Download Strategies to maintain redox homeostasis during photosynthesis

Journal of Experimental Botany, Vol. 56, No. 416, pp. 1481–1489, June 2005
doi:10.1093/jxb/eri181 Advance Access publication 25 April, 2005
FOCUS PAPER
Strategies to maintain redox homeostasis during
photosynthesis under changing conditions
Renate Scheibe*, Jan E. Backhausen, Vera Emmerlich and Simone Holtgrefe
Department of Plant Physiology, Faculty of Biology and Chemistry, University of Osnabrueck,
D-49069 Osnabrueck, Germany
Received 18 February 2005; Accepted 5 April 2005
Abstract
Introduction
Plants perform photosynthesis and assimilatory processes in a continuously changing environment. Energy production in the various cell compartments and
energy consumption in endergonic processes have to
be well adjusted to the varying conditions. In addition,
dissipatory pathways are required to avoid any detrimental effects caused by over-reduction. A large number of short-term and long-term mechanisms interact
with each other in a flexible way, depending on intensity
and the type of impact. Therefore, all levels of regulation are involved, starting from energy absorption and
electron flow events through to post-transcriptional
control. The simultaneous presence of strong oxidants
and strong reductants during oxygenic photosynthesis
is the basis for regulation. However, redox-dependent
control also interacts with other signal transduction
pathways in order to adapt metabolic processes and
redox-control to the developmental state. Examples are
given here for short-term and long-term control following changes of light intensity and photoperiod, focusing on the dynamic nature of the plant regulatory
systems. An integrating network of all these mechanisms exists at all levels of control. Cellular homeostasis
will be maintained as long as the mechanisms for
acclimation are present in sufficiently high capacities.
If an impact is too rapid, and acclimation on the level of
gene expression cannot occur, cellular damage and cell
death are initiated.
Plants operate well between the extreme situations of overoxidation and over-reduction, as caused by the presence of
oxygen, and the simultaneous generation of strong reductants in the photosynthetic electron transport chain. There is
a close relationship between over-reduction of the electron
transport chain and generation of oxygen radicals when the
Mehler reaction is occurring during active photosynthesis.
Antioxidants and redox buffers are therefore present in
order to minimize the risk of detrimental effects. Energydissipating pathways are also functioning at the level of
photosystem II to reduce energy input.
Since electron transport is coupled with ATP production,
further reactions are required to adjust the ratio between
NADPH and ATP to the actual demand. D1 turnover, state
transitions, non-photochemical energy quenching, xanthophyll cycle, chlororespiration, cyclic electron transport, and
the Mehler reaction are some of the poising systems of the
chloroplast, frequently described as flexible ways to maintain a balanced electron flow and the required rate of ATP
production under changing conditions. Further pathways,
involving cell compartments, such as the malate valve,
alternative oxidase (AOX), Q cycles in the electron transport chains, and photorespiration, also contribute and give
rise to flexible ATP/eÿ ratios.
Living cells as open systems require a constant flux of
energy for continuous biomass production and consumption, and depend on cellular homeostasis to maintain all
functions. This, in turn, can only be achieved when the
relatively small pools of ATP/ADP, NAD(P)H/NAD(P)
and other redox carriers, as well as cellular pH, remain at
balanced ratios. This will allow the input and withdrawal of
energy and reducing equivalents at the required rates and
keep the system at homeostasis.
Key words: Light acclimation, malate valve, over-reduction,
oxidative stress, photosynthesis, poising mechanisms, redox
control, redox homeostasis, regulatory networks.
* To whom correspondence should be addressed. Fax: +49 541 969 2265. E-mail: [email protected]
Abbreviations: AOX, alternative oxidase; Fd, ferredoxin, GSH/GSSG, reduced/oxidized glutathione; LHC, light-harvesting complex; NADP-MDH, NADPdependent malate dehydrogenase; PS, photosystem; ROS, reactive oxygen species.
ª The Author [2005]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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1482 Scheibe et al.
