Novel regulators in photosynthetic redox control of plant metabolism and gene expression.

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ates compensatory responses either to readjust redox
homeostasis or to repair oxidative damage. Basically,
Update on Photosynthetic Redox Control
½AQ1?
Novel Regulators in Photosynthetic Redox Control of
Plant Metabolism and Gene Expression1
Karl-Josef Dietz2* and Thomas Pfannschmidt2
Biochemistry and Physiology of Plants, Bielefeld University, 33615 Bielefeld, Germany (K.-J.D.); and
Lehrstuhl fu ¨r Pflanzenphysiologie, Friedrich-Schiller-Universita ¨t Jena, 07743 Jena, Germany (T.P.)
Reduction-oxidation (redox) reactions are an essen-
tial part of cell metabolism and represent a major
fraction of all catabolic and anabolic reactions. Their
dominant characteristic is that they generate and con-
sume compounds with in part highly negative redox
potential. Redox reactions occur at many sites in the
cell, e.g. in membranes such as thylakoids, plastid
envelope, and plasma membrane and in aqueous cell
phases such as the stroma, thylakoid lumen, and
cytosol. Electron transport systems in cell membranes,
particularly in the photosynthetic and respiratory
electron transport chains, employ diverse redox cofac-
tors such as iron-sulfur (FeS) clusters and quinones
and also excitable systems in photosynthesis that all
can generate reactive oxygen species (ROS). The redox
state of the aqueous phase is dominated by soluble
redox metabolites, which include NAD(P)H, glutathi-
one, and e.g. metabolite pairs such as malate and
oxaloacetate, and in addition thiol/disulfide proteins
(Foyer and Noctor, 2009). The redox potential of a
compound is a relative attribute and defines its pro-
pensity to donate electrons to another compound
within a given redox couple. The process of electron
transfer is directed from the compound with more to
that with the less negative redox potential. By this
means redox reactions largely determine the thermo-
dynamics of the energetic fluxes in living cells. How-
ever, the electron transfer within a redox couple needs
to be strictly controlled to avoid the unintended elec-
tron transfer to other substrates with a relative positive
redox potential. Oxygen represents such a compound
and electron transfer to it can generate potentially
harmful ROS.
To balance redox metabolism and minimize ROS or
reactive nitrogen species (RNS) formation, cells oper-
ate a redox signaling network. The network senses
environmentally induced redox imbalances and initi-
the network consists of redox input elements, redox
transmitters, redox targets, and redox sensors (Dietz,
2008). The basic structure and many components of
the thiol-disulfide redox regulatory network are con-
served among all cells and most cell compartments.
The significance of this network is well established for
some pathways, but still emergent for additional
functions due to the ongoing identification of novel
redox targets. Lindahl and Kieselbach provided a
comprehensive inventory of the experimentally iden-
tified disulfide proteomes of the chloroplast (Lindahl
and Kieselbach, 2009). As part of the Plant Physiology
Focus Issue on Plastid Biology, this Update focuses on
plastid redox regulation as an example for the basic
principles of redox regulation in metabolism. In addi-
tion, the function of recently identified new players in
plastid redox regulation is described.
SUPPLY OF REDUCTION POWER BY
PHOTOSYNTHETIC LIGHT REACTIONS AND
ITS DISTRIBUTION
All reducing power in plant cells ultimately origi-
nates from the light-driven electron transfer from
water to NADP+, which is performed by the photo-
synthetic apparatus in the thylakoid membrane sys-
tem of chloroplasts (Fig. 1). In linear electron transport,
the reaction center of PSII is excited, creating a high
energetic potential form of it (P680 / P680*). This is
strong enough to initiate a charge separation that
allows an electron to move via pheophytin to the first
stable electron acceptor QA, a plastoquinone bound to
the PSII subunit D2. The resulting electron gap within
the reaction center is closed by electrons from water
delivered by the manganese cluster of the water-split-
ting complex. As the by-product of this reaction,
molecular oxygen (O2) is released. The electrons of
QAare then transferred to a second plastoquinone (QB)
bound to the reaction center protein D1. After receiv-
ing two electrons the reduced plastoquinone (PQH2) is
released from PSII and carries them to another mem-
brane complex, the cytochrome b6f complex (Cytb6f).
Here, PQH2becomes oxidized. This is the slowest step
within photosynthetic electron transport turning the
PQ pool into an ideal sensor for unbalanced excitation
of the two photosystems. The electrons are transferred
by the Cytb6f complex to a lumenal electron carrier,
plastocyanin, which transports the electrons to PSI. In
½AQ4?
