Jumat, 22 April 2011


Ann. appl. Biol. (2004), 144:273-283
Printed in UK
*Corresponding Author E-mail: jaume.flexas@uib.es
© 2004 Association of Applied Biologists
Understanding down-regulation of photosynthesis under water stress: future
prospects and searching for physiological tools for irrigation management
Laboratori de Fisiologia Vegetal, Departament de Biologia, Universitat de les Illes Balears, Carretera de
Valldemossa Km 7.5, 07122 Palma de Mallorca, Balearic Islands, Spain
(Accepted 2 March 2004; Revised version received 10 December 2003)
Photosynthetic down-regulation and/or inhibition under water stress conditions are determinants for
plant growth, survival and yield in drought-prone areas. Current knowledge about the sequence of
metabolic events that leads to complete inhibition of photosynthesis under severe water stress is
reviewed. An analysis of published data reveals that a key regulatory role for Rubisco in photosynthesis
is improbable under water stress conditions. By contrast, the little data available for other Calvin cycle
enzymes suggest the possibility of a key regulatory role for some enzymes involved in the regeneration
of RuBP. There are insufficient data to determine the role of photophosphorylation.
Several important gaps in our knowledge of this field are highlighted. The most important is the
remarkable scarcity of data about the regulation/inhibition of photosynthetic enzymes other than Rubisco
under water stress. Consequently, new experiments are urgently needed to improve our current
understanding of photosynthetic down-regulation under water stress. A second gap is the lack of
knowledge of photosynthetic recovery after irrigation of plants which have been subjected to different
stages of water stress. This knowledge is necessary in order to match physiological down-regulation
by water stress with controlled irrigation programmes.
Key words: Drought, photosynthesis, stomatal limitations, metabolic limitations, C3
 plants, Rubisco
Water stress is considered to be the main
environmental factor limiting plant growth and yield
worldwide, especially in semi-arid areas (Boyer,
1982). It is well known that one of the primary
physiological targets of water stress is
photosynthesis (Chaves, 1991; Cornic, 1994;
Lawlor, 1995). However, there is a long-standing
controversy as to whether water stress mainly limits
photosynthesis through stomatal closure (Sharkey,
1990; Chaves, 1991; Cornic, 1994; Ort et al., 1994)
or metabolic impairment (Boyer, 1976; Lawlor,
1995). The suggestion that impaired ATP synthesis
is the main factor limiting photosynthesis even under
mild water stress (Tezara et al., 1999), has further
stimulated debate in recent years (Cornic, 2000;
Cornic & Fresneau, 2002; Flexas & Medrano, 2002;
Lawlor & Cornic, 2002; Tezara et al., 2002).
It has been argued that stomatal closure is the main
limitation to photosynthesis, since maximum values
can be recovered by supplying large amounts of CO2
to the leaves (Cornic, 2000; Cornic & Fresneau,
2002). Other reports, however, have suggested that
maximum photosynthesis is not totally recovered by
high CO2
 in water stressed plants (Graan & Boyer,
1990; Quick et al., 1992). This has been attributed
to the inhibition of key metabolic processes, such
as photophosphorylation (Younis et al., 1979; Tezara
et al., 1999), the capacity for ribulose-1,5-
bisphosphate (RuBP) regeneration (GimÈnez et al.,
1992; Gunasekera & Berkowitz, 1993) and Rubisco
activity (Medrano et al., 1997; Maroco et al., 2002;
Parry et al., 2002). Each of these processes has been
proposed to be the main limitation to photosynthesis
under water stress. Other authors, by contrast, have
stated that photophosphorylation (Ortiz-LÛpez et al.,
1991), the capacity for RuBP regeneration (Lal  et
al., 1996) or the activity of Rubisco (Lal et al., 1996;
Pankovic et al., 1999; Delfine et al., 2001; Pelloux
et al., 2001) remained unaffected during water stress.
Sometimes even the same authors have observed
discrepancies among their own studies. For instance,
Tezara et al. (1999) attributed water stress-induced
decreases of photosynthesis to impaired
photophosphorylation in sunflower, but they stated
in a later study using the same species that ìdecrease
in net photosynthesis with water deficiency was274 JAUME FLEXAS ET AL.
related to lower Rubisco activity rather than to ATP
and RuBP contentsî (Tezara et al., 2002). Similarly,
we have recently highlighted that decreased RuBP
contents matched decreased photosynthetic capacity
under water stress in field-grown grapevines
(Medrano et al., 2003), but we have also observed
decreased photosynthetic capacity with constant
RuBP contents under water stress in other
experiments with the same species (Bota  et al.,
2004). These apparent discrepancies could arise from
the fact that different studies may have been made
under different degrees of water stress, since the
assessment of severity of water stress is a complex
matter. The two most commonly used water status
parameters to assess water stress intensity are leaf
water potential (Ψ) and relative water content
(RWC). However, the precise response of stomatal
conductance and photosynthesis to Ψ and RWC
depends on the genotype (Tardieu & Simonneau,
1998), the environmental conditions during water
stress (Schulze & Hall, 1982) and the velocity of
water stress imposition (Flexas et al., 1999), among
other factors. Alternatively, to discriminate stress
severities leading to metabolic limitations to
photosynthesis, the water stress-induced variations
of sub-stomatal CO2
 concentration (Ci) have been
proposed as a reference parameter (Lawlor, 1995,
2002). However, it is now doubtful that this kind of
analysis is reliable under drought. Two main
problems have been described in relation to Ci
calculations: patchy stomatal closure (Laisk, 1983;
Buckley et al., 1997) and changes in the cuticular
conductance of water vapour (Boyer et al., 1997).
In addition, drought-induced changes in the
mesophyll conductance of CO2
 may also invalidate
the interpretation of AN-Ci analysis (where AN is net
photosynthesis) (Flexas et al., 2002 a,b; Centritto et
al., 2003).