Apart from the need for homeostasis of the redox
systems, there is, however, a need for redox signals that
induce the acclimation of metabolism to sustained
changes, i.e. during development and upon stress situations. Various factors have already been suggested to
initiate such signal transduction pathways leading to the
changed expression of certain genes. Reactive oxygen
species (ROS) such as hydrogen peroxide (H2O2), oxygen
radicals (Oÿ
2 ), nitric oxide (NO), or the redox-state of any
of the intersystem or soluble redox components could act
as signals (Foyer and Noctor, 2003; Laloi et al., 2004).
Therefore, although balanced pools of reductants/oxidants
are essential as redox buffers (ascorbate/dehydroascorbate
and GSH/GSSG) in order to accommodate (or dampen)
very rapid changes without affecting homeostasis, glutathione, ascorbate, and other redox components have also
been implicated in redox signalling (May et al., 1998;
Dietz, 2003; De Gara, 2004).
In this paper, the hypothesis put forward is that a large
number of factors are able to induce acclimation reactions
well before any damage becomes apparent, and that the
major redox pools are kept relatively constant over a
wide range of conditions. Therefore, it is assumed that
over-reduction is sensed by the plant before any major
imbalances of the redox components occur and, moreover,
before any oxidative damage occurs. This is achieved by
the presence of flexible mechanisms that interact in
multidimensional networks of regulation at many levels
of cellular activities. On the other hand, once a certain
limit has been reached or the developmental fate has been
programmed previously, cell death will occur within a
short time (Fig. 1).
Developmental state / pretreatment of plant
Sustained change
Long-term control:
Gene expression
Irreversible damage
Fluctuating conditions
Short-term control:
Poising mechanisms
Redox-signal transduction
Optimizing photosynthesis
Antioxidant defence
Redox homeostasis
Reducing conditions
Cell death
Oxidizing conditions
Fig. 1. Flexible system of redox-control in plants during photosynthesis.
Depending on the metabolic, environmental, and developmental situation
as well as on the type of change of conditions (light, temperature, CO2)
redox-signals lead to readjustment of the components of photosynthesis,
poising, and antioxidant systems. Both short-term (by direct effects) and
long-term (at the level of gene expression) control interact with further
metabolic and hormonal signals. When pre-acclimation has failed to
occur in time or senescence has started already, irreversible breakdown
processes will start (cell death).
Short-term adjustment of the redox state in the
electron transport chain
Photosynthesis takes place under continuously changing
conditions, as light, temperature, internal CO2, and nutrient
supply are concerned. Plants are therefore adapted to
a dynamic environment by multiple regulatory systems
that provide high flexibility. Transient short-term changes
can be balanced by a set of protection systems which
stabilize the redox poise in the electron transport chains and
modulate light-use efficiency. Mechanisms that demonstrate the high flexibility of electron transfer and the
concomitant formation of a proton motive force have been
summarized recently, showing the multiple ways that each
of these processes can be adjusted in a varying environment
(Kramer et al., 2004; Nedbal et al., 2003; Holt et al., 2004;
Avenson et al., 2005). For dissipation of excess excitation
energy, various mechanisms have been described that are
able either to avoid the generation of reductants giving rise
to the production of ROS, or to serve as alternative electron
acceptors in order to avoid over-reduction and, potentially,
the formation of toxic intermediates (Mullineaux and
Karpinski, 2002; Niyogi, 1999). Imbalance between the
light energy distribution of PSII and PSI can be regulated
and controlled by state transitions (Allen, 2002). There is
strong experimental evidence that under increasing light
intensities the LHCII kinase is inactivated by reduction via
a membrane-bound thioredoxin-like protein, which itself is
controlled by the thiol-redox potential of the chloroplast
stroma (Martinsuo et al., 2003). The mode of action of the
state transitions indicates that they act primarily to compensate for differential excitation of the two photosystems,
as occurs when the spectral composition of the light
changes, for example, during dawn and sunset or shading
by other leaves during the day.
Light energy is primarily converted into electron flow
that concomittantly results in the generation of the pH
gradient which again is the driving force for ATP synthesis.