1This work was supported by the German Science Foundation
(grant nos. DFG, FOR804, TP1, and TP3
2These authors contributed equally to the article.
* Corresponding author; e-mail karl-josef.dietz@uni-bielefeld.de.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Karl-Josef Dietz (karl-josef.dietz@uni-bielefeld.de
www.plantphysiol.org/cgi/doi/10.1104/pp.110.170043
½AQ2?
).
½AQ3?
).
Plant Physiology?, April 2011, Vol. 155, pp. 1–9, www.plantphysiol.org ? 2010 American Society of Plant Biologists1

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carbohydrates in the Benson-Calvin cycle or reduced
intermediates in sulfur or nitrogen metabolism. Re-
duced substrates can be used to generate reduction
equivalents in the dark or in nonphotosynthetic tis-
sues, thereby allowing the plant to uncouple redox-
dependent reactions from a direct connection to the
light reactions of photosynthesis (see below).
Reduction of soluble antioxidants such as ascorbate
or glutathione is used to defend cellular components
against oxidative damage by ROS. These are unavoid-
able by-products of oxygenic photosynthesis and are
PSI the electrons are excited to a reduction potential
sufficient to reduce (via a number of redox steps) ferre-
doxin (Fd) on the stromal side of PSI. From here most
electrons are transferred by the enzyme Fd-NADP-
oxido-reductase to NADP+generating NADPH + H+.
By this means the electrons cross a redox potential
difference of about 1.13 V in total that is strong enough
to fuel all subsequent redox-dependent reactions in the
cell.
The reducing power delivered from the electron
transport chain is distributed to mainly three cate-
gories of processes that are connected to each other.
These are (1) anabolic reactions of metabolism, (2) the
antioxidant systems, and (3) redox regulatory systems.
In metabolism the reduction equivalent NADPH is
often directly used as cofactor in enzymatic reactions,
mainly in anabolic reactions synthesizing molecules of
higher complexity or energetic content, for instance
generated by uncontrolled electron leakage mainly at
PSI. ROS can generate deleterious effects and must be
detoxified. Thereby, ROS function as important sinks
for reducing power. But ROS also perform important
signaling functions and trigger cellular processes in-
cluding stress responses, pathogen defense, and tar-
geted cell death (Apel and Hirt, 2004; Foyer and
Noctor, 2005; Mullineaux and Baker, 2010) that repre-
sent important research fields in plant science of its
own, however, due to space constraints are not cov-
ered in this Update here.
The redox regulatory system of a plant cell appears
to be the most complex one among the three given
categories of redox metabolism, antioxidant defense,
and redox regulation. The redox system consists of a
great number of various components that generate a
hierarchical and highly interconnected network. The
components and their relationships and functional
connections are described in more detail in the fol-
lowing section.
THE STRUCTURE OF THE REDOX REGULATORY
DITHIOL-DISULFIDE NETWORK
Four main routes of redox regulation exist in chlo-
roplasts, namely via (1) Fd directly, (2) NADPH, (3)
thioredoxin (Trx) system, and (4) glutathione/gluta-
redoxin (Fig. 2; Dietz, 2008). Trxs are reduced by a
specific enzyme, the Fd-Trx-oxido-reductase (FTR)
Figure 1. Redox chemistry of photosynthetic electron transport chain and associated redox regulators. The sketch displays
thylakoidmembrane compartments, intrinsicandextrinsicproteincomplexesofthe photosynthetic electrontransportchain, and
associated redox mediators (green) and various redox transmitters (yellow; red contours mark novel ones described in the text in
more detail) coupled to it. The yellow and red flashes indicate light-dependent charge separation in the reaction centers of PSII
and PSI as well as their different absorption maxima (680 and 700 nm, respectively). Thick black arrows represent the main
electron flow from water to NADPand the subsequent cellular processes (blue). Thin black arrows indicate the flow of a minor
proportion of electrons used for regulatory redox reactions. Blue arrows mark the influence of regulators on distinct cellular
processes as well as potential interactions. For detailed explanations, see the text.
Dietz and Pfannschmidt
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four members, respectively, namely Trxf1, f2, m1 to
m4, x, y1, y2, z, and CDSP32(Meyer et al., 2005), and
GrxC5, S12, S14, and S16 (Rouhier, 2010). The redox
network basically represents an electron flow system
where photosynthesis or nonphotosynthetic metabolic
reactions provide electrons to input elements such as
Fd, NADPH, and GSH. Transmitters mediate electron
transfer from input elements to the targets or fulfill
special functions. Electrons finally are drained by ROS,
RNS, peroxides, and O2. Generation of ROS is stimu-
lated upon metabolic imbalances that usually are
catalyzing the reduction of a conserved dithiol group
in the Trx that is then used by the Trx molecule to
transfer the reductive power to its specific target
protein(s). Thus, they function as redox transmitters.
