Download Tansley Review No. 112 Oxygen processing in photosynthesis

REVIEW
New Phytol. (2000), 146, 359–388
Tansley Review No. 112
Oxygen processing in photosynthesis :
regulation and signalling
C H R I S T I N E H. F O Y E R*    G R A H A M N O C T O R
Department of Biochemistry and Physiology, IACR-Rothamsted, Harpenden,
Herts AL5 2JQ, UK
Received 20 July 1999 ; accepted 7 January 2000

I. I
II. P   
III. O    
1. Oxygen ‘ poises ’ electron transport and
carbon assimilation
2. The role of oxygen in ATP synthesis
3. How fast is O reduction at PSI ?
#
4. Chloroplastic processing of H O
# #
IV. R   

1. The thioredoxin system
2. Manipulating the expression of thiolregulated enzymes
3. Modifying sensitivity to thiol regulation
V. P
1. The pathway and its genetic manipulation
2. Engineering plants that photorespire less ?
3. Is photorespiration important in energy
dissipation ?
4. Production and processing of
photorespiratory H O
# #
360
360
362
362
364
364
366
368
368
369
369
369
369
371
372
5. Catalase and foliar H O levels
# #
6. Catalase and non-photorespiratory H O
# #
generation
VI. R
1. ‘ Photosynthetic ’ respiration
2. AOS in the mitochondrion
3. AOX : regulation and significance to
photosynthesis
VII. P   

1. The need for sensors, signals and
transducers
2. Signal transduction at the local level
3. Remote signalling and responses leading to
acclimation of photosynthesis ?
4. Interactions between AOS, NO), and
antioxidants
VIII. C
Acknowledgements
References
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
The gradual but huge increase in atmospheric O concentration that followed the evolution of oxygenic
#
photosynthesis is one consequence that marks this event as one of the most significant in the earth’s history. The
high redox potential of the O \water couple makes it an extremely powerful electron sink that enables energy to
#
be transduced in respiration. In addition to the tetravalent interconversion of O and water, there exist a plethora
#
of reactions that involve the partial reduction of O or photodynamic energy transfer to produce active oxygen
#
species (AOS). All these redox reactions have become integrated during evolution into the aerobic photosynthetic
cell. This review considers photosynthesis as a whole-cell process, in which O and AOS are involved in reactions
#
at both photosystems, enzyme regulation in the chloroplast stroma, photorespiration, and mitochondrial electron
transport in the light. In addition, oxidants and antioxidants are discussed as metabolic indicators of redox status,
acting as sensors and signal molecules leading to acclimatory responses. Our aim throughout is to assess the
insights gained from the application of mutagenesis and transformation techniques to studies of the role of O and
#
related redox components in the integrated regulation of photosynthesis.
*Author for correspondence (tel j44 1582 763133 ; fax j44 1582 763010 ; e-mail christine.foyer!bbsrc.ac.uk).
Abbreviations : AOS, active oxygen species ; AOX, alternative oxidase ; APX, ascorbate peroxidase ; CF -ATPase, reversible chloroplast
"
ATPase\ATP synthase ; DHA, dehydroascorbate ; DHAR, DHA reductase ; FBPase, fructose-1,6-bisphosphatase ; Fd, ferredoxin ;
FNR, ferredoxin : NADP+ oxidoreductase ; GSH, reduced glutathione ; GSSG, oxidized glutathione ; GOGAT, glutamate synthase ;
GR, glutathione reductase ; GS, glutamine synthetase ; MDHA, monodehydroascorbate ; NADP-MDH, NADP+-dependent malate
dehydrogenase ; 2-OG, s-oxoglutarate ; PEPC, phosphoenolpyruvate carboxylase ; PQ, plastoquinone ; PSI, photosystem I ; PSII
photosystem II ; Q cycle, cyclic mechanism of electron transfer involving plastoquinone and the cytochrome b f complex : RPP,
'
reductive pentose phosphate ; Rubisco, RuBP carboxylase\oxygenase ; RuBP, ribulose 1,5-bisphosphate ; SOD, superoxide dismutase ;
Td, thioredoxin.
REVIEW
360
C. H. Foyer and G. Noctor
I. 
Light, water, CO and nutrients are the fluctuating
#
driving forces and substrates of photosynthesis, but
O is invariant. However, oxygen is a dangerous ally
#
whose reactivity must be controlled if dangerous
side-reactions are to be avoided. The capacity of
metabolism to use light energy is constrained by the
environment, which has necessitated the evolution of
an integrated process of great flexibility, able to
operate over a broad window of environmental
conditions. At low light levels, in unstressed conditions, photosynthesis operates with maximal quantum yield but low capacity. The photosynthetic
process accelerates as light intensity increases but
becomes less light-efficient, eventually reaching a
ceiling rate. Walker (1992) considered that genetic
attempts to enhance photosynthesis should concentrate on raising this ceiling, because there is less
scope for improvement in quantum yield. In field
conditions, maximal photosynthetic rate (the ceiling)
is frequently depressed by environmental constraints
(stress). Many attempts to prevent depression of the
ceiling under conditions of stress have focused on
protecting photosynthesis from oxidative damage by
the manipulation of enzymes involved in the detoxification of active oxygen species (AOS). The aim of
these manipulations has been to control the products
of the damaging side-reactions that depress photosynthesis.
Glycollate
oxidase
Mitochondrial
terminal
oxidases
Superoxide
dismutase
+0.94 V
Ascorbate
peroxidase
O22– (H2O2)
+e– –e–
During photosynthesis, water is oxidized by the
manganese-containing oxygen-evolving complex
–0.33 V
O2• – (HO2)
+e– –e–
II.    
PSI
acceptors
O2
+e– –e–
The multiplicity of roles played by O in metab#
olism is a reflection of its distinctive chemistry.
Molecular oxygen is highly unusual in that its two
least strongly bound electrons are unpaired and have
parallel spins. This property means that groundstate O is triplet but that excitation leads to the
#
formation of the highly reactive singlet state. The
tetravalent interconversion of O and water is a
#
highly specialized reaction that can be catalysed by
relatively few enzymes. Oxygen chemistry dictates
that reduction is preferentially univalent although
some enzymes can achieve bivalent O reduction to
#
produce H O (Fig. 1). The AOS produced by
# #
partial reactions vary greatly in their reactivity but
are all less stable than O or water.
#
This review concentrates on recent developments
in the regulation of oxygenic photosynthesis. We
emphasize reactions involving singlet O , reduced
#
intermediates such as superoxide or H O , and
# #
enzyme systems associated with photosynthesis that
interconvert O and water. Special attention is paid
#
to developments made possible by mutagenesis and
transformation techniques that have confirmed,
extended or challenged knowledge derived from
purely biochemical and biophysical studies.
Catalase
+0.46 V
OH• + OH–(H2O)
+e– –e–
OH– (H2O)
+2.18 V
PSII
Fig. 1. Interconversion of O and water, showing intermediate active oxygen species. Protonated forms of
#
anions are shown in parentheses. Numbers give approximate redox potentials. The redox potential (per
electron) for the tetravalent interconversion of oxygen and water is approx. j0n81 V. The arrows show some
of the numerous reactions involving oxygen species. The reactions shown are key processes in the
photosynthetic cell ; the most important enzymes or reactants involved in these reactions are shown in boxes.
The oxygen species acting as substrate in each reaction is indicated by the vertical position of the box. Oxidation
of water is in green, blue shows disproportionation reactions and reactions involving net electron transfer to
oxygen are in red.
REVIEW
Oxygen processing in photosynthesis : regulation and signalling
(Wydrzynski et al., 1996) without appreciable release
of the unstable partly reduced oxygen species shown
in Fig. 1 (superoxide, H O and hydroxyl radical).
# #
Although partial water oxidation can lead to H O
# #
formation (Fine & Frasch, 1992), this is not a
predominant reaction in physiological situations.
Some AOS formation linked to PSII activity may
occur on the acceptor side either through electron
flow from phaeophytin or through the reduction of
O by semiquinone species (Ananyev et al., 1994).
#
Hydroxyl radicals generated from H O in the PSII
# #
reaction centre have been shown to be potent
inhibitors of PSII function (Tschiersch & Ohmann,
1993 ; Jakob & Heber, 1996). In addition, excess
light increases the probability of formation of highly
reactive singlet O , through energy transfer from
#
excited triplet chlorophyll to ground-state triplet O .
#
Photoinhibition involves the light-induced inactivation of photosynthetic electron transport
(Powles, 1984 ; Osmond, 1994). The principal site of
photoinhibition is the D1 reaction-centre protein of
PSII. Light-induced damage to D1 is followed by its
removal from the membrane and turnover of the
apoprotein at a rate much more rapid than any of the
other PSII proteins (Mattoo et al., 1981). For
continued PSII function, excised D1 must be
replaced by a new functional peptide in a process
known as the repair cycle. The photoinhibition of
PSI has also been demonstrated in a number of
species (Havaux & Davaud, 1994 ; Terashima et al.,
1994). The PSI reaction centre complex is composed
of two subunits which are the products of the psaA
and psaB genes. Suppression of the degradation of
these subunits and protection of PSI activity are
observed if photoinhibitory treatments are applied
under anaerobic conditions, implicating AOS in PSI
photoinhibition (Sonoike, 1996).
Because the photoinhibition of PSII occurs both
under aerobic and anaerobic conditions, an obligatory role for O \AOS has not been established.
#
Studies of the role of O are complicated by two
#
possible opposing effects : although active O can
#
directly damage proteins such as D1, O can also
#
alleviate photoinhibition by acting as an electron
acceptor (Krause, 1994).
Two types of PSII photoinhibition have been
described : donor-side inhibition (Vass et al., 1992)
and acceptor-side inhibition (Jegerscho$ ld & Styring,
1990). Although both types can be observed in
isolated thylakoids, acceptor-side inhibition is considered to be the predominant process in physiological situations. In this scenario excessive
irradiance leads to the formation within PSII of
highly reactive species that inevitably generate AOS,
notably singlet oxygen and the hydroxyl radical,
which are capable of initiating damage to D1. Singlet
oxygen, hydroxyl radical, superoxide and H O can
# #
be generated within PSII complexes (Richter et al.,
1990 ; Jakob & Heber, 1996). Although several
361
components associated with PSII have antioxidant
activity (Ananyev et al., 1994), the reactivity of
singlet O and hydroxyl radicals means that avoid#
ance strategies that prevent their formation are the
best form of defence. The mechanisms involved in
these strategies have been discussed at length
recently (Foyer & Harbinson, 1999).
Degradation of the D1 protein might begin with
damage caused by hydroxyl radicals or singlet O but
#
it also involves the activation of a specific protease
(Mattoo et al., 1989). Although a large body of data
suggests that photoinhibition is favoured by the
over-reduction of PSII electron acceptors, the
results of a study of tobacco with genetically
decreased cytochrome b \f indicates that this is not
'
the sole factor triggering photoinhibition (Hurry et
al., 1996).
Oxidative degradation of thylakoid proteins and
pigments probably occurs simultaneously with
acclimatory responses in pigment-protein composition, signalled by changes in the redox state of key
components (Pfannschmidt et al., 1999). Redox
control of mRNA abundance and mRNA-binding
proteins involved in D1 synthesis, and redox regulation of the expression of light-harvesting antenna
proteins, have been reported (Danon & Mayfield,
1994 ; Escoubas et al., 1995 ; Maxwell et al., 1995).
Hence, changes in the turnover of D1 might be a
general response to changes in the redox poise of the
chloroplasts (Huner et al., 1996 ; Giardi et al., 1997).
As discussed in section VII, redox poise also
regulates the expression of several genes coding for
chloroplast proteins (Pearson et al., 1993 ; Escoubas
et al., 1995 ; Henkow et al., 1996).
Although the involvement of AOS in D1 damage
and breakdown in vivo remains to be established,
protection of the thylakoid membrane in vitro is
afforded by a number of energy-quenching and
antioxidant compounds. Prerequisites for survival of
excessive irradiation during stress are carotenoids
and α-tocopherol, which are synthesized on the inner
chloroplast envelope membranes (Audran et al.,
1998 ; Joyard et al., 1998). As a lipophilic scavenger
of singlet O , hydroxyl radicals and lipid peroxy
#
radicals, the membrane-bound antioxidant α-tocopherol protects the photosynthetic pigments from
oxidation (Fryer, 1992). The carotenoid pigments
associated with PSII and the light-harvesting antennae quench triplet states directly and have
antioxidant activity (Siefermann-Harms, 1987 ;
Miller et al., 1996). The D1 protein is partly
protected against the harmful effects of singlet O by
#
β-carotene bound to the reaction centre (Telfer et
al., 1994). All carotenoids can quench triplet chlorophyll or singlet oxygen and, in so doing, are
themselves converted to the triplet state. As the
probability of energy transfer from triplet carotenoids to ground-state O is relatively low, singlet
#
O formation is minimized by this mechanism and
#
362
REVIEW
C. H. Foyer and G. Noctor
the carotenoid decays to the ground state, releasing
energy harmlessly as heat. Thermal dissipation by
direct quenching of singlet excited chlorophyll is
also possible and is manifested as non-photochemical
quenching of chlorophyll a fluorescence quenching
(Horton et al., 1996). This process also involves the
carotenoid pigments that comprise the ‘ xanthophyll
cycle ’ (Demmig-Adams, 1990).
