Thioredoxin m4 Controls Photosynthetic Alternative
Electron Pathways in Arabidopsis1[C][W]
Agathe Courteille 2, Simona Vesa 2, Ruth Sanz-Barrio, Anne-Claire Cazalé, Noëlle Becuwe-Linka,
Immaculada Farran, Michel Havaux, Pascal Rey, and Dominique Rumeau*
Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7265, Biologie Végétale et
Microbiologie Environnementales, 13108 Saint-Paul-lez-Durance, France (A.C., S.V., N.B.-L., M.H., P.R., D.R.);
Commissariat à l’Energie Atomique, Institut de Biologie Environnementale et Biotechnologie, Service de
Biologie Végétale et Microbiologie Environnementales, Laboratoire d’Ecophysiologie Moléculaire des Plantes,
13108 Saint-Paul-lez-Durance, France (S.V., N.B.-L., M.H., P.R., D.R.); Université Aix-Marseille, 13108 SaintPaul-lez-Durance, France (A.C., S.V., N.B.-L., M.H., P.R., D.R.); Instituto de Agrobiotecnologia, Universidad
Pública de Navarra-Consejo Superior de Investigaciones Científicas-Gobierno de Navarra, 31006 Pamplona,
Spain (R.S.-B., I.F.); Institut National de la Recherche Agronomique, Laboratoire des Interactions PlantesMicroorganismes, Unité Mixte de Recherche 441, F-31326 Castanet-Tolosan, France (A.-C.C.); and Centre
National de la Recherche Scientifique, Laboratoire des Interactions Plantes-Microorganismes, Unité Mixte de
Recherche 2594, F-31326 Castanet-Tolosan, France (A.-C.C.)
In addition to the linear electron flow, a cyclic electron flow (CEF) around photosystem I occurs in chloroplasts. In CEF, electrons
flow back from the donor site of photosystem I to the plastoquinone pool via two main routes: one that involves the Proton
Gradient Regulation5 (PGR5)/PGRL1 complex (PGR) and one that is dependent of the NADH dehydrogenase-like complex.
While the importance of CEF in photosynthesis and photoprotection has been clearly established, little is known about its
regulation. We worked on the assumption of a redox regulation and surveyed the putative role of chloroplastic thioredoxins
(TRX). Using Arabidopsis (Arabidopsis thaliana) mutants lacking different TRX isoforms, we demonstrated in vivo that TRXm4
specifically plays a role in the down-regulation of the NADH dehydrogenase-like complex-dependent plastoquinone reduction
pathway. This result was confirmed in tobacco (Nicotiana tabacum) plants overexpressing the TRXm4 orthologous gene. In vitro
assays performed with isolated chloroplasts and purified TRXm4 indicated that TRXm4 negatively controls the PGR pathway as
well. The physiological significance of this regulation was investigated under steady-state photosynthesis and in the pgr5 mutant
background. Lack of TRXm4 reversed the growth phenotype of the pgr5 mutant, but it did not compensate for the impaired
photosynthesis and photoinhibition sensitivity. This suggests that the physiological role of TRXm4 occurs in vivo via a
mechanism distinct from direct up-regulation of CEF.
In plant thylakoids, photosynthesis involves a linear
electron flow (LEF) from water to NADP+ via PSII,
cytochrome b6/f, PSI, and soluble carriers. LEF produces NADPH and generates a transthylakoidal electrochemical proton gradient that drives the synthesis
of ATP. Besides LEF, cyclic electron flow (CEF) can
also occur, involving only PSI (for review, see Johnson,
2011; Kramer and Evans, 2011). These additional reactions include two main distinct pathways involving
1
This work was supported by the Centre National de la Recherche
Scientifique (grants to A.C., S.V., and A.-C.C.).
2
These authors contributed equally to the article.
* Corresponding author; e-mail [email protected].
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:
Dominique Rumeau ([email protected]).
[C]
Some figures in this article are displayed in color online but in
black and white in the print edition.
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.112.207019
508
either the Proton Gradient Regulation5 (PGR5)/PGRL1
complex (Munekage et al., 2002; DalCorso et al., 2008) or
the NADH dehydrogenase-like complex (NDH; for
review, see Battchikova et al., 2011; Ifuku et al., 2011).
The functioning of either CEF pathway, which generates a pH gradient DpH without any accumulation of
NADPH, is thought to achieve the appropriate ATP/
NADPH balance required for the biochemical needs of
the plant, especially under certain environmental conditions such as low CO2 (Golding and Johnson, 2003),
heat (Clarke and Johnson, 2001), cold (Clarke and
Johnson, 2001), drought (Golding and Johnson, 2003;
Kohzuma et al., 2009), high light (Munekage et al.,
2004), or dark-to-light transitions (Joliot and Joliot, 2005;
Fan et al., 2007). CEF-generated DpH is also involved in
photoprotection owing to the down-regulation of PSII
via nonphotochemical quenching (Munekage et al., 2004;
Takahashi et al., 2009). Very recently, the role of the PGR5
protein as a regulator of LEF has been established. It has
proved to be essential in the protection of PSI from
photodamage (Suorsa et al., 2012).
