doi:10.1093/aob/mcf110, available online at www.aob.oupjournals.org
Limitation to Photosynthesis in Water-stressed Leaves: Stomata vs. Metabolism
and the Role of ATP
DAVID W. LAWLOR*
IACR-Rothamsted, Harpenden, Herts., AL5 2AQ, UK
Received: 8 November 2001 Returned for revision: 5 January 2002 Accepted: 20 February 2002
Decreasing relative water content (RWC) of leaves progressively decreases stomatal conductance (g
s
), slowing
CO
2
assimilation (A) which eventually stops, after which CO
2
is evolved. In some studies, photosynthetic poten-
tial (A
pot
), measured under saturating CO
2
, is unaffected by a small loss of RWC but becomes progressively
more inhibited, and less stimulated by elevated CO
2
, below a threshold RWC (Type 1 response). In other stud-
ies, A
pot
and the stimulation of A by elevated CO
2
decreases progressively as RWC falls (Type 2 response).
Decreased A
pot
is caused by impaired metabolism. Consequently, as RWC declines, the relative limitation of A
by g
s
decreases, and metabolic limitation increases. Causes of decreased A
pot
are considered. Limitation of ribu-
lose bisphosphate (RuBP) synthesis is the likely cause of decreased A
pot
at low RWC, not inhibition or loss of
photosynthetic carbon reduction cycle enzymes, including RuBP carboxylase/oxygenase (Rubisco). Limitation of
RuBP synthesis is probably caused by inhibition of ATP synthesis, due to progressive inactivation or loss of
Coupling Factor resulting from increasing ionic (Mg
2+
) concentration, not to reduced capacity for electron or
proton transport, or inadequate trans-thylakoid proton gradient (DpH). Inhibition of A
pot
by accumulation of
assimilates or inadequate inorganic phosphate is not considered signi®cant. Decreased ATP content and imbal-
ance with reductant status affect cell metabolism substantially: possible consequences are discussed with refer-
ence to accumulation of amino acids and alterations in protein complement under water stress.
ã 2002 Annals of Botany Company
Key words: Photosynthesis, relative water content (RWC), ATP synthesis, stomata, amino acid metabolism, ribulose
bisphosphate synthesis, protein synthesis, chaperones.
INTRODUCTION
The photosynthetic rate (A) of leaves of both C
3
and C
4
plants decreases as their relative water content (RWC) and
water potential (y) decrease (Chaves, 1991; Cornic, 1994;
Kramer and Boyer, 1995; Lawlor, 1995; Cornic and
Massacci, 1996) (Fig. 1). However, the relative importance
of stomatal conductance (g
s
) in restricting the supply of CO
2
to metabolism (stomatal limitation), and of metabolic
impairment which decreases the potential rate of A (A
pot
,
non-stomatal limitation), is unclear and discussion has
become polarized. Stomatal limitation is considered to
decrease both A and CO
2
concentration in the intercellular
spaces of the leaf (C
i
), which inhibits metabolism (Kaiser,
1987; Downton et al., 1988; Cornic, 2000) (Fig. 2). Thus,
Kaiser (1987) and Kaiser and Foster (1989) concluded that
nitrate reductase (NR) activity, and Vassey et al. (1991) that
sucrose phosphate synthase (SPS) activity, were inhibited
by low C
i
or the associated low rate of A. Limited A
decreases consumption of electrons released from water as a
consequence of the light reactions: the ensuing excess of
excitation energy is dissipated by non-photochemical
quenching by the xanthophyll cycle in the photosystem
antennae (see Lawlor, 2001). A key feature in this analysis
is that the CO
2
concentration in the atmosphere (C
a
) can be
increased so that the CO
2
concentrations in the intercellular
spaces and in the chloroplast stroma (C
c
) rise, and restore A
to A
pot
. This occurs over a range of RWC from 100 to 80 %
(note that RWC values are generalizations for mesophytes),
but at more severe stress A
pot
is decreased (eventually
ceasing at approx. 40 % RWC) and cannot be restored by
elevated C
a
. The decrease in A
pot
at low RWC (Chaves,
1991) has been considered to be a consequence of low C
i
.
Another point of view (Keck and Boyer, 1974; Lawlor,
1995; Escalona et al., 1999; Flexas et al., 1999; PankovicÏ
et al., 1999) considers that A
pot
and metabolism decrease
progressively, i.e. elevated C
a
is less and less able to restore
A
pot
to the unstressed rate as RWC falls. Thus, metabolism
is inhibited. This is not regarded as a consequence of the
effect of low C
i
or C
c
on NR or SPS. Rather, decreased ATP
synthesis by the enzyme ATP synthase (Coupling Factor,
CF) is considered to be the primary effect of decreasing
RWC (Keck and Boyer, 1974; Tezara et al., 1999), due
largely, but not exclusively, to the effects of increasing ion
(speci®cally Mg
2+
) concentrations in the chloroplast as
RWC falls (Younis et al., 1979); CO
2
depletion is not the
primary effect (Tang et al., 2002). Metabolic limitation is
often observed and correlates with loss of ATP content,
which starts to decrease with mild water stress (Tezara et al.,
1999; Flexas and Medrano, 2002). Thus, the limitation to
A
pot
is caused by inadequate ATP not CO
2
, with major
consequences for metabolism; excess excitation energy is
dissipated via non-photochemical quenching.
