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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
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- that brings the electron transport capacity into balance
iological responses, which may lead to some adapta- with carbon metabolism (Epron et al., 1992). Under
tion to drought stress, may vary considerably among natural conditions, however, where high light and high
species. In general, strategies of drought-avoidance or temperatures are often associated with water deficits,
drought-tolerance can be recognized, both involving these or other protective mechanisms (Krause, 1988)
diverse plant mechanisms that provide the plants the may be insufficient to dissipate the excess of excita-
ability to respond and survive drought (Levitt, 1980). tion energy and a chronic photoinhibition, character-
Cowpea [Vigna unguiculata (L.) Walp.] is an im- ized by a slow-reversible reduction in the photosyn-
portant crop largely grown in warm and hot regions of thetic efficiency (Osmond, 1994), may result.
Africa, Asia and the Americas and is often regarded as The maintenance of high cell water potentials in
being well-adapted to high temperatures and drought species with a drought-avoidance strategy avoids di-
compared with other crop species (Ehlers and Hall, rect metabolic injuries (Levitt, 1980) and minimizes
1997). Drought adaptation in cowpea has been related the necessity of metabolic adjustments, which are usu-
to the minimization of water losses by the control ally found in drought-tolerant species (Levitt, 1980;
of stomatal aperture (de Carvalho et al., 1998). Ac- Shackel and Hall, 1983). In these latter species, a net
cordingly, studies have demonstrated that cowpea is increase in solute concentrations, known as osmotic
able to maintain high leaf water potential or high leaf adjustment, allows, by promoting lowering of the cell
relative water content during water stress (Bates and osmotic potential, the maintenance of leaf turgor un-
Hall, 1981; Shackel and Hall, 1983; Küppers et al., der water-limited conditions, with beneficial effects
1988; de Carvalho et al., 1998), thus avoiding tis- over turgor-dependent processes, such as cell enlarge-
sue dehydration. However, this strategy, by involving ment and stomatal opening (Morgan, 1984). Accord-
stomatal closure, may lead to decreases in CO2 as- ing to its drought-avoidance characteristics, cowpea
similation (Chaves, 1991) and hence in growth and has been shown to present no osmotic adjustment un-
yield. der drought conditions (Shackel and Hall, 1983; Petrie
Stomatal regulation of photosynthesis during water and Hall, 1992). Nonetheless, the stomatal and also
shortage has been well documented (Chaves, 1991). At the non-stomatal, if existent, limitation of photosyn-
least under mild drought conditions, it has been shown thesis may lead to a drought-induced starvation injury
that stomata play the dominant role in controlling the (Levitt, 1980) and consequently also metabolic alter-
decline of net CO2 uptake, by leading to decreases ations.
in leaf internal CO2 concentrations (Cornic and Bri- The aim of this study was to evaluate the responses
antais, 1991; Cornic, 2000). Nonetheless, the limita- of photosynthesis to water stress in cowpea plants,
tions to CO2 assimilation imposed by stomatal closure both in terms of CO2 assimilation, as measured by
may promote an imbalance between photochemical leaf gas exchanges, and of the functionality of the
activity at photosystem II (PSII) and electron require- photosynthetic apparatus, as assessed by chlorophyll
ment for photosynthesis, leading to an overexcitation a fluorescence measurements. Additionally, carbohy-
and subsequent photoinhibitory damage of PSII reac- drate and proline content was simultaneously analyzed
tion centers (Krause, 1988; Long et al., 1994). In this in order to detect possible metabolic alterations in
way, damages to the photosynthetic machinery may water-stressed cowpea leaves. The underlying hypoth-
eventually occur, imposing an additional non-stomatal esis is that photosynthesis in water-stressed cowpea
limitation to the process. Investigations based on as- plants is limited by stomatal and non-stomatal factors,
sessments of chlorophyll a fluorescence have shown both contributing to the occurrence of metabolic alter-
that PSII is quite resistant to water deficits, being ei- ations at the leaf level.