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Redox Regulation of the NDH and PGR Pathways
The two cyclic pathways are redundant (Munekage
et al., 2004), sharing ferredoxin (Fd) as a common
stromal electron donor (Yamamoto et al., 2011) and
electron carriers from plastoquinone (PQ) to PSI with
LEF. Thus, LEF and either of the CEF pathways may be
in competition. The molecular events that allow CEF to
challenge LEF remain enigmatic, particularly when
considering that the conditions that require CEF are
also those under which LEF is in excess. Efforts to
understand the appropriate functioning of CEF have
led to the proposition of several models segregating
cyclic and linear pathways at a structural level (for
review, see Eberhard et al., 2008; Cardol et al., 2011;
Johnson, 2011; Rochaix, 2011). According to the restricted diffusion model, founded on the uneven distribution of the photosynthetic protein complexes in
the thylakoids, there is little competition between CEF
and LEF, as CEF occurs in stroma lamellae where PSI is
concentrated while LEF takes place in the grana stacks.
In line with the supercomplex model, whose relevance
was demonstrated in the microalga Chlamydomonas
reinhardtii, CEF happens within tightly bound supercomplexes containing PSI, with its own light-harvesting
complex (LHCI), the PSII light-harvesting complex
(LHCII), cytochrome b 6/f, Fd, Fd NADP reductase
(FNR), and the integral membrane protein PGRL1
(Iwai et al., 2010). In higher plants, an association between NDH and PSI subunits suggests the formation
of such supercomplexes (Peng et al., 2009). The availability of FNR, found either free in the stroma or
bound to the thylakoids (Zhang et al., 2001), has also
been proposed to modulate partitioning between LEF
and CEF (Joliot and Joliot, 2006; Joliot and Johnson,
2011). In addition, more dynamic models that illustrate
competitive processes involved in the distribution of
electrons between the cyclic and linear flows have been
proposed. The competition between cytochrome b6/f
and FNR for electrons from Fd could regulate the
segregation between LEF and CEF (Breyton et al., 2006;
Yamamoto et al., 2006; Hald et al., 2008). A few years
ago, Joliot and Joliot (2006) suggested that the ATP/
ADP ratio was one of the parameters that triggered
on the transition between LEF and CEF. It was also
established that the redox poise of chloroplast stroma
contributed to the regulation of the photosynthetic
pathway and played an important role in defining the
extent of CEF. Breyton et al. (2006) scrutinized this
redox regulation and established that the fraction of
PSI complexes engaged in CEF could be modulated
by changes in the stromal redox state. Overreduction
of the NADPH pool was involved in the repartition
between LEF and CEF (Joliot and Joliot, 2006). The
NADPH/NADP+ ratio was proposed as a regulator of
PGR-dependent CEF in vivo (Okegawa et al., 2008).
All the published data supporting a role for the redox status in the regulation of CEF urged us to investigate a putative role of thioredoxins (TRX) in the
regulation of CEF. TRX are ubiquitous disulfide reductases regulating the redox status of target proteins
(for review, see Lemaire et al., 2007; Meyer et al., 2009).
In chloroplast, TRX mediate the light regulation of numerous enzymes, among which some belong to the
Calvin cycle (for review, see Schürmann and Buchanan,
2008; Montrichard et al., 2009; Lindahl et al., 2011).
Global proteomic approaches have revealed that wellknown photosynthetic complex subunits may be partners of TRX, such as PsbO in PSII, plastocyanin, Rieske
Fe-S protein in cytochrome b6/f, and PsaK and PsaN in
PSI (for review, see Montrichard et al., 2009; Lindahl
et al., 2011). Furthermore, regarding the regulation of
photosynthesis, TRX have also been involved in state
transitions (Rintamäki et al., 2000; Buchanan and
Balmer, 2005), and their participation in the control of
the redox poise of the electron transport chain has also
been suggested (Johnson, 2003).
In this work, we have investigated the possible role of
TRX in the regulation of CEF. Using Arabidopsis (Arabidopsis thaliana) mutants with altered expression of genes
encoding different plastid TRX, we have established
in vivo the inhibitor activity of TRXm4 on the NDHdependent pathway for plastoquinone reduction. This
result was confirmed in transplastomic tobacco (Nicotiana
tabacum) plants overexpressing the TRXm4 orthologous
gene. Moreover, in vitro assays performed with isolated chloroplasts indicated that TRXm4 negatively controls the PGR-dependent electron flow as well.
RESULTS
NDH-Dependent Electron Flow Is Enhanced in the
Presence of Thiol Reagents
As a preliminary approach to investigate the putative role of TRX in the regulation of CEF, we examined
the effect of N-ethylmaleimide (NEM), a thiol-alkylating
agent, on the NDH-dependent alternative pathway.