The direct addition of exogenous antioxidants and
antioxidative enzymes in the aqueous phase offers
less direct protection because these agents do not
have ready access to the sites of photodamage within
the lipid phase of the membrane. However, increasing the concentrations of these antioxidants can
indirectly protect PSII from photoinhibition by
protecting the thiol-modulated enzymes (section IV)
from oxidation and inactivation, thus maintaining
maximal electron transport rates, or by providing an
alternative electron sink in the Mehler-peroxidase
reaction, as discussed in section III.
The ability to acclimate rapidly to fluctuations in
irradiance is essential to minimize photodamage but
relatively little is known about the acclimatory and
protective processes that maintain high photosynthetic efficiency during stress. Important in
limiting oxidative damage within the thylakoid
membrane might be the induction of enzymes such
as glutathione peroxidases (Eshdat et al., 1997 ;
Mullineaux et al., 1998) and two-cysteine peroxiredoxins (Baier & Dietz, 1999) whose expression is
minimal under stress-free conditions. These
enzymes reduce organic peroxides to their corresponding alcohols and might be crucial in controlling chain-type reactions that follow the initiation
of lipid peroxidation by singlet O or hydroxyl
#
radical. Intensive study has been devoted to modification of D1 by the use of mutagenetic techniques
(Gleiter et al., 1992 ; Perewoska et al., 1994 ; Nixon et
al., 1995 ; Minagawa et al., 1996 ; Lardans et al.,
1997 ; Ma$ enpa$ a$ et al., 1998 ; Mulo et al., 1998). None
of these has elucidated the role of AOS at specific
sites within PSII.
The presence of photodynamic pigments in an
oxygen atmosphere is potentially dangerous ; chlorophyll metabolism is therefore rigorously controlled.
The requirement for coordinated enzyme activities
during chlorophyll synthesis has been emphasized
by a study in which the activity of uroporphyrinogen
II decarboxylase was decreased in tobacco by antisense technology (Mock & Grimm, 1997). Relatively
minor decreases in enzyme activity were associated
with a huge accumulation of the substrate and
necrotic lesions whose severity was dependent on
light intensity. These effects were accompanied by
the induction of several components of the antioxidative system and were explained in terms of
photosensitization reactions in which tetrapyrrole
precursors generate AOS, in particular singlet O
#
(Mock & Grimm, 1997 ; Mock et al., 1998).
Acclimation of the photosynthetic apparatus to
environmental change involves a battery of regulatory processes such as, in the short term, reversible
protein phosphorylation and, in the longer term,
changes in the saturation state and composition of
thylakoid lipids, and activation of thylakoidassociated proteases (Wada et al., 1994 ; Yang et al.,
1998). An important response might be the production of stress-induced proteins such as ELIPs
(early light-induced proteins), which seem to function as transient pigment-binding proteins because
they bind both chlorophyll and lutein (Adamska,
1997). As discussed in section VII, there is growing
evidence that the perturbation of redox homeostasis
and the generation of AOS are central to acclimatory
processes. These species are also implicated in the
activation of stromal proteases and the mechanism of
degradation of proteins such as Rubisco (GarciaFerris & Moreno, 1994), but the binding of inhibitors
of low molecular mass, such as carboxyarabinitol 1phosphate, prevents protein degradation when CO
#
assimilation is inhibited under conditions of stress
(Khan et al., 1999). AOS mediate damage to proteins
through direct interaction with specific amino acid
residues (lysine, arginine, proline and threonine).
This oxidation provides a type of ‘ tagging ’ that
renders peptide chains more susceptible to protease
attack and that can be measured as increased
carbonyl group formation on polypeptides
(Kingston-Smith & Foyer, 2000).
III.     
1. Oxygen ‘ poises ’ electron transport and carbon
assimilation
All PSI acceptors, including Fd, have sufficiently
negative redox potentials to reduce O to superoxide
#
(Fig. 2), the primary product of the Mehler reaction
(Mehler, 1951 ; Asada et al., 1974). However, it must
be noted that many components associated with the
thylakoid electron transport system can either have
pro-oxidant or antioxidant actions, depending on the
redox poise of the chloroplasts. For example,
ferredoxin : NADP+ oxidoreductase (FNR) can act
both as a pro-oxidant that generates superoxide
(Goetze & Carpentier, 1994) or an antioxidant
protecting PSII (Krapp et al., 1997). Similarly, O
#
can promote photoinhibition or protect against it,
depending on the prevailing conditions (Krause,
1994).
Whole-chain electron transport from water to O
#
is known as ‘ pseudocyclic electron flow ’. Electron
flow to O at PSI prevents over-reduction of the
#
electron transport chain (Arnon & Chain, 1975 ;
Egneus et al. 1975 ; Heber et al. 1978 ; Polle, 1996)
and ‘ poises ’ the electron carriers for more efficient
functioning (Heber et al., 1978 ; Ziem-Hanck &
REVIEW
Oxygen processing in photosynthesis : regulation and signalling
363
–0.5
Fdox /Fdred
1
–0.4
3
2
•–
O2 /O2
NADP+/NADPH
5
–0.3
ESS /ESH
Redox
potential
(V)
Tdox / Tdred
4
GSSG / GSH
–0.2
6
–0.1
Dehydroascorbate / ascorbate
0
Fig. 2. Stromal redox couples important in oxygen processing in the chloroplast. The vertical position indicates
approximate redox potential, also indicated by the deepness of blue. The redox potential span of
thiol\disulphide couples on thiol-regulated enzymes is a likely mean value. This redox potential can vary
significantly between target enzymes (see the text, section IV(1)). The sizes of the ellipses represent the
approximate relative pool sizes. Red arrows denote the direction of net electron flow in important redox
reactions discussed in the text. These are : 1, superoxide generation via reduction of O by Fd (primary PSI
#
acceptors, which have lower redox potentials than Fd, are also able to reduce O to superoxide (O )−)) ; 2,
#
#
thioredoxin (Td) reduction, catalysed by ferredoxin : thioredoxin reductase ; 3, NADPH formation, catalysed
by ferredoxin : NADP+ oxidoreductase ; 4, reduction of glutathione, catalysed by glutathione reductase ; 5,
activation of stromal enzymes by reduced Td ; 6, ascorbate regeneration from dehydroascorbate, occurring
chemically or catalysed by dehydroascorbate reductase. ESH\ESS, thiol-modulated enzymes ; Fd, ferredoxin ;
GSH, reduced glutathione ; GSSG, oxidized glutathione.
Heber, 1980 ; Levings & Siedow, 1995). The chloroplast stroma remains largely oxidized at high irradiance, even at the CO compensation point
#
(Harbinson & Foyer, 1991). In these circumstances
electron drainage to O might serve three functions :
#
(1) to provide the appropriate redox poise necessary
for cyclic electron flow, (2) to keep the stromal redox
state relatively oxidized, and (3) to maintain the
trans-thylakoid ∆pH to allow dissipative processes in
the thylakoid membrane to continue (Harbinson &
Foyer, 1991).
The hypothesis that electron flow to O is
#
increased when the NADP+ pool is depleted is
widely accepted, but very little evidence supports
this view because the coupled regulation of electron
transport rates prevents NADP+ depletion even in
the absence of CO (Takahama et al., 1981). As the
#
light incident on a leaf is increased, the primary
electron donor of PSI, P , becomes progressively
(!!
more oxidized (Harbinson & Foyer, 1991). This
occurs because the rate-limiting step of electron
transport, the oxidation of PQ by the cytochrome
b \f complex, lies between PSII and PSI (Haehnel,
'
1984). The oxidation of PQ involves the release of
protons into the thylakoid lumen. The progressive
inhibition of this reaction as the lumen pH decreases
in the light (Hope et al., 1994) limits the rate of
electron transport between the photosystems, a
phenomenon that is known as ‘ photosynthetic
control ’ (Foyer et al., 1990). It is unknown whether
such control of the redox state of the PQ pool is
responsive to the redox poise of PSI electron
acceptors such as Fd. However, both Fd and
superoxide can transfer electrons back to the cytochrome b \f complex, either through ferredoxin :
'
plastoquinone oxidoreductase activity or, for superoxide, via plastocyanin (Takahashi & Asada, 1988 ;
Hormann et al., 1993 ; Asada, 1994 ; Bendall &
Manasse, 1995). In addition, the thylakoid membrane contains complexes that are analogous to the
mitochondrial complex I and which might be involved in redox-poising PSI electron transport in a
process termed ‘ chlororespiration ’ (Bennoun, 1982).
Multiple systems of control match the supply of
electrons to PSI with the rate of Fd oxidation such
that NADP+ availability rarely limits electron transport. The activities of the two photosystems are
coordinated so that the rate of electron flow to PSI
increases only when electron flow away from PSI is
increased. Most of the electrons from PSI are passed
to NADP+ from Fd via FNR, but the Fd pool also
acts as a feed-forward regulator of the thiol-
364
REVIEW
C. H. Foyer and G. Noctor
modulated enzymes of the reductive pentose phosphate (RPP) pathway through thioredoxin (Td) (Fig.
2).
2. The role of oxygen in ATP synthesis
The trans-thylakoid ∆pH has a central role in
photosynthesis. It drives ATP synthesis, is involved
in the regulation of stromal enzyme activities
(Leegood et al., 1985) and, as already discussed,
regulates electron flow by affecting both PSII
photochemical efficiency and the rate of plastoquinol
oxidation. The ∆pH has to be low enough to allow
Fd reduction to continue but high enough to support
energy dissipation and ATP synthesis.
According to our current understanding, any
whole-chain electron transport pathway should be
coupled to ATP synthesis. This has been experimentally demonstrated for O reduction and
#
associated reactions (Egneus et al., 1975), which
yield ATP : 2e− ratios that are not much lower than
those observed during the reduction of NADP+
(Furbank & Badger, 1983 ; Forti & Elli, 1995). This
suggests that photosynthetic control must check O
#
reduction in the same way as it averts over-reduction
of the NADP+ pool, by preventing excessive reduction of PSI (Foyer & Harbinson, 1999).
Discussion of the relationship between the ∆pH
and interacting processes is still clouded by uncertainty over the precise nature of the proton
gradients involved ; that is, whether they are predominantly ‘ bulk-phase ’ in nature or are more
localized. For example, it is possible that protons
derived from water-splitting and those produced at
the cytochrome b \f complex are not kinetically
'
equivalent in terms of their activity in ATP synthesis, or that proton gradients driving ATP synthesis and ensuring photosynthetic control are not in
equilibrium (Laasch & Weis, 1988). These possibilities complicate the interpretation of experimental
results. One factor alleviating photosynthetic control
during the Mehler-peroxidase cycle could be the
consumption of intrathylakoidal protons that might
accompany O reduction, proposed to explain the
#
low pH optimum observed for O uptake (Hormann
#
et al., 1993). Proton deposition inside the thylakoid
during NADP+ reduction can be represented as
follows :
H OjNADP+j3H+ (stroma)
#
4 "O jNADPHj4H+ (lumen)
# #
(no protonmotive Q cycle)
H OjNADP+j5H+ (stroma)
#
4 "O jNADPHj6H+ (lumen)
# #
(obligatory protonmotive Q cycle)
Because O reduction occurs predominantly on the
#
acceptor side of PSI, similar proton stoichiometries
should also hold during pseudocyclic electron transport. However, if superoxide is protonated inside the
thylakoid but dismutated in a phase energetically
continuous with the stroma,
2O j2e− 4 2O )−
#
#
(superoxide formation)
2O )−j2H+ (lumen) 4 2HO
#
#
(superoxide protonation)
2HO 4 H O (stroma)jO
#
# #
#
(superoxide dismutation)
Such a phenomenon would mean that, of the protons
deposited during electron transfer, between 50% (no
Q cycle) and 33% (Q cycle) would flow back to the
stroma through a route that does not involve ATP
synthesis. In the steady state, however, two electrons
are also required to reduce H O to water (Fig. 3).
# #
Electron transfer to H O should support the luminal
# #
deposition of the same number of protons as electron
transfer to NADP+, so that the proportion of protons
dissipated via superoxide protonation during steadystate reduction of O would be approx. 25% (no Q
#
cycle) or 17% (Q cycle). Backflow of protons through
superoxide protonation on the interior of the
thylakoid, if it occurred, would therefore be significant but nevertheless unlikely to remove photosynthetic control completely, although it could
contribute to the small discrepancy between
ATP : 2e− ratios observed in spinach thylakoids
reducing O and NADP+ (Furbank & Badger, 1983).