Since the demand for reducing equivalents and ATP is
varying, uncoupling of this primary process is required to
provide flexibility. Poising mechanisms act principally in
three different ways: (i) by transfer of electrons back into
the photosynthetic electron transport chains (cyclic electron
transport), (ii) by reduction of O2 and subsequent metabolism of ROS (pseudocyclic electron flow or water–water
cycle), and (iii) by indirect export of reducing equivalents
(malate valve, described in more detail below). Although
the underlying physiological and biochemical mechanisms
are completely different, all three pathways are regulated in
such a way that they do not compete for electrons required
for reductive assimilatory reactions in the chloroplasts
(Backhausen et al., 2000).
From many in vitro measurements, it can deduced that C3
plants are capable of performing cyclic electron flow, but the
problem is to estimate the flux through this cyclic process in
Redox homeostasis during changing conditions
intact leaves (Heber, 2002). Using different techniques,
contradictary results were obtained. Thus the discussion is
ongoing as to whether C3 plants are, in fact, using this
pathway in vivo (discussed in detail by Johnson, 2005).
Cyclic electron flow around PSI in the presence of ‘active’
PSII can only be measured under stress conditions, when the
linear electron transport is saturated either in high light, at
low temperature, or under conditions when carbon fixation is
limited. Under these conditions a bulk of previously inactive
PSI seems to be activated (Golding et al., 2004), and there is
experimental evidence that this activation is linked to
a decreasing stromal NADP/NADPH ratio (Rajagopal
et al., 2003). In addition, it has been proposed that the cyt
b6 f complex is regulated by the stromal redox potential of
the chloroplast stroma via a thioredoxin-mediated mechanism (Johnson, 2003). Nevertheless, one important piece of
evidence concerning the in vivo relevance of cyclic electron
transport came from Arabidopsis double mutants impaired
in NDH (NADH dehydrogenase) and PGR5 (proton
gradient regulation) as part of the Fd-dependent pathway
(Munekage et al., 2004). These authors suggested that the
PGR5 pathway contributes to the generation of a proton
gradient inducing thermal dissipation when Calvin-cycle
activity is reduced. An additional advantage might be to limit
the over-reduction of the acceptor side of PSI, thus preventing PSI inhibition. In conclusion, down-regulation of linear
electron flow and activation of cyclic electron flow seem to
respond to a common signal: the stromal redox poise
(Johnson, 2003).
Various environmental conditions, i.e. light, cold, and
drought stress, as well as pathogen attack, lead to a limitation of linear electron transport due to an over-reduction
of stromal electron acceptors. Under these conditions the
formation of different kinds of ROS is accelerated either by
transfer of electrons to O2, generating superoxide radicals
(Oÿ
2 ), hydrogen peroxide (H2O2) or hydroxyl radicals
(HOÿ), respectively. In addition, singlet oxygen (O12 ) can
be formed by energy transfer from triplet P680. The
stroma-exposed centres of PSI act as electron donors
(Asada, 2000), as long as none of the physiological
electron acceptors are available in the oxidized state.
Additional sites such as plastosemiquinone, where O2
reduction may occur, are still under debate (Ivanov and
Khorobrykh, 2003). The subsequent reactions (disproportionation of O12 into H2O2, its detoxification by ascorbate,
and ascorbate regeneration by GSH at the expense of
NADPH) have been reviewed in much detail (Foyer and
Noctor, 2000). The two scavenging systems, one microcompartmentalized with the PSI complex and one present
in the stroma, exhibit high affinities, and all enzymes are
present with high activities (Asada, 2000). Thus, the
concentration of H2O2, the most stable intermediate, is
kept below 1 lM under non-stressing conditions. Even
under photon-stress conditions, the flux through the water–
water cycle increases, but no accumulation of O12 and H2O2
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is detectable when the supply with reductant is sufficient
(Polle, 1996; Asada, 2000). It is interesting to note that
peroxiredoxins have been found to have a regulatory
function in this process (König et al., 2002).