Arabidopsis leaves were infiltrated with NEM, and the
NDH-dependent electron flow was probed in vivo
using chlorophyll fluorescence (Fig. 1A) and chlorophyll thermoluminescence (Fig. 1B). Control leaves
infiltrated with water displayed the transient increase
in the basal level of chlorophyll fluorescence after a
light-to-dark transition attributable to PQ and the
primary electron-accepting plastoquinone of PSII reduction specifically mediated by the NDH complex
(Shikanai, 2007). Accordingly, the postillumination
fluorescence transient was not observed in the ndho1 mutant lacking NDH (Fig. 1A). Infiltration of control
leaves with NEM (0.25 or 0.5 mM; Fig. 1A) induced a
strong and sustained increase in the chlorophyll fluorescence transient signal. Far-red (FR) light irradiation,
which preferentially energizes PSI, rapidly quenched
the fluorescence signal to the F0 level, and this effect
was reversible, as fluorescence rapidly returned to the
previous level when FR was switched off (Fig. 1A).
This indicates that the NEM-induced increase in the
fluorescence transient is due to a reversible reduction
of the PSII acceptors. A similar increase in the postillumination chlorophyll fluorescence transient was
obtained using leaves infiltrated with iodoacetamide,
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Courteille et al.
In Arabidopsis, NDH-mediated electron transport in
the dark can also be probed by measuring the afterglow (AG) luminescence signal (Havaux et al., 2005;
Lintala et al., 2009; Peeva et al., 2012). Following FR
illumination at 0°C, an AG thermoluminescence signal
peaking at approximately 40°C was detected in control
leaves in addition to the B band appearing at approximately 18°C to 20°C (Fig. 1B, WT). This signal
reflects a heat-induced electron flow from the stroma
to the PQ pool and the PSII centers (Miranda and
Ducruet, 1995). In Arabidopsis, and in contrast to other
plant species, the AG band is associated mainly with
the NDH activity (Havaux et al., 2005; Lintala et al.,
2009). This is confirmed by the strong reduction of the
AG band in the ndho-1 mutant and its low sensitivity
to antimycin A (AA), a specific inhibitor of the PGRdependent pathway (Fig. 1B, bottom panel). In leaves
infiltrated with NEM, the AG thermoluminescence
band shifted to lower temperature (from approximately 40°C to approximately 27°C; Fig. 1B, top
panel). The shifted AG emission was weakly inhibited
in leaves treated with AA (Fig. 1B, top panel, WT+
NEM+AA) and could not be detected in ndho1 leaves, indicating that NDH mediates this phenomenon (Fig. 1B, bottom panel, ndho-1+NEM). This band
shift indicates a stimulated electron transfer rate from
stroma to the PQ pool: electrons are rapidly transferred to the secondary quinone acceptor at room
temperature, leading to charge recombinations within
PSII and to an AG thermoluminescence band merged
with the B band (Ducruet et al., 2005; Apostol et al.,
2006; Peeva et al., 2012). In previous works (Ducruet
et al., 2005; Peeva et al., 2012), the downward shift of
the peak temperature of the AG band was correlated
with an acceleration of CEF, as measured by the rate of
rereduction of the oxidized primary electron donor in
PSI (P700+) in the dark following a FR light illumination. Leaf absorbance measurements at 820 nm, which
Figure 1. In vivo detection of the NDH-dependent electron flow by
chlorophyll fluorescence and chlorophyll thermoluminescence measurements in Arabidopsis leaves infiltrated with NEM. A, Chlorophyll
fluorescence of Arabidopsis leaves either infiltrated with water or with
NEM at 0.5 and 0.25 mM was monitored with a pulse amplitude-modulated fluorometer. After a 5-min illumination with white light (250
mmol photons m22 s21), light was switched off and the chlorophyll
fluorescence transient increase was recorded under low nonactinic light.
FR indicates the pulse of FR light (1 s). Measurements were performed
1 h following infiltration. ndho-1, Arabidopsis mutant lacking the NDH
complex. B, Chlorophyll thermoluminescence was measured in Arabidopsis leaf discs by increasing leaf temperature from 0˚C to 60˚C after a
20-s illumination with FR light at 0˚C. Heat-induced electron flux from
the stroma to PSII acceptors appears as a band (AG) peaking at approximately 40˚C. The shoulder appearing at approximately 18˚C to 20˚
C is the B band corresponding to charge recombinations between preexisting plastoquinone and S2/S3 states. AA indicates 5 mM AA. A.U.,
Absorbance units; T, temperature; WT, wild type.
another alkylating reagent (data not shown). In contrast, no transient signal could be detected in leaves of
the ndho-1 and pgr5 mutants infiltrated with NEM
(Supplemental Fig. S1).
Figure 2. Half-times (t1/2) of dark rereduction of P700+ in leaves of Arabidopsis plants after a FR light period. WT+H2O or WT+NEM, Leaves of
wild-type plants were infiltrated with water or NEM (0.25 mM), respectively. Leaves were irradiated with FR light (more than 715 nm, 120 W
m22) and half-time was determined subsequently, after turning off the FR
light. Data expressing half-times are means 6 SD of four experiments.