It is necessary to know the precise sequence of
events leading to photosynthetic inhibition under
progressive water stress because photosynthesis is
one of the key determinants for plant productivity
and survival, and water stress is thought to be an
increasing problem for plant performance in the
present climate change scenario (Chaves  et al.,
2003). The response of respiration to water stress,
the other basic component of plant productivity, is
beyond the scope of the present review. However,
the lack of knowledge about respiratory responses
to water stress is remarkable, and this is an important
issue that needs to be addressed in the near future
(Flexas et al., 2004). Regarding photosynthesis, the
presence of non-stomatal limitations constrains the
validity of canopy conductance models for the
estimation of canopy photosynthesis under severe
water stress conditions, thus reducing the accuracy
of estimations of canopy productivity (Moriana  et
al., 2002; Reichstein  et al., 2002). Moreover, the
appearance of non-stomatal limitations usually
coincides with decreasing water-use-efficiency at the
leaf level, which may have a repercussion for water-
use-efficient irrigation programs (Flexas  et al.,
2002a,b; GulÌas et al., 2002; Medrano et al., 2002).
The aim of the present paper is to critically review
the data in the literature and our own experimental
results, in relation to the effects of water stress on
photosynthesis. The present analysis supports the
view that light saturated, daily maximum, stomatal
conductance (gs
) is a useful parameter for comparing
the response of photosynthesis to water stress
between different species and experiments. Water
stress-induced depression of photosynthetic
metabolism is reviewed using gs
 as the reference
parameter for water stress intensity. In all the
experiments reviewed here, water stress was
imposed gradually by withholding watering. Plants
were grown at saturating or near-saturating light, and
optimal temperatures (except in those experiments
under field conditions, where plants could have
eventually endured periods of excessive
temperature). Measurements of photosynthesis were
made at saturating light and atmospheric CO2
Looking for a Water Stress Reference Resulting
in the Most Generalised Response Pattern
In an attempt to solve the observed discrepancies
among different studies, Lawlor & Cornic (2002)
have proposed that two different patterns or
syndromes of photosynthetic responses to water
stress could occur (Type I and Type II). Definitions
of these syndromes  use leaf relative water content
(RWC) as the indicator of water stress intensity at
the leaf level. In both Type I and Type II plants, net
photosynthesis (AN) decreases as RWC becomes
smaller due to increasing water stress. However, in
Type I plants, the rate of photosynthesis under light-
and CO2
-saturated conditions (ASAT) remains
unaffected until RWC drops to very low values. By
contrast, in Type II plants, ASAT declines in parallel
to net photosynthesis as RWC decreases.
Such classification of plant responses to water
stress presents some limitations. First, the same
species can behave as either Type I or Type II,
depending on the experimental conditions (Lawlor
& Cornic, 2002), thus indicating that the type of
response to water stress is not fully determined by
the genotype. Second, in some species like
grapevines, it has been shown that ASAT progressively
declines during water stress with very low (if any)
reductions in RWC. Such isohydric behaviour does
not match either the Type I or the Type II response,
so it could possibly be classified as a Type III (Fig.
We have shown that, among C3
 plants, water275 Photosynthetic down-regulation under water stress
allow a direct comparison of the regulation of
different metabolic processes in response to
progressive water stress, even when data from
different species are included. From here on, gs
be used as the indicator of water stress severity in
order to follow photosynthetic metabolic variations.
Down-Regulation of Photosynthetic
Metabolism Along a Stomatal Conductance
Gradient During Progressive Water Stress
An initial review of the literature, using gs
 as the
indicator of the severity of water stress, used
chlorophyll fluorescence and gas exchange
parameters to indicate key photosynthetic points
(Flexas et al., 2002 a,b; Medrano et al., 2002). Such
analysis suggested that photosynthetic metabolism
is progressively down regulated as water stress
intensifies, and three stages of inhibition of
photosynthesis were described:
stress-induced changes in photosynthetic rate can
be more generally related to variations in light-
saturated stomatal conductance (gs
) than to RWC or
leaf water potential (Flexas & Medrano, 2002; Flexas
et al., 2002 a,b; Medrano et al., 2002). The use of gs
(stomatal conductance) as an indicator of the
intensity of water stress has revealed a more general
pattern of photosynthetic responses to progressive
water stress that is somewhat independent of a) the
velocity of water stress imposition, b) the
environmental conditions and c) the genotype. As
shown in Figure 1B, examples of the three different
response Types show a rather similar pattern of
response of ASAT to gs
. Even so, there is still some
variability in the response of different photosynthetic
parameters to gs
. This may come from other
important factors affecting photosynthesis, such as
life-form, leaf habit, canopy characteristics, intrinsic
photosynthetic capacity of a given species, etc.
In spite of this, comparison of the response of
photosynthesis to water stress on the basis of gs
seems to be more appropriate when comparing
between different studies. In fact, gs
 responds to
many internal and external factors involved in plant
water stress signalling (i.e. xylem and leaf water
status, xylem pH and abscisic acid (ABA) content,
and possibly others presently unknown), which
makes gs
 an integrative parameter of all the signals
associated with the plant responding to water stress.
In supporting of this idea, for instance, Correia  et
al. (1995) demonstrated that the maximum daily
stomatal conductance, but not its diurnal
fluctuations, was determined by the xylem ABA
concentration in field-grown grapevines.
On the other hand, it remains to be elucidated
whether the fact that relationships between gs
photosynthetic parameters are largely independent
of genotype and environmental conditions is due to
(i) a direct effect of water stress-induced stomatal
closure on down-regulation of photosynthetic
metabolism, possibly through internal CO2
signalling, as already suggested (Sharkey, 1990; Ort
et al., 1994; Cornic & Fresneau, 2002), or (ii) a
strong co-regulation of both stomatal closure and
photosynthetic metabolism, possibly reflecting that
both processes share one or several common agents
that are elicited by water stress, as suggested by
Cornic (1994). In view of the different patterns of
relationship between RWC and photosynthesis
observed, it is likely that decreased cell water content
and/or increased concentration of certain ions is the
general cause of metabolic impairment under natural
conditions, as proposed by Lawlor (2002).
Nevertheless, to fully understand the mechanism by
which photosynthetic metabolism is impaired or
down regulated at severe water stress would merit a
detailed and profound analysis. For the moment, the
robustness of the above mentioned relationships may
Fig. 1. (A) The dependence of photosynthetic capacity
on RWC in three different species presenting three
different syndromes: Phaseolus vulgaris L., Type I
(circles); Rhamnus ludovici-salvatoris R. Chodat, Type
II (squares) and Vitis vinifera L., Type III (triangles).
(B) Response of photosynthetic capacity to stomatal
conductance in these three species. Data on Phaseolus
are from Brestic et al. (1995), and data on the other
two species from J Bota et al. (unpublished results).