The redox targets also possess intra- or intermolecular
dithiol groups and their reduction most commonly
leads to the activation of the enzyme. By this means
many Benson-Calvin cycle enzymes are activated in
the light when the photosynthetic light reaction is
running while they are inactivated in the dark. Gluta-
thione reductase links reduction of oxidized glutathi-
one to NADPH oxidation to yield reduced glutathione
(GSH). Each of the four pathways fuels, targets, or
regulates specific processes. Recently, novel players in
the plastid redox regulatory network were identified,
namely NADPH-dependent Trx reductase C (NTRC)
in metabolic control and antioxidant defense (Serrato
et al., 2004; Michalska et al., 2009), Trx-z in chloroplast
development and transcription (Arsova et al., 2010;
Schro ¨ter et al., 2010 ), and Grxs in disulfide reduction,
deglutathionylation, and FeS-cluster assembly (Rouh-
ier, 2010). The plastid Trx and Grx families in Arabi-
dopsis (Arabidopsis thaliana
½AQ5?
½AQ6?
½AQ7?
½AQ8?
) are comprised of 11 and
induced by sudden environmental changes. Un-
quenched excited states of chlorophyll and overreduc-
tion of specific energetic redox couples may cause ROS
production as described above. In chloroplasts high-
reduction states of the plastoquinone pool, Fd, and
NADP systems indicate excess excitation energy and
thus an imbalance between energy supply and de-
mand. Under such conditions singlet oxygen, O2
hydrogen peroxide are released. Within the network
O2and ROS maintain electron flow by either con-
trolled reaction with Prx or Gpx
uncontrolled reaction by oxidizing sensitive protein
thiols that in many cases display regulatory function.
Well-known examples are the thiol-disulfide activa-
tion of Benson-Calvin cycle enzymes such as Fru-1,6-
bisphosphatase (FBPase) and the reductive activation
of the malate valve (Scheibe et al., 2005). It is important
to note that in addition to photosynthetic electron
transport, other routes also provide reductive power
to plastids and the thiol/disulfide network, e.g. the
oxidative pentose phosphate pathway and the ma-
late/oxaloacetate shuttle (malate valve) across the
plastid envelope. These pathways are especially im-
portant at night and in nonphotosynthetic plastids.
The oxidative pentose phosphate pathway operates
within chloroplasts and, in principle, reverses the
reductive pentose phosphate pathway. Since both
pathways share essential metabolites and even a num-
ber of enzymes this would create a futile cycle. Reg-
ulation via reduced Trx prevents such a waste of
energy by activation of FBPase and seduheptulose-
bisphosphatase in the reductive cycle and parallel
inactivation of the Glc-6-P dehydrogenase in the oxi-
dative cycle. This directs FBP into the reductive cycle
2, and
½AQ9?
proteins or in an
Figure 2. Schematics of the dithiol-
disulfide network of the chloroplast.
The top layer depicts the supply path-
ways feeding electrons to the input
elements Fd, NADPH, and GSH. In
competition with other electron-drain-
ing reactions these input elements do-
nate electrons to a set of redox
transmitters. These adjust the redox
state of a large set of target proteins,
here represented by FBPase, malate
dehydrogenase (MDH), FLN, and AG-
Pase or donate electrons to thiol-de-
pendent peroxidases (Prx, Gpx). ROS,
RNS, peroxides, and O2serve as final
electron acceptors maintaining elec-
tron flux through the network by reox-
idizingprotein thiols.
function in deglutathionylation, FeS-
cluster assembly/delivery, or as redox
sensors and seem to lack disulfide re-
duction activity.
Some Grx
Redox Regulation in Plastids
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drogen peroxide, have been employed to identify
redox proteins. To assess the physiological signifi-
cance, fine-scaled redox buffers must be used to iden-
tify the redox environment that activates the redox
switch of the redox proteins.
in the light. In the dark Trx becomes oxidized and the
opposite situation becomes predominant. By this
means the reduction state of Trx creates a conditional
separation of metabolic fluxes within the same com-
partment. It should be noted that the dominant Trx
regulation is supported by fine-tuning mechanisms
including other parameters such as ATP/ADP ratio,
Mg2+ion, and pH gradients as well as the NADPH/
NADP ratio.