#
3. How fast is O reduction at PSI ?
#
It is widely accepted that the Mehler reaction acts as
an alternative sink for electrons. Oxygen reduction is
tightly coupled to the production and destruction of
H O by ascorbate peroxidase (APX) (Fig. 3)
# #
(Groden & Beck, 1979 ; Anderson et al., 1983a) in the
Mehler-peroxidase cycle (Neubauer & Schreiber,
1989 ; Neubauer & Yamamoto, 1992). This consists
of four reactions occurring at the surface of the
membrane in the region of PSI (Fig. 3a) : (1) electron
transfer from water to O , producing superoxide ; (2)
#
dismutation of the superoxide radical catalysed by a
membrane-bound superoxide dismutase (SOD) isoform, producing H O ; (3) reduction of H O to
# #
# #
water by APX ; and (4) the regeneration of ascorbate
from monodehydroascorbate (MDHA) (Miyake &
Asada, 1992 ; Grace et al., 1995). MDHA is a
powerful electron acceptor that oxidizes reduced Fd
(Miyake & Asada, 1994). On the thylakoid membrane these reactions constitute a closed thylakoidbound scavenging system acting within a 5–10 nm
layer on the surface of the membrane (Miyake &
Asada, 1994 ; Asada, 1996). The addition of H O to
# #
thylakoid membranes or to isolated intact chloroplasts causes a rapid increase in the photochemical
REVIEW
Oxygen processing in photosynthesis : regulation and signalling
H2O
(a)
H2O
(b )
H2O
(c )
MDHA
MDHA
MDHA
APX
APX
APX
AA
AA
H2O2
365
AA
MDHAR
H2O2
H2O2
DHA
DHAR
GSSG
SOD
SOD
O2• –
O2
NADP+
O2• –
O2
NADPH
SOD
O2• –
GR
NADPH
O2
Fd
Fd
PSI
PSI
FNR
GSH
NADP+
Stroma
Fd
FNR
PSI
Intrathylakoid space
Fig. 3. Oxygen reduction at PSI and processing of chloroplastic H O . The predominant path producing H O
# #
# #
from O is denoted by red arrows. The initiating reaction of the reduction of O to water involves the generation
#
#
−
of superoxide (O ) ) by Fd or other PSI acceptors. Membrane-associated and soluble superoxide dismutase
#
(SOD) catalyse the conversion of superoxide to H O (superoxide can also be reduced to H O by PSI
# #
# #
acceptors). H O is reduced to water through the action of ascorbate peroxidase (APX), found in both
# #
thylakoid-bound and stromal forms. The blue arrows show three distinct paths of ascorbate (AA) regeneration.
(a) The Mehler-peroxidase cycle, a sequence closely associated with the thylakoid membrane in which the
primary product of ascorbate peroxidation, monodehydroascorbate (MDHA), is photochemically reduced to
ascorbate by Fd. SOD and APX are in coloured ellipses to indicate association with the thylakoid membrane.
(b) Regeneration of ascorbate from MDHA by the NADPH-dependent enzyme MDHA reductase (MDHAR).
(c) Regeneration of ascorbate via the ascorbate–glutathione cycle. Dehydroascorbate (DHA) is formed from
MDHA by spontaneous dismutation, and is reduced by reduced glutathione (GSH), a reaction catalysed by
DHA reductase (DHAR). Oxidized glutathione (GSSG) is reduced to GSH by glutathione reductase (GR), at
the expense of NADPH. In (b) and (c), enzymes are in white ellipses to denote location primarily in the soluble
phase. Reactions are not shown stoichiometrically : whichever pathways operate, the net reduction of one
molecule of O to two water molecules requires four electrons from the photosynthetic electron transport chain
#
and supports the generation of a trans-thylakoid pH gradient. FNR, ferredoxin : NADP+ oxidoreductase.
component of chlorophyll a fluorescence quenching,
supporting the conclusion that H O generation and
# #
degradation act as alternative electron sinks that
maintain the oxidized state of the primary stable
PSII electron acceptor, QA (Neubauer & Schreiber,
1989 ; Foyer et al., 1994).
The Mehler-peroxidase cycle performs an essential protective function in preventing the accumulation of superoxide and H O that could
# #
otherwise lead to hydroxyl radical formation. In
cyanobacteria, the absence of one component, SOD,
provokes photoinhibition (Herbert et al., 1992).
Enhanced protection of PSII has been shown in
transformed plants with increased chloroplastic
SOD activity (Arisi et al., 1998). Overexpression of
Fe-SOD altered the regulation of photosynthesis at
low partial pressures of CO , indicating that the
#
Mehler reaction can prevent QA over-reduction
under conditions that cause a decline in intercellular
CO concentration, which might be relevant to
#
physiological conditions that result in stomatal
closure. These observations are somewhat surprising
because the dismutation of superoxide is not con-
sidered to be a major factor limiting the overall flux
through the pathway. However, with only one
exception, overexpression of any of the SOD isoforms has been repeatedly shown to enhance the
protection of photosynthesis and leaf metabolism
against oxidative damage (reviewed by Foyer, 1997).
The extent to which O can be used to sustain high
#
rates of electron transport requires careful consideration because the contribution of pseudocyclic
electron flow to ATP synthesis means that ATP
must be consumed at a rate commensurate with its
production to ensure continued electron flux through
the chain. In Scenedesmus, however, electron transport rates are similar regardless of whether CO or
#
O is used as an electron acceptor, even at saturating
#
light intensities (Radmer & Kok, 1976). This implies
either a lack of photosynthetic control in these
organisms or the existence of an unidentified ATP
sink able to regenerate ADP and phosphate consumed during O reduction. In certain algae, the
#
relative insensitivity of thiol-regulated photosynthetic enzymes (see section IV) permits the
accumulation of H O and its excretion into the
# #
366
REVIEW
C. H. Foyer and G. Noctor
surrounding medium. The excretion of H O might
# #
partly explain the high rates of O reduction, because
#
this would allow comparable rates of O uptake to be
#
sustained by electron transport rates only half as fast
as those necessary to reduce O to water via the paths
#
shown in Fig. 3. However, as pointed out previously,
an ATP sink or uncoupling mechanism has to be
invoked to support rates of O reduction that are
#
comparable to rates of CO fixation (Radmer & Kok,
#
1976).
In leaves, numerous studies with chlorophyll
fluorescence analysis have shown that electron flow
is largely non-cyclic over a wide range of irradiances
and that donor-side limitation of PSI predominates,
because the primary site of control over thylakoid
electron transport resides between PSII and PSI
(Foyer et al., 1992). This implies that, even if it
occurs at the high rates that have been suggested
(Osmond & Grace, 1995), O reduction is con#
strained by the proton gradient, as discussed in the
previous section.
Precise estimates of Mehler flux are difficult to
obtain because there is no direct method of measurement. Likely values for flux through the Mehlerperoxidase cycle in planta are not known and
estimated rates vary enormously (Marsho et al.,
1979 ; Steiger & Beck, 1981 ; Badger, 1985 ;
Robinson, 1988). Mass spectrometric measurements
are complicated by concomitant O uptake in
#
photorespiration and mitochondrial respiration
(sections V and VI). The use of metabolic inhibitors
or high [CO ] to remove these processes is not
#
without problems. Inhibitors might lead either to an
underestimation of the Mehler reaction (because of
the removal of ATP-consuming reactions) or, possibly, an overestimation (because of the absence of
NADP+ regeneration). Similarly, effects of high
[CO ] on either stromal redox state or adenylate
#
status might mean that estimates of the Mehler
reaction under these conditions do not necessarily
reflect rates that occur in air. Although it is often
considered that the Mehler reaction is unaffected
when photorespiration is minimized by low O , it is
#
worth noting that half-maximal rates of O reduction
#
are obtained at O concentrations varying from 2 to
#
60 µM (Robinson, 1988) which corresponds to an
atmospheric concentration of approx. 0n15–5% O at
#
25mC.
In many cases, concentrations of CO intended to
#
eliminate photorespiration have probably not been
sufficiently high, and it is likely that ongoing lightdependent O uptake at these ‘ high ’ CO concen#
#
trations mainly reflects continuing photorespiration
(Canvin et al., 1980 ; Osmond & Grace, 1995). A
complementary approach has exploited transformed
plants with greatly decreased Rubisco activities
(Ruuska et al., 2000). These plants have lowered
capacities for CO fixation and photorespiration but
#
their capacity for electron transport is much less
affected. As predicted from considerations of photosynthetic control, electron transport to O (pseudo#
cyclic electron flow) was modified in parallel with
overall non-cyclic electron flow, confirming the
notion that significant electron flow to O is possible
#
only in the presence of an ATP sink. The Mehler
reaction is therefore unable to replace CO as#
similation and photorespiration as a sink for electrons
(Ruuska et al., 2000). Rates of electron transport to
O might increase transiently during light flecks,
#
when rapid adjustment of regulation between the
processes of electron transport and CO assimilation
#
is required (Pearcy, 1990). This situation is analogous to the induction period of photosynthesis
(Marsho et al., 1979 ; Foyer et al., 1992). Although it
has frequently been considered that high light
intensities promote higher rates of O reduction
#
(Canvin et al., 1980 ; Badger, 1985 ; Grace & Logan,
1996), it has also been suggested that O reduction
#
accounts for a greater proportion of electron flow at
low light intensities (Heber et al., 1978). If electrons
do leak to O at a fixed proportion of overall electron
#
transport rates, then absolute rates of O reduction
#
will obviously increase with light intensity but will
not increase further at light intensities above those
saturating for photosynthesis (see an analogous
discussion of photorespiration in section V(3)).
Lastly, it can be noted that abundant data from
chlorophyll fluorescence analysis argue against a
high proportion of electrons flowing to O in vivo
#
(Genty et al., 1989 ; Harbinson et al., 1990 ; Cornic &
Briantais, 1991).
These considerations suggest that electron transport rates to O are very low or that they are
#
proportional to the requirement for additional ATP
production. The physiological relevance of steadystate estimates of the rate of electron flow to O
#
might be virtually meaningless because pseudocyclic
electron flow will switch on and off according to
energy and ATP requirements. At any point it might
be lower or higher than the value of 10% of the total
electron transport rate that is often discussed
(Egneus et al., 1975 ; Gerbaud & Andre, 1980 ;
Robinson, 1988 ; Tourneux & Peltier, 1995). A
photoprotective role for the Mehler reaction in
preventing photoinhibition cannot be discounted,
notably because pseudocyclic electron flow increases
the trans-thylakoid pH gradient and promotes
energy dissipation (Osmond & Grace, 1995 ; Biehler
& Fock, 1996 ; Lovelock & Winter, 1996). In theory,
in the absence of a corresponding ATP sink, this
could be achieved by relatively low rates of electron
transport (Noctor & Foyer, 2000).
4. Chloroplastic processing of H O
# #
The principal H O -scavenging enzymes in leaves
# #
are APX and catalase (Groden & Beck, 1979 ; Kelly
& Latzko, 1979 ; Willekens et al., 1995). The modes
REVIEW
Oxygen processing in photosynthesis : regulation and signalling
of action of these two enzymes are essentially
different. Catalases catalyse the dismutation of two
molecules of H O to water and molecular O at near
# #
#
diffusion-controlled rates, whereas peroxidases use
substrates to reduce H O to water. APX has a much
# #
higher affinity for H O than catalase but catalase has
# #
a much higher Vmax (Asada, 1992). Peroxisomal
APX might prevent catalase inactivation that can
occur at low concentrations of H O . As discussed in
# #
section V, the CAT-1 isoform has a major role in
H O scavenging during photorespiration.
# #
Analysis of gene sequences in Arabidopsis suggests
the existence of up to seven APX isoforms, two
soluble in the cytosol and three associated with
microbodies, as well as the two chloroplastic isoforms (Jespersen et al., 1997). The significance of
chloroplastic APX, which is highly active and shows
a high specificity for ascorbate as reductant, might
reside in the need to keep stromal H O levels low, to
# #
protect the thiol-regulated enzymes. Such a role
requires the rapid regeneration of reduced ascorbate,
to sustain its antioxidative function, and this can
occur via several pathways (Fig. 3). At neutral pH
values, the primary product of ascorbate peroxidation, MDHA, has a short lifetime : if not rapidly
reduced via the paths shown in Fig. 3a,b, it will
disproportionate spontaneously to ascorbate and the
oxidized form, dehydroascorbate (DHA), at a rate
similar to that observed for the disproportionation
of superoxide (Bielski et al., 1971). Ascorbate must
then be regenerated from DHA (Fig. 3c).
The paths shown in Fig. 3b,c will be important in
scavenging H O that escapes the thylakoid# #
associated Mehler-peroxidase cycle. Equally, these
stromal reactions could act to scavenge H O
# #
entering the chloroplasts from the cytosol. As the
peroxide anion exists almost entirely in the
uncharged form (H O ) at physiological pH, it can
# #
diffuse freely through biological membranes. Indeed, chloroplasts are capable of rapidly detoxifying
H O of external origin (Nakano & Asada, 1980 ;
# #
Anderson et al., 1983a). It seems that catalase and
APX act cooperatively to remove H O in leaves,
# #
ensuring maximal H O destruction at a minimal
# #
cost in terms of reducing power (for related discussion, see section V(6)). In some species, however,
this simple picture can be complicated by intercellular compartmentation. For instance, maize
shows pronounced sensitivity to oxidative damage
induced by exposure to low temperature. It has been
proposed that this might be partly due to the
differential compartmentation of antioxidants in
maize leaves, which necessitates the transport of
reduced forms of ascorbate and glutathione from the
mesophyll to the bundle sheath cells, processes that
are slowed at low temperatures (Doulis et al.,
1997). This hypothesis receives support from the
observation that bundle sheath proteins are much
more susceptible to oxidative damage than those of
367
the mesophyll (Kingston-Smith & Foyer, 2000),
suggesting that stress conditions provoke a deficit in
antioxidant capacity in the bundle sheath.