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Redox Regulation of the NDH and PGR Pathways
reflects changes in the redox state of P700, were performed on Arabidopsis leaves (Fig. 2). Leaves were
first illuminated with FR light, leading to the oxidation
of P700, then rereduction by stromal reductants was
recorded in the dark after switching off the FR light.
The half-time of the dark rereduction measured in
leaves of control (wild type) and NDH mutant (ndho-1)
plants was not significantly different (Fig. 2). In contrast, following NEM (0.25 mM) infiltration, the halftime decreased substantially as compared with leaves
infiltrated with water, indicating an acceleration of the
electron donation from the stroma to PSI via PQ.
Taken together, the chlorophyll fluorescence, P700+
reduction kinetics, and AG data suggested that NDHmediated electron flow is enhanced in the presence of
thiol-alkylating reagents.
NDH Activity Is Increased in an Arabidopsis Mutant
Lacking TRXm4
Because TRX are key actors in thiol-mediated redox
regulation of chloroplastic enzymes, we examined NDH
activity in Arabidopsis mutants lacking chloroplastic
TRX. When available, candidate T-DNA insertion lines
were obtained from the Nottingham Arabidopsis Stock
Center. Genomic PCR and reverse transcription (RT)
analyses were performed to select the knockout mutants
(Laugier et al., 2012; Supplemental Figs. S2 and S3).
trxm1, trxm3, trxf1, trxx, and trxy2 mutant lines displayed
the typical NDH-dependent transient increase in chlorophyll fluorescence reported in wild-type plants (Fig. 3A).
Interestingly, the two trxm4 lines (trxm4-1 and trxm4-2)
exhibited a highly stimulated transient (Fig. 3B). PSI
excitation using a FR flash rapidly switched off the signal
in a reversible way. The stimulated fluorescence transients in trxm4-1 and trxm4-2, which appeared similar to
the signal recorded following NEM infiltration (Fig. 1A),
indicated that the NDH-dependent electron pathway
was up-regulated in plants specifically lacking TRXm4.
AG luminescence analysis confirmed the chlorophyll
fluorescence transient measurements (Fig. 3C). The
downshifted band peaking at approximately 27°C was
detected in trxm4-1 (Fig. 3C) and trxm4-2 (data not
shown), as observed previously in leaves infiltrated with
NEM. Neither the postillumination chlorophyll fluorescence transient nor the shifted AG band was detected in
the double mutant ndho-1 3 trxm4-1 (Fig. 3, B and C).
The NDH-dependent increase in PQ reduction recorded in the trxm4 mutants could be interpreted as
the result of an inhibition of the PQ pool reoxidation
by the plastid terminal oxidase (PTOX) rather than a
stimulation of the NDH activity. However, infiltration
of wild-type leaves with n-propyl gallate, a powerful
inhibitor of PTOX (Josse et al., 2003), does not induce
any change in the chlorophyll fluorescence transient or
in the AG signal (data not shown) ascribed to NDHmediated PQ reduction, suggesting that an inhibitory
effect of TRXm4 on the PTOX-dependent oxidation
pathway is very unlikely. Thus, we concluded that
TRXm4 negatively regulates NDH activity.
Figure 3. Characterization of the NDH-dependent electron flow in
Arabidopsis mutants lacking TRXm1, TRXm3, TRXm4, TRXf1, TRXy2,
and TRXx. A, Transient chlorophyll fluorescence rise measured under
nonactinic light following a 5-min illumination with white light (250
mmol photons m22 s21) in the wild type (WT), trxm1, trxm3, trxf1, trxy2,
and trxx. B, Transient chlorophyll fluorescence rise measured as above in
the wild type, trxm4-1, trxm4-2, and the double mutant ndho-1 3 trxm41. FR indicates the pulse of FR light (1 s). C, AG chlorophyll thermoluminescence measurements from leaf discs of wild-type, trxm4-1, and
ndho-1 3 trxm4-1 plants after a 20-s FR excitation. A.U., absorbance
units; T, temperature. [See online article for color version of this figure.]
The NDH-Dependent Electron Pathway Is Inhibited in
Tobacco Plants Overexpressing the TRXm4
Orthologous Gene
The NDH pathway was also measured in transplastomic tobacco plants overexpressing endogenous
TRXf and TRXm genes (NtTRXf and NtTRXm, respectively; Sanz-Barrio et al., 2012) from the plastid
genome under the control of the strong psbA promoter.
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Courteille et al.
Interestingly, alignment of the NtTRXm complementary DNA sequence with the four Arabidopsis TRXm
gene sequences indicated that NtTRXm displayed the
highest level of similarity to TRXm4 (data not shown),
suggesting that NtTRXm and Arabidopsis TRXm4 are
orthologs. While in the tobacco plants overexpressing
NtTRXf (o/exTRXf) the postillumination chlorophyll
fluorescence increase indicative of NDH activity was
similar to the wild type, in the tobacco plants overexpressing NtTRXm (o/exTRXm) it was not detected,
indicating that the NDH pathway was inhibited in
these plants (Fig. 4). Following infiltration with NEM
(0.25 mM; Fig. 4), control tobacco plants (wild type)
displayed a highly stimulated chlorophyll fluorescence
rise as observed in Arabidopsis (Fig. 1A). In o/exTRXm
plants, NEM treatment contributed to the recovery of
the NDH-specific chlorophyll fluorescence signal. These
results were in agreement with the phenotype of the
Arabidopsis mutant trxm4-1, reinforcing our conclusion that, in vivo, the NDH-dependent electron flow is
specifically regulated by TRXm4.