RWC (%)
30 40 50 60 70 80 90 100 Photosynthetic capacity (mmol CO2 m-2
Stomatal conductance (mol H2O m-2
0.00 0.04 0.08 0.12 0.16 0.20 Photosynthetic capacity (mmol CO2 m-2
Vitis vinifera L.
Type III
Rhamnus ludovici-salvatoris 
Type II
Phaseolus vulgaris L.
- Stage 1. As gs
 decreases from its maximum value
(remarkably, irrespective of the actual value, which
was largely variable depending on the species) to
about 0.15 mol H2
O m-2
, net photosynthesis (AN)
decreased slowly with very small variations of ASAT
and no variation of the photosynthetic electron
transport rate (ETR). Intrinsic water-use-efficiency
) progressively increased at this stage.
- Stage 2. When gs
 drops between 0.15 and 0.05
mol H2
O m-2
, AN and ASAT further decrease, and
so does ETR, suggesting increased metabolic
limitations. However, a continuous decline of the
sub-stomatal CO2
 concentration (Ci) suggests that
stomatal closure is still the dominant limitation to
photosynthesis. The mesophyll conductance to CO2
is also suggested to start decreasing at this stage.
Intrinsic water-use-efficiency still increased at this
stage, reaching maximum values at gs
 around 0.05
mol H2
O m-2
- Stage 3. When gs
 declines below 0.05 mol H2
, Ci commonly increases sharply, reflecting
either erroneous estimations of Ci or impaired
photosynthetic metabolism. Intrinsic water use
efficiency usually decreases at this stage, and both
ASAT and ETR become very low.
Therefore, the analysis of gas exchange and
chlorophyll fluorescence variations along a gs
gradient suggests that photosynthetic metabolism is
not impaired under water stress until gs
 is quite low,
and AN is already severely reduced, as already stated
(Cornic & Fresneau, 2002). However, as mentioned
previously there are questions about whether
assessments of metabolic limitations based on Ci
analysis are reliable under drought. Two main
problems have been described related to Ci
calculations in stressed leaves: patchy stomatal
closure (Laisk, 1983; Buckley et al., 1997) and the
increase of the relative importance of cuticular
transpiration when stomata are closing in drying
leaves (Boyer et al., 1997). Moreover, even if
correctly estimated, Ci may not represent the actual
 concentration in the chloroplasts (Cc), since the
mesophyll conductance to CO2
 is finite and decreases
in response to water stress (Flexas  et al., 2002a;
Centritto  et al., 2003). Therefore, the previous
analysis based on  in vivo measurements is
questionable and, to assess metabolic limitations to
photosynthesis, it would be better to rely on
biochemical analysis. In fact, Flexas & Medrano
(2002) and Medrano  et al. (2002), have already
highlighted that biochemical evidence does not
always match gas exchange and fluorescence data.
Flexas & Medrano (2002) presented a preliminary
analysis of some biochemical data regarding
photosynthetic metabolism, using discrete gs
intervals as a reference of water stress intensity. In
the present review, we analyse only metabolic
parameters for which a sufficient amount of data
including simultaneous gs
 measurements is available,
so that a clear picture of variations along an entire
 gradient can be drawn.
A large number of simultaneous measurements of
 and Rubisco activity under water stress conditions
has accumulated during the last 20 yr or so, which
has been enlarged with recent data by Castrillo  et
al. (2001), Delfine et al. (2001), Maroco et al. (2002)
and Tezara et al. (2002), as well as from unpublished
results from our group in up to eleven new species
(Bota et al., 2004; J GalmÈs, unpublished). Figure
2A shows a pool of all these results. It is very clear
that Rubisco activity remains largely unaffected by
water stress at gs
 higher than 0.10 - 0.15 mol
O m-2
, and this is irrespective of the maximum
 attained by a given species. Below that threshold,
Rubisco activity steeply declines, as stomata close
further. The strength of the observed gs
 threshold is
remarkable, since the plot includes data from a large
number of different species that present substantially
different ecological strategies and water stress
resistance. Therefore, the relationship presented
strongly supports the notion that Rubisco activity is
highly stable and resistant to water stress.
By contrast, the data on water stress-induced
regulation of the activity of photosynthetic enzymes
other than Rubisco are scarce. To the best of our
knowledge there is only one study where the activity
of several enzymes involved in RuBP regeneration
has been measured concurrently with gs
 under more
than two different water stress intensities
(Thimmanaik et al., 2002, see Fig. 2B). The same
applies for the activity of sucrose phosphate synthase
(SPS), which was analysed in maize (a C4
 plant) by
Pelleschi  et al. (1997, see Fig. 2C), and nitrate
reductase (NR), which was analysed in Ziziphus
rotundifolia by Arndt  et al. (2001, see Fig. 2D).
Thimmanaik  et al. (2002) studied the activity of
several photosynthetic enzymes under progressive
water stress in two different cultivars of Morus alba
(Fig. 2B). Unlike Rubisco, the activity of all those
enzymes started declining at early steps of water
stress, when gs
 was still high. By contrast, the
activities of SPS (Fig. 2C) and NR (Fig. 2D)
remained substantially unaffected at gs
 higher than
0.10-0.15 mol H2
O m-2
, as did Rubisco. The high
stability of the activity of the latter two enzymes
under water stress is remarkable, since both have
been considered to be down regulated by low CO2
availability under mild water stress (Kaiser &
Fˆrster, 1989; Vassey  et al., 1991). Therefore, it
seems that the activities of many enzymes related to
photosynthesis remain unaffected by moderate water
stress, being impaired only when gs
 is lower than
0.10- 0.15 mol H2
O m-2
, whereas some enzymes
involved in the regeneration of RuBP are
progressively impaired or down regulated from very
early stages of water stress (Fig. 2). Thus, these277 Photosynthetic down-regulation under water stress
results present the possibility that some enzymes
involved in the regeneration of RuBP could play a
key regulatory role in photosynthesis under water
stress, although this role should be confirmed in a
larger number of species. Also, photosynthesis could
be impaired by reduced photophosphorylation under
water stress (Tezara  et al., 1999; Lawlor, 2002).