As a third option for balancing the stromal NADPH/ATP
ratio, the malate valve was put forward as a system to
export excess reducing equivalents as malate while continuing ATP production (Scheibe, 2004). This highly
flexible mechanism results in strong crosstalk with cytosol,
mitochondria, and peroxisomes, and will be discussed later
on in more detail.
Ferredoxin-thioredoxin system for regulation of
the stromal redox-state
During photosynthesis, electrons become available as reduced ferredoxin, and NADPH and ATP are generated.
Various chloroplast enzymes are regulated by the
ferredoxin-thioredoxin system (Buchanan, 1984; Scheibe,
1991; Schürmann and Jacquot, 2000). Electrons are transferred from ferredoxin to the thioredoxins which are
present in the chloroplast in various isoforms. Finally,
electrons are transferred to the target proteins (Dai et al.,
2000; Marchand et al., 2004). Among the many targets
identified and proposed to date, proteins with different
functions such as Calvin-cycle enzymes, and other metabolic enzymes, as well as proteins involved in the stress
response, can be found.
Four steps in the Calvin cycle are subject to such
regulation. Unique regulatory sequences containing disulphide bridges are reduced in the target enzymes in the
light. Continuous oxidation by O2 reverts the reducing
step, thus leading to a ‘futile cycle’ for the sake of
regulation, consuming electrons for the continuous reduction of the re-oxidized regulatory cysteine residues. Light/
dark-modulated enzymes are unique with their very
negative redox potentials of their regulatory cysteines
(Hirasawa et al., 2000). A differential fine-regulation of
the activation of the target enzymes ensures that all steps of
triose-phosphate production and ribulose 1,5-bisphosphate
regeneration are adjusted to the required actual fluxes
(Faske et al., 1995). So, during photosynthesis, fluxes are
adjusted continuously at each step depending on the
metabolic situation. While redox-cycles are the basis,
fine-tuning by metabolites is acting individually in each
case as a feed-forward or a feed-back mechanism. Thus
there is a common principle in the basis of these fast and
flexibly responding regulatory systems (Scheibe, 1991).
The ferredoxin-thioredoxin system is also involved in the
regulation of the malate valve (see below) and ATP
synthase, as well as in the light-induced inactivation of
the plastidic isoform of glucose 6-phosphate dehydrogenase (Scheibe, 1991). It is interesting to note that chloroplasts possess a large number of thioredoxin isoforms
1484 Scheibe et al.
more or less specifically interacting with the various target
enzymes. However, more isoforms of thioredoxin and
related proteins such as glutaredoxins, peroxiredoxins,
and cyclophilins, are also localized in the cytosol and
mitochondria (Dietz, 2003), probably functioning in other
redox-dependent processes (Meyer et al., 1999; Marchand
et al., 2004).
Crosstalk between chloroplast and cytosol: the
malate valve
One of the chloroplast enzymes which is also reduced via
the ferredoxin-thioredoxin system in the light is NADPmalate dehydrogenase (NADP-MDH) which serves as
a redox valve. It uses excess NADPH to convert OAA to
malate in order to regenerate the electron acceptor NADP.
NADP-MDH activation is inhibited by its product NADP,
so NADP-MDH switches off its own activity when NADPH
is consumed for assimilatory processes in the chloroplast,
and therefore no reducing equivalents should be exported as
malate (Scheibe, 1991). Since ATP production continues
while electrons are transferred to malate, the malate valve as
an indirect export system for reducing equivalents is a useful
means to balance the ATP/NADPH ratio in the chloroplast
(Backhausen et al., 1998).
Malate can easily be transported across shuttle systems
of the cellular membranes. Plastidic dicarboxylate translocators have recently been identified which could fulfil the
malate-oxaloacetate shuttle function (Taniguchi et al.,
2002; Renné et al., 2003). In the cytosol, malate can serve
(i) to provide NADH for nitrate reduction, (ii) to generate
ATP in the mitochondria, (iii) to support photorespiration,
or (iv) to be stored in the vacuole. Recently, the vacuolar
malate transporter from Arabidopsis (At tDT) has been
identified (Emmerlich et al., 2003).