Electron Transport within PSI Is Increased in the
Arabidopsis Mutant Lacking TRXm4
The flow of electron donation from stromal reductants to PSI in the absence of PSII activity was studied
in leaves of trxm4 mutants using the kinetics of P700
oxidation in FR light and P700+ rereduction in the dark.
In trxm4-1, the rate of P700+ rereduction after FR light
was noticeably accelerated compared with that of
wild-type and ndho-1 plants (Fig. 5A). The kinetics of
P700 photooxidation induced by FR illumination was
also monitored in dark-adapted leaves of the wild type
and mutants lacking NDH activity or TRXm4 (Fig. 5B,
top and bottom panels, respectively). In wild-type
leaves, FR light induced a rapid oxidation of P700,
reaching the maximum level after about 2 s There was
almost no difference between the kinetic curves of
the wild type and ndho-1, as observed previously by
Shikanai et al. (1998) and Nandha et al. (2007). Conversely, in trxm4-1, P700 oxidation was much slower,
indicating a more important flow of electrons coming
from the stroma to the intersystem chain able to
maintain PSI in a reduced state. To characterize further
the effect of TRXm4 on electron transport, we examined the P700+ level during steady-state photosynthesis
in white light (Fig. 6). No difference in the P700 oxidation ratio could be recorded between the wild type,
ndho-1, and trxm4 during steady-state photosynthesis
at two photon flux densities, 100 and 1,000 mmol
photons m22 s21.
Taken together, the P700 redox state measurements suggest that TRXm4 inhibits electron transport when PSI is probed with FR light, but this effect
appears to have no significant repercussion on PSI
photochemistry during steady-state photosynthesis
in white light.
TRXm4 Exerts a Negative Control on Both the PGR- and
NDH-Dependent Electron Flows
Figure 4. In vivo detection of NDH activity by chlorophyll fluorescence measurements in tobacco leaves. Transient chlorophyll fluorescence rise was measured under nonactinic light after a 5-min
illumination in tobacco leaves from wild-type (WT) plants and plants
overexpressing the gene encoding TRXm4. NEM (0.25 mM) was infiltrated 1 h before measurements. FR indicates the pulse of FR light (1 s).
Nonphotochemical PQ reduction can be determined
in vitro using lysed chloroplasts (Munekage et al.,
2002, 2004). PQ reduction activity is monitored as an
increase in chlorophyll fluorescence under a weak
measuring light (1.0 mmol photons m22 s21) upon Fd
and NADPH addition (Fig. 7A). In this assay, reduced
Fd, the mediator of CEF, is generated by NADPH via
the reverse activity of FNR.
In the presence of Arabidopsis TRXm4, which was
produced in Escherichia coli and purified (Supplemental
Fig. S4), PQ reduction activity was partly impaired (Fig.
7B). The inhibition was specific of TRXm4, since the
addition of a commercial preparation of reduced TRX
from E. coli to wild-type chloroplasts induced no change
in the PQ reduction activity (Fig. 7B; WT+TRX E. coli).
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Redox Regulation of the NDH and PGR Pathways
both complexes in CEF around PSI (Fig. 7B). AA,
which is a specific inhibitor of the PGR-dependent
pathway, mimicked the phenotype of pgr5 (Fig. 7B,
WT+AA). When added to an extract of chloroplasts
from ndho-1, AA almost fully inhibited PQ reduction
(Fig. 7B, ndho-1+AA), indicating that, under our experimental conditions, the full activity recorded in the
wild type is the sum of the NDH and PGR activities.
PQ reduction activity was measured in isolated chloroplasts following the addition of reduced TRXm4. PQ
reduction activity was impaired when TRXm4 was
added to chloroplasts of ndho-1 plants lacking the
NDH-dependent pathway. Conversely, the addition of
TRXm4 had no effect on the reaction with ruptured
chloroplasts from the pgr5 mutant. Similarly, incubation of TRXm4 with chloroplasts from wild-type plants
premixed with AA did not change the PQ reduction
activity. These results show that, contrary to its effect
in vivo, TRXm4 lost its capacity to modulate the NDH
pathway in vitro, suggesting that the target of TRXm4
may be either lost during the preparation of ruptured
chloroplasts or not accessible in the assay.