Although Fig. 3c shows DHA reduction occurring
enzymically, it should be noted that DHA reductase
(DHAR) activity is frequently undetectable in
chloroplasts (Foyer & Halliwell, 1976 ; Morell et al.,
1997). Non-enzymic reduction of DHA by reduced
glutathione (GSH) is, however, increased at alkaline
pH values, such as those occurring in the illuminated
stroma (Foyer & Halliwell, 1976). Although DHA is
always found in leaves and other plant tissues (Foyer
et al., 1983 ; Robinson, 1997), it is possible that it
does not accumulate in chloroplasts (Morell et al.,
1997). One observation that provides some evidence
against this idea has come from transformed poplars
overexpressing glutathione reductase (GR) (Foyer et
al., 1995). These plants have increased ascorbate
levels, suggesting that glutathione is important in
ascorbate regeneration via DHA reduction. Furthermore, it has been experimentally demonstrated that
chloroplasts are capable of sustaining ascorbate regeneration from DHA : the addition of DHA drives
turnover of the NADPH pool and leads to the
evolution of O by the photosynthetic electron
#
transport system (Anderson et al., 1983b). When
H O is added to isolated chloroplasts, glutathione
# #
and ascorbate pools show significant turnover,
suggesting that both antioxidants contribute to H O
# #
detoxification (Anderson et al., 1983a, b). Interestingly, decreased electron transport resulting from
photoinhibition leads to the inactivation of thiolmodulated enzymes such as NADP+-dependent
malate dehydrogenase (NADP-MDH) and fructose1,6-bisphosphatase (FBPase), yet the ascorbate and
glutathione pools remain reduced (Dujardyn &
Foyer, 1989 ; Foyer et al., 1989). These observations
might reflect the relative redox potentials of each
component (Fig. 2) and indicate that, under photoinhibitory conditions, electrons are partitioned to
stromal antioxidant pools in preference to carbon
assimilation.
Several studies have found that GR shows the
lowest extractable activity of the enzymes of the
ascorbate–glutathione cycle. For example, APX
activity is often 10–20 times that of GR (Gillham &
Dodge, 1986), although it can be noted that APX
activity is typically measured at 0n1 mM H O ,
# #
which is probably 10–100-fold higher than the
stromal concentration in vivo. It is conceivable that
GR activity might limit flux through the ascorbate–
glutathione cycle under certain conditions, explaining why the overexpression of this enzyme leads
to a more reduced glutathione pool and resistance to
photoinhibition, as well as increases in total glutathione and ascorbate contents (Foyer et al., 1995). By
contrast, the activities of GR (and DHAR) might not
need to be as high as that of APX because the former
enzymes participate in only one of the possible paths
368
REVIEW
C. H. Foyer and G. Noctor
for ascorbate regeneration (Fig. 3c) and additional
mechanisms are able to regenerate ascorbate from
MDHA (Fig. 3a,b). Because ascorbate is produced
in the mitochondria and glutathione is synthesized in
the chloroplasts and cytosol (Noctor & Foyer,
1998a), it is possible that the capacity of the
ascorbate–glutathione cycle is limited by the transport of these antioxidants across the chloroplast
envelope. Clarification of this question requires more
knowledge of the likely rates of antioxidant degradation in the illuminated chloroplast. Virtually no
information is available on these important steps of
transport and turnover.
IV.   
 
1. The thioredoxin system
Several stromal enzymes are light-regulated via the
Td system, which transfers reducing equivalents
from PSI, via Fd, ferredoxin : thioredoxin reductase
and Td, to disulphide bridges on target enzymes
(Fig. 4) (Buchanan, 1991). Td is a small protein
(approx. 12 kDa) of which three types have been
found in plants (Tdm, Tdf and Tdh). Chloroplastic
target enzymes are reduced by Tdf, Tdm, or both
(Buchanan, 1991). The list of enzymes subject to this
regulation is ever-growing : recent evidence suggests
that Td functions to activate acetyl-CoA carboxylase, which catalyses the first committed step in the
synthesis of fatty acids (Sasaki et al., 1997), and also
the regulatory protein Rubisco activase (Zhang &
Portis, 1999).
Intrathylakoid
space
The control of many of the thiol-regulated
enzymes is complex, with reductive activation itself
and the significance of reduction both depending on
the concentrations of substrates and\or co-factors
(reviewed by Leegood et al., 1985, Scheibe, 1987 and
Buchanan, 1991). Interestingly, it has been shown
that two of the thiol-regulated enzymes of the RPP
pathway, phosphoribulokinase and glyceraldehyde3-phosphate dehydrogenase, might also be regulated
by another small protein, termed CP12 (Wedel &
Soll, 1998). When simultaneously bound to this
protein, the enzymes are inactive. They become
active when dissociated, a process mediated by
NADPH (Wedel & Soll, 1998).
It is generally considered that the predominant
function of the Td system is to enable active
assimilatory metabolism in the light while preventing
futile cycling of metabolites and ATP hydrolysis in
the dark. Dark inactivation is probably due to autooxidation of thiol groups on Td and target enzymes
in the absence of ongoing reduction (Leegood &
Walker, 1982 ; Jacquot et al., 1984). Oxidation is
mediated either by O or by H O (Fig. 4). Because
#
# #
oxidative species are formed at higher rates in the
light than in the dark, continuous reduction of the
target enzymes is necessary to maintain active states.
The reduction state of enzymes in the illuminated
leaf therefore reflects the balance between oxidative
and reductive fluxes, enabling plastic regulation of
enzyme activation state (Leegood & Walker, 1982 ;
Leegood et al., 1985). One exception is probably the
membrane-bound CF -ATPase, which is activated
"
at much lower electron pressures than the soluble
thiol-regulated enzymes (Quick & Mills, 1986 ;
Enzyme more active in oxidized form:
Glucose-6-phosphate dehydrogenase
Stroma
hv
FTR
S
FeS
S
SH
Td
SH
Target
enzyme
(oxidized
form)
S
2 O2• –
S
H2O
Photosystem I
Ferredoxin
e–
Fes
FTR
FeS
hv
SH
SH
S
Td
S
Target
enzyme
(reduced
form)
SH
SH
H2O2
2 O2
Enzymes more active in reduced form:
NADP-malate dehydrogenase
Fructose-1, 6-bisphosphatase
Sedoheptulose-1, 7-bisphosphatase
Glyceraldehyde-3-phosphate dehydrogenase
Phosphoribulokinase
CF1-ATPase
Acetyl CoA carboxylase
Fig. 4. Reduction of chloroplastic enzymes by the ferredoxin–thioredoxin system. Oxidation of enzymes can
occur, as shown, through direct oxidation of enzyme thiol groups by molecular oxygen or H O : these species
# #
can also oxidize the redox-active thiols of thioredoxin (Td), which can in turn oxidize target enzymes through
a reversal of the reducing process. The ultimate product of O reduction by thiol groups is water. The list of
#
target enzymes is not intended to be exhaustive. FTR, ferredoxin : thioredoxin reductase ; O )−, superoxide.
#
REVIEW
Oxygen processing in photosynthesis : regulation and signalling
Kramer et al., 1990) and is much more resistant to
oxidation in the light (Noctor & Mills, 1988). It has
become clear that, even among the soluble enzymes
regulated by the Td system, there might be considerable heterogeneity in enzyme activation state
under given conditions owing to significant divergence in the redox potentials of the key thiols\
disulphides between target enzymes (D. Ort, pers.
comm.).
H O is a potent oxidant of enzyme thiol groups ;
# #
its inhibitory effect on CO fixation is due to the
#
inactivation of thiol-regulated enzymes (Kaiser,
1979 ; Charles & Halliwell, 1980). Reported total
foliar concentrations of H O (100–300 µM) (Rao et
# #
al., 1997 ; Dat et al., 1998) often exceed those
required to inhibit photosynthesis in isolated chloroplasts through oxidation of the thiol-regulated
enzymes (Kaiser, 1979 ; Charles & Halliwell, 1980).
The chloroplastic H O concentration is maintained
# #
much lower than these total tissue levels (Polle,
1997), principally by chloroplastic APX. Some
contribution of peroxisomal catalase to control of the
stromal H O level cannot be completely excluded
# #
(for related discussion see section V(6)).
In addition to H O , DHA and oxidized gluta# #
thione (GSSG) are able to inactivate thiol-regulated
enzymes (Wolosiuk & Buchanan, 1977 ; Morell et al.,
1997). In the illuminated chloroplast, therefore, as
well as flux from PSI to H O , DHA and GSSG via
# #
the Mehler-peroxidase and ascorbate–glutathione
cycles, some flux must also occur via thiol groups on
Td and Td-reduced enzymes. This view emphasizes
the plurality of routes through which electrons can
be transferred from water cleaved at PSII to AOS
and antioxidant pools. Electron transfer from Td
to peroxides might also be possible via the activity
of chloroplastic 2-cysteine peroxiredoxins (Baier &
Dietz, 1999), although the precise nature of the
reductant used by these enzymes is not yet clear.
2. Manipulating the expression of thiol-regulated
enzymes
The sensitivity of CO fixation in higher plants to
#
H O (Kaiser, 1979) demonstrates the need for
# #
reductive activation of the thiol-regulated enzymes
of the RPP pathway during photosynthesis. Because
three enzymes of the pathway (FBPase, sedoheptulose-1,7-bisphosphatase and phosphoribulokinase) catalyse reactions that are irreversible under
physiological conditions, they have often been considered as potential rate-limiting points in CO
#
fixation (Buchanan, 1991). Little support for stringent control by individual enzymes has come from
studies with anti-sense technology : CO fixation is
#
affected only when total enzyme capacity is markedly
decreased (Kossmann et al., 1994 ; Paul et al., 1995 ;
Price et al., 1995 ; Harrison et al., 1998). However,
the effect of decreases in enzyme amounts might be
369
countered by compensatory increases in the activation state of the remaining enzyme pool, as
observed for phosphoribulokinase (Banks et al.,
1999). Similar effects were observed in plants
transformed for another thiol-regulated enzyme,
NADP-MDH. In this case, decreased maximal
capacities were associated with a more active pool of
enzymes in vivo, whereas the reduction state of
NADP-MDH was lower in plants overexpressing
the enzyme (Trevanion et al., 1997 ; Backhausen et
al., 1998). Anti-sense studies have highlighted the
ability of the RPP pathway to operate at similar rates
at different internal substrate concentrations, so that
decreased activities of one enzyme can perturb
metabolite pools significantly without affecting overall rates of CO fixation (Price et al., 1995).
#
3. Modifying sensitivity to thiol regulation
In addition to manipulation of the expression of
native enzymes, site-directed mutagenesis is a
powerful tool for elucidating the control of thiol
reduction and its significance in foliar metabolism. A
chloroplastic FBPase coding sequence has been
modified by the selective mutation of cysteine
residues to serine : the loss of one of the cysteine
residues yielded an enzyme that was constitutively
active when expressed in Escherichia coli (Jacquot et
al., 1997). A similar approach for NADP-MDH
showed that the regulatory properties of the enzyme
could be markedly modified, but also revealed that
the reduction of more than one disulphide bridge
was necessary for activation (Issakidis et al., 1992).
Considerable insight into the significance of light–
dark regulation by the Td system might come from
studies in which constitutively active enzymes are
introduced into plants (for further discussion, see
Miginiac-Maslow et al., 1997).
V .               
1. The pathway and its genetic manipulation
The term photorespiration describes the lightdependent uptake of O and the associated release of
#
CO . The initiating reaction of photorespiration is
#
the oxygenation of ribulose 1,5-bisphosphate
(RuBP) at Rubisco, yielding 3-phosphoglycerate and
2-phosphoglycollate. Approximately 75% of the
carbon entering 2-phosphoglycollate is recycled to 3phosphoglycerate through a complex series of
reactions involving enzymes localized in the chloroplasts, peroxisomes and mitochondria (Berry et al.,
1978). These reactions, which represent the major
metabolic fate of glycollate carbon, together constitute the ‘ photorespiratory pathway ’ (Fig. 5).
In addition to enzymes directly involved in carbon
recycling, other enzymes have important roles in
370
REVIEW
C. H. Foyer and G. Noctor
H2O
Chloroplast
O2
RuBP
O2
2-Phosphoglycollate
H2O2
2
1
Glyoxylate
Glycollate
Hydroxypyruvate
Rubisco
6
7
8
PGA
Glycerate Serine
Glutamate
NH3
5
NH3
Glutamine
2-OG
GOGAT
Glutamate
Glutamate
4
3
2-OG
Glycine
Glycine
Serine
GS
Peroxisome
Catalase
GDC
CO2
Mitochondrion
Cytosol
Fig. 5. The principal features of photorespiratory metabolism. Solid arrows show metabolic conversions,
dotted arrows show movement between compartments. Blue arrows show reactions involved in carbon
recycling, green arrows reactions involved in amino cycling, and red arrows reactions involving the production
and processing of H O . Key enzymes discussed in the text are in white ellipses. Other reactions (numbered)
# #
are catalysed by phosphoglycollate phosphatase (1), glycollate oxidase (2), serine : glyoxylate aminotransferase
(3), glutamate : glyoxylate aminotransferase (4), serine hydroxymethyl transferase (5), hydroxypyruvate
reductase (6), glycerate kinase (7) and reductive pentose phosphate pathway enzymes (8). GDC, glycine
decarboxylase ; GOGAT, glutamate synthase ; GS, glutamine synthetase ; 2-OG, 2-oxoglutarate ; PGA, 3phosphoglycerate.
photorespiratory metabolism. As shown in Fig. 5,
these include glutamine synthetase (GS) and
glutamate synthase (GOGAT), which reincorporate
the ammonia released in glycine oxidation (Keys et
al., 1978), and catalase, which processes H O
# #
formed during the oxidation of glycollate in the
peroxisome (Willekens et al., 1995). The oxidation
of glycollate by molecular oxygen means that
photorespiration consumes at least two molecules of
O for each C unit passing through the pathway
#
#
(Fig. 5). Further O consumption might be linked to
#
glycine oxidation in the mitochondrion (section VI).