Malate is a very common organic acid found in every
plant tissue that plays a central role in plant metabolism
(Lance and Rustin, 1984) since it is intermediate in the
Krebs and glyoxylate cycles and serves as a mobile storage
molecule for CO2 and reducing equivalents in C4 and CAM
plants. It has been suggested that part of the malate is used
directly for fatty acid synthesis in chloroplasts after being
converted into pyruvate (Krömer and Scheibe, 1996).
Malate accumulation as a means to vary the osmotic
pressure is involved in the regulation of stomatal aperture.
Apart from its role as carrier for CO2 and reducing
equivalents, as well as in the carbon skeleton for anabolic
metabolism, malate as an organic acid is one of the major
compounds capable of influencing the acid/base equilibrium of the cell. Acidification of plant cells often results in
a rapid decrease in their malate content, and the consumption of protons associated with the decarboxylation of
malate is proposed to counteract acidity (Mathieu et al.,
1986; Sakano et al., 1998). The control of the cellular pH
value is essential since most enzymes exhibit a narrow pH
optimum. It is thought that the pH of plant cells is regulated
in two main ways: by proton-coupled pumps at the plasma
membrane and by a biochemical pH stat (Davies, 1986;
Sakano, 1998). It is proposed that PEP carboxylase is
responsible for the synthesis of malate and NAD(P)-malic
enzyme for its decarboxylation (Davies, 1986). In addition,
the malate valve could contribute with additional malate
accumulation. Finally, it has been suggested that a pH shift
could serve as a signal to induce systems to counteract
imbalances (Felle, 2001).
Mitochondrial and peroxisomal contributions to
photosynthetic efficiency and redox
homeostasis
Apart from the mechanisms outlined above, there are also
strong contributions to redox homeostasis coming from
mitochondrial and peroxisomal reactions in the light.
Malate exported from the chloroplast can serve as a substrate
for light-enhanced dark respiration (LEDR) (Padmasree
et al., 2002), and AOX was shown to function as an
‘antioxidant enzyme’ by dissipating electrons without the
generation of ATP, thus preventing the formation of ROS
due to over-reduction of the mitochondrial electron transport chain (Vanlerberghe and McIntosh, 1992). Interestingly, AOX is a target of redox-modification by the
mitochondrial thioredoxin system (Gray et al., 2004), and
induction of AOX transcription and translation is caused by
multiple stress factors such as cold (Vanlerberghe and
McIntosh, 1992), or over-reduction (Zhang et al., 2003).
The role of leaf mitochondria for cellular redox homeostasis
was shown when the cytoplasmic male-sterile mutant
CMSII of tobacco was used (Dutilleul et al., 2003). These
plants do not possess a functional complex I, but due to
multiple adjustments of their redox-balancing systems no
oxidative stress becomes apparent, and an increased tolerance to ozone and pathogens was observed (Noctor et al.,
2004). In this context, it is of interest to mention the recent
findings showing that knock-out plants lacking the mitochondrial type II peroxiredoxin F possess a strong phenotype, especially during stress and when AOX was inhibited
(Finkemeier et al., 2005). Finally, disruption of the TCA
cycle by decreasing the amount of mitochondrial MDH had
dramatic effects on photosynthesis and growth (Nunes-Nesi
et al., 2005).
Most importantly, photoautotrophic plant cells are
unique in possessing various pathways of energy production that are flexibly linked and interact in different ways in
order to maintain redox homeostasis. The essential contribution of mitochondria for efficient photosynthesis has
been stressed repeatedly. The supporting role of photorespiration for redox balancing during photosynthesis, especially under stress conditions should be also mentioned
Redox homeostasis during changing conditions
here, as well as its contribution to acclimation (Gardeström
et al., 2002; Noctor et al., 2002; Padmasree et al., 2002;
Fernie et al., 2004; van Lis and Atteia, 2004).