Interestingly, the in vitro experiments revealed the
inhibitory effect of TRXm4 on the PGR-dependent
pathway. Whether this effect also occurred in vivo was
investigated in tobacco plants overexpressing TRXm4
using the AG thermoluminescence technique (Fig. 8,
o/exTRXm). Contrary to Arabidopsis, the AG luminescence signal in tobacco leaves has been shown to
rely mainly on the AA-sensitive pathway (Havaux
et al., 2005). Figure 8 showed that the amplitude of the
thermoluminescence band was drastically reduced in
o/exTRXm, indicating that the PGR-dependent pathway was inhibited in these plants. Fd-dependent PQ
reduction activity was also probed in vitro using
ruptured tobacco chloroplasts (Fig. 9). As reported
Figure 5. Characterization of electron transport within PSI in the
trxm4-1 mutant. A, Half-times (t1/2) of the dark rereduction of P700+ in
leaves of plants after a FR light period. Data expressing half-times are
means 6 SD of four experiments. B, Kinetics of P700 oxidation by FR
light in dark-adapted leaves of Arabidopsis wild-type (WT) and mutant
plants. Leaves were maintained in the dark for 10 min before FR illumination (more than 715 nm, 120 W m22). a.u., Absorbance units.
When extensively oxidized by dithiothreitol (DTT)
treatment (see “Materials and Methods”), TRXm4 was
no longer an inhibitor of PQ reduction (Fig. 7B, ox
TRXm4).
In this assay, the contributions of the PGR- and
NDH-dependent pathways to the total PQ reduction
activity can be discriminated using chloroplasts from
mutants deficient in either pathway or in the presence
of AA, a specific inhibitor of the PGR-dependent pathway. Thus, the effect of TRXm4 on either pathway was
investigated. As described previously by Munekage
et al. (2002, 2004), in chloroplasts isolated from
Arabidopsis mutants lacking either the NDH complex
(ndho-1) or PGR5 (pgr5), the PQ reduction activity
was decreased, demonstrating the participation of
Figure 6. P700 oxidation ratio in leaves of Arabidopsis mutants lacking
NDH or TRXm4 irradiated at two different photon flux densities. Oxidized P700 was recorded during illumination with actinic white light at
100 or 1,000 mmol photons m22 s21. The maximum P700 oxidation
level was recorded with a pulse of intense white light in the presence
of FR light. WT, Wild type.
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Enhancement of NDH Activity in Plants Lacking
the PGR-Dependent Pathway Compensates the
Light-Dependent Growth Phenotype But Not
Photosynthesis Inhibition or Photoinhibition
NDH appeared essential only in the pgr5 mutant
background (Munekage et al., 2004). While single
mutants lacking either PGR5 or NDH did not display
visible phenotypes when grown under limited light
conditions (50 mmol photons m22 s21), the double
mutant exhibited severe growth inhibition. Correlated
with this phenotype, a drastic inhibition of photosynthesis was recorded in double mutant plants lacking
both pathways at low light, which also occurred in the
pgr5 single mutant under normal- or high-light conditions. As an attempt to reveal TRXm4 function,
trxm4-2 and pgr5 mutants were crossed. Double mutant trxm4-2 3 pgr5 plants were grown under normallight conditions (200 mmol photons m22 s21). Under
these conditions, pgr5 plants displayed delayed growth
as compared with the wild type (Fig. 10A) but trxm4-2 3
pgr5 exhibited an improved growth as compared with
pgr5 plants, similar to that of wild-type plants (Fig.
10A). The photosynthetic activity and thermal dissipation, both impaired in pgr5 (Munekage et al., 2004;
Figure 10, C and D), appeared similarly impaired in
the double mutant trxm4-2 3 pgr5 (Fig. 10, B and C),
demonstrating that although TRXm4-mediated NDH
enhancement (Fig. 10B) did improve the growth of
plants lacking the main CEF pathway, the effect was
not mediated by improving photosynthesis and/or
thermal energy dissipation.
Figure 7. Electron donation to PQ in ruptured chloroplasts isolated
from Arabidopsis leaves. Increase in chlorophyll fluorescence by the
addition of NADPH (250 mM) and Fd (5 mM) under weak illumination
(1.0 mmol photons m22 s21) was recorded in ruptured chloroplasts (1.5
mg chlorophyll mL21) from wild-type (WT) and mutant (pgr5 and
ndho-1) leaves. The maximum PSII fluorescence in the dark-adapted
state (Fm) level was recorded following the application of a saturating
flash (SF). The maximum photochemical efficiency of PSII in the darkadapted state values from the different samples were from 0.7 to 0.8.
A, Typical trace of a chlorophyll fluorescence change in chloroplasts
from wild-type leaves. B, Magnified traces from the boxed area in A
obtained using chloroplasts from different mutants incubated with AA
(5 mM), TRX E. coli (200 mM), or TRXm4 (200 mM). [See online article
for color version of this figure.]
previously (Endo et al., 1998; Munekage et al., 2004)
and supporting thermoluminescence measurements,
we confirmed that the chlorophyll fluorescence increase upon the addition of Fd and NADPH to tobacco
chloroplasts was mostly due to PGR activity and, accordingly, was fully impaired by AA. This activity was
also fully inhibited in o/exTRXm4 and partly inhibited
in the wild type following the addition of TRXm4.
Taken together, the experiments performed on Arabidopsis and tobacco chloroplasts show that the NDH
activity and also the PGR-dependent electron flow are
regulated by TRXm4.