NOVEL TARGETS OF REDOX REGULATION IN
PLASTID METABOLISM
Proteomics-Based Identification of Novel Targets
Redox proteomics is an emerging technology aimed
at defining the redox protein inventory of the cells and
cell compartments and analyzing the redox state of
target proteins on a broad scale. Both gel- and chro-
matography-based redox protein screening systems
have been applied to plant and chloroplast protein
fractions and resulted in lists of thylakoid lumenal,
stromal, and chloroplast membrane-bound candidate
redox proteins that undergo thiol modifications, most
commonly dithiol-disulfide transitions (Meyer et al.,
2005; Rouhier et al., 2005; Stro ¨her and Dietz, 2008;
Lindahl and Kieselbach, 2009; Hall et al., 2010). With
increasing sensitivity these approaches allow for pro-
teome-wide identification of proteins potentially sub-
jected to thiol-disulfide or nitrosothiol transitions in
vivo but face some drawbacks: (1) Low abundance
proteins still are underrepresented in the target lists,
thus mainly dominant and metabolic proteins are
identified, while regulatory proteins escape from the
identification. (2) Despite specific methods such as Trx
and Grx trapping or Trx-dependent reduction of pre-
viously oxidized targets, the results from in vitro
methods lack specificity and cannot be translated
into mechanisms in vivo. (3) The functional signifi-
cance of intramolecular or intermolecular thiol-disul-
fide transitions or nitrosylation of proteins needs to be
explored in each case by time-intensive biochemical
and molecular biological studies. Only in few and
selected cases, the experimental verification has been
attempted. (4) Information is needed on the critical cell
redox conditions that activate the regulatory redox
switches. Often, rather extremely reducing, with di-
thiothreitol (DTT), or oxidizing conditions, with hy-
NTRC
FTR accepts electrons from photosynthetic electron
transport and donates them to the various Trxs as
redox transmitters. In addition to this conventional
pathway, NTRC was identified in a genome-wide
screen for Trx reductase (NTR)-like proteins that are
enzymes found in the cytosol and mitochondrion. The
chloroplast NTRC consists of an N-terminal NTR
domain with NADPH and FAD binding as well as
double Cys active sites and a C-terminal Trx domain.
When separately expressed in Escherichia coli, both
domains reveal the respective activity (Serrato et al.,
2004). Homologous genes are found in plants, algae,
and some cyanobacteria. The 2-CysPrx was identified
as the first substrate that is efficiently reduced by
NTRC. Recently ADP-Glc pyrophosphorylase (AG-
Pase) was recognized as another target of NTRC-
dependent redox regulation (Michalska et al., 2009).
Thus NTRC is a novel player in the thiol-dependent
plastid redox network and allows for reduction of
disulfides at the expense of NADPH as electron donor
and also functions in darkness and nonphotosynthetic
plastids. NTRC and Trx activate starch synthesis un-
der reducing conditions (see below).
Redox Regulation of Metabolism
Thiol regulation of Benson-Calvin cycle enzyme
activities links light-dependent electron pressure in
photosynthetic light reactions to ATP and NADPH
consumption in reductive carbohydrate metabolism.
The regulatory mechanism may be considered as a
prototypic feed-forward activation loop. In fact thiol
state-dependent regulation of carbon fluxes through
the Benson-Calvin cycle and their link to Trx (Trx-f)-
mediated activation of chloroplast FBPase marked the
starting point of more than 30 years of successful
research on redox regulation in metabolism (Buchanan
and Balmer, 2005). In addition to FBPase, sedoheptu-
lose-1,7-bisphosphatase, activities of ribulose-5-P ki-
nase, glyceraldehyde-3-P dehydrogenase, and Rubisco
activase are controlled by Trx. Trx-f donates electrons
to target proteins that have a broad range of redox
midpoint potentials Em(Hutchison et al., 2000). Dif-
ferential inactivation of target proteins, e.g. in the
Calvin cycle, is unrelated to the value of Embut highly
relevant for photoinhibition under nonoptimal envi-
ronmental conditions such as chilling temperatures
(Hutchison et al., 2000). This complexity is partly
explained by the fact that thiol modulation is tied
into additional metabolic control systems, e.g. the
presence of Fru-1,6-BP is needed for FBPase thiol
activation (Reichert et al., 2000). Two main carbon
pathways drain carbon from the Benson-Calvin cycle,
namely Suc synthesis following export of triose phos-
phate to the cytosol and starch synthesis in the plas-
tids. The committed step of starch synthesis is
catalyzed by AGPase (Fig. 3). AGPase is activated by
reduction of a disulfide bridge between the two
slightly smaller subunits of the tetrameric holenzyme
in vitro (Ballicora et al., 2000) and in vivo (Tiessen
et al., 2002). Reduction is achieved by Trx-f and Trx-m
in vitro and allows for a 4-fold stimulation of ADP-Glc
synthesis (Ballicora et al., 2000). A good correlation
exists between Suc concentration, reduction state of
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4 Plant Physiol. Vol. 155, 2011

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ylase, biotin carboxyl carrier protein, transcarboxylase
a-subunit, and transcarboxylase b-subunit with three,
one, two, and five Cys residues, respectively (Sasaki
and Hatano, 1997). One of the a- or b-subunits is
suggested to mediate the redox regulation (Kozaki
and Sasaki, 1999). Biotin carboxyl carrier subunit of
ACCase in Chlamydomonas reinhardtii is subjected to
S-thiolation with glutathione (Michelet et al., 2008).