Long-term acclimation to changed light
intensities
Considering the various changes imposed on the cells
performing photosynthesis under changing conditions, it is
obvious that sustained imbalances in the redox situation can
also cause long-term acclimation, i.e. restructuring of the
cellular systems at the level of gene expression. Therefore,
it has to be assumed that a small shift in the redox balance
should be sensed in the nucleus, after translocation of
a redox signal to the nucleus, possibly mediated by
phosphorylation cascades, has occurred. As a well studied
example, acclimation to increased light intensities is described. In contrast to ‘poising’ or short-term acclimation,
as described earlier, that is characterized by reaction times
of seconds and minutes, long-term acclimation includes
changed gene transcription and responds in the range of
hours or days.
Moderate, but longer lasting changes of light intensity
and temperature cause large alterations in leaf ultrastructure, and of protein and pigment composition (Anderson
and Osmond, 1987; Chow et al., 1990). Chloroplasts of
leaves acclimated to low light are characterized by a high
content of grana thylakoids, and a relatively high number
of photosystems and light-harvesting complexes (LHCII),
but they possess only low quantities of stromal proteins.
Such low-light plants respond to an increase in light
intensity with a very characteristic alteration of gene
expression. As a consequence of high-light acclimation,
the light saturation of photosynthesis is reached at higher
light intensities, the Chl a/b ratio decreases due to a loss of
LHCII, and the content of ATPase and of Calvin-cycle
enzymes increases. The relative amount of stroma thylakoids is also increased, while the portion of grana
thylakoids is lowered (Anderson and Osmond, 1987). In
the case of Arabidopsis, it was shown that the response
towards different light intensities is not linear, but follows
a complex pattern with separate low-light and high-light
responses (Bailey et al., 2001).
There is evidence that, in the case of fully developed
plants, the alterations in gene expression occurring during
light acclimation are strongly influenced by redox signals
that are released by the chloroplasts. Imbalances between the
input of light energy and the capacities to use this energy for
metabolism are immediately detected in the photosynthetic
apparatus (Fujita et al., 1987; Huner et al., 1998), although it
is not yet clear from where such redox signals originate.
Inside the thylakoid membranes, the cyt b6 f complex, the
plastoquinone pool, and the phosphorylation status of PSII
are possible sources and soluble, redox-active components,
1485
such as thioredoxins or glutathione, are also considered as
signals in the current discussions.
The influence of photoreceptors (phytochromes, cryptochromes, and phototropins), which control the expression
of many target genes of redox regulation in the early stages
of development or during greening (e.g. LHCII, RubisCO),
seems to disappear with increasing plant age. This is
experimentally difficult to confirm with wild-type plants,
since an increase in light intensity will not only increase the
redox state of the chloroplasts, but will also lead to an
increased input of photons into the photoreceptors. However, there is some evidence for this assumption: At first,
transgenic potato plants underexpressing Fd1, the major
ferredoxin isoform for photosynthetic electron distribution
inside the chloroplasts, when grown under high light
intensities, suffer permanently from elevated redox states
of plastoquinone and other intersystem carriers. Results
obtained with the mutant plants indicate that they display an
enhanced light-acclimation response (Holtgrefe et al.,
2003). Arabidopsis plants which lack functional photoreceptors are able to acclimate to a changed light intensity.
Only the det1-signal transduction mutant showed some
differences (Walters et al., 1999), and it may be possible
that the final steps during redox-mediated light acclimation
use the same mechanisms that are used by the photoreceptors in seedlings during greening, with only the signal
input having switched from phytochromes and cryptochromes towards the photosynthetic apparatus.
Finally, changes of gene expression as they occur during
light acclimation cannot be regarded as simple events, with
one signal leading directly to expression changes of only
one gene or of a group of genes. Firstly, the signal is delayed
by short-term acting mechanisms inside the chloroplasts
which buffer imbalances as much as possible. Secondly,
type and extent of the response are further influenced in
many ways by factors which link it to the actual status of the
cell (e.g. sugars, pH, redox-state, and phytohormones).