Photorespiratory O uptake is masked by catalase
#
activity, which results in evolution of approximately
half of the O consumed by glycollate oxidase, and
#
by O evolution at PSII. From this it is inferred,
#
first, that photorespiration cannot be quantified by
simple gas exchange measurements and, second, that
the process is a sink for photosynthetic assimilatory
power.
Likely rates of photorespiration in leaves have
recently been reviewed elsewhere (Keys, 1999). In
brief, flux through the photorespiratory pathway
depends on the ratio of carboxylation to oxygenation
of RuBP (C : O ratio). This ratio reflects, first, the
specificity factor of Rubisco, which indicates the
enzyme’s affinity and turnover rate for CO relative
#
to those for O , and second, the ratio of the
#
chloroplastic concentration of CO to that of O
#
#
(Sharkey, 1988). Decreased C : O ratios with increasing temperature probably reflect changes in
both of these factors (Ku & Edwards, 1977 ; Hall &
Keys, 1983 ; Chen & Spreitzer, 1992). Photorespiration is attenuated by increasing the availability of CO relative to that of O , as can be
#
#
observed in the laboratory and as occurs naturally in
many algae and in the bundle sheath cells of many C
%
leaves. If one can reasonably talk of typical C : O
ratios, then these might be 2–3 in well-watered C
$
leaves at 20–35mC (Sharkey, 1988 ; De Veau &
Burris, 1989 ; Peterson, 1989). Such ratios mean
that, for each carbon atom fixed at Rubisco, 0n67–1
atoms pass through the glycollate pool.
Mutants have been instrumental in confirmation
REVIEW
Oxygen processing in photosynthesis : regulation and signalling
of the photorespiratory pathway (Somerville &
Ogren, 1979 ; Blackwell et al., 1988 ; Lea & Forde,
1994 ; Leegood et al., 1995). Most of these mutants
are unable to survive in air, which emphasizes the
need for an effective photorespiratory pathway and
leads to the inevitable conclusion that the elimination
of RuBP oxygenation is likely to be the most
succesful approach to attenuating photorespiration
(section V(2)). The inability to grow and photosynthesize in air can be overcome by crossing
mutants with wild-type plants to produce heterozygous plants that have intermediate enzyme activities (Hall et al., 1987 ; Havir & McHale, 1988 ;
Ha$ usler et al., 1994). Recent studies with barley
mutants heterozygous for glycine decarboxylase or
serine : glyoxylate aminotransferase suggest that
enzymes involved in photorespiratory carbon
recycling exert significant control over photosynthesis only when photorespiratory rates are very high
relative to net CO fixation (Wingler et al., 1997,
#
1999a). Minimal control in these plants implies
excess activities in the wild type, consistent with the
notion that the principal function of the complex
recycling pathway is to reclaim the carbon diverted
into glycollate. Nevertheless, work with mutants and
transformed plants suggests that the fate of photorespiratory metabolites has some flexibility. In
Euglena, serine is formed via an alternative pathway
in which methylene-tetrahydrofolate is synthesized
with formate derived from glyoxylate. Work with
barley mutants with very low glycine decarboxylase
activity has shown that this pathway, which allows
the conversion of glycine to serine without the
release of ammonia, can also occur in higher plants
(Wingler et al., 1999b). Genetic transformation has
provided good evidence that photorespiratory amino
acids can be used in biosyntheses. In poplars with an
enhanced capacity for glutathione synthesis, maximal accumulation of this tripeptide (γ-Glu-Cys-Gly)
requires the production of glycine via the photorespiratory pathway (Noctor et al., 1999). Glycine
and serine can also be withdrawn from the photorespiratory pathway for export (Madore &
Grodzinski, 1984).
Manipulation of photorespiratory enzymes
through genetic transformation is likely to add
considerably to the knowledge gained through the
production of photorespiratory mutants. Plants have
been transformed to contain altered levels of GS
(Lea & Forde, 1994 ; Kozaki & Takeba, 1996 ;
Temple et al., 1998), Fd-GOGAT (Ferrario-Me! ry
et al., 2000) and hydroxypyruvate reductase (Oliver
et al., 1993, 1995 ; Sloan et al., 1993). Recently, antisense technology has been used to produce potato
plants with decreased glycine decarboxylase (H.
Bauwe & D. Heineke, pers. comm.). Like barley
mutants with very low enzyme capacity (Blackwell et
al., 1990), the transgenic potatoes accumulate glycine
to high levels when illuminated in air, even though
371
their ability to grow in air and the capacity of their
mitochondria to metabolize glycine resemble the
heterozygous barley mutants more closely (Wingler
et al., 1997). Much useful information should be
generated by comparison of the anti-sense transformants with studies of the full and heterozygous
barley mutants.
2. Engineering plants that photorespire less ?
Because photorespiration decreases net CO fixation,
#
there is considerable interest in engineering plants
that photosynthesize without photorespiring. There
are many reports in the literature of plants
engineered to contain less Rubisco (Quick et al.,
1991) and, as discussed in section III, these have
been used effectively to dissect the contributions
made by photorespiration and the Mehler reaction to
photosynthetic O uptake (Ruuska et al., 2000). Such
#
manipulations have also received attention with a
view to decreasing the nitrogen requirement of crop
species (Quick et al., 1992 ; Masle et al., 1993).
Importantly, however, a striking and unexpected
side-effect of decreased Rubisco is greatly increased
sensitivity to the air pollutant ozone (Wiese & Pell,
1997). This seems to be caused by an effect of
decreased Rubisco on the volume of air space within
the leaf, exemplifying the unforeseen effects that can
result from even simple manipulations.
In theory, the most attractive approach to
decreasing the rate of photorespiration is by manipulation of the catalytic properties of Rubisco.
Although it has been argued that oxygenation is an
unavoidable consequence of the Rubisco reaction
mechanism (Andrews & Lorimer, 1978), the observation that higher plants have generally higher
specificity factors than green algae and photosynthetic bacteria (Jordan & Ogren, 1981) shows
that the initial appearance of Rubisco should not be
considered an evolutionary fait accompli. Even
within phylogenetic groups, there is some variation
in Rubisco specificity factors (Parry et al., 1989 ;
Read & Tabita, 1994 ; Delgado et al., 1995). It is
therefore clear that, in principle, considerable scope
exists for decreasing photorespiratory rates by
increasing the enzyme’s specificity factor. This
might be achieved either by site-directed mutagenesis (Chen & Spreitzer, 1992 ; Parry et al., 1992 ;
Read & Tabita, 1994 ; Kostov et al., 1997 ; Madgwick
et al., 1998) or by gene replacement, allowing the
introduction of Rubisco with naturally high
specificity factors into agriculturally important
species (Kanevski et al., 1999).
Aside from attempts to manipulate Rubisco,
photorespiration could be decreased in C plants by
$
introducing metabolic features that are currently
unique to plants that photorespire less, such as algae
or C plants (Panstruga et al., 1997 ; Ku et al., 1999 ;
%
Ha$ usler et al., 1999). It is perhaps worth noting here
372
REVIEW
C. H. Foyer and G. Noctor
that, although independent of temperature in C
%
plants, the quantum yield of CO uptake decreases
#
with increasing temperature in C plants (Ehleringer
$
& Bjo$ rkman, 1977). There is a crossover point at
which the quantum yields of C and C photo$
%
synthesis are equivalent, suggesting that the energetic costs of abolishing photorespiration and those
of processing photorespiratory metabolites are more
or less equal at this temperature (30mC in the study of
Ehleringer & Bjo$ rkman, 1977). From an energetic
point of view, therefore, this transgenic approach is
only likely to be cost-effective if ambient
temperatures are high enough to make the energetic
price worth paying or if light is sufficiently plentiful
that quantum yield never becomes relevant.
Recent work with mutants of Amaranthus suggests
that photorespiratory rates in C plants, although
%
undoubtedly lower than in most C plants, are far
$
from negligible (Lacuesta et al., 1997). Indeed,
under appropriate conditions, mutants lacking the
C -type phosphoenolpyruvate carboxylase (PEPC)
%
display photorespiratory rates that, as a proportion
of net photosynthesis, are similar to those observed
in many C plants (Lacuesta et al., 1997). This work
$
shows that C plants contain high activities of the
%
enzymes necessary to process phosphoglycollate,
even if flux through the pathway is attenuated by
relatively high CO concentrations in the bundle
#
sheath chloroplasts and the CO evolved in the
#
bundle sheath mitochondria escapes less readily
from the leaf than in C plants.
$
In the long term, severe depletion of enzymes
involved in the photorespiratory recycling pathway
is not an effective way of reducing photorespiration,
because many such lesions are lethal when plants are
grown in air (Somerville & Ogren, 1979 ; Blackwell et
al., 1988). Nevertheless, interesting effects of moderate decreases in GS activity have been reported
(Ha$ usler et al., 1994). Heterozygous barley mutants,
in which foliar enzyme capacities were about half
those in the wild-type, showed a decrease in the
number of electrons transported per molecule of
CO fixed. This effect was most marked under
#
conditions favouring a low C : O ratio and high
absolute rates of photorespiration, and has been
discussed in detail elsewhere (Ha$ usler et al., 1994 ;
Leegood et al., 1995). A similar effect has been
obtained by overexpression of PEPC (Ha$ usler et al.,
1999 ; and references therein). The interesting explanation of this effect was that increased PEPCdependent CO uptake led to higher, not lower, CO
#
#
availability, because the increased production of
respiratory substrates stimulated CO release
#
through malic enzyme and the tricarboxylic acid
cycle (Ha$ usler et al., 1999).
Decreases in photorespiratory carbon loss might
be achieved by metabolic engineering to allow
adequate processing of photorespiratory metabolites
via novel pathways with modified stoichiometries of
CO evolution. One possibility is the introduction of
#
enzymes that enable the conversion of glycollate to
glycerate by a route that does not include a
decarboxylation step. The rate of CO evolution
#
might also be modified by catalase activity, as
discussed in section V(5).
Would decreased photorespiratory CO release
#
necessarily entail a sustained increase in net photosynthesis and improved growth ? Slower rates of
photorespiration will only increase photosynthetic
rates when the latter is not limited by utilization of
photosynthate and\or the associated release of Pi
(Leegood & Furbank, 1986 ; Sharkey et al., 1986).
Longer-term studies of the effect of CO enrichment
#
have revealed that the initial stimulation of photosynthesis is often followed by decreases in rates
(Stitt, 1991). These decreases might to some extent
reflect stomatal closure but are probably mainly due
to the down-regulation of photosynthetic enzymes as
a result of increased foliar carbohydrate. We can
therefore surmise that improved growth due to
decreased photorespiration might not be achieved
unless increased strength of carbohydrate sinks is
also engineered. The availability of key nutrients
such as nitrogen will also influence the plant’s
capacity to exploit any carbon gain resulting from
decreased photorespiration.
3. Is photorespiration important in energy
dissipation ?
Photorespiration uses energy in at least three processes : the recycling of glycollate carbon, the
reincorporation of ammonia, and the turnover of the
RPP pathway resulting from oxygenation (Fig. 5). If
one calculates energy utilization per RuBP
metabolized, the process of photorespiration probably demands only slightly more energy than CO
#
fixation. However, the oxygenation of RuBP does
significantly increase the energy required per molecule of CO fixed. This fact has led to the concept
#
that the physiological significance of photorespiration lies in its wastefulness (Osmond &
Bjo$ rkman, 1972 ; Heber & Krause, 1980 ; Osmond &
Grace, 1995). According to this view, the processes
initiated by oxygenation can be considered, in
metabolic terms, to be a futile cycle that uses ATP
and reductant to avert potentially deleterious excitation energy densities in the photosynthetic apparatus. One effect of the photorespiratory pathway
could therefore be to prevent reaction-centre damage
when light energy is in excess, consistent with the
effect of O in preventing photoinhibition (Krause,
#
1994).
We can ask how this postulated function of
dissipation should be most usefully considered. Is it
a metabolic counterpart of dissipation mechanisms
in light-harvesting that are induced by increasing
irradiance ? Because photorespiration also uses as-
REVIEW
Oxygen processing in photosynthesis : regulation and signalling
similatory power when light strongly limits photosynthesis (Ehleringer & Bjo$ rkman, 1977), it seems
that there is a fundamental difference between this
process and those that operate in the pigment beds.
As the light intensity increases, there is an increasing
probability of a photon being dissipated as heat,
whereas the probability that it will serve to power the
turnover of the photorespiratory pathway decreases.
Nevertheless, the photorespiratory pathway might
represent a physiologically desirable and inherent
‘ inefficiency ’ in the phototosynthetic process
(Osmond & Grace, 1995). This idea receives apparent support from a transgenic study in tobacco
underexpressing or overexpressing chloroplastic GS
(Kozaki & Takeba, 1996). Underexpression of the
enzyme was shown to lead to an enhanced susceptibility to photoinhibition (Kozaki & Takeba,
1996). However, the conclusion drawn from this
effect was somewhat simplistic because, as discussed
above, work with mutants has made it clear that a
decreased capacity to recycle photorespiratory
products leads to a disruption of the overall photosynthetic process. It is difficult to manipulate
photorespiratory flux without affecting overall rates
of photosynthesis. Data obtained with plants overexpressing GS offer a more persuasive argument :
severe photo-oxidation observed in a wild-type leaf
exposed to saturating light for 24 h was not observed
as a result of similar treatment of a leaf in which GS
was overexpressed (Kozaki & Takeba, 1996). However, no indication was given of the reproducibility
of this startling effect ; the significance of the study as
a whole is somewhat difficult to evaluate in the
absence of more detailed data on the characterization
of the transformants.