Biotin carboxylase is target of glutathionylation in
Arabidopsis cell culture (Dixon et al., 2005). Thus, each
of the subunits of ACCase is potentially controlled by
the chloroplast, and starch synthesis (Tiessen et al.,
2002; Geigenbergeret al., 2005). NTRC also reductively
activates AGPase. NTRC-deficient Arabidopsis show
less redox-dependent stimulation of AGPase activity
and lower starch synthesis rates in high light and upon
external feeding of Suc. Inhibition in ntrc knockout
plants ranges between 40% and 60% in leaf chloro-
plasts and reaches 90% in nonphotosynthetic amylo-
plasts (Michalska et al., 2009).
In addition of redox regulation in carbohydrate
metabolism, proteomic and biochemical data indicate
that thiol modifications also control other major met-
abolic pathways such as nitrogen assimilation, tetra-
pyrrole synthesis, and lipid synthesis (see above;
Lindahl and Kieselbach, 2009). Here we only discuss
recent advance in understanding regulation of lipid
metabolism.
Lipid synthesis that occurs in the plastids is a strong
sink for electrons. Synthesis of palmitic acid (C16)
from acetyl-CoA requires 14 molecules of NADPH
and seven molecules of ATP. Plastid redox state affects
lipid metabolism (Fig. 3). Acetyl-CoA carboxylase
(ACCase) catalyzes the committed step of malonyl-
CoA production in plastid lipid synthesis. Isolated
ACCase in vitro is inactive without reductant and
activated after addition of DTT or reduced Trx-f1 or
Trx-m (Sasaki and Hatano, 1997). Reductive activation
is supported by pH shift to alkalinization and by
increasing Mg2+concentrations. The chloroplast AC-
Case consists of four polypeptides, the biotin carbox-
redox regulation using diverse mechanisms. This fact
underlines the link between redox state and lipid
metabolism. Envelope-bound monogalactosyldiacyl-
glycerol synthase (MGD) synthesizes monogalactosyl-
diacylglycerol from diacylglycerol and UDP-Gal.
Monogalactosyldiacylglycerol is a major lipid compo-
nent of chloroplasts. In vitro MGD activity depends on
the presence of reductants such as DTT, is inhibited by
thiol-alkylating agents, and is modulated by Trx acting
on intramolecular disulfide bonds (Yamaryo et al.,
2006). Plant MGD possesses nine conserved Cys res-
idues. Its regulation by thiol redox state is suggested to
enable galactolipid synthesis along with photosyn-
thetic activity and to foster replacement of eventually
oxidized lipids under conditions that cause oxidative
stress (Yamaryo et al., 2006).
Control of Lumenal Redox Environment
Redox information is intensively used in metabolic
regulation in the stroma. Recent evidence reveals that
also lumenal proteins undergo dithiol-disulfide tran-
sitions and that lumenal redoxprocesses are critical for
normal development. Thus, a set of 15 lumenal pro-
teins has been found in the compilation from pub-
lished data of the chloroplast disulfide proteome
(Lindahl and Kieselbach, 2009). The hcf164 mutant
was identified in a screen for high chlorophyll fluo-
rescence phenotype. HCF164 encodes a lumenal Trx-
like protein involved in functional assembly of Cytb6f
complex (Lennartz et al., 2001) and targets proteins
such as subunit N of PSI (PsaN). Thylakoid-bound
cytochrome c defective A (CcdA) is a homolog of
prokaryotic thiol-disulfide transporters and required
for efficient electron transfer from the stroma to the
lumen (Motohashi and Hisabori, 2010). The tentative
model suggests that thylakoid-associated CcdA re-
ceives electrons from stromal Trx-m and donates them
to HCF164 that in turn reduces target proteins. Elec-
tron transfer processes are not only involved in com-
plex assembly but also in lumenal antioxidant defense
since the Prx Q that is a thiol-dependent peroxidase
Figure 3. Redox regulation in plastid
starch and lipid synthesis. AGPase as
committed step of starch synthesis, and
ACCase and MGD in plastid lipid syn-
thesis are activated by reduction of
regulatory thiols, either by Trx or in
case of AGPase by NTRC as described
in the text in more detail.