Acclimation of metabolism to photoperiod and
developmental needs
The interrelationship between the various metabolic needs
during distinct phases of development and the control of the
expression of components required for energy metabolism
will be demonstrated using another example. Not only light
quality and quantity, but also the duration of the photoperiod is decisive for many responses in plants. Unfortunately, few data concerning the influence of daylength on
metabolic processes are available, because the majority of
the published data deals with the morphogenic aspect of
flowering induction, i.e. the shift in the primordia from the
vegetative state to the formation of flower organs.
Arabidopsis is a facultative long-day plant, i.e. flowering
is promoted in long days (in the ecotype Columbia),
1486 Scheibe et al.
especially when the light period lasts 16 h or longer
(Koornneef et al., 1998). Arabidopsis will also form
flowers in shorter days, but then the vegetative phase lasts
longer, with the consequence that these plants accumulate
more biomass before flowering. Even in the extreme case of
complete darkness, when enough energy is supplied as
sucrose, Arabidopsis plants will finally form flowers. As far
as the metabolic strategies are concerned, there are big
differences between short-day and long-day-grown plants,
long before flower formation is started. During vegetative
growth, biomass production is of prime importance. Flowering redirects the assimilates from the leaves to inflorescene growth and seed formation. Although photoperiod
and temperature act as strong inducers of flowering, other
factors have been identified which are also involved in
flower induction. Light, temperature, sucrose, glutamine, as
well as gibberellins and cytokinins, act together to induce
floral morphogenesis (for a recent review see Corbesier and
Coupland, 2005).
The need for light acclimation and for optimization of the
assimilatory processes is apparent in the vegetative stage of
the plant and in young leaves. There are already various
examples of a changed level of NADP-MDH in situations
such as growth under cold stress or drought at high light,
when photosynthesis and carbon metabolism are affected to
a different extent (Huner et al., 1996; Savitch et al., 2000,
2001). Under elevated CO2, expression of NADP-MDH in
tobacco is decreased (Backhausen and Scheibe, 1999). In
short-day grown Arabidopsis plants, a treatment with low
temperature and high light induces expression of NADPMDH, while this is not the case in leaves of long-day-grown
plants which apparently start to become source organs due
to floral induction, and simply increase their antioxidant
systems (B Becker, S Holtgrefe, JE Backhausen, R Scheibe,
unpublished results).
In tobacco, the function of the individual leaves changes
with age and developmental stage. Young leaves are sink
organs initially, then they develop into source leaves. In
later stages, tobacco leaves are often used as storage tissues,
mostly for starch. The capacity of NADP-MDH is highest
in young leaves, and starts to decrease after sink leaves
have turned into source leaves (Faske et al., 1997). The
ability of the leaf cells to change the capacity of NADPMDH after acclimation to altered CO2 concentrations is
also much higher in young leaves, while in sink leaves, it
remains more or less uninfluenced by the CO2 level
(Backhausen and Scheibe, 1999). It has been suggested
by Walters (2005) that a difference in the carbohydrate
status of the leaves may influence light acclimation. The
overall pattern of gene expression changes dramatically in
senescent leaves, including the induction of senescencespecific transcription factors (Zentgraf et al., 2004). Again,
the natural ageing process appears to be correlated with
a shift of the cellular redox state to more oxidized
conditions, and is subject to a combination of redox and
hormone signalling as shown recently for legume nodules
(Puppo et al., 2004).
Networks of signal transduction pathways
When acclimation to long-term changes of the environment
is induced at the level of gene expression, a plethora of
different signal molecules has been implicated. Beyond the
harmful action of ROS, these molecules play a central role
in many signalling pathways (for reviews see Mullineaux
and Karpinski, 2002; Apel and Hirt, 2004). For redoxrelated effects, ROS (H2O2, O12 ), NO, ascorbate, glutathione, and many others were shown to induce gene expression
of many more-or-less specific enzyme systems (Wingate
et al., 1988; Arrigoni and De Tullio, 2002; Neill et al., 2002;
Mittler, 2002; Wendehenne et al., 2004). An interesting
question is the source of the signal, the transduction
pathway across cellular compartments and the integration
of various types of information. Finally, the translocation of
a messenger molecule to the nucleus has to be assumed. In
this respect, the large gene family of thioredoxins and
glutaredoxins could be important, since more functions and
targets of the large number of isoforms present in plants are
yet to be discovered (Meyer et al., 1999; Marchand et al.,
2004). Most metabolic pathways are closely linked with
primary energy production or consumption. Since enzymes
and cofactors involved in these processes can apparently
also function in modifying gene expression, an interesting
hypothesis has been put forward for their role as a direct link
between metabolism and transcription (Shi and Shi, 2004).