One condition in which theory dictates an important role for photorespiration is when the intercellular [CO ] : [O ] ratio drops owing to stomatal
#
#
closure, for example in drought stress. In this case,
flux through the photorespiratory pathway will be
enhanced (Lawlor, 1976), maintaining photosynthetic electron transport rates while changing the
allocation of ATP and reductant between photorespiration and CO fixation (Cornic & Briantais,
#
1991). Drought stress is likely to be observed at high
ambient temperatures, which in themselves increase
the ratio of photorespiration to net CO fixation
#
(Hanson & Peterson, 1986). Despite the evidence
that photorespiratory activity is stimulated during
drought stress, it remains to be clearly demonstrated
that an increased proportion of absorbed energy
flows to photorespiration under these conditions (for
further discussion see Osmond & Grace (1995)).
Photorespiration is unlikely to be a significant energy
sink at low temperatures because of slow overall
metabolic rates and high C : O ratios.
Can changes in light intensity by themselves alter
the proportion of energy allocated to photorespiration ? If the fraction of fixed carbon flowing
373
through the photorespiratory pathway depends
solely on the C : O ratio at Rubisco, absolute rates of
photorespiration will increase with light intensity in
proportion to CO fixation. This would entail both
#
processes ’ reaching a ceiling at the same light
intensities, and photorespiration would seem to be a
poor adaptive mechanism for the dissipation of
supra-optimal light. One factor that would increase
the amount of energy required per molecule of CO
#
fixed, independently of the C : O ratio at Rubisco, is
an increased peroxidative decarboxylation of photorespiratory oxo-acids (Hanson & Peterson, 1986)
(see section V(5)). However, no marked change was
observed in the proportion of electrons allocated to
photorespiration as a function of light intensity
(Hanson & Peterson, 1986 ; Peterson, 1990). Variation in the C : O ratio with light intensity remains
doubtful and in any case must be much less
significant than effects caused by changes in temperature or stomatal resistance.
4. Production and processing of photorespiratory
HO
# #
In a C leaf under conditions favouring high rates of
$
oxygenation, such as a warm summer’s day, the
photorespiratory pathway will probably be the
fastest process generating H O (Fig. 5). Unequivo# #
cal evidence that catalase is crucial in removing
photorespiratory H O first came from the isolation
# #
of a barley mutant, with very low catalase activity,
that was unable to photosynthesize and grow well in
air, particularly at high light intensity and temperature (Kendall et al., 1983). More recently,
similar effects have been reported for tobacco plants
in which the major form of catalase was decreased by
anti-sense technology (Chamnongpol et al., 1996 ;
Takahashi et al., 1997 ; Willekens et al., 1997 ;
Brisson et al., 1998). These observations conclusively demonstrate the necessity of sufficient catalase
activity to cope with photorespiratory H O pro# #
duction. Thus, although APX and other components
of the ascorbate–glutathione pathway might be
associated with peroxisomes (Yamaguchi et al., 1995 ;
Bunkelmann & Trelease, 1996 ; Jime! nez et al., 1997 ;
Zhang et al., 1997), these enzymes seem incapable of
coping with the abundant H O production that
# #
accompanies high photorespiratory flux. The tight
link between catalase and photorespiration is further
evidenced by the relatively low activities in C
%
species (Tolbert et al., 1969) and the absence of a
phenotype in maize mutants deficient in catalase
(Scandalios, 1994).
In eukaryotes, catalase is a haem protein with a
very high maximal activity but a very low affinity for
H O : activity increases linearly with substrate up to
# #
concentrations well above those found in living cells.
It is now clear that both C and C plants contain
$
%
multiple isoforms of catalase (Willekens et al., 1995 ;
374
REVIEW
C. H. Foyer and G. Noctor
Scandalios et al., 1997). Apart from a mitochondrial
isoform in maize, catalase seems to be localized
predominantly, if not exclusively, in microsomes
(Willekens et al., 1995). Some activity in maize can
be found in the cytosol (Scandalios et al., 1997). The
principal difference between the microsomal isoforms seems to lie in their expression patterns,
although significant enzymological differences might
also exist (Havir & McHale, 1989b).
The effects obtained by engineering decreases in
catalase activity might have considerable physiological relevance because, like the D1 protein of
PSII, catalase undergoes continuous turnover in the
light. Ongoing protein synthesis is required to
maintain catalase activities : under conditions in
which degradation exceeds resynthesis, catalase
activities can decrease (Feierabend & Engel, 1986).
In stress conditions that impair protein synthesis,
such as low or high temperature, or salt stress, a
light-dependent decrease in total catalase protein
and activity is observed (Volk & Feierabend, 1989 ;
Hertwig et al., 1992).
The ready permeability of membranes to H O
# #
might enable enzyme systems ouside the peroxisome
to contribute to the scavenging of photorespiratory
H O . However, although the chloroplast can
# #
metabolize external H O at high rates (section
# #
III(4)), it seems from the studies of plants with
decreased catalase activities that other routes of
H O detoxification are unable to take the place of
# #
catalase effectively when photorespiration is rapid.
The extent to which H O leaks from the peroxisome
# #
during glycollate oxidation in plants with sufficient
catalase activity is unclear. There is evidence that
photorespiratory metabolites are efficiently channelled within the peroxisome (Heupel & Heldt,
1994 ; Raghavendra et al., 1998). The peroxisome’s
ultrastructure might therefore hinder H O efflux,
# #
maintaining high concentrations in the vicinity of
catalase. Because catalase has a high Km for H O ,
# #
high substrate concentrations would substantially
increase the efficiency of H O removal, perhaps
# #
preventing the oxidation of other compounds in
peroxidatic activity, which can occur at lower H O
# #
concentrations (Scandalios et al., 1997).
5. Catalase and foliar H O levels
# #
Even under conditions in which foliar metabolism is
severely disrupted, low catalase activity rarely leads
to increases in total foliar H O levels. This seems to
# #
be true whether catalase activity is decreased by
cold-induced light inactivation (MacRae &
Ferguson, 1985 ; Volk & Feierabend, 1989), chemical
inhibition (Ferguson & Dunning, 1986) or transgenic
manipulation (Willekens et al., 1997). The administration of aminotriazole to pea seedlings via the
transpiration stream caused an increase in H O
# #
levels only when conditions favoured very high
photorespiratory rates (Amory et al., 1992). One
clear response to lower catalase activities is an
increase in foliar glutathione content (Smith et al.,
1984, 1985 ; Smith, 1985 ; Ferguson & Dunning,
1986 ; Volk & Feierabend, 1989 ; Willekens et al.,
1997). In the barley mutant (Smith et al., 1984) and
in transgenic tobacco (Willekens et al., 1997),
transfer to ‘ photorespiratory ’ conditions caused a
striking increase in total glutathione (approx. 6-fold
within 2–4 d), almost all of which was due to
accumulation of the oxidized form. By contrast, the
redox state of the ascorbate pool was little affected
(Willekens et al., 1997). These effects were accompanied by the induction of both GSH-dependent
peroxidase and APX (Willekens et al., 1997),
although no increase in APX activity was observed
after the chemical inhibition of catalase activity in
pea (Amory et al., 1992). The latter study also found
no change in extractable GR activity, similar to
results obtained after decreases in catalase activity by
high light and low temperature (Volk & Feierabend,
1989). However, in the barley mutant, extractable
foliar GR activity approximately doubled on transfer
to air, with similar kinetics to the accumulation and
oxidation of the glutathione pool (compare Azevedo
et al., 1998 and Smith et al., 1984).
Tight functional linkage between the ascorbate
and glutathione redox couples (Foyer & Halliwell,
1976) is indicated by the preferential oxidation of the
glutathione pool when catalase activity is decreased
(Willekens et al., 1997). Because no enzymes have
been reported in leaves that are capable of catalysing
the H O -dependent peroxidation of GSH at rates
# #
comparable to that of ascorbate, oxidation of the
glutathione pool probably occurs primarily via
ascorbate peroxidation. A small contribution might
be made by the GSH-dependent reduction of organic
hydroperoxides catalysed by GSH-peroxidases. A
correlation between decreased catalase activity and
accelerated ascorbate peroxidation was evidenced by
an increased MDHA electron spin resonance signal
on the treatment of bean leaves with sulphite
(Veljovic-Jovanovic et al., 1998). If the glutathione
pool is oxidized via ascorbate peroxidation, its
oxidation state will depend on the relative rates of
reaction of GSH with DHA and the reduction of
GSSG by GR. The net oxidation of the glutathione
pool therefore suggests that GR activity, whether
increased or not, is unable to keep pace with the
rapid redox cycling of the ascorbate pool (although
effects due to a limiting supply of reductant cannot
be discounted). Because this is consistent with the
improved protection of the ascorbate pool observed
in plants overexpressing GR in the chloroplast
(Foyer et al., 1995), decreased catalase activity might
result in increased metabolism of photorespiratory
H O in this organelle. In the barley catalase mutant,
# #
however, GSSG accumulation was found to be more
or less equally distributed between the chloroplast
REVIEW
Oxygen processing in photosynthesis : regulation and signalling
and the rest of the cell (Smith et al., 1985). Factors
likely to be responsible for the increases in total
glutathione have been discussed elsewhere (Noctor
& Foyer, 1998a).
H O can also be reduced to water by non-enzymic
# #
reaction with oxo-acids, including glyoxylate and
hydroxypyruvate (Zelitch, 1972 ; Elstner & Heupel,
1973). Because these reactions result in the release of
CO , they increase the proportion of glycollate
#
carbon evolved in the photorespiratory pathway,
thereby increasing the quantum requirement of CO
#
fixation and decreasing the rate of net photosynthesis. Although their quantitative significance is
controversial, these reactions probably occur to some
extent in vivo and are increased by inhibition of
catalase activity in leaf peroxisomes (Walton & Butt,
1981). In pea, the inhibition of catalase caused an
increase in formate content, presumably due to the
decarboxylation of glyoxylate (Amory et al., 1992).
Evidence has been presented that the percentage of
glycollate carbon respired through the photorespiratory pathway is variable ; at the CO com#
pensation point this can be double that of the widely
accepted minimum of 25% (Hanson & Peterson,
1986). If this is so, then a genetic approach to
reducing H O -dependent decarboxylation is a strat# #
egy worth pursuing in the quest to attenuate
photorespiration. Indeed, tobacco mutants have
been isolated that show improved rates of photosynthesis, particularly at high O levels, and that
#
have increased catalase activities (Zelitch, 1989).
More recently, in transformed tobacco, the underexpression of catalase has been shown to be
associated with an increased CO compensation
#
point, whereas the reverse was observed in plants
overexpressing the enzyme (Brisson et al., 1998). By
contrast, labelling experiments in the catalasedeficient barley mutant produced no evidence for
increased CO release in conditions promoting high
#
rates of photorespiration (Kendall et al., 1983), even
though increased CO release can occur in barley
#
lacking sufficient aminotransferase activity to keep
pace with glyoxylate production (Murray et al.,
1987). Further work is required to resolve the
apparent discrepancy between the results in barley
and tobacco (Kendall et al., 1983 ; Brisson et al.,
1998).
6. Catalase and non-photorespiratory H O
# #
generation
Considerable attention has recently been focused on
the question of whether catalase is important in
metabolizing H O of non-photorespiratory origin,
# #
particularly in C plants, which have very high foliar
$
catalase activities. There is controversy over whether
catalase has an important role in the hypersensitive
response and systemic acquired resistance that can
follow pathogen attack (Chen et al., 1993 ; Rao et al.,
375
1997 ; Dat et al., 1998). In maize seedlings, which
have higher photorespiratory rates than mature
plants (De Veau & Burris, 1989), enhanced catalase
transcripts, protein and activities were associated
with decreased chilling-induced oxidative damage
(Prasad, 1996). However, this effect was due to induction of the maize-specific mitochondrial isoform :
markedly decreased peroxisomal catalase activities
did not increase sensitivity to chilling in tobacco
(Willekens et al., 1997). Nevertheless, leaf discs
from the same tobacco plants metabolized exogenous
H O less effectively and showed increased ion
# #
leakage after illumination in the presence of methyl
viologen, whose primary site of action is in the
chloroplast. Moreover, the symptoms developed by
leaves from these plants in supra-optimal light could
be prevented by the simple yet ingenious step of
infiltrating the leaf with a solution of bovine catalase, whose action was presumably extracellular
(Willekens et al., 1997). In apparent contrast to
these results, which suggest that catalase has important roles in processes other than photorespiration, total foliar catalase activities can be decreased
substantially, without detriment to the leaf, by
growth at high [CO ] for 1–2 d (Havir & McHale,
#
1989a ; Volk & Feierabend, 1989). Similarly, barley
mutants with very low catalase activity show normal
growth at high [CO ] (Kendall et al., 1983).