Redox Regulation in Plastids
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pools was investigated after 1 h (Kolbe
et al., 2006). The diagrams compare
the distribution patterns under control
conditions (A) and after DTT treatment
(B). The numbers give the relative dis-
tribution in percent of total. Green,
Up-regulation; red, down-regulation.
was at least partly assigned to the lumenal proteome
(Petersson et al., 2006). Prxs detoxify a broad range of
peroxide substrates but must be regenerated by thiol
donors in each catalytic cycle. Other lumenal thiol
targets include the components of the water-splitting
complex PsbO1, O2, P1, and Q, peptidyl prolyl cis/
trans isomerases of the FKBP type (FK506 binding
protein) and the Deg1 protease (Stro ¨her and Dietz,
2008; Lindahl and Kieselbach, 2009).
Adjustment of Plastid and Leaf Cell Metabolism by
Redox Stimuli
Exogenous and endogenous factors such as light
quantity and quality, CO2availability in combination
with low or high temperature, and other stresses
modify the balance between energy supply and de-
mand, alter cellular and chloroplast redox milieu, and
affect the state of the dithiol/disulfide redox network.
The significance of redox-dependent readjustment of
global metabolism under nonstressed conditions has
been revealed by two types of experiments. Kolbe et al.
(2006) manipulated the redox state of Arabidopsis leaf
discs by applying the reductant DTT in the light. A
combination of transcript profiling, metabolome, and
14C-flux analyses revealed a profound redirection of
metabolism upon shifting from control conditions in
light to light plus DTT.14C-Glc uptake and flux into
Suc decreased by25% and 56%,respectively. Incontrast
synthesis of cell wall constituents increased to 860%
that of proteins to 430%, amino acids 280%, starch
260%, and organic acid 230% (Fig. 4). The changes in
flux were accompanied by congruent changes in me-
tabolite levels. Metabolites of central carbohydrate
metabolism decreased while end products such as
the amino acids Asn, Cys, Ile, Pro, Tyr, and Val
accumulated (Kolbe et al., 2006). Redox proteomics
has identified several enzymes of amino acid synthesis
as potential targets of Trx-dependent regulation, e.g.
Ala aminotransferase, Asp aminotransferase, argini-
nosuccinate synthase, dihydroxyacid dehydratase, ke-
tolactid reductoisomerase, branched chain ketoacid
decarboxylase, 3-isopropylmalate dehyrogenase, and
Cys synthase (Lindahl and Kieselbach, 2009).
In a physiological approach it could be demon-
strated that such readjustments of metabolism are of
direct relevance for plant responses to environmental
cues. Arabidopsis plants grown on soil were subjected
to defined light quality shifts known to generate
distinct redox signals within the PQ pool by uneven
excitation of the two photosystems. The acclimation
responses of the plants that counterbalance this un-
even excitation were observed by transcriptomics and
metabolomics as well as further physiological exper-
iments including the acclimation mutant stn7 (Fey
et al., 2005; Wagner et al., 2008; Bra ¨utigam et al., 2009).
Besides the expected structural and functional recon-
figuration of the photosynthetic apparatus a strong
impact on metabolism genes and metabolites was
observed. Bioinformatic analyses uncovered that as
part of the light acclimation response the plants redi-
rect their metabolism between two distinct states and
that these have great importance for plant growth
efficiency. One metabolic state is characterized by
decreases in primary photosynthetic metabolite levels
and increases in important intermediates of subse-
quent metabolic pathways, while the second metabolic
state is characterized by a down-regulation of many
subsequent metabolites, including amino acids and
organic acids. Thus, environmentally induced redox
signals within the photosynthetic electron transport
chain trigger two adjustment loops that coordinate
metabolic states and/or fluxes with the efficiency of
the photosynthetic light reaction. This is in line with
the above-mentioned study and underlines the impor-
tance of redox signaling networks for the global ad-
justment of plant metabolism.
REDOX REGULATION OF PLASTID
GENE TRANSCRIPTION
Plastids possess their own genome, the plastome,
and a complete machinery to express the genetic
Figure 4. Redirection of leaf metabo-
lism under highly reducing conditions.