DISCUSSION
TRXm4 Controls the NDH- and PGR-Dependent PQ
Reduction Pathways
Supposedly essential for balancing the ATP/NADPH
production ratio (Allen, 2003) and photoprotection
Figure 8. AG chlorophyll thermoluminescence measurements in tobacco leaves overexpressing the gene encoding TRXm4. Chlorophyll
thermoluminescence was measured in tobacco leaf discs by increasing
leaf temperature from 0˚C to 60˚C after a 20-s illumination with FR
light at 0˚C. Heat-induced electron flux from the stroma to PSII acceptors appears as an AG band peaking at approximately 40˚C. A.U.,
Absorbance units; WT, wild type.
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Redox Regulation of the NDH and PGR Pathways
Figure 9. Electron donation to PQ in ruptured chloroplasts isolated from
tobacco leaves. Increase in chlorophyll fluorescence by the addition of
NADPH (250 mM) and Fd (5 mM) under weak illumination (1.0 mmol
photons m22 s21) was recorded in ruptured chloroplasts (1.5 mg chlorophyll mL21) from wild-type (WT) plants and plants overexpressing the gene
encoding TRXm4. Chloroplast extracts were incubated with AA (5 mM) or
TRXm4 (200 mM). [See online article for color version of this figure.]
(Munekage et al., 2004; Takahashi et al., 2009), the
electron flow through the NDH- and PGR-dependent
pathways must be accurately regulated (Kramer et al.,
2004). To date, little is known about factors regulating
the electron flow through these pathways, mostly because investigation of the CEF rate has remained a
difficult task, since no reliable method able to discriminate the electron flux through the LEF and CEF is
available (Johnson, 2005; Baker et al., 2007). In this
work, using a combination of in vivo and in vitro
assays, we explored the role of plastid TRX in the redox
control of the photosynthetic alternative electron pathways and established the inhibitory role of TRXm4 on
the NDH- and PGR-dependent electron flows.
The regulatory action of TRXm4 on the NDH-dependent pathway was first detected using chlorophyll
fluorescence and AG measurements, since these
methods enable specific investigation of NDH activity
in vivo in Arabidopsis leaves (Havaux et al., 2005;
Shikanai, 2007). These experimental approaches revealed that NDH activity was increased when TRXm4
was lacking in Arabidopsis mutants and fully extinguished in tobacco plants overexpressing TRXm4.
Thus, TRXm4 negatively regulates the NDH-mediated
electron pathway in vivo. Further information on the
role of TRXm4 on CEF required recording P700 oxidation under FR illumination and subsequent P700+ dark
reduction kinetics. Indeed, these parameters have been
widely used to gain information on the activation of
cyclic pathways (Joët et al., 2002; Johnson, 2005;
Nandha et al., 2007). In trxm4 leaves, P700 oxidation
and reduction kinetics indicated that TRXm4 mutants
were able to perform CEF at a higher rate than the wild
type (Fig. 5), which could indicate a highly reduced
pool of chloroplast stroma donors in trxm4.
All the above-mentioned methods enable detecting a
dark electron flow from the stroma and do not allow
exploring the contribution of TRXm4 to CEF regulation
in vivo under steady-state photosynthesis. Because it
has been established that the operation of CEF under
limiting light conditions is unlikely (Heber et al., 1978;
Cornic et al., 2000) and PGR-dependent PSI CEF requires adequate light to be fully activated (Shikanai,
2007), a pertinent assessment of the regulating role of
TRXm4 needed to be performed under light conditions.
Thus, the P700+ level indicative of the electron acceptance
capacity from PSI was monitored during steady-state
photosynthesis. No difference between the wild type,
ndho-1, and trxm4 was recorded under these conditions,
suggesting that NDH-dependent flow up-regulation
has no effect on photosynthesis. These results are in
agreement with the features of trxm4 plants, which displayed no visible phenotype when grown under standard conditions (Supplemental Fig. S3) and no alteration
in the light intensity dependence of electron transfer
reactions and nonphotochemical quenching (NPQ;
Supplemental Fig. S5, A and B). No difference in the
NPQ transiently generated in dark-adapted leaves from
the wild type, ndho-1, and trxm4-1 exposed to low light
could be recorded (Supplemental Fig. S5C), indicating
that transient generation of the transthylakoidal pH
gradient (Kalituho et al., 2007) was similar in wild-type
and mutant plants. Knocking out genes encoding NDH
subunits either in tobacco (Burrows et al., 1998; Kofer
et al., 1998; Shikanai et al., 1998) or in Arabidopsis
(Rumeau et al., 2005; Shikanai, 2007) did not affect
overall photosynthetic electron transport, and plants did
not display any visible phenotype. Thus, it appears
logical that up-modulation of NDH activity did not
impact noticeably on photosynthetic activity. Only a
subtle NPQ change was recorded in trxm4, in agreement
with the discrete NPQ increase detected in NDH mutants (Hashimoto et al., 2003; Kamruzzaman Munshi
et al., 2005; Rumeau et al., 2005). This is in agreement
with previous results that demonstrated that the NDH
contribution to photosynthetic electron transport is minor in Arabidopsis, at least when the plants are grown
under standard conditions (Shikanai, 2007).