#
Because cyanobacteria possess catalase (Tel-Or et
al., 1986), we might wonder why this enzyme is not
present in the higher plant chloroplast. Some, but
not all, cyanobacteria contain APX (Miyake et al.,
1991), suggesting that this enzyme appeared during
cyanobacterial evolution and eventually replaced
catalase in processing H O produced by the photo# #
synthetic electron transport chain. One possibility is
that catalase would simply not be able to maintain
sufficiently low H O levels in the higher-plant
# #
chloroplast. In animals, however, in which catalase
and glutathione peroxidase are the major activities
scavenging H O , and where H O levels are gen# #
# #
erally lower than in leaves, catalase activities in
different organs are inversely correlated with H O
# #
concentration (Scandalios et al., 1997). Because
enzyme regulation by the Td system occurs among
all major taxonomic groups that perform oxygenic
photosynthesis (Buchanan, 1991), some insight can
be gained from a comparison of the interspecific
characteristics and distribution of this regulation
with the occurrence of catalase and APX in cyanobacteria. It is worth noting here that photosynthesis
and the thiol-regulated enzymes might be less
sensitive to oxidants in both unicellular algae and
cyanobacteria, perhaps allowing these species to
tolerate relatively high stromal H O concentrations
# #
(Takeda et al., 1995). Moreover, the recent cloning
and purification of catalase from one cyanobacterium
showed the enzyme to be very similar to catalases
from non-photosynthetic bacteria, with higher
376
REVIEW
C. H. Foyer and G. Noctor
peroxidatic activity and affinity for H O than plant
# #
catalases (Mutsada et al., 1996). These properties
might have contributed to the protection against
drought conferred on tobacco plants by overexpression of an E. coli catalase in the chloroplast
(Shikanai et al., 1998).
VI. 
significant compared with photosynthetic O evol#
ution. Respiration in the light and possible interactions with carbon and nitrogen assimilation have
been reviewed recently (Kro$ mer, 1995 ; Noctor &
Foyer, 1998b). Here we shall confine ourselves to a
brief discussion of mitochondrial AOS generation
and removal, and the related question of the role of
the alternative oxidase (AOX).
1. ‘ Photosynthetic ’ respiration
Respiratory activity in the light can be considered
part of the photosynthetic process for at least three
reasons (Fig. 6). First, the oxidation of photorespiratory glycine might be linked to mitochondrial
electron transport activities. Second, some oxidative
flow of carbon must occur through parts of the
glycolytic pathway and the tricarboxylic acid cycle,
to generate biosynthetic precursors, in particular
those required for photosynthetic nitrogen assimilation. Third, the mitochondria might be important
in adjustment of the stromal redox state during
photosynthesis, by burning off excess reducing
equivalents. The inability to determine respiratory
CO release and O uptake in the light with any
#
#
degree of precision complicates the interpretation of
photosynthesis measurements, particularly those of
maximum quantum yield performed at low light
intensities, in which respiratory O uptake might be
#
Photorespiration
2. AOS in the mitochondrion
Superoxide production occurs at two principal sites
in plant mitochondria (Fig. 6) : NAD(P)H dehydrogenases and the cytochrome bc complex (Rich &
"
Bonner, 1978 ; and references therein), the reductant
in the latter case probably being ubisemiquinone
(Turrens et al., 1985). These processes result in the
formation of H O , primarily through the action of a
# #
mitochondrion-specific
Mn-dependent
SOD
(Jackson et al., 1978).
The metabolism of H O of mitochondrial origin
# #
is controversial. Extramitochondrial enzymes, particularly catalase and extracellular guaiacol-type
peroxidases, might have important roles (Puntarulo
et al., 1991). A study in maize reported that
mitochondria contain cytochrome c-peroxidase activity, but that associated APX and guaiacol-type
peroxidase activities are cytosolic contaminants
Rest of cell
Respiratory C flow Export of reductant
for biosyntheses
from chloroplast
NAD(P)+ NAD(P)H
Glycine
Reductant
Inner
mitochondrial
membrane
Glycine
decarboxylase
complex
CO2
NH3
CH2-THF
O2• –
O2
I
NADH
NAD+
O2
Q
Intermembrane space
H2 O
AOX
IV
III
O2
O2• –
O2
H2 O
Matrix
Fig. 6. Diagram of mitochondrial electron transport showing potential sites of superoxide (O d−) generation and
#
likely important interactions with photosynthetic processes (in green). Electron transfer within the
mitochondrial chain is depicted by red arrows. Reducing equivalents from NAD(P)H can also enter the chain
at other dehydrogenases associated with the inner membrane. AOX, alternative oxidase ; I, complex I (NADH
dehydrogenase), III, complex III (cytochrome bc complex) ; IV, complex IV (cytochrome oxidase) ; Q,
"
ubiquinone ; CH -THF, methylene tetrahydrofolate.
#
REVIEW
Oxygen processing in photosynthesis : regulation and signalling
(Prasad et al., 1995). Other investigators have
concluded that APX activity might be integral to
plant mitochondria (Dalton et al., 1993 ; Yamaguchi
et al., 1995 ; Jime! nez et al., 1997), although immunogold labelling failed to localize APX in this compartment (Dalton et al., 1993) and mitochondrial
cDNA or gene sequences have not yet been identified. Mitochondria contain GR activity (Edwards et
al., 1990 ; Rasmusson & Møller, 1990), which might
be attributable to the same protein as chloroplastic
GR, with both activities being encoded by a common
gene (Creissen et al., 1995). If so, the biochemical
and kinetic differences between the activities
(Edwards et al., 1990) could perhaps reflect
organellar differences in modification of the protein.
How significant is the mitochondrial contribution
to the total AOS production of the photosynthetic
cell in the light ? During electron transport in
mitochondria from capsicum fruit (Purvis, 1997),
superoxide production accounted for just over 1% of
total electron flow to O . This proportion of electron
#
flow to superoxide is broadly similar to that estimated in PSII membrane fragments (Ananyev et al.,
1994) but is smaller than many estimates of the
fraction of electrons engaged in the univalent
reduction of O at PSI (section III(3)). Because total
#
electron transport rates in the chloroplast can easily
exceed total mitochondrial electron transport rates,
absolute rates of superoxide production in the
mitochondria are presumably much lower than those
in the chloroplast. Similarly, in C species, peroxi$
somal H O generation is likely to far oustrip that
# #
occurring in the mitochondria. Nevertheless, the
mitochondria might make a more significant contribution to AOS generation in the illuminated
photosynthetic cell under certain stress conditions,
such as chilling.
3. AOX : regulation and significance to
photosynthesis
One reason that plant mitochondria do not produce
more superoxide could be the presence of an AOX
that catalyses the tetravalent reduction of O by
#
ubiquinone. Although the importance of this enzyme
in thermogenesis is established, its role in most plant
tissues is less well defined. The long-held view that
the AOX is important only when reductant is in
excess has been challenged by the recent elucidation
of various factors controlling the activity of the
enzyme and its dependence on the reduction state of
the ubiquinone pool. Activity is increased by certain
organic acids, notably pyruvate, and by reduction of
the protein (Millar et al., 1993 ; Umbach & Siedow,
1993 ; Vanlerberghe et al., 1995). In this active state,
the AOX is likely to compete with the cytochrome
pathway for electrons (Day et al., 1994). Effects
similar to those of added thiols were observed in
377
the presence of organic acids whose oxidation can
be NADP+-linked ; it was therefore proposed that a
mitochondrial NADPH-Td system is responsible
for reduction (Vanlerberghe et al., 1995). Recent
studies using site-directed mutagenesis to identify
regulatory cysteine residues have reported somewhat
contrasting results for the Arabidopsis and tobacco
proteins (Rhoads et al., 1998 ; Vanlerberghe et al.,
1998). In addition to these controls over activity,
various factors, including H O , induce expression
# #
of the tobacco Aox1 gene (Wagner & Krab, 1995 ;
Vanlerberghe & McIntosh, 1996). Because it has
been shown that AOX activity is associated with
lower rates of superoxide generation in isolated
mitochondria, it seems that an important function of
this enzyme might be in keeping rates of univalent
O reduction comparatively low (Purvis et al., 1995 ;
#
Purvis, 1997). Despite this, severe underexpression
of the tobacco enzyme led to marked visible
symptoms only when the cytochrome pathway was
inhibited (Vanlerberghe et al., 1995).
In isolated mitochondria, the rate of superoxide
production is decreased by addition by ADP or
uncouplers (Purvis et al., 1995 ; Purvis, 1997).
Because AOX activity supports little or no ATP
synthesis, it might be important in regulating ATP
yields and the extent to which ATP : ADP ratios feed
back on electron transport and favour superoxide
formation. New evidence has been presented that the
AOX is important in allowing high rates of oxidation
of photorespiratory glycine by the mitochondrial
electron transport chain (Igamberdiev et al., 1997).
Although isolated leaf mitochondria oxidize glycine
in preference to other respiratory substrates (Dry et
al., 1983), it has often been considered improbable
that the electron transport chain is the major route
regenerating NAD+ for ongoing glycine decarboxylase activity (Fig. 6). There are two principal reasons
for this. First, carbon recycling via the photorespiratory pathway requires NAD+ (in the mitochondrion, to oxidize 50% of the glycine formed)
and NADH (to reduce hydroxypyruvate in the
peroxisome) in equal measure, and shuttle systems
have been characterized that can compete effectively
with the electron transport chain (Journet et al.,
1981 ; Ebbighausen et al., 1985). Second, high rates
of oxidative phosphorylation linked to glycine oxidation could potentially cause a very high ATP : ADP
ratio in the mitochondria and cytosol of photorespiring leaf cells, which has not generally been
observed (Gardestro$ m & Wigge, 1988). This
anomaly might be resolved in part by AOX-linked
glycine oxidation (Igamberdiev et al., 1997),
although this would necessitate another source of
reductant for peroxisomal glycerate synthesis. More
generally, variable electron flow through the AOX
might contribute to the adjustment of cellular
adenylate status during photosynthesis, allowing
flexibility in the rate of necessary respiratory flows
378
REVIEW
C. H. Foyer and G. Noctor
linked to processes such as nitrogen assimilation
(Noctor & Foyer, 1998b, 2000).
VIII.    

1. The need for sensors, signals and transducers
As semi-autonomous organelles with a wide range of
assimilatory functions and unique biochemical pathways, chloroplasts are able to transcribe and translate
the limited amount of genetic information contained
within the plastid genome but are strongly dependent on imported proteins that are encoded in
the nuclear genome and translated in the cytoplasm.
The photosynthetic apparatus has to respond rapidly
to fluctuations in essential inputs, such as light and
CO , and this frequently requires the concerted
#
activation of processes occurring in several cellular
compartments. Leaves at the top or the very bottom
of the canopy can experience conditions approximating steady-state but most leaves are subject to
alternating periods of high and low light. In
understorey plants, sunflecks are a vital resource and
maximum use of available energy is made possible
by appropriate regulation (Pearcy, 1990). However,
light energy input can be far in excess of that
required by metabolism, leading to potentially
dangerous imbalances in supply and demand that
provoke photoinhibitory damage. The supply of
light energy therefore has to be carefully managed to
maximize the efficiency of capture and minimize the
risk of damage.
Current evidence suggests that, in addition to the
relatively rapid regulation of light harvesting,
changes in supply and demand are accommodated
through short-term and long-term adaptation
mediated by transduction of signals sensed by the
redox state of key components (Fig. 7). At least two
interrelated redox components seem to be important.
First, certain stimuli are transduced with AOS
(particularly H O ) as signalling messengers. Se# #
cond, considerable evidence points to a crucial role
for the redox state of at least one electron transport
component, PQ. The past few years have seen the
accumulation of considerable evidence supporting
the existence of local (intracellular) sensor-response
systems. Very recently, results have been obtained
that suggest the intriguing possibility of the remote
acclimation of photosynthesis.
2. Signal transduction at the local level
As the rate-limiting step of photosynthetic electron
transport, the rate of PQ oxidation is central to the
control of photosynthesis. Although it has been
known for many years that the redox state of the PQ
pool influences the rate of electron transfer between
the photosystems, it is only relatively recently that
the impacts on transcriptional, translational and
post-translational regulation have been recognized.
The first indications that the PQ pool functions as
a sensor to initiate adaptive responses date back
almost 20 yr. Short-term changes in redox state (of
the order of seconds to minutes) lead to the posttranslational adaptive changes known as state
Environmental
Developmental
Changes in:
H2O2
PQ redox state
Nitric oxide
Antioxidants
Oxidative load
Electron pressure
Membrane
potentials
Signal
transport
Intracellular
Intercellular
Long distance
Acclimatory
responses
Induction of
antioxidative system,
acclimation of photosynthesis
Fig. 7. Conceptual relationships between sensors, transmitters and responses in signal transduction leading to
the acclimation of photosynthesis. Information relating to developmental and environmental conditions is
detected through changes in bioenergetic parameters such as electron pressure, entailing the modulation of
specific components such as oxidant concentrations and the redox state of quinones involved in electron
transport chains. Emerging evidence indicates that signal transduction can operate locally (within cells) or
remotely (between different cells and tissues).
REVIEW
Oxygen processing in photosynthesis : regulation and signalling
transitions, involving oligomeric changes in the
organization of the photosystems (Bennett, 1979).
Control over state transitions is mediated by the
redox-dependent activation of a protein kinase as the
PQ pool becomes reduced (Horton et al., 1981). This
kinase phosphorylates the major PSII lightharvesting component (LHCII), causing LHCII to
become detached from the PSII complex, thereby
contributing to the regulation of the distribution of
excitation energy between the photosystems.