Although this possibility remains open as well, a
recent report by Tezara et al. (2002) has shown that
when gs 
was strongly reduced under water stress,
from 0.8 to 0.1 mol H2
O m-2
, the observed
decrease in leaf ATP content was only marginally
significant. Further studies are needed to clarify
whether photophosphorylation is generally impaired
under water stress and at what gs
Summarising, both gas-exchange/chlorophyll
fluorescence data and biochemical data, analysed
using gs
 as a reference for water stress intensity,
strongly support the idea that photosynthetic
metabolism is quite resistant to water stress, and does
not limit photosynthesis until the water stress is
severe (i.e. gs
 below 0.10 - 0.15 mol H2
O m-2
However, there is still an important lack of
knowledge about the variations in many metabolic
components affecting photosynthesis along a gs
gradient during water stress. Among these, the
following must be considered: (1) all the enzymes
involved in the regeneration of RuBP in the Calvin
cycle; (2) water stress-induced oxidative stress and
protective antioxidant responses; (3)
photophosphorylation; and (4) agents possibly
involved in CO2
 diffusion inside leaves (carbonic
anhydrase, aquaporins). Therefore, it would be
necessary to study these aspects properly for a better
Fig. 2 (left). (A) Relationship between initial Rubisco
activity and gs
. We selected data on Rubisco activity
from the available literature in which gs
 was given.
Data are average values for three to 12 replicates,
depending on the reference (standard errors have been
remove to facilitate visualising general trends). These
data are from the following species and references:
Capsicum annuum L. (Delfine et al., 2001), Casuarina
equisetifolia L. (S·nchez-RodrÌguez  et al., 1999);
Cistus albidus L. (J GalmÈs, unpublished results);
Helianthus annuus L. (Pancovic et al., 1999; Tezara
et al., 1999, 2002), Hordeum vulgare L. (Lal  et al.,
1996; Wingler  et al., 1999, 2000); Hypericum
balearicum L. (J GalmÈs, unpublished results);
Lycopersicon esculentum Mill. (Castrillo et al., 2001);
Lysimachia minoricensis J. J. Rodr. (J GalmÈs,
unpublished results); Medicago sativa L. (AntolÌn &
S·nchez-DÌaz, 1993), Mentha aquatica L. (J GalmÈs,
unpublished results); Nicotiana sylvestris L. (Bota et
al., 2004); Phaseolus vulgaris L. (Bota et al., 2004;
Brestic  et al., 1995; Castrillo  et al., 2001); Phlomis
italica L. (J GalmÈs, unpublished results); Pistacia
lentiscus L. (J GalmÈs, unpublished results); Rhamnus
alaternus L. (Bota et al., 2004); Rhamnus ludovici-
salvatoris R. Chodat (Bota  et al., 2004); Trifolium
subterraneum L. (Medrano  et al., 1997),  Triticum
aestivum L. (Holaday et al., 1992), Vicia faba L. (Lal
et al., 1996) and Vitis vinifera L. (Bota et al., 2004;
Maroco et al., 2002). (B) Effect of progressive water
stress on the activity of several photosynthetic
enzymes:  RuBP kinase (filled circles), 3-PGA kinase
(empty circles), NAD dehydrogenase (filled triangles)
and NADP dehydrogenase (filled squares) in two
different cultivars of Morus alba  L.,  studied by
Thimmanaik et al. (2002). (C) Effect of progressive
water stress on SPS activity in maize studied by
Pelleschi et al. (1997). (D) Effect of progressive water
stress on NR activity, analysed in Ziziphus rotundifolia
Lam. by Arndt et al. (2001). Data are expressed as %
of maximum values for comparison.
gs (mol H2O m-2
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
NR activity
(% of maximum)
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
SPS Activity
(% of maximum)
gs (mol H2O m-2
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
Initial Rubisco Activity
(% of maximum)
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
RuBP regeneration 
enzyme activity
(% of maximum)
understanding of the metabolic impairment under
severe water stress. Moreover, even if 0.10 - 0.15
mol H2
O m-2
 is assumed to be the gs
below which non-stomatal limitations to
photosynthesis appear, two important questions need
to be addressed:
1. How frequent is the occurrence of low stomatal
conductance under semi-arid conditions, such as
those in the Mediterranean area?
2. How fast is photosynthesis in recovering from
low gs
 after re-watering?
How Frequent is the Occurrence of Low
Stomatal Conductance under Semi-Arid
It has been shown that photosynthetic metabolism
seems little impaired under water stress whenever
 is higher than 0.10-0.15 mol H2
O m-2
. This
could lead to the misinterpretation that water stress-
induced metabolic impairment is rare under natural
conditions. However, gs
 values lower than 0.10-0.15
mol H2
O m-2
 are often met by plants living in
semi-arid areas, like the Mediterranean.
To illustrate the variability that can be found on
seasonal patterns of gs
 under Mediterranean
conditions, two examples are given in Fig. 3. Fig.
3A shows the seasonal variation of gs
 in natural
stands of holm oak (Quercus ilex) during 2000 in
two localities in Mallorca, which are separated by
less than 50 Km (J GulÌas et al., unpublished results).
While in BinifaldÛ gs
 was below 0.15 mol H2
O m-2
 only during a few months in summer, in
Puigpunyent gs
 was below this value during most of
the year. Similarly, important differences can be
observed among years, caused by the large inter-
annual precipitation variability usually observed
under Mediterranean conditions. For instance, field-
grown Vitis vinifera cv. Tempranillo showed gs
values lower than 0.15 mol H2
O m-2
 during most
of the summer in 1999, but these values were never
reached during the rainy summer of 2002, where gs
progressively increased to atypically high values
along the summer (Fig. 3B, J Flexas  et al.
unpublished results). Also in 1999, at the same
vineyard, Vitis vinifera cv. Manto Negro was able
to maintain gs
 values above 0.15 mol H2
O m-2
2-3 wk more than Tempranillo (Fig. 3B).
Therefore, it seems that gs
 lower than 0.10-0.15
mol H2
O m-2
 is a condition which is often met
and consequently plants frequently endure metabolic
impairment, at least in semi-arid areas. These
conditions may impose restrictions to photosynthetic
recovery by leaves after rainfall or irrigation, with
its consequent losses for plant productivity.
However, do we really know how rapidly
photosynthesis recovers from low gs
 after re-
How Fast does Photosynthesis Recover from
Low gs
 After Re-watering?