Leaf discs were illuminated in the ab-
sence or presence of the reductant DTT
and supplied with
tion of14C label in different metabolite
14C-Glc. Distribu-
Dietz and Pfannschmidt
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information on it. Although this plastome encodes just
approximately 120 genes (in vascular plants) the ex-
pression mechanisms appear to be rather complex and
highly regulated. This includes a number of redox
control mechanisms that influence regulatory proteins
at all important levels of gene expression, i.e. tran-
scription, posttranscriptional mechanisms, and trans-
lation initiation (Pfannschmidt and Liere, 2005).
A major target of photosynthetic redox signals is the
plastid-encoded RNA polymerase (PEP; Fig. 5). Un-
balanced excitation of the two photosystems generates
either a reduced or oxidized PQ pool that act as signals
that control the phosphorylation of the light-harvest-
ing complexes of PSII via the thylakoid-associated
kinase STN7 (Pesaresi et al., 2009
chaix, 2010). The same signals also trigger a phospho-
rylation cascade towardthe PEP enzyme that results in
changes of photosynthesis gene expression (Allen and
Pfannschmidt, 2000). Both processes aim to counteract
the unbalanced excitation to maintain photosynthesis
efficiency as high as possible. The phosphorylation
cascade likely includes the action of a number of
further kinases (STN8, an ortholog of STN7; CSK, the
chloroplast sensor kinase; PTK, the plastid transcrip-
tion kinase), generating a phosphorylation network. In
a simplified view the reduction of the PQ pool acti-
vates STN7, which provides an input signal for the
subsequent kinase network. This controls the phos-
phorylation state of the sigma factor Sig1 that in turn
½AQ10?
; Lemeille and Ro-
regulates the relative transcription of the photosyn-
thesis reaction center genes psbA (encoding the D1
protein of PSII) and psaA/B (encoding the P700 apo-
protein of PSI; Shimizu et al., 2010). This view coincides
with the observation that CSK, PTK, and Sig1 were
able to interact with each other in the yeast (Saccharo-
myces cerevisiae) two-hybrid system (Puthiyaveetil
et al., 2010). In organello transcription experiments in
presence of kinase inhibitors and/or the reductant
DTT, however, indicated that this phosphorylation-
dependent signal interacts with a second, thiol-depen-
dent signal (Steiner et al., 2009). PTK, a casein-kinase 2
type enzyme has been reported to be under control of
the redox state of glutathione (Ogrzewalla et al., 2002),
but its activity could not be modulated with DTT. This
suggested the involvement of a further regulator.
Recently, two independent studies identified a novel
Trx-like protein that likely represents this additional
player (Arsova et al., 2010; Schro ¨ter et al., 2010). The
novel Trx was named Trx-z because of its distinct
evolutionary position in relation to Trx-x and Trx-y. In
a yeast two-hybrid screen it was identified as interact-
ing protein of two chloroplast-located phosphofructo-
kinase-like proteins called
Arabidopsis knockout mutant line of Trx-z exhibited
pale-white leaves and was viable only on Suc-supple-
mented medium, a unique phenotype since the Trx
system is highly redundant and can easily compensate
for the loss of single components. Gene expression
FLN1and 2.The
Figure 5. Protein factors and signaling pathways involved in photosynthetic redox control of plastid transcription. The
photosynthetic electrontransport chainand its proteincomplexesaredepicted asin Figure 1. The sketch refers to lightconditions
which preferentially excite PSII. Under these conditions the PQ pool becomes reduced, hence, activating the thylakoid-bound
kinase STN7. This kinase is oppositely regulated at its lumenal side by a reduction signal from the Trx system that is not active
under these conditions (Bra ¨utigam et al., 2009). STN7 provides an input signal that presumably is integrated within a subsequent
phosphorylation network targeted to the control of the PEP. PEP-associated proteins (PAPs) are given in yellow and described in
text. One group of PAPs is mediating the phosphorylation signal, another one appears to integrate a second signal originating
from the Trx system. Both signals together contribute to the transcriptional activation of the psaA/B operon (a mixed operon
encoding a ribosomal subunit at its end [rps14, given in light pink]). The pathway by which Trx-z is reduced is unknown to date
(question mark).
Redox Regulation in Plastids
Plant Physiol. Vol. 155, 20117

Page 8

Uncorrected Proof
future research: The predictions on redox targets from
proteomics approaches need to be addressed by bio-
chemical studies (Stro ¨her and Dietz, 2008; Lindahl and
Kieselbach, 2009). Most indicated linkages in the net-
work are still hypothetical, e.g. it is not clear whether
FTR is able to reduce all Trxs or how Trx-z as integral
component of the transcriptional complex is reduced.
