Keywords: Carbohydrates; Chlorophyll fluorescence; Gas exchange; Photosynthesis; Proline; Water stress

1. Introduction

∗ Corresponding author. Tel.: +55-19-3231-5422;

Water availability is an important factor affecting
fax: +55-19-3231-4943. plant growth and yield, mainly in arid and semi-arid
E-mail address: caruso@iac.sp.gov.br (E.C. Machado). regions, where plants are often subjected to periods of

0098-8472/$ – see front matter © 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S0098-8472(03)00059-5
46 R.P. Souza et al. / Environmental and Experimental Botany 51 (2004) 45–56
drought. The occurrence of morphological and phys-
iological responses, which may lead to some adapta-
tion to drought stress, may vary considerably among
species. In general, strategies of drought-avoidance or
drought-tolerance can be recognized, both involving
diverse plant mechanisms that provide the plants the
ability to respond and survive drought (Levitt, 1980).
Cowpea [Vigna unguiculata (L.) Walp.] is an im-
portant crop largely grown in warm and hot regions of
Africa, Asia and the Americas and is often regarded as
being well-adapted to high temperatures and drought
compared with other crop species (Ehlers and Hall,
1997). Drought adaptation in cowpea has been related
to the minimization of water losses by the control
of stomatal aperture (de Carvalho et al., 1998). Ac-
cordingly, studies have demonstrated that cowpea is
able to maintain high leaf water potential or high leaf
relative water content during water stress (Bates and
Hall, 1981; Shackel and Hall, 1983; Küppers et al.,
1988; de Carvalho et al., 1998), thus avoiding tis-
sue dehydration. However, this strategy, by involving
stomatal closure, may lead to decreases in CO2 as-
similation (Chaves, 1991) and hence in growth and
yield.
Stomatal regulation of photosynthesis during water
shortage has been well documented (Chaves, 1991). At
least under mild drought conditions, it has been shown
that stomata play the dominant role in controlling the
decline of net CO2 uptake, by leading to decreases
in leaf internal CO2 concentrations (Cornic and Bri-
antais, 1991; Cornic, 2000). Nonetheless, the limita-
tions to CO2 assimilation imposed by stomatal closure
may promote an imbalance between photochemical
activity at photosystem II (PSII) and electron require-
ment for photosynthesis, leading to an overexcitation
and subsequent photoinhibitory damage of PSII reac-
tion centers (Krause, 1988; Long et al., 1994). In this
way, damages to the photosynthetic machinery may
eventually occur, imposing an additional non-stomatal
limitation to the process. Investigations based on as-
sessments of chlorophyll a fluorescence have shown
that PSII is quite resistant to water deficits, being ei-
ther unaffected (Lu and Zhang, 1998; Shangguan et al.,
2000) or affected only under very severe drought con-
ditions (Saccardy et al., 1998). In fact, photodamage
may be prevented through processes of thermal de-
activation, downregulating PSII photochemistry, in a
so-called dynamic photoinhibition (Osmond, 1994),
that brings the electron transport capacity into balance
with carbon metabolism (Epron et al., 1992). Under
natural conditions, however, where high light and high
temperatures are often associated with water deficits,
these or other protective mechanisms (Krause, 1988)
may be insufficient to dissipate the excess of excita-
tion energy and a chronic photoinhibition, character-
ized by a slow-reversible reduction in the photosyn-
thetic efficiency (Osmond, 1994), may result.
The maintenance of high cell water potentials in
species with a drought-avoidance strategy avoids di-
rect metabolic injuries (Levitt, 1980) and minimizes
the necessity of metabolic adjustments, which are usu-
ally found in drought-tolerant species (Levitt, 1980;
Shackel and Hall, 1983). In these latter species, a net
increase in solute concentrations, known as osmotic
adjustment, allows, by promoting lowering of the cell
osmotic potential, the maintenance of leaf turgor un-
der water-limited conditions, with beneficial effects
over turgor-dependent processes, such as cell enlarge-
ment and stomatal opening (Morgan, 1984). Accord-
ing to its drought-avoidance characteristics, cowpea
has been shown to present no osmotic adjustment un-
der drought conditions (Shackel and Hall, 1983; Petrie
and Hall, 1992). Nonetheless, the stomatal and also
the non-stomatal, if existent, limitation of photosyn-
thesis may lead to a drought-induced starvation injury
(Levitt, 1980) and consequently also metabolic alter-
ations.
The aim of this study was to evaluate the responses
of photosynthesis to water stress in cowpea plants,
both in terms of CO2 assimilation, as measured by
leaf gas exchanges, and of the functionality of the
photosynthetic apparatus, as assessed by chlorophyll