Considering the inhibitory effect of TRXm4 on the
PGR-dependent pathway monitored using lysed chloroplasts, it appeared limited, since 75% of chlorophyll
fluorescence was recorded when TRXm4 was added
(Fig. 7). We ruled out a nonspecific effect of TRXm4,
since the inhibition was not detected when E. coli TRX
was added to the ruptured chloroplast preparations.
However, recorded under very low light (i.e. when the
PGR pathway is not fully activated), its physiological
significance must be questioned. In planta, the TRXm4mediated partial inhibition of the PGR-dependent flow
probably plays a minor role, since mutants lacking PGR
display significant changes in photosynthesis and the
role of NDH appeared essential for photosynthesis only
in the pgr5 mutant (Munekage et al., 2004). Indeed,
mutants lacking both the NDH- and PGR-dependent
pathways exhibited severe growth phenotypes, and
photosynthesis was impaired more severely than in the
single mutant pgr5. Therefore, it appeared important to
investigate the effect of enhancing NDH activity in the
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Courteille et al.
Figure 10. Characterization of the
photosynthetic activity of the trxm4-2
3 pgr5 double mutant. A, Phenotype
of double mutant trxm4-2 3 pgr5 as
compared with the wild type (WT) and
the pgr5 mutant. B, Transient chlorophyll fluorescence rise measured under nonactinic light following a 5-min
illumination with white light (250
mmol photons m22 s21) in the wild
type and in trxm4-2 3 pgr5. C, Light
intensity dependence of the quantum
yield of PSII photochemistry (FPSII) in
trxm4-2 3 pgr5, trxm4-2, pgr5, and
wild-type leaves. Values shown are
means 6 SD (n = 3). D, Light intensity
dependence of NPQ of chlorophyll
fluorescence in trxm4-2 3 pgr5,
trxm4-2, pgr5, and wild-type leaves.
Values shown are means 6 SD (n = 3).
[See online article for color version of
this figure.]
pgr5 background. Double mutants lacking PGR5 and
TRXm4 were created. Under standard growth conditions, pgr5 3 trxm4-2 plants display a favorable phenotype similar to the wild type (Fig. 10), indicating that
NDH activity enhancement represents an advantage for
plant development when the main alternative electron
pathway is inhibited. However, the positive effect was
not mediated via an improvement of photosynthetic
electron flow (Fig. 10B) or NPQ (Fig. 10C), which
remained similarly impaired in pgr5 3 trxm4-2 and pgr5
(Munekage et al., 2004). Thus, although TRXm4 plays
an important role in regulating nonphotochemical PQ
reduction in chloroplasts, the physiological significance
of this regulation still remains to be determined. Possibly, redox regulation of alternative electron flows could
be important for some metabolic reactions, as shown
previously for carotenoid biosynthesis and the alternative electron transport mediated by the PTOX (Kuntz,
2004).
A Specific Function for TRXm4
Chloroplast TRX are currently subdivided into four
types, TRXf, TRXm, TRXy, and TRXx, which are
encoded in Arabidopsis by two, four, two, and one
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Redox Regulation of the NDH and PGR Pathways
genes, respectively. The biochemical characterization
of the four TRXm family members has been extensively performed in vitro (Duek and Wolosiuk, 2001;
Issakidis-Bourguet et al., 2001); however, the roles of
the different isoforms have not been clearly established
yet. Here, we report, to our knowledge for the first
time, a specific function for the TRXm4 isoform in vivo.
In the past, the characterization of Arabidopsis plants
lacking the different TRXm isoforms was attempted.
No visible phenotype could be attributed to the
TRXm1-, TRXm2-, and TRXm4-deficient plants, at least
under standard growth conditions. Only the mutant
lacking TRXm3 (named gat1) displayed growth impairment and turned out to be affected in symplastic
trafficking (Benitez-Alfonso et al., 2009). It was suggested that the lack of phenotype of the trxm1, trxm2,
and trxm4 mutants could reflect compensation by other
TRXm family members and/or functional redundancy.
Our results, on the contrary, suggest that TRXm4 could
perform a nonredundant and specific function (Fig. 3).
An intriguing question concerns the target(s) of
TRXm4 by which NDH- and PGR-mediated electron
pathway regulation occurs. In vitro experiments using
isolated and ruptured chloroplasts suggest that there
are two different targets at least, distinctively mediating NDH and PGR regulation, respectively (Fig. 7).
Many studies have been performed in order to identify
TRX targets. Most of them were performed in vitro,
leading to a list of about 500 candidates in oxygenic
photosynthetic organisms, including soluble and
membrane proteins (for review, see Montrichard et al.,
2009). It must be highlighted that among the numerous
targets, neither proteins belonging to the NDH complex nor PGR5 or PGRL1 have been found. Specifically, considering TRXm, it was demonstrated that
NADP malate dehydrogenase (Collin et al., 2