Several studies have analysed photosynthetic
recovery upon re-watering after a water stress period
(Larcher et al., 1981; Castrillo & Calcagno, 1989;
van Rensburg & Kr¸ger, 1993; Giardi et al., 1996;
Flexas et al., 1999; Marron et al., 2002; Thimmanaik
et al., 2002). However, a systematic analysis,
including amount and velocity of recovery starting
at different water stress intensities and in different
plant species, is still lacking.
It is usually assumed that the presence of non-
stomatal limitations or metabolic impairment
imposes restrictions to photosynthetic recovery by
leaves after rainfall or irrigation (Quick et al., 1992).
However, this is not always the case. In grapevines,
a complete recovery of the maximum AN occurred
after just one night upon irrigation, even though
previous gs
 was below 0.1 mol H2
O m-2
 and some
metabolic down-regulation was evidenced by
decreased ETR (Flexas  et al., 1999). In the same
species under severe water stress, however, when gs
was lower than 0.05 mol H2
O m-2
, photosynthesis
did not reverse one day after irrigation (Quick et al.,
1992). Therefore, under mild to moderate water
stress, photosynthetic recovery after re-watering is
quite fast, at least in a well-adapted species such as
grapevines. However, how rapid is recovery from
severe stress? Figure 4 shows a time course for
photosynthetic recovery upon irrigation of grapevine
plants growing outdoors during summer in the
Mediterranean, and subjected to very severe water
stress. Clearly, there was about a 60% recovery only
one night after irrigation, but it took up to 4 days to
reach almost full recovery (prior to water stress
imposition, AN values were 11 µmol CO2
, not
shown). The extent of recovery of AN, gs
 and ETR
was similar (Fig. 4), although this could be species-
dependent. These results are similar to those obtained
by Marron et al. (2002) in two different poplar clones
subjected to similar water stress intensity and also
presenting very low gs
. The poplars showed about
50% recovery of gs
 during the first day upon re-
watering, and they took 4 more days to achieve full
recovery. Intervals of several days (usually less than
one week) for full photosynthesis recovery after very
low gs
 would also be consistent with the present
evidence about the recovery of different metabolic
processes. Castrillo & Calcagno (1989), for instance,
showed in two cultivars of tomato that Rubisco
activity was recovered from 50-60% of controls to
100% 4 days after re-watering. Similarly,
Thimmanaik  et al. (2002) showed an almost
complete recovery of several Calvin cycle enzymes
only 2 days after re-watering, even when their
activities were strongly depressed (see Fig. 2).
However, these authors considered as ësevere water279 Photosynthetic down-regulation under water stress
0 20 40 60 80 100 120 140
AN (mmol CO2 m-2
0 20 40 60 80 100 120 140
gs (mol H2O m-2
Time after rewatering (h)
0 20 40 60 80 100 120 140
ETR (mmol e
Fig. 4. Effect of re-watering on photosynthetic
parameters, AN (A), gs
 (B) and ETR (C) in grapevine
plants (Vitis vinifera L.) growing under outdoors
conditions during summer 1999 in Mallorca. Results
are means ± SE of six replicates.
0 50 100 150 200 250 300 350
(mol H2O m-2 s-1)
150 160 170 180 190 200 210 220 230
(mol H2O m-2 s-1)
Fig. 3. Evolution of gs
 along the year in different
species and studies: (A) Quercus ilex L. in two different
localities in Mallorca, BinifaldÛ (open circles) and
Puigpunyent (closed circles) during 2000 (J GulÌas,
unpublished results). (B) Two cultivars of field grown
grapevine (Vitis vinifera L.): Tempranillo during
summer 1999 (open circles) and summer 2002 (open
triangles), and Manto Negro during summer 1999
(closed circles) (J Flexas, unpublished results).
photosynthesis during summer even when
supplemental water is added (NoguÈs & Alegre,
2002), and some have been shown to require several
weeks or months for complete photosynthetic
recovery after re-hydration (Harley  et al., 1987;
MunnÈ-Bosch & Alegre, 2000).
In addition, van Rensburg & Kr¸ger (1993), using
four different tobacco cultivars, clearly showed that
the time required for full recovery was strongly
dependent upon the different water stress-tolerance
capacity. Moreover, Marron et al. (2002) stated that
stressí a situation where gs
 was still higher than 0.2
mol H2
O m-2
. Finally, Mittler et al. (2001) have
described that photosynthetic metabolism can be
recovered within 24 h in Retama raetam, a desert
plant that maintains very low levels of most
photosynthetic and photosynthesis-related enzymes
during the summer, which has been called a
ëdormantí canopy. The reason for such a rapid
capacity to recover seems to be due to the continuous
presence during the summer of the RNA transcripts
encoding for the abolished proteins (Mittler et al.,
By contrast, we observed in field-grown Rhamnus
ludovici-salvatoris plants, an evergreen sclerophyll
shrub endemic of the Balearic Islands, that almost
no photosynthetic recovery occurred 1 wk after
frequent irrigation in mid summer, even when leaf
RWC was fully recovered (J GulÌas and J Flexas,
unpublished). Frequently, field-grown woody plants
from arid and semi-arid areas do not improve280 JAUME FLEXAS ET AL.
in their study the recovery was only observed in
young but fully matured leaves, while older leaves
did not recover upon re-watering and entered a
senescence and oxidative process. Also, Giardi  et
al. (1996) showed that photosystem II structure and
function recovered by only 50% two days after
irrigation in severely water stress stressed Pisum
plants, but no recovery was observed when water
stress was accompanied by a high light treatment.
Unfortunately, these authors did not provide gs
values. Finally, it has also been shown that recovery
is quicker and higher after a first drying cycle than
after subsequent cycles (Larcher et al., 1981; Flexas
et al., 1999).
Therefore, recovery may depend not only on the
severity of stress and the species analysed, but also
on a complex interaction with plant or leaf age, light
intensity, number of consecutive drying cycles, and
many other possible factors. In summary, current
knowledge about the implication of stomatal and
non-stomatal limitations in recovery of
photosynthesis upon re-watering after different water
stress intensities imposed in different plants and
conditions is extremely scarce. This knowledge
would be of importance for the development of
irrigation programmes that maximise water use
efficiency, as well as for improving the accuracy of
the predictions of ecosystem productivity from
climate data.