a fluorescence measurements. Additionally, carbohy-
drate and proline content was simultaneously analyzed
in order to detect possible metabolic alterations in
water-stressed cowpea leaves. The underlying hypoth-
esis is that photosynthesis in water-stressed cowpea
plants is limited by stomatal and non-stomatal factors,
both contributing to the occurrence of metabolic alter-
ations at the leaf level.
2. Material and methods
The study was carried out with cowpea plants [V.
unguiculata (L.) Walp. cultivar Vita 7] cultivated
 R.P. Souza et al. / Environmental and Experimental Botany 51 (2004) 45–56
from seeds on 2.8 l pots (one plant per pot) contain-
ing silica:vermiculite 1:2 in a greenhouse situated at
the Experimental Station of Instituto Agronômico de
Campinas, SP, Brazil (22◦ 54 S and 47◦ 05 W, 674 m
altitude). Plants received 250 ml of Hoagland and
Arnon’s (Hoagland and Arnon, 1938) nutrient solu-
tion twice a week, being irrigated with distilled water
in the remaining days. Three water stress experiments,
hereafter named experiment A, B and C, were carried
out with 28–35 days old plants. Four or five plants per
treatment (control and water-stressed) were sampled
for the measurements. In all experiments, water stress
was imposed by withholding water.
2.1. Experiment A
After water stress imposition, gas-exchange and
chlorophyll a fluorescence measurements were
performed under controlled conditions in a growth
chamber (28 ◦ C, 70% RH, 270 ␮mol m−2 s−1 ), to
which the plants were daily transferred at 07:00 h.
These measurements were daily repeated until the CO2
assimilation rates approached zero, which was con-
sidered the point of maximum stress. The plants were
then rewatered and the measurements were continued
until recovery of the assimilation rates to the control
level. A different set of both control and water-stressed
plants was used each day, since the photosynthe-
sis measurements were followed by destructive leaf
harvests.
Gas-exchange measurements were done with a
portable photosynthesis system (LCi, ADC, Herts,
UK), using the youngest completely expanded leaf of
each plant, under a saturating photosynthetic photon
flux density (PPFD) of 1800 ␮mol m−2 s−1 provided
by an external halogen lamp. Fluorescence mea-
surements were done with a modulated fluorometer
(FMS 1, Hansatech, Norfolk, UK) using the satura-
tion pulse method (Schreiber et al., 1994), prior to
the gas-exchange measurements on the same sampled
leaves. Chlorophyll fluorescence measurements were
initially taken on dark-adapted leaves for 30 min and
then for the same leaves after a 30 min acclimation pe-
riod to the growth chamber PPFD. Immediately before
measurements under light, leaves received a 10 s expo-
sure of an actinic light of 270 ␮mol m−2 s−1 provided
by the fluorometer’s fiber optic. The following param-
eters were assessed (Schreiber et al., 1994): the maxi-
47
mum quantum yield of PSII [Fv/Fm = (Fm−Fo)/Fm],
the excitation capture efficiency of open centers
[Fv /Fm = (Fm −Fo )/Fm ], the effective quantum
yield of PSII [F/Fm = (Fm −Fs)/Fm ], the apparent
electron transport rate (ETR = F/Fm ×PPFD×0.5
×0.84) and the coefficients of photochemical quench-
ing [qP = (Fm −Fs)/(Fm −Fo )] and non-photochemi-
cal quenching [NPQ = (Fm−Fm )/Fm ], where Fm
and Fo are, respectively, maximum and minimum
fluorescence of dark-adapted leaves, Fm and Fs are,
respectively, maximum and steady-state fluorescence
in the light-adapted state and Fo is minimum fluo-
rescence after far-red illumination of the previously
exposed leaves. For the calculation of ETR, 0.5 was
assumed as the fraction of the excitation energy
distributed to PSII and 0.84 as the fractional light
absorption of the leaf.
Subsequent to these measurements, the sampled
leaves were collected in liquid N2 , lyophilized and
later submitted to carbohydrate and proline analysis.
The extraction was performed in methanol:chloro-
form:water (12:5:3 v/v/v), followed by the separation
of the aqueous fraction (Bieleski and Turner, 1966)
containing the soluble compounds. The residue was
extracted with a 30% percloric acid solution for ob-
tainment of the hydrolyzed starch fraction (Yemm
and Willis, 1954). Quantification of total soluble
sugars and starch were done using anthrone method
(McReady et al., 1950). Sucrose determinations were
also done by anthrone method, being carried out
according to van Handel (1968) with modifications.
Reducing sugars were estimated by the difference be-
tween total soluble sugars and sucrose. Proline quan-
tification was done according to Rena and Masciotti
(1976).
2.2. Experiment B
After water stress imposition, gas-exchange and
chlorophyll a fluorescence measurements were per-
formed as in experiment A. Here, however, a same set
of plants (five control and five water-stressed plants)