The interactions between the network components
need to be qualitatively and quantitatively assessed
by in vitro and in vivo methods such as isothermal
titration calorimetry, kinetic assays, and fluorescence
resonance energy transfer similar as done for the
analyses indicated the same plastid gene expression
profiles as in PEP-deficient mutants, pointing to an
important role of Trx-z in plastid development and
gene expression (Arsova et al., 2010). These observa-
tions were complemented by mass spectrometry re-
sults that demonstrated that both the Trx-like protein
and the FLN2 kinase are intrinsic subunits of the PEP
enzyme of chloroplasts (Schro ¨ter et al., 2010). This
provides an immediate explanation for the phenotype
and the expression profiles in the knockout mutant. A
lack in Trx-z prevents a proper assembly of the PEP
enzyme and, consequently, the developmental transi-
tion from the nuclear-encoded RNA polymerase-
driven transcription to the PEP-dependent transcrip-
tion does not take place. The precise functional role of
Trx-z within the PEP complex, its relation to FLN1 and
2, as well as its regulatory impact remains to be
elucidated. Furthermore, it is completely open how it
relates to the other known redox regulators mentioned
above. In summary, our understanding of photosyn-
thetic redox signal transduction toward the level of
gene expression is still at the beginning. The increasing
number of identified regulatory components unravels
step by step a complex mechanistic redox tool box
enabling chloroplasts to respond to a wide range of
environmental conditions in a dynamic and flexible
manner.
CONCLUSION AND OUTLOOK
The cellular redox environment has global signifi-
cance in regulating most plastid processes, namely
carbohydrate, lipid, amino acid, and tetrapyrrole me-
tabolism as well as gene transcription, protein synthe-
sis, and also e.g. protein import via modulating the
activity of the translocons of the inner and outer
chloroplast membranes (TIC, TOC
2010). Tentative experimental evidence and theoretical
considerations (Fig. 2) suggest that redox regulation in
plastids is a tightly interlinked phenomenon. How-
ever, it is still characterized by dispersed knowledge
on (1) redox effects on only single processes that have
been characterized in detail, (2) unsurpassed complex-
ity of involved players particularly redox transmitters,
(3) limited knowledge on linkages among the compo-
nents, and (4) poor quantitative understanding of
network function. These shortcomings direct us to
½AQ11?
; Balsera et al.,
chloroplast 2-Cys peroxiredoxin (Barranco-Medina et al.,
2008; Muthuramalingam et al., 2009). Models of partial
networks and simulations of electron fluxes and redox
states will help to test our knowledge and predict
regulatory states that can be validated in further
experiments (Kemp et al., 2008). In the end this will
help to understandhow the fluctuating environment is
reflected by distinct changes in the cellular redox
signaling networks and paves the avenue for a sys-
tematic research of plant acclimation in response to
environmental challenges.
Sequence data from this article can be found in the GenBank/EMBL data
libraries under accession numbers nnn
½AQ12?
.
ACKNOWLEDGMENT
We regret that we had to omit much relevant literature due to the strict
space constraints.
Received November 26, 2010; accepted December 23, 2010; published nnn.
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QUERIES -pp170043
[AQ1] Please confirm/correct this subtitle: "Update on Photosynthetic Redox Control." If amended, the subtitle must
start with "Update on..."
[AQ2] Please confirm or amend editing of funding footnote. Also, please note that per journal style, only one
corresponding author is permitted. Thank you.
[AQ3] Inserted corresponding author’s name and e-mail address in the distribution of materials statement. Please
confirm or revise. Thank you.
[AQ4] If appropriate, please define PQ here at first use. Thank you.
[AQ5] Changed to Schro ¨ ter here and twice later in the text to match PubMed and the reference list.
[AQ6] If appropriate, please define Grx here at first use. Thank you.
[AQ7] Journal style is to cite the full Latin name at first use in the text. Please confirm or amend insertions throughout
article. Thank you.
[AQ8] When appropriate, please define all gene, protein, and mutant names at first use in the text. Thank you.
[AQ9] Please define Prx and Gpx here at first use. Thank you.
[AQ10] The in-text citation Pesaresi et al. (2009) is not in the reference list. Please correct the citation, add the
reference to the list, or delete the citation.
[AQ11] If appropriate, please define TIC and TOC here at first and only uses. Thank you.
[AQ12] Journal style requires that accession numbers be listed in this manner. Please provide appropriate number or
delete if necessary. Thank you.
[AQ13] The reference Pesaresi et al. (2010) is not cited in the text. Please add an in-text citation or delete the reference.
[AQ14] Please insert volume number and page range in Puthiyaveetil et al. (2010) reference. Thank you.
[AQ15] Please check Sasaki and Hatano (1997) reference and verify its accuracy (including the page range). Thank
you.