Concluding Remarks and Future Prospect
In summary, using light-saturated, daily maximum
stomatal conductance (gs
) as the indicator for the
intensity of water stress reveals a pattern of
photosynthetic response to water stress that is
common to all C3
 species analysed. Analysing
different components of photosynthesis along a gs
range suggests that photosynthetic metabolism is
substantially resistant to water stress until gs
 is below
0.10-0.15 mol H2
O m-2
. Nevertheless, these low
 values are usually observed under natural
conditions in semi-arid areas, suggesting that non-
stomatal limitations are an important component of
over-season photosynthetic inhibition in these areas
at the global scale. The gs
 threshold usually coincides
with a changing pattern of variation of intrinsic water
use efficiency (AN/gs
) in response to water stress, so
that maximum AN/gs
 is achieved at the limit between
diffusional and metabolic limitations to
photosynthesis (Fig. 5). This should be taken into
account when developing water use efficient
irrigation programmes.
Besides these findings, a number of important gaps
of knowledge have been highlighted in the present
review. Here we propose what, to our point of view,
should be the research priorities in order to advance
in the understanding of photosynthesis response to
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
Intrinsic water-use efficiency (AN/gs) (mmol CO2 
gs (mol H2O m-2
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
Fig. 5. Relationship between the intrinsic water use
efficiency (AN/gs
) and the stomatal conductance (gs
in three deferent studies: (A) Study including endemic
(Hypericum balearicum L., Lysimachia minoricensis
J. J. Rodr., Phlomis italica L.) and non-endemic (Cistus
albidus L., Mentha aquatica L., Pistacia lentiscus L.)
species from the Balearic Islands by J GalmÈs
(unpublished results). (B) Study of 13 Mediterranean
sclerophyll woody species: Arbutus unedo L., Cistus
albidus L., Cistus monspeliensis L., Cistus salvifolius
L., Hypericum balearicum L., Cneorum tricoccon L.,
Olea europaea L., Phyllirea latifolia L., Pistacia
lentiscus L., Quercus coccifera L., Quercus ilex L.,
Rhamnus alaternus L., Rhamnus ludovici-salvatoris
R. Chodat, by J GulÌas (unpublished results). (C) Study
in Vitis vinifera L. plants by J Flexas (unpublished
results).281 Photosynthetic down-regulation under water stress
Boyer J S, Wong S C, Farquhar G D. 1997. CO2
 and water
vapour exchange across leaf cuticle (epidermis) at various
water potentials. Plant Physiology 114:185-191.
Brestic M, Cornic G, Fryer M J, Baker N R. 1995. Does
photorespiration protect the photosynthetic apparatus in
French bean leaves from photoinhibition during drought
stress? Planta 196:450-457.
Buckley T N, Farquhar G D, Mott K A. 1997. Qualitative
effects of patchy stomatal conductance distribution features
on gas-exchange calculations. Plant Cell and Environment
Castrillo M, Calcagno A M. 1989. Effects of water stress and
rewatering on ribulose 1,5-bisphosphate carboxylase activity,
chlorophyll and protein contents in two cultivars of tomato.
Journal of Horticultural Science 64:717-724.
Castrillo M, Fern·ndez D, Calcagno A M, Trujillo I, Guenni
L. 2001. Responses of ribulose-1,5-bisphosphate carboxylase,
protein content, and stomatal conductance to water deficit in
maize, tomato, and bean. Photosynthetica 39:221-226.
Centritto M, Loreto F, Chartzoulakis K. 2003. The use of
low [CO2
] to estimate diffusional and non-diffusional
limitations of photosynthetic capacity of salt-stressed olive
saplings. Plant, Cell and Environment 26:585-594.
Chaves M M. 1991. Effects of water deficits on carbon
assimilation. Journal of Experimental Botany 42:1-16.
Chaves M M, Maroco J P, Pereira J S. 2003. Understanding
plant responses to drought - from genes to the whole plant.
Functional Plant Biology 30:239-264.
Cornic G. 1994. Drought stress and high light effects on leaf
photosynthesis. In Photoinhibition of Photosynthesis: From
Molecular Mechanisms to the Field. pp. 297-313. Eds. N R
Baker and J R Bowyer. Oxford BIOS Scientific Publishers.
Cornic G. 2000. Drought stress inhibits photosynthesis by
decreasing stomatal aperture ñ not by affecting ATP synthesis.
Trends in Plant Sciences  5:187-188.
Cornic G, Fresneau C. 2002. Photosynthetic carbon reduction
and carbon oxidation cycles are the main electron sinks for
Photosystem II activity during a mild drought. Annals of
Botany 89:887-894.
Correia M J, Pereira J S, Chaves M M, Rodrigues M L,
Pacheco C A. 1995. ABA xylem concentrations determine
maximum daily leaf conductance of field-grown Vitis vinifera
L. plants. Plant Cell and Environment 18:511-521.
Delfine S, Loreto F, Alvino A. 2001. Drought-stress effects on
physiology, growth and biomass production of rainfed and
irrigated Bell Pepper plants in the Mediterranean region.
Journal of American Society of Horticultural Sciences
Flexas J, Medrano H. 2002. Drought-Inhibition of
photosynthesis in C3
 plants: stomatal and non-stomatal
limitation revisited. Annals of Botany 89:183-189.
Flexas J, Escalona J M, Medrano H.  1999. Water stress
induces different levels of photosynthesis and electron
transport rate regulations in grapevines. Plant, Cell and
Environment 22:39-48.
Flexas J, GalmÈs J, Ribas-CarbÛ M, Medrano H. 2004. The
effects of drought in plant respiration. In  Advances in
Photosynthesis and Respiration XX. Plant Respiration. Eds
H Lambers and M Ribas-CarbÛ. Belgium: Kluwer Academic
Publishers B.V. (In press).
Flexas J, Bota J, Escalona J M, Sampol B, Medrano H.
2002a. Effects of drought on photosynthesis in grapevines
under field conditions: an evaluation of stomatal and
mesophyll limitations. Functional Plant Biology 29:461-471.
Flexas J, Escalona J M, Evain S, GulÌas J, Moya I, Osmond
C B, Medrano H. 2002b. Steady-state chlorophyll
fluorescence (Fs) measurements as a tool to follow variations
of net CO2
 assimilation and stomatal conductance during
water-stress in C3
 plants. Physiologia Plantarum  114:231-
water stress and irrigation scheduling:
1. The variations along a gs
 gradient during water
stress of many metabolic components affecting
photosynthesis are presently unknown, and should
be evaluated. Among these, the most important
would be: (1) all the enzymes involved in
regeneration of RuBP in the Calvin cycle; (2) water
stress-induced oxidative stress and protective
antioxidant responses; (3) photophosphorylation;
and (4) agents possibly involved in CO2
inside leaves (carbonic anhydrase, aquaporins).