was accompanied during the whole water stress and
recovery period. So, after the daily measurements
in the growth chamber, the plants were brought
back to the greenhouse. This experiment was carried
out in a greenhouse at higher air relative humidity
and lower temperatures as compared with experiment
48 R.P. Souza et al. / Environmental and Experimental Botany 51 (2004) 45–56
A, thus conditioning a slower rate of stress imposi-
tion.
A quenching analysis during photosynthetic induc-
tion in previously dark-adapted leaves was performed,
according to Bolhàr-Nordenkampf and Öquist (1993),
in plants at the maximum stress and after recovery.
The measurements were done under a continuous ac-
tinic light (500 ␮mol m−2 s−1 provided by the fluo-
rometer’s fiber optic), by means of application of 20
sequential saturating light pulses at 15 s intervals, fol-
lowed by a final far-red pulse. Following the preced-
ing measurements, at the point of maximum stress and
after recovery, measurements of leaf water potential
were also carried out, using a pressure chamber (Plant
Water Status Console 3005, Soil Moisture, Santa Bar-
bara, CA, USA).
2.3. Experiment C
In this experiment, gas-exchange and chlorophyll
a fluorescence measurements were performed during
the water stress period in plants transferred outdoors
under natural irradiance and prevalent environmen-
tal conditions. In this case, a PAM-2000 fluorometer
(Walz, Effeltrich, Germany) was used for fluorescence
measurements. These measurements were taken for
both exposed and 30 min dark-adapted leaves. The
same fluorescence parameters described in experiment
A were assessed.
2.4. Experimental design and statistical
analyses
In all the experiments, a completely randomized
experimental design was used. Control and water
stress treatments were statistically compared. Prior to
statistical analysis, data were tested for homogeneity
of variances and for normal distribution by Bartlett’s
test and Kolmogorov–Smirnov test as modified by
Lilliefors (1967). Where necessary, log or square-root
transformation of data was used. In all cases where the
above assumptions of parametric analysis were met
or achieved by means of data transformation, com-
parison of means were done by the Student’s t-test.
Otherwise, data were analyzed by the non-parametric
Wilcoxon’s test. Both tests were done at the 5% level
of probability.
3. Results
Water-stressed cowpea plants presented reductions
in CO2 assimilation rates paralleled by reductions in
stomatal conductance and transpiration rates. Under
the controlled conditions of measurements in experi-
ment A, values began to decline, in relation to control
plants, 2 or 3 days after the suspension of irrigation.
The minimum values were attained on the sixth day
from water withholding, when the plants were rewa-
tered (Fig. 1). Following the reduction in stomatal
conductance, declines in the intercellular CO2 con-
centration were also observed; at the maximum stress,
the intercellular CO2 decreased to around 140 ␮mol
mol−1 , while for control plants it was around 250
␮mol mol−1 (Fig. 1). Upon rewatering, the recovery
of plants was complete within 2 days (Fig. 1). In
experiment B, which was characterized by a slower
imposition of water stress, the day of maximum stress
was attained only on the 11th day after withholding
water (results not shown). Nonetheless, the general
response pattern was similar.
Decreases of leaf water potential in water-stressed
plants were not very marked, although the stressed
plants reached, at maximum stress, a significant
(P < 0.05) lower leaf water potential (mean value of
−1.27 MPa) in relation to controls (mean value of
−0.71 MPa). After rewatering and a recovery period,
leaf water potential of water-stressed plants returned
to comparable control levels.
Daily recordings of fluorescence parameters in
experiment A showed that the maximum quantum
yield of PSII, as revealed by Fv/Fm ratio, was only
slightly affected after the fifth day from water with-
holding (Fig. 2A), being paralleled by increases in
the minimum values of basal fluorescence (Fo) (re-
sults not shown). This slight decrease of maximum
quantum yield in stressed plants was confirmed in
experiment B, in which the Fv/Fm ratio was signifi-
cantly (P < 0.05) lower at the maximum water stress
in relation to controls (Table 1). A tendency of higher
Fo values and lower Fm (maximum fluorescence)
was also observed in water-stressed plants (Table 1).
After rewatering and a recovery period, Fv/Fm values
of water-stressed plants returned back to the control
level (Table 1). However, the daily recordings in ex-
periment A showed that, despite the final complete
recovery, the decreases of Fv/Fm values persisted or
 R.P. Souza et al. / Environmental and Experimental Botany 51 (2004) 45–56 49