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 2. Material and methods
may be prevented through processes of thermal de-
activation, downregulating PSII photochemistry, in a The study was carried out with cowpea plants [V.
so-called dynamic photoinhibition (Osmond, 1994), unguiculata (L.) Walp. cultivar Vita 7] cultivated
R.P. Souza et al. / Environmental and Experimental Botany 51 (2004) 45–56 47
from seeds on 2.8 l pots (one plant per pot) contain- mum quantum yield of PSII [Fv/Fm = (Fm−Fo)/Fm],
ing silica:vermiculite 1:2 in a greenhouse situated at the excitation capture efficiency of open centers
the Experimental Station of Instituto Agronômico de [Fv /Fm = (Fm −Fo )/Fm ], the effective quantum
Campinas, SP, Brazil (22◦ 54 S and 47◦ 05 W, 674 m yield of PSII [F/Fm = (Fm −Fs)/Fm ], the apparent
altitude). Plants received 250 ml of Hoagland and electron transport rate (ETR = F/Fm ×PPFD×0.5
Arnon’s (Hoagland and Arnon, 1938) nutrient solu- ×0.84) and the coefficients of photochemical quench-
tion twice a week, being irrigated with distilled water ing [qP = (Fm −Fs)/(Fm −Fo )] and non-photochemi-
in the remaining days. Three water stress experiments, cal quenching [NPQ = (Fm−Fm )/Fm ], where Fm
hereafter named experiment A, B and C, were carried and Fo are, respectively, maximum and minimum
out with 28–35 days old plants. Four or five plants per fluorescence of dark-adapted leaves, Fm and Fs are,
treatment (control and water-stressed) were sampled respectively, maximum and steady-state fluorescence
for the measurements. In all experiments, water stress in the light-adapted state and Fo is minimum fluo-
was imposed by withholding water. rescence after far-red illumination of the previously
exposed leaves. For the calculation of ETR, 0.5 was
2.1. Experiment A assumed as the fraction of the excitation energy
distributed to PSII and 0.84 as the fractional light
After water stress imposition, gas-exchange and absorption of the leaf.
chlorophyll a fluorescence measurements were Subsequent to these measurements, the sampled
performed under controlled conditions in a growth leaves were collected in liquid N2 , lyophilized and
chamber (28 ◦ C, 70% RH, 270 mol m−2 s−1 ), to later submitted to carbohydrate and proline analysis.
which the plants were daily transferred at 07:00 h. The extraction was performed in methanol:chloro-
These measurements were daily repeated until the CO2 form:water (12:5:3 v/v/v), followed by the separation
assimilation rates approached zero, which was con- of the aqueous fraction (Bieleski and Turner, 1966)
sidered the point of maximum stress. The plants were containing the soluble compounds. The residue was
then rewatered and the measurements were continued extracted with a 30% percloric acid solution for ob-
until recovery of the assimilation rates to the control tainment of the hydrolyzed starch fraction (Yemm
level. A different set of both control and water-stressed and Willis, 1954). Quantification of total soluble
plants was used each day, since the photosynthe- sugars and starch were done using anthrone method
sis measurements were followed by destructive leaf (McReady et al., 1950). Sucrose determinations were
harvests. also done by anthrone method, being carried out
Gas-exchange measurements were done with a according to van Handel (1968) with modifications.
portable photosynthesis system (LCi, ADC, Herts, Reducing sugars were estimated by the difference be-
UK), using the youngest completely expanded leaf of tween total soluble sugars and sucrose. Proline quan-
each plant, under a saturating photosynthetic photon tification was done according to Rena and Masciotti
flux density (PPFD) of 1800 mol m−2 s−1 provided (1976).