More recent work has also implicated the redox
state of the PQ pool in mediating longer-term
adaptation that requires alterations in the rate of
transcription and\or translation of plastid and nuclear genes (Danon & Mayfield, 1994 ; Escoubas et
al., 1995 ; Maxwell et al., 1995 ; Vener et al., 1995,
1998 ; Karpinski et al., 1997 ; Pfannschmidt et al.,
1999). In some cases, these effects seem to occur via
by a redox-activated protein kinase. Analogous
signalling systems have been described in other
organisms. In E. coli, for example, a protein, Aer,
acts as a signal transducer sensing the availability of
intracellular energy. Together with the serine chemoreceptor, Tsr, Aer senses the proton motive force (or
the cellular redox state), enabling the bacterium to
locate environments in which maximal energy is
available for growth (Rebbapragada et al., 1997).
In the photosynthetic bacterium, Rhodobacter
sphaeroides, a two-component regulatory system is
well characterized that is involved in the redox
control of photosynthetic gene expression in response to O availability. The prr loci afford positive
#
regulation of photosynthetic genes during anaerobiosis (Eraso & Kaplan, 1994 ; Mosley et al., 1994).
prrB encodes a sensor histidine kinase that is
responsive to the removal of O and functions
#
through the response regulator, prrA, which
activates the expression of genes involved in photosynthesis. A similar system involving RegA has been
described in Rhodobacter capsulatus (Mosley et al.,
1994). Mutations in prrA lead to a loss of photosynthetic gene expression, whereas the deletion of
prrB has no effect, perhaps owing to the availability
of an alternative phosphate donor (Eraso & Kaplan,
1996). The two-component prrA\B system might
also integrate the control of photosynthesis and
nitrogen fixation (Joshi & Tabita, 1996), the need to
remove excess reducing power generally being of
paramount importance in the orchestration of photosynthetic carbon and nitrogen metabolism.
In higher plants the redox state of the photosynthetic electron transport chain and PSII excitation pressure seem to be key elements in redox
sensing. For instance, the PQ redox state controls
the rate of transcription of PSI and PSII reaction
centre proteins (Pfannschmidt et al., 1999). Changes
in the rate of transcription seem to arise from longterm light-induced perturbations of the PQ redox
state that ultimately lead to changes in the stoi-
379
chiometry of PSI and PSII. When the flux through
either photosystem limits the rate of electron transport, the transcription of genes encoding components
of the other photosystem is induced. Conversely,
genes encoding proteins comprising the photosystem
whose activity is in excess are repressed. This
indicates that extensive metabolic\redox cross-talk
couples the redox state of the PQ pool to the
transcription of specific chloroplast genes.
The PQ pool might also influence the transcription
of antioxidant genes. Exposure of low-light-adapted
Arabidopsis plants to high light induced the induction of genes encoding cytosolic APX (Karpinski
et al., 1997). Although the biochemical significance
of a cytosolic response to a stress of chloroplastic
origin remains to be determined, this does imply the
existence of signals that relay information on the
redox state of the photosynthetic electron transport
chain to the nucleus. The APX protein family also
seems to be associated with a second type of signal
transduction system in plants, involving 14–3-3
proteins, a highly conserved, relatively small and
functionally diverse protein family. In plants the
best characterized group of 14–3-3 proteins is
involved in the control of nitrate reductase activity
(Moorhead et al., 1996), whereas a second group
interacts directly with the C-terminal region of the
plant plasmalemma H+-ATPase in a fusicoccindependent manner (Jahn et al., 1997). In general,
these proteins are considered to coordinate carbon–
nitrogen interactions in leaves and to be involved in
the transcriptional regulation of genes in stress
situations (Brandt et al., 1992 ; Jarillo et al., 1994 ;
Ferl, 1996). APX3 interacts directly with GF14λ (H.
Zhang, pers. comm.), suggesting that this
Arabidopsis 14–3-3 protein could have a role in signal
transduction involving antioxidants.
3. Remote signalling and responses leading to
acclimation of photosynthesis ?
One of the earliest responses of plants to pathogen
attack is the elicited production of AOS by plasmalemma-associated systems such as NADPH
oxidases. H O produced in this way is implicated in
# #
eliciting local responses such as cell-wall crosslinking and induction of antioxidant defences, as
well as long-range defence responses in the process
known as systemic acquired resistance (Levine et al.,
1994 ; Draper, 1997 ; Durner et al., 1997 ; Delledonne
et al., 1998). It has only recently been considered
that AOS might also act as signalling molecules
mediating systemic acclimation of the photosythetic
system (Foyer & Noctor, 1999 ; Karpinski et al.,
1999). Responses induced in Arabidopsis subjected
to high levels of light were not confined to the
exposed leaf areas, but were also observed in adjacent
leaves on the same rosette that had not been exposed
(Karpinski et al., 1999). The adjacent leaves showed
380
REVIEW
C. H. Foyer and G. Noctor
increases in H O levels after the remote stress,
# #
coupled with increased expression of the luciferase
reporter gene under control of the promoter from
APX2, a gene encoding a cytosolic APX. Most
intriguingly of all, subsequent light stress of adjacent
leaves that had not been directly exposed led to
smaller decreases in photosynthetic efficiency than in
leaves from plants on which no leaves had been
prestressed (Karpinski et al., 1999). These results
indicate the presence of a systemic signalling system
for excess irradiation that responds to changes in
cellular redox state. Once again, key components
seem to be H O and signals arising from the PQ
# #
pool (Karpinski et al., 1999). Although the advantage
of this system to plants exposed to a relatively
constant light regime is not clear, it might be of
potential benefit to species subject to rapid alterations in light intensity ; for instance, plants whose
major source of light is sunflecks. The induction of
protective systems in leaves remote from the excess
light stimulus would perhaps give such plants a preemptive advantage in preventing damage while
allowing them to make maximal use of transiently
abundant light energy.
4. Interactions between AOS, NO) and antioxidants
Whereas AOS produced by the chloroplasts and
mitochondria can act alone as signals to trigger
changes in gene expression, it is clear from studies in
both animal and plant systems that signalling via
AOS is integrated with a second signalling pathway
involving active nitrogen species. Oxidative stress in
mammalian cells is enhanced by the production of
active nitrogen species, notably nitric oxide (NO)).
This secondary messenger induces signalling
cascades involving cyclic GMP and Ca#+. It is also a
potent inhibitor of cytochrome c oxidase, and
increases the production of superoxide by mammalian mitochondria (Poderoso et al., 1996). This
increase in AOS production initiates a reaction
between NO) and superoxide to form hydroxyl
radicals and peroxynitrite. AOS modulate NO)
signalling, leading to apoptosis in cells attacked by
pathogens (Delledonne et al., 1998).
NO) is synthesized in plants, probably either from
arginine via a nitric oxide synthase or by nitrite
reduction, and has been shown to be an integral
component of secondary messenger cascades and to
induce defence systems (Cueto et al. 1996 ; Leshem,
1996 ; Laxalt et al., 1997 ; Beligni & Lamattina,
1999). A potent inhibitor of nitrogenase, NO) forms
a stable complex with iron (II) leghaemoglobin in
developing and mature root nodules but is less
abundant in older nodules and is absent from
senescent ones (Trinchant & Rigaud, 1982 ; Mathieu
et al., 1998). This observation presents an intriguing
possibility regarding the interpretation of studies on
the expression of haemoglobin-like proteins in plants
(Bulow et al., 1999). Although effects observed have
been discussed in terms of altered O transport or
#
storage, they could also reflect the modification of
signal transduction cascades involving NO). Like the
mammalian enzyme, the plant cytochrome oxidase is
inhibited by NO), whereas the AOX is unaffected
(Millar & Day, 1996), contrasting with the stimulatory effect of AOS on AOX gene expression
discussed in section VI(3).
Of the complex array of antioxidants found in
plant cells, only glutathione and ascorbate have
documented multiple functions, as discussed previously (Noctor & Foyer, 1998a). The millimolar
concentrations of these antioxidants in photosynthetic cells might be necessary for roles other
than AOS scavenging. The maintenance of ascorbate
and glutathione homeostasis, in the face of the
multiple demands placed on the pools, involves a
complex interplay between synthesis, degradation,
transport, storage, oxidation–reduction, further
metabolism, and catabolism, because plants respond
to changing environmental conditions. Although the
ascorbate : DHA and GSH : GSSG ratios are rigorously controlled, these redox couples are ideally
suited to signal transduction, first because they are
relatively stable and second because they are mobile
compounds that are transported within cells and
between cells. Each component might itself be
involved in signal transduction but it is more likely
that the redox state of each couple is the most
influential factor. In particular, GSSG readily
interacts with proteins to form mixed disulphides in
the process known as thiolation (Thomas et al.,
1995). Thiolation modifies metabolism, protein
turnover and gene transcription, because it protects
enzymes from irreversible inactivation and degradation. It might protect the thiol-modulated
enzymes from proteolysis under conditions of oxidative stress. Therefore, in addition to changes in
AOS
concentrations,
fluctuations
in
the
ascorbate : DHA and GSH : GSSG ratios in photosynthetic cells might have important consequences
not only for defence metabolism but also for the
regulation of genes associated with adaptive
responses.
VIII. 
The above discussion has treated photosynthesis as a
whole-cell process, with special emphasis on the
interactions between the different pathways that
involve O . It has long been considered that
#
photosynthetic cells are particularly susceptible to
oxidative damage, because photosynthesis is a photodynamic process producing O . During evolution,
#
the photosynthetic process has encompassed
chemically unavoidable reactions with O such as the
#
Mehler reaction and the oxygenase activity of
Rubisco. Despite the potentially damaging nature of
REVIEW
Oxygen processing in photosynthesis : regulation and signalling
the side-products of these reactions, it is now evident
that much of the responses to increased concentrations of AOS, even those that are apparently
deleterious, do not result from simple physicochemical damage, but reflect acclimatory changes in
gene expression and metabolism. This means that
the manipulation of photosynthetic oxygen metabolism offers great potential both because of the
pivotal functions of oxygen species in energy metabolism and their pivotal roles in signal transduction
and the control of gene expression. Although AOS
are always present in plant cells, accumulation above
a certain threshold provides an early signal of
perturbations in energy metabolism. In the extreme
case, the accumulation of AOS triggers apoptosis
and premature senescence in leaves, effects that
require the antioxidative system to be perturbed or
temporarily overwhelmed.
Mutants provide an incisive tool for dissecting
mechanistic processes, but this approach is complicated by a compensatory engagement of alternative pathways. We emphasize that mechanisms that
can operate are not necessarily those that do operate
under physiological conditions, and the relative flux
through different electron transport pathways remains controversial. A key example is electron
partitioning to the Mehler reaction. Although this
subject will no doubt continue to evoke much debate,
recent work with transformed plants has provided
evidence in vivo to corroborate concepts derived
from studies in vitro of isolated systems that suggest
that in the absence of an ATP sink the rate of this
pathway is relatively low (Ruuska et al., 2000). The
attractive idea that the Mehler peroxidase pathway is
capable of functioning as an unrestricted overflow
for electrons is therefore challenged, as a result of the
ability to target specific processes in vivo by using
molecular approaches. This work also highlights the
multiple, often unforeseen, advances that are enabled
by the production of transformed plants. Plants with
decreased Rubisco were initially used to explore the
fractional control of photosynthesis (Quick et al.,
1991), but in addition to an evaluation of the
importance of the Mehler reaction, such transformants have been used to study other questions
such as nitrogen use efficiency and ozone tolerance
(Quick et al., 1992 ; Masle et al., 1993 ; Wiese & Pell,
1997).
No doubt there exist many components, particularly stress-inducible proteins, that remain to be
discovered. Recent examples of such discoveries are
2-cysteine peroxiredoxins and glutathione-dependent peroxidases, which are thylakoid-associated and
are induced by stress conditions but whose functions
remain uncharacterized. Where components with
known functions have been manipulated, some
success in raising the ceiling of photosynthesis under
stress conditions has been achieved. Further success
is likely to require a much deeper understanding of
381
the integrated regulation of gene expression, protein
stability and enzyme activity, as well as the interactions between the various components. Although
many studies have shown that overexpression of
antioxidative enzymes such as SOD can protect
against photoinhibition, the mechanism of protection
remains obscure. Parameters such as PSII efficiency
or overall rates of photosynthesis provide a useful
global estimate of physiological function but do not
reveal which components are modified by oxidative
stress and in which order. For example, a diagnostic
marker of oxidative damage to proteins (such as D1)
is essential to enable the design of appropriate
engineering by site-directed mutagenesis.
Optimal protection is likely to require the targeting
of enzymes such as SOD and APX to the thylakoid
membrane. So far, there have been no reports of
specifically engineered decreases in the activities of
the thylakoid isoforms of these enzymes. Like high
activities of antioxidative enzymes, high chloroplastic concentrations of antioxidants such as
ascorbate, glutathione and α-tocopherol should have
beneficial effects on photosynthesis, as demonstrated
in numerous studies on isolated chloroplasts. Plants
with modified amounts of these components will
enable us to probe the relationship between photosynthesis and signal transduction under conditions
of stress. Perhaps the most striking example so far of
the success of this approach is provided by plants
with decreased catalase activity, which show a classic
hypersensitive response arising solely from H O
# #
produced in photorespiration. These plants are likely
to be instrumental in elucidating the relationships
between antioxidant synthesis, redox state and
stress-induced gene expression. New technologies
continue to provide new and interesting insights into
the questions discussed in this review : the rapidity of
further progress is likely to depend on the success
with which imminent technological advances and
improved diagnostics can be coupled to a rigorous
investigation of function.
              
We thank Jeremy Harbinson, Peter Lea, Martin Parry,
Matthew Paul and Bill Rutherford for discussion and
comments on sections of the manuscript in preparation.
We are grateful to Peter Horton for his critical appraisal
and constructive comments.

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