Fig. 1. Gas-exchange responses of cowpea plants to an imposed water stress period and recovery upon rewatering (experiment A). A, CO2
assimilation rates; gs, stomatal conductance; E, transpiration rates; Ci, intercellular CO2 concentration. Arrow indicates rewatering. Values
are means ( ± S.E.) for four plants.

even became more marked on the first and second day
subsequent to rewatering. Here again, Fo increases
paralleled the Fv/Fm decreases (results not shown).
The effective quantum yield of PSII (Fig. 2C) and
the apparent electron transport rate (Fig. 2D) also de-
clined from the fifth day of water withholding, while
significant differences (P < 0.05) in photochemical
quenching between control and stressed plants were
only observed on the day of maximum stress (Fig. 2E).
Increases in non-photochemical quenching (Fig. 2F)
and decreases in the excitation capture efficiency of
open centers (Fig. 2B) were observed from the third
day of the beginning of the water stress treatment. The
alterations in all these parameters, except photochem-
ical quenching, here again, persisted after rewatering
for at least 1 day. Nonetheless, the recovery was

Table 1
Fluorescence data presented for cowpea plants at maximum water stress and after recovery
Maximum stress After recovery

Control Water-stressed Control Water-stressed

Fo 623.2 ( ± 8.1) 638.6 ( ± 15.5) 441.6 ( ± 12.4) 433.0 ( ± 7.8)

Fm 4014.0 ( ± 38.6) 3934.0 ( ± 123.2) 2617.0 ( ± 64.2) 2572.2 ( ± 31.3)
Fv/Fm 0.845 ( ± 0.002) 0.838 ( ± 0.002) 0.831 ( ± 0.004) 0.832 ( ± 0.002)
Maximum stress corresponds to the 11th day after water withholding in experiment B, in which a slower water stress imposition occurred;
recovery was assessed after 5 days from rewatering. Fo, minimum fluorescence values; Fm, maximum fluorescence values; Fv/Fm, PSII
maximum quantum yield. Values are means ( ± S.E.) for five plants.
50 R.P. Souza et al. / Environmental and Experimental Botany 51 (2004) 45–56