by an external halogen lamp. Fluorescence mea-
surements were done with a modulated fluorometer 2.2. Experiment B
(FMS 1, Hansatech, Norfolk, UK) using the satura-
tion pulse method (Schreiber et al., 1994), prior to After water stress imposition, gas-exchange and
the gas-exchange measurements on the same sampled chlorophyll a fluorescence measurements were per-
leaves. Chlorophyll fluorescence measurements were formed as in experiment A. Here, however, a same set
initially taken on dark-adapted leaves for 30 min and of plants (five control and five water-stressed plants)
then for the same leaves after a 30 min acclimation pe- was accompanied during the whole water stress and
riod to the growth chamber PPFD. Immediately before recovery period. So, after the daily measurements
measurements under light, leaves received a 10 s expo- in the growth chamber, the plants were brought
sure of an actinic light of 270 mol m−2 s−1 provided back to the greenhouse. This experiment was carried
by the fluorometer’s fiber optic. The following param- out in a greenhouse at higher air relative humidity
eters were assessed (Schreiber et al., 1994): the maxi- and lower temperatures as compared with experiment
48 R.P. Souza et al. / Environmental and Experimental Botany 51 (2004) 45–56
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 only observed on the day of maximum stress (Fig. 2E).
subsequent to rewatering. Here again, Fo increases Increases in non-photochemical quenching (Fig. 2F)
paralleled the Fv/Fm decreases (results not shown). and decreases in the excitation capture efficiency of
The effective quantum yield of PSII (Fig. 2C) and open centers (Fig. 2B) were observed from the third
the apparent electron transport rate (Fig. 2D) also de- day of the beginning of the water stress treatment. The
clined from the fifth day of water withholding, while alterations in all these parameters, except photochem-
significant differences (P < 0.05) in photochemical ical quenching, here again, persisted after rewatering
quenching between control and stressed plants were 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
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- the three parameters significantly differed (P < 0.05)
yses during photosynthetic induction, performed at between control and stressed plants. After rewater-
the maximal stress in experiment B, showed clearly ing and recovery, the photochemical quenching and
the development of a higher non-photochemical the PSII effective quantum yield of stressed plants
quenching in water-stressed plants, accompanied by returned back to control values (Fig. 3C, D). The
a lower development of photochemical quenching development of non-photochemical quenching in
and lower values of effective quantum yield of PSII water-stressed plants after recovery was even lower
(Fig. 3A, B). At the end of the induction curve, all 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 Leaf carbohydrate determinations during the stress
(P < 0.05). and the recovery period revealed that the content of
Measurements of gas exchanges and fluorescence total soluble sugars almost doubled at the maximum
parameters under natural environmental conditions of stress (Fig. 4A). This accumulation response was de-
high irradiance, high temperature and low relative hu- tected from the third day of the beginning of water
midity, in experiment C, revealed an earlier response withholding in experiment A. Two days after rewater-
to water stress than the observed in the preceding ing, there was a decrease in the content of total soluble
experiments (Table 2). On the second day after water sugars (Fig. 4A). The alterations in total soluble sugars
suspension, great and significant (P < 0.05) reductions matched the alterations in reducing sugars (Fig. 4C).
in assimilation and transpiration rates, stomatal con- Leaf sucrose levels were low and did not vary sig-
ductance and intercellular CO2 concentrations were nificantly during the experiment (Fig. 4B). On the
observed. In relation to fluorescence parameters, sig- other hand, leaf starch content was high and declined
nificant differences (P < 0.05) were observed between upon water stress. Starch values in water-stressed
control and stressed plants only for the electron trans- plants did not return to the control level after re-
port rates and photochemical quenching, although a watering (Fig. 4D). Proline content in leaves varied
tendency of increase in non-photochemical quench- only slightly during stress imposition and recovery.
ing and of decrease in the other parameters—PSII Differences (P < 0.05) between control and stressed
maximum and effective quantum yield and excitation plants were only observed around the maximum stress
capture efficiency—was also observed (Table 2). (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
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 53
conditions of restricted CO2 assimilation associated be not very important, since assimilation rates were
with a high irradiance, the photochemical system, recovered on the second day after the end of the water
even in an initial phase of stress, became overex- stress, independently of the decreased Fv/Fm values
cited as revealed by significant decreases in electron (see Figs. 1 and 2). So, although some impairment of
transport rates and in photochemical quenching (see photochemical activity may be present in an advanced
Table 2). The observed tendencies of increases in stage of stress, it did not limit the overall process.