Similarly, the responses of plant respiration and
respiratory components should also be evaluated,
provided that respiration is, together with
photosynthesis, the other important component of
plant production.
2. The analysis of the recovery of different
photosynthetic components upon re-watering from
different water stress intensities in different plants
and conditions. This knowledge would be of
importance for the development of deficit irrigation
programmes, as well as for improving the accuracy
of ecosystem productivity predictions from climate
3. From a more practical point of view, the
improved physiological knowledge should induce
the development of physiologically based indicators
to improve irrigation programs in semi-arid areas,
which should be tested extensively under real crop
conditions. In this sense, scaling up from
physiological to more agronomical programs would
be required.
Drs M Centritto, M M Chaves, D W Lawlor, C
Lovisolo, M S·nchez-DÌaz, and H R Schultz
provided useful comments on the present review
during the Association of Applied Biologistsí
meeting ìOptimisation of Water Use by Plants in
the Mediterraneanî, held in Cala Bona (Mallorca),
from 24-28 March 2003. These are gratefully
AntolÌn M C, S·nchez-DÌaz M. 1993. Effects of temporary
droughts on photosynthesis of alfalfa plants.  Journal of
Experimental Botany 44:1341-1349.
Arndt S K, Clifford S C, Wanek W, Jones H G, Popp M.
2001. Physiological and morphological adaptations of the fruit
tree Ziziphus rotundifolia in response to progressive drought
stress. Tree Physiology 21:705-715.
Bota J, Medrano H, Flexas J. 2004. Is photosynthesis limited
by decreased Rubisco activity and RuBP content under
progressive water stress? The New Phytologist 162:671-682.
Boyer J S. 1976. Photosynthesis at low water potentials.
Philosophical Transactions of the Royal Society B  273:501-
Boyer J S. 1982. Plant productivity and environment. Science
218:443-448.282 JAUME FLEXAS ET AL.
Regulation of photosynthesis of C3
 plants in response to
progressive drought: the interest of stomatal conductance as
a reference parameter. Annals of Botany 89:895-905.
Medrano H, Escalona J M, Cifre J, Bota J, Flexas J. 2003. A
ten-year study on the physiology of two Spanish grapevine
cultivars under field conditions: effects of water availability
from leaf photosynthesis to grape yield and quality.
Functional Plant Biology 30:607-619.
Mittler R, Merquiol E, Hallak-Herr E, Rachmilevitch S,
Kaplan A, Cohen M. 2001. Living under a ëdormantí canopy:
a molecular acclimation mechanism of the desert plant
Retama raetam. The Plant Journal 25:407-416.
Moriana A, Villalobos F J, Fereres E. 2002. Stomatal and
photosynthetic responses of olive (Olea europaea L.) leaves
to water deficits. Plant Cell and Environment 25:395-405.
MunnÈ-Bosch S, Alegre L. 2000. Changes in carotenoids,
tocopherols and diterpenes during drought and recovery, and
the biological significance of chlorophyll loss in Rosmarinus
officinalis plants. Planta 210:925-931.
NoguÈs S, Alegre L. 2002. An increase in water deficit has no
impact on the photosynthetic capacity of field-grown
Mediterranean plants. Functional Plant Biology 29:621-630.
Ort D R, Oxborough K, Wise R R. 1994. Depressions of
photosynthesis in crops with water deficits. In Photoinhibition
of Photosynthesis: From Molecular Mechanisms to the Field.
Eds N R Baker and J R Bowyer. Oxford: BIOS Scientific
Ortiz-LÛpez A, Ort D  R, Boyer J S. 1991.
Photophosphorylation in attached leaves of Helianthus
annuus at low water potentials. Plant Physiology 96:1018-
Pankovic D, Sakac Z, Kevresan S, Plesnicar M. 1999.
Acclimation to long-term water deficit in the leaves of two
sunflower hybrids: photosynthesis, electron transport and
carbon metabolism. Journal of Experimental Botany 50:127-
Parry M A J, Andralojc P J, Khan S, Lea P J, Keys A J.
2002. Rubisco activity: effects of drought stress. Annals of
Botany 89:833-839.
Pelleschi S, Rocher J-P, Prioul J-L. 1997. Effect of water
restriction on carbohydrate metabolism and photosynthesis
in mature maize leaves. Plant, Cell and Environment 20:493-
Pelloux J, Jolivet Y, Fontaine V, Banvoy J, Dizengremel P.
2001. Changes in Rubisco and Rubisco activase gene
expression and polypeptide content in Pinus halepensis M.
subjected to ozone and drought. Plant, Cell and Environment
Quick W P, Chaves M M, Wendler R, David M, Rodrigues
M L, Passaharinho J A, Pereira J S, Adcock M D, Leegood
RC, Stitt M.  1992. The effect of water stress on
photosynthetic carbon metabolism in four species grown
under field conditions. Plant, Cell and Environment 15:25-
Reichstein M, Tenhunnen J D, Roupsard O, Ourcival J-M,
Rambal S, Miglietta F, Peressotti A, Pecchiari M, Tirone
G, Valentini R. 2002. Severe drought effects on ecosystem
 and H2
O fluxes at three Mediterranean evergreen sites:
revision of current hypotheses?. Global Change Biology
S·nchez-RodrÌguez J, PÈrez P, MartÌnez-Carrasco R. 1999.
Photosynthesis, carbohydrate levels and chlorophyll
fluorescence-estimated intercellular CO2
 in water-stressed
Casuarina equisetifolia Forst. and Forst. Plant, Cell and
Environment 22:867-873.
Schulze E-D, Hall A E. 1982. Stomatal responses, water loss
and CO2
 assimilation rates of plants in contrasting
environments. In Encyclopedia of Plant Physiology, Vol. 12B.
Physiological Plant Ecology II. Eds O L Lange, P S Nobel
and C B Osmond. Berlin, Heidelberg, New York: Springer-
Giardi M T, Cona A, Geiken B, Kucera T, MasojÌdek J,
Mattoo A K. 1996.  Long-term drought stress induces
structural and functional reorganization of photosystem II.
Planta 199:118-125.
GimÈnez C, Mitchell V J, Lawlor D W. 1992. Regulation of
photosynthesis rate of two sunflower hybrids under water
stress. Plant Physiology  98:516-524.
Graan T, Boyer J S. 1990. Very high CO2
 partially restores
photosynthesis in sunflower at low water potentials. Planta
GulÌas J, Flexas J, Abadia A, Medrano H. 2002.
Photosynthetic responses to water deficit in six Mediterranean
sclerophyll species: possible factors explaining the declining
distribution of Rhamnus ludovici-salvatoris, an endemic
Balearic species. Tree Physiology 22:687-697.
Gunasekera D, Berkowitz G A. 1993. Use of transgenic plants
with Rubisco antisense DNA to evaluate the rate limitation
of photosynthesis under water stress.  Plant Physiology
Harley P C, Tenhunen J D, Lange O L, Beyschlag W. 1987.
Seasonal and diurnal patterns in leaf gas exchange of Phillyrea
angustifolia growing in Portugal. In Plant response to stress:
Functional analysis in Mediterranean ecosystems, NATO ASI
Series, Vol. G15, pp. 329-337. Eds J D Tenhunen, F M
Catarino, O L Lange and W C Oechel. Berlin: Springer Verlag.
Holaday A S, Ritchie S W, Nguyen H T. 1992. Effects of water
deficit on gas-exchange parameters and ribulose 1,5-
bisphosphate carboxylase activation in wheat. Environmental
and Experimental Botany 32:403-410.
Kaiser W M, Fˆrster J. 1989. Low CO2
 prevents nitrate
reduction in leaves. Plant Physiology 91:970-974.
Laisk A. 1983. Calculation of leaf photosynthetic parameters
considering the statistical distribution of stomatal apertures.
Journal of Experimental Botany 34:1627-1635.
Lal A, Ku M S B, Edwards G E. 1996. Analysis of inhibition
of photosynthesis due to water stress in the C3
Hordeum vulgare and Vicia faba: electron transport, CO2
fixation and carboxylation capacity. Photosynthesis Research
Larcher W, de Moraes J A P V, Bauer H. 1981. Adaptive
responses of leaf water potential, CO2
-gas exchange and water
use efficiency of Olea europaea during drying and rewatering.
In Components of Productivity of Mediterranean-Climate
region ñ Basic and Applied Aspects,  pp. 77-83. Eds N S
Margaris and H A Mooney. The Hague, Boston, London: Dr
W Junk Publishers.
Lawlor D W. 1995. The effects of water deficit on
photosynthesis. In  Environment and Plant Metabolism.
Flexibility and Acclimation, pp. 129-160. Ed. N Smirnoff.
Oxford: BIOS Scientific Publisher.
Lawlor D W. 2002. Limitations to photosynthesis in water-
stressed leaves: stomata vs. metabolism and the role of ATP.
Annals of Botany 89:871-885.
Lawlor D W, Cornic G. 2002. Photosynthetic carbon
assimilation and associated metabolism in relation to water
deficits in higher plants. Plant, Cell and Environment 25:275-
Maroco J P, Rodrigues M L, Lopes C, Chaves M M. 2002.
Limitations to leaf photosynthesis in field-grown grapevine
under drought ñ metabolic and modelling approaches.
Functional Plant Biology 29:451-459.
Marron N, Delay D, Petit J-M, Dreyer E, Kahlem G,
Delmotte F M, Brignolas F. 2002. Physiological traits of
two Populus x  euramericana clones, Luisa Avanzo and
Dorskamp, during a water stress and re-watering cycle. Tree
Physiology 22:849-858.
Medrano H, Parry M A J, Socias X, Lawlor D W. 1997. Long
term water stress inactivates Rubisco in subterranean clover.
Annals of Applied Biology 131:491-501.
Medrano H, Escalona J M, Bota J, GulÌas J, Flexas J. 2002.283 Photosynthetic down-regulation under water stress
Sharkey T D. 1990. Water stress effects on photosynthesis.
Photosynthetica 24:651.
Tardieu F, Simonneau T. 1998. Variability among species of
stomatal control under fluctuating soil water status and
evaporative demand: modelling isohydric and anisohydric
behaviours. Journal of Experimental Botany 49:419-432.
Tezara W, Mitchell W J, Driscoll S D, Lawlor D W. 1999.
Water stress inhibits plant photosynthesis by decreasing
coupling factor and ATP. Nature 401:914-917.
Tezara W, Mitchell W J, Driscoll S D, Lawlor D W. 2002.
Effects of water deficit and its interaction with CO2
on the biochemistry and physiology of photosynthesis in
sunflower. Journal of Experimental Botany 53:1781-1791.
Thimmanaik S, Giridara Kumar S, Jyothsna Kumari G,
Suryanarayana N, Sudhakar C. 2002. Photosynthesis and
the enzymes of photosynthetic carbon reduction cycle in
mulberry during water stress and recovery. Photosynthetica
van Rensburg L, Kr¸ger G H J. 1993. Comparative analysis
of differential drought stress-induced suppression of and
recovery in carbon dioxide fixation: stomatal and non-
stomatal limitation in Nicotiana tabacum. Journal of Plant
Physiology 142:296-306.
Vassey T L, Quick W P, Sharkey T D, Stitt M. 1991. Water
stress, carbon dioxide and light effects on sucrose phosphate
synthase activity in  Phaseolus vulgaris.  Physiologia
Plantarum 81:37-44.
Wingler A, Quick W P, Bungard R A, Bailey K J, Lea P J,
Leegood R C. 1999. The role of photorespiration during
drought stress: an analysis utilizing barley mutants with
reduced activities of photorespiratory enzymes. Plant, Cell
and Environment 22:361-373.
Wingler A, Lea P J, Quick W P, Leegood R C. 2000.
Photorespiration: metabolic pathways and their role in stress
protection. Philosophical Transactions of the Royal Society
B 355:1517-1529.
Younis H M, Boyer J S, Govindjee. 1979. Conformation and
activity of chloroplast coupling factor exposed to low
chemical potential of water in cells. Biochimica et Biophysica
Acta 548:328-340.

Tidak ada komentar:

Poskan Komentar