Fig. 2. Fluorescence parameters responses of cowpea plants to an imposed water stress period and recovery upon rewatering (experiment
A). Fv/Fm, PSII maximum quantum yield; Fv /Fm , excitation capture efficiency of open centers; F/Fm , PSII effective quantum yield;
ETR, apparent electron transport rate; qP, photochemical quenching coefficient; NPQ, non-photochemical quenching coefficient. Arrow
indicates rewatering. Values are means ( ± S.E.) for four plants.

complete within 2 or 3 days (Fig. 2). Quenching anal-
yses during photosynthetic induction, performed at
the maximal stress in experiment B, showed clearly
the development of a higher non-photochemical
quenching in water-stressed plants, accompanied by
a lower development of photochemical quenching
and lower values of effective quantum yield of PSII
(Fig. 3A, B). At the end of the induction curve, all
the three parameters significantly differed (P < 0.05)
between control and stressed plants. After rewater-
ing and recovery, the photochemical quenching and
the PSII effective quantum yield of stressed plants
returned back to control values (Fig. 3C, D). The
development of non-photochemical quenching in
water-stressed plants after recovery was even lower
than in controls (Fig. 3C, D), with final values, at
 R.P. Souza et al. / Environmental and Experimental Botany 51 (2004) 45–56 51

Fig. 3. Quenching analyses of chlorophyll a fluorescence during photosynthetic induction in control and water-stressed cowpea plants.
Measurements performed at the maximum stress (A, B) and after recovery (C, D). Maximum stress corresponds to the 11th day after water
withholding in experiment B, in which a slower water stress imposition occurred; recovery was assessed after 5 days from rewatering.
F/Fm , PSII effective quantum yield; qP, photochemical quenching coefficient; NPQ, non-photochemical quenching coefficient. Values
are means ( ± S.E.) for five plants.

the end of the induction curve, differing significantly
(P < 0.05).
Measurements of gas exchanges and fluorescence
parameters under natural environmental conditions of
high irradiance, high temperature and low relative hu-
midity, in experiment C, revealed an earlier response
to water stress than the observed in the preceding
experiments (Table 2). On the second day after water
suspension, great and significant (P < 0.05) reductions
in assimilation and transpiration rates, stomatal con-
ductance and intercellular CO2 concentrations were
observed. In relation to fluorescence parameters, sig-
nificant differences (P < 0.05) were observed between
control and stressed plants only for the electron trans-
port rates and photochemical quenching, although a
tendency of increase in non-photochemical quench-
ing and of decrease in the other parameters—PSII
maximum and effective quantum yield and excitation
capture efficiency—was also observed (Table 2).
Leaf carbohydrate determinations during the stress
and the recovery period revealed that the content of
total soluble sugars almost doubled at the maximum
stress (Fig. 4A). This accumulation response was de-
tected from the third day of the beginning of water
withholding in experiment A. Two days after rewater-
ing, there was a decrease in the content of total soluble
sugars (Fig. 4A). The alterations in total soluble sugars
matched the alterations in reducing sugars (Fig. 4C).
Leaf sucrose levels were low and did not vary sig-
nificantly during the experiment (Fig. 4B). On the
other hand, leaf starch content was high and declined
upon water stress. Starch values in water-stressed
plants did not return to the control level after re-
watering (Fig. 4D). Proline content in leaves varied
only slightly during stress imposition and recovery.
Differences (P < 0.05) between control and stressed
plants were only observed around the maximum stress
(Fig. 5).
52 R.P. Souza et al. / Environmental and Experimental Botany 51 (2004) 45–56

Table 2
Photosynthetic parameters in control and water-stressed cowpea plants under natural environmental conditions, around 15:00 h, on the
second day after water withholding in experiment C (average PPFD, temperature and relative humidity of 1700 ␮mol m−2 s−1 ; 37 ◦ C and
53%, respectively)
Parameters Treatments Percentage
of control
Control Water-stressed

A (␮mol m−2 s−1 ) 16.99 (± 0.94) 4.00 (± 0.10) 23.5

gs (mol m−2 s−1 ) 0.32 (± 0.00) 0.06 (± 0.01) 18.8
E (mmol m−2 s−1 ) 6.02 (± 1.16) 1.86 (± 0.13) 30.9
Ci (␮mol mol−1 ) 260.5 (± 5.38) 201.0 (± 7.90) 77.2
Fv/Fm 0.732 (± 0.005) 0.663 (± 0.053) 90.6
Fv /Fm 0.568 (± 0.037) 0.484 (± 0.074) 85.2
F/Fm 0.504 (± 0.030) 0.362 (± 0.076) 71.8
ETR 95.92 (± 10.19) 69.02 (± 11.16) 72.0
qP 0.888 (± 0.022) 0.726 (± 0.045) 81.8
NPQ 4.311 (± 0.692) 5.296 (± 1.120) 122.9
A, CO2 assimilation rates; gs, stomatal conductance; E, transpiration rates; Ci, intercellular CO2 concentration; Fv/Fm, PSII maximum
quantum yield; Fv /Fm , excitation capture efficiency of open centers; F/Fm , PSII effective quantum yield; ETR, apparent electron transport
rate; qP, photochemical quenching coefficient; NPQ, non-photochemical quenching coefficient. Values are means (± S.E.) for five plants.

Fig. 4. Leaf carbohydrate content in cowpea plants submitted to an imposed water stress period and recovery upon rewatering (experiment
A). Arrow indicates rewatering. Values are means (± S.E.) for four plants.
 R.P. Souza et al. / Environmental and Experimental Botany 51 (2004) 45–56
Fig. 5. Leaf proline content in cowpea plants submitted to an
imposed water stress period and recovery upon rewatering (exper-
iment A). Arrow indicates rewatering. Values are means (± S.E.)
for four plants.
4. Discussion
The reductions in CO2 assimilation rates in water-
stressed cowpea plants were largely dependent on
stomatal closure, which decreased available internal
CO2 and restricted water loss through transpiration.
This response seemed to be effective in preventing
large decreases in leaf water potential and, thus, ap-
peared to be the basis for the dehydration avoidance
in cowpea.
The recovery of CO2 assimilation rates upon re-
watering, in experiment A, indicated that water stress
did not persistently affect photosynthetic activity. Ac-
cording to Cornic (2000) a rapid increase in the assim-
ilation rates of water-stressed plants upon rehydration
indicates that the basic mechanism of photosynthetic
biochemistry and photochemistry is not impaired
by the lack of water, leading to the conclusion that
decreases in net CO2 uptake are only the result of
stomatal closure. However, in cowpea, the recovery
of intercellular CO2 concentrations observed 1 day
after rewatering was not accompanied by immediate
recovery of assimilation rates. This suggests the oc-
currence of some transient non-stomatal limitation of
photosynthesis. Mesophyll impairment of photosyn-
thetic activity in some cowpea cultivars has already
been suggested, based on gas-exchange measure-
ments performed at high CO2 concentrations (Lopez
et al., 1987) or on assessments of photosynthetic
53
capacity, as measured by photosynthetic O2 pro-
duction (Campos et al., 1999). Lopez et al. (1987)
suggested that the non-stomatal limitation of pho-
tosynthesis observed in water-stressed cowpea may
have occurred by some impairment either of pho-
tochemical activity or of some biochemical activity
other than Rubisco (ribulose-1,5-bisphosphate car-
boxylase/oxygenase). The results of this study, based
on chlorophyll a fluorescence measurements, showed
that, during the initial phase of stress (i.e. the first
fourth days in experiment A), the photochemical ac-
tivity was not affected, while, at an advanced phase
(i.e. the fifth and sixth day) a significant effect of
water stress was observed.
In the initial phase of stress, no decreases in maxi-
mum and in effective quantum yield of PSII, in elec-
tron transport rate and in photochemical quenching
could be observed. From the third day on, however,
coinciding with great reductions in CO2 assimilation
rates, decreases in efficiency of excitation capture
and increases in non-photochemical quenching were
present, indicating the occurrence of a non-radiative
energy dissipation mechanism (Schreiber et al., 1994),
in which a higher proportion of absorbed photons is
lost as thermal energy instead of being used to drive
photosynthesis (Shangguan et al., 2000). Such ther-
mal deactivation of the excess of excitation energy is a
protective mechanism, which maintains high oxidative
state of primary electron acceptors of PSII, reducing
the probability of photodamage (Epron et al., 1992).
It is important to note, however, that these results
were obtained by measurements performed in growth
chambers under mild conditions of temperature and
relative humidity associated with low irradiances. The
susceptibility of PSII to photoinhibition may increase
by the interaction of substrate water depletion with
other environmental stresses normally associated with
water deficits (Kaiser, 1987; Epron et al., 1992). In
fact, early and accentuated responses to water stress
could be evidenced in cowpea by the measurements
performed in experiment C under natural environ-
mental conditions. In this case, the combination of a
mild water stress (i.e. that provided by 2 days with-
out irrigation) with elevated temperatures and lower
air relative humidity led to accentuated reductions in
stomatal conductance, with consequent strong restric-
tions in the internal CO2 availability and assimilation
and transpiration rates (see Table 2). Under these
54 R.P. Souza et al. / Environmental and Experimental Botany 51 (2004) 45–56
conditions of restricted CO2 assimilation associated
with a high irradiance, the photochemical system,
even in an initial phase of stress, became overex-
cited as revealed by significant decreases in electron
transport rates and in photochemical quenching (see
Table 2). The observed tendencies of increases in
non-photochemical quenching and decreases in the
excitation capture efficiency revealed the develop-
ment of protective mechanisms, downregulating PSII
activity with decreases in both its effective and maxi-
mum quantum yield. Since no alterations in morning
values of Fv/Fm occur in the initial stage of stress, as
demonstrated in experiment A, the diurnal decreases
of Fv/Fm ratio under environmental conditions re-
vealed the occurrence of a dynamic photoinhibition,
which seemed to be effective in protecting the photo-
chemical apparatus from the high risk of photodamage
occasioned by the superimposed stresses to which the
plants were subjected.
In the late stage of stress, the effects of water stress
upon the photochemical system were revealed by sig-
nificant decreases in the maximum quantum yield of
PSII accompanied by increases in the levels of mini-
mum fluorescence. These alterations possibly reflect