non-photochemical quenching and decreases in the Anyway, the mismatch between assimilation rates and
excitation capture efficiency revealed the develop- intercellular CO2 concentrations on the first day after
ment of protective mechanisms, downregulating PSII rewatering seemed to be associated with the mainte-
activity with decreases in both its effective and maxi- nance, at this time, of alterations in photochemical
mum quantum yield. Since no alterations in morning activity. These alterations, which involved transient
values of Fv/Fm occur in the initial stage of stress, as reductions in effective quantum yield of PSII, elec-
demonstrated in experiment A, the diurnal decreases tron transport rates, excitation capture efficiency and
of Fv/Fm ratio under environmental conditions re- increases in non-photochemical quenching, revealed
vealed the occurrence of a dynamic photoinhibition, a short-time lasting down-regulation of PSII activity.
which seemed to be effective in protecting the photo- Metabolic changes were also evidenced in water-
chemical apparatus from the high risk of photodamage stressed cowpea plants. The occurrence of starch hy-
occasioned by the superimposed stresses to which the drolysis and a build-up of reducing sugars in leaf
plants were subjected. tissue were evident from the third day of stress im-
In the late stage of stress, the effects of water stress position, coinciding with important decreases in as-
upon the photochemical system were revealed by sig- similation rates. Increases in proline level were only
nificant decreases in the maximum quantum yield of observed around maximum stress. Both soluble sug-
PSII accompanied by increases in the levels of mini- ars and proline can be used in osmotic adjustment as
mum fluorescence. These alterations possibly reflect compatible solutes (Ingram and Bartels, 1996). How-
a disorder in PSII (Osmond, 1994). So, perhaps, in ever, in cowpea, osmotic adjustment is not expected
a more severe phase of water stress in cowpea, the to occur due to the drought avoidance strategy and, in
capacity of protective mechanisms was exceeded. fact, no evidences of osmotic adjustment have been
The observed decreases in effective quantum yield of found (Shackel and Hall, 1983; Petrie and Hall, 1992;
PSII, in electron transport rates and in photochemical Campos et al., 1999). So, the observed metabolic
quenching indicated an overexcitation of the photo- alterations may have been a consequence of stress
chemical system. When this is the case, accumulation rather than adaptive responses with beneficial effects
of reduced electron acceptors may increase the prob- on plant physiological functioning. Despite its known
ability of generation of reactive radicals, which can role in osmotic adjustment, proline has been consid-
damage PSII components (Barber and Andersson, ered, in some studies, a symptom of injury (Irigoyen
1992). Great values of non-photochemical quenching et al., 1992), probably resulting from an excessive pro-
in cowpea were observed around maximum stress tein breakdown during water deficits (Levitt, 1980).
and they may have resulted from the occurrence In water-stressed cowpea, the late and small increases
of both photoprotective and photodamaging mecha- in proline levels strongly suggest that they had no role
nisms (Maxwell and Jonhson, 2000), in spite of the in osmotic adjustment, being, thus, related with some
drought avoidance strategy. Upon rewatering, the ini- stress-imposed injury. In other cowpea cultivars, sig-
tial accentuation of decreases in Fv/Fm ratio, as was nificant increases in proline were also observed only
observed in experiment A, indicated the occurrence under severe drought (Campos et al., 1999), which re-
of possibly membrane-related damages affecting thy- inforces their implication as a potential sign of injury.
lakoid function. In fact, membrane damages are more Starch depletion and accumulation of soluble sugars
likely to occur during rehydration than during partial during water stress have also been reported for V. un-
dehydration (Kaiser, 1987). Nevertheless, the occur- guiculata cv.1183 (Campos et al., 1999) and they may
rence of damages either during the development of reflect a shift on metabolism as consequence of some
stress or upon rewatering in cowpea plants seemed to starvation injury imposed by water stress. For the
R.P. Souza et al. / Environmental and Experimental Botany 51 (2004) 45–56 55
cowpea cultivar 1183, Campos et al. (1999) suggested Bieleski, R.L., Turner, N.A., 1966. Separation and estimation of
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