From the large number of studies presenting genomewide expression analyses and the availability of mutants for
each gene, it now becomes clear that upon one type of
imposed stress there is no single pathway or any isolated
response, but a sophisticated network of signal transduction
pathways. These are connected in multiple ways, as can be
assumed from the large overlap of genes expressed upon
different kinds of stress (Takahashi et al., 2004). The
expression profiles of transcription factors involved in
stress responses also suggest a complex network for signalling pathways and co-ordinated transcriptional regulation
(for review see Chen and Zhu, 2004). Again, the crosstalk
with phytohormone-induced processes becomes apparent,
as was shown recently for the det-2 mutants with a gene
defect of brassinosteroid biosynthesis that causes an early
acclimation to overcome this deficiency, resulting in an
increased resistance to oxidative stress (Cao et al., 2005).
However, even with the same stress applied to a plant,
there must still be differential sets of responses depending
on the superimposed developmental programme that is
active at a certain time or in a particular part of the plant.
Therefore, genes involved in energy metabolism and redox
homeostasis would be expected to respond in different
ways, depending on the developmental state of the plant
Redox homeostasis during changing conditions
(B Becker, S Holtgrefe, JE Backhausen, R Scheibe, unpublished results). In a dynamic light environment, the temporal pattern of the changes (in terms of length and direction
either up or down in intensity) will influence the type and
extent of the responses at the different levels of regulation.
This means that a specific response can be differently
modulated depending on the additional factors acting on the
system. This is also true when the individual history of each
plant is considered. Different types of responses can be
expected if, in the past, vernalization or pathogen attack, for
example, have occurred.
The role of the large number of small, thiol-containing
redox-active proteins in plants, such as thioredoxins,
glutaredoxins, peroxiredoxins, and cyclophilins as well as
protein-disulphide isomerases might well be within this
network, interconnecting the different parts of the hierarchical system, acting at a borderline between redox poise,
redox signalling, and repair. Due to the unique situation in
plant cells, oxidants are easily generated when reductants
accumulate as can happen during photosynthesis in the
absence of electron acceptors. When the impact on the
system is too strong and too rapid, the poising systems
might not be sufficient for short-term acclimation. If then
long-term acclimation cannot take place in time, oxidative
damage will occur, although the redox-balance is buffered
by various systems as described above. S-glutathionylation
could serve as a protective mechanism for protein thiols
(Ito et al., 2003), as shown earlier for vertebrates where it
had been suggested as a link between stress protection
and regulation of cell cycle (Cotgraeve and Gerdes, 1998).
A typical pattern of response for a living organism,
possessing all the mechanisms of control as discussed
previously, is a dampened oscillation using transiently the
full capacity of the respective system. This is true for the
photosynthetic electron transport system upon rapid
changes of light (Scheibe and Stitt, 1988). In the gene
expression pattern observed a short time after a stress
condition has been applied, such oscillation patterns in the
kinetics for different gene clusters might reflect the flexible
adjustment to a new homeostatic state.
Recent studies of protein–protein interactions have started
to draw a picture of microcompartmentation as an additional
level of control. Such transient associations of enzymes to
membranes and cytoskeleton might help to channel certain
pathways to subcellular sites. Such a concept has been put
forward for the glycolytic pathway of animals (Masters
et al., 1987) and the synthesis of several secondary products
in plants (Winkel, 2004), and might well turn out to be of
importance for complex signal transduction pathways.
Acknowledgement
The recent work from the author’s laboratory and the basic idea for
this paper originates from the collaborative work in FOR 387,
financially supported by the DFG.
1487
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