a disorder in PSII (Osmond, 1994). So, perhaps, in
a more severe phase of water stress in cowpea, the
capacity of protective mechanisms was exceeded.
The observed decreases in effective quantum yield of
PSII, in electron transport rates and in photochemical
quenching indicated an overexcitation of the photo-
chemical system. When this is the case, accumulation
of reduced electron acceptors may increase the prob-
ability of generation of reactive radicals, which can
damage PSII components (Barber and Andersson,
1992). Great values of non-photochemical quenching
in cowpea were observed around maximum stress
and they may have resulted from the occurrence
of both photoprotective and photodamaging mecha-
nisms (Maxwell and Jonhson, 2000), in spite of the
drought avoidance strategy. Upon rewatering, the ini-
tial accentuation of decreases in Fv/Fm ratio, as was
observed in experiment A, indicated the occurrence
of possibly membrane-related damages affecting thy-
lakoid function. In fact, membrane damages are more
likely to occur during rehydration than during partial
dehydration (Kaiser, 1987). Nevertheless, the occur-
rence of damages either during the development of
stress or upon rewatering in cowpea plants seemed to
be not very important, since assimilation rates were
recovered on the second day after the end of the water
stress, independently of the decreased Fv/Fm values
(see Figs. 1 and 2). So, although some impairment of
photochemical activity may be present in an advanced
stage of stress, it did not limit the overall process.
Anyway, the mismatch between assimilation rates and
intercellular CO2 concentrations on the first day after
rewatering seemed to be associated with the mainte-
nance, at this time, of alterations in photochemical
activity. These alterations, which involved transient
reductions in effective quantum yield of PSII, elec-
tron transport rates, excitation capture efficiency and
increases in non-photochemical quenching, revealed
a short-time lasting down-regulation of PSII activity.
Metabolic changes were also evidenced in water-
stressed cowpea plants. The occurrence of starch hy-
drolysis and a build-up of reducing sugars in leaf
tissue were evident from the third day of stress im-
position, coinciding with important decreases in as-
similation rates. Increases in proline level were only
observed around maximum stress. Both soluble sug-
ars and proline can be used in osmotic adjustment as
compatible solutes (Ingram and Bartels, 1996). How-
ever, in cowpea, osmotic adjustment is not expected
to occur due to the drought avoidance strategy and, in
fact, no evidences of osmotic adjustment have been
found (Shackel and Hall, 1983; Petrie and Hall, 1992;
Campos et al., 1999). So, the observed metabolic
alterations may have been a consequence of stress
rather than adaptive responses with beneficial effects
on plant physiological functioning. Despite its known
role in osmotic adjustment, proline has been consid-
ered, in some studies, a symptom of injury (Irigoyen
et al., 1992), probably resulting from an excessive pro-
tein breakdown during water deficits (Levitt, 1980).
In water-stressed cowpea, the late and small increases
in proline levels strongly suggest that they had no role
in osmotic adjustment, being, thus, related with some
stress-imposed injury. In other cowpea cultivars, sig-
nificant increases in proline were also observed only
under severe drought (Campos et al., 1999), which re-
inforces their implication as a potential sign of injury.
Starch depletion and accumulation of soluble sugars
during water stress have also been reported for V. un-
guiculata cv.1183 (Campos et al., 1999) and they may
reflect a shift on metabolism as consequence of some
starvation injury imposed by water stress. For the
 R.P. Souza et al. / Environmental and Experimental Botany 51 (2004) 45–56
cowpea cultivar 1183, Campos et al. (1999) suggested
that the accumulation of soluble sugars was a result
of some metabolic impairment that affected sugar
composition in the leaf or its translocation. They also
suggested that this response may have contributed
to the observed inhibition of photosynthesis during
water stress. In fact, it has been demonstrated that the
accumulation of sugars in source leaves may inhibit
photosynthesis by feedback processes (Goldschmidt
and Huber, 1992).
We verified in cowpea that, soon after stress relief
(i.e. 1 day after rewatering in experiment A), the as-
similation rates did not recover to control levels in
spite of the internal CO2 availability and the soluble
sugars level remained as high as at the point of max-
imum stress. Therefore, we raised the hypothesis that
an end-product inhibition of photosynthesis may have
been present, contributing to a minor non-stomatal
limitation during stress and also during the initial stage
of recovery. With the progress of recovery and dissi-
pation of accumulated soluble sugars, the end-product
inhibition may have been relieved, making possible
the complete recovery of assimilation rates. So, in
water-stressed cowpea plants, whereas stomatal clo-
sure played a major role in limiting photosynthesis,
a non-stomatal limitation seemed also to be present,
confirming our working hypothesis. Although the sug-
gested transient feedback inhibition of photosynthe-
sis needs to be investigated in future research, it may
be consistent and explain the previously discussed
short-time lasting downregulation of photochemical
activity upon rewatering.
Acknowledgements
R.P. Souza, E.C. Machado and J.A.G. Silveira thank
Conselho Nacional de Desenvolvimento Cientı́fico e
Tecnológico (CNPq) for financial support.
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