Whilst emphasizing the overall loss of photosynthetic
potential, this is not to deny that the initial decrease in A in
ã 2002 Annals of Botany Company
* For correspondence. E-mail david.lawlor@bbsrc.ac.uk
Annals of Botany 89: 871±885, 2002

air can be due to lower g
s
, although metabolic effects
become increasingly dominant (e.g. Tezara et al., 1999). It
is essential to understand how A
pot
is affected because the
nature and sensitivity of metabolic processes determine the
responses of plants to water de®cits, and the processes
required to prevent damage. These protective mechanisms
allow plants to function in terms of productivity, reproduc-
tion and ecological ®tness in different environments and
under varying water balance. Development of a generally
acceptable model (Fig. 2) of what may be called the `water
de®ciency syndrome', to emphasize the complex nature of
the problem and the wide range of responses to changing
conditions, is an important but rather distant goal. Despite
many years of effort to understand the causes of the myriad
changes in cellular biochemistry, e.g. accumulation of
`stress metabolites' such as amino acids including proline
(Samaras et al., 1995) and citrulline (Yokota et al., 2002),
and changes in gene expression and protein synthesis (see
Bray, 2002), resulting from decreased RWC, conditions
within the cell responsible for altered metabolism are poorly
known. Understanding of the interactions between photo-
synthesis and other metabolic processes, illustrated in
Fig. 2, is advanced; ultimately, a more quantitative approach
will be required. Changes in gene expression and protein
synthesis are being studied intensively, as mechanisms of
cellular adaptation to drought, and major attempts made to
genetically modify plants to be more drought tolerant or
resistant. The long-term goals of stabilizing and increasing
yields under drought may be more successful given clearer
understanding of conditions within cells at decreased RWC.
This review considers the evidence for the stomatal and
non-stomatal regulation of A, and inhibition of A
pot
in leaves
of C
3
plants in relation to RWC. Changes in photosynthetic
metabolism and the role of ATP synthesis are discussed.
Conditions in stressed cells and consequences of changed
ATP and A are related to metabolism, particularly amino
acid and protein synthesis.
PHOTOSYNTHESIS UNDER STRESS
The `classical' response of A to decreasing RWC and water
potential, whether induced by decreasing the water supply
to the roots and thence leaves by soil drying or application
of osmotica, is shown in Fig. 1 for leaves of mesophytes. It
is assumed, for discussion, that incident photosynthetically
useful photon ¯ux (PPF, wavelength 400±700 nm) saturates
A, and that excess energy captured is dissipated non-
photochemically. Between 100 and 90 % RWC (the control,
unstressed state), g
s
is maximal but with C
a
of 350 ml l
±1
CO
2
and 210 ml l
±1
O
2
, A is not saturated with CO
2
. Raising C
a
to
>1000 ml l
±1
saturates A, giving A
max
, the maximum
metabolic capacity, or potential (A
pot
) of the system. If
metabolic processes are inhibited then A
pot
is decreased.
When the tissue is unstressed at large RWC, A
pot
= s A
max
,
but at small RWC, A
pot
< A
max
of unstressed leaves; A and
A
pot
approach or become zero at approx. 40 % RWC.
Decreasing RWC causes g
s
and A to decrease, approxi-
mately in parallel, although at small values of RWC, g
s
reaches a minimum but Amay continue to decrease. In some
experiments (see Cornic, 2000), when A decreases as RWC
falls initially, elevated C
a
(50 up to 150 ml l
±1
, equivalent to
5 to 15 %) restores A to A
max
, i.e. A
pot
is unaffected.
Thereafter A drops, and stimulation by elevated CO
2
is
smaller, showing that A
pot
is limited by metabolic factors at
low RWC. This has been called a Type 1 response (Lawlor
and Cornic, 2002). In other studies, A falls progressively as
RWC falls, but is also less stimulated by elevated CO
2
. This
shows that A
pot
is progressively inhibited, and the effect of
g
s
diminished, with increasing stress. This is called a Type 2
response (Lawlor and Cornic, 2002).
What causes the differences in response of A
pot
to RWC
between experiments? Are differences in techniques respon-
sible? Are there fundamental differences between species?
F IG . 1. A, Schematic of the basic responses of actual photosynthetic rate
(A) in air (360 mmol CO
2
m
±2
s
±1
) and potential photosynthetic rate (A
pot
)
measured at elevated CO
2
concentration, to relative water content
(RWC). Type 1 and 2 responses of A
pot
are shown. In the Type 1
response, A
pot
is unaffected until a 20±30 % decrease in RWC occurs,
when it becomes metabolically limited. In Type 2, the change is linear,
showing progressive metabolic limitation. In both types in well-watered
leaves, photosynthetic rate (A) is stimulated by elevated CO
2
. Elevated
CO
2
maintains A at the potential rate (A
pot
) in the Type 1 response as
RWC decreases; but at RWC below approx. 80 % A
pot
decreases in Type
1. Elevated CO
2
simulates A progressively less as RWC decreases in
Type 2, showing that A
pot
is inhibited. B, Scheme of the changes in CO
2
inside the leaf (C
i
) during steady-state A, as stomatal conductance (g
s
)
decreases with falling RWC, associated with Type 1 or Type 2
photosynthetic response (1 with C
i
decreasing to compensation point; 2
with C
i
decreasing but not to compensation point). The equilibrium
compensation point, G, associated with Type 1 response is indicated.
There are differences between experiments, with C
i
not decreasing, or
decreasing somewhat, or substantially. This may re¯ect different methods
of assessing C
i
.
872 LawlorÐWater Stress Limitation of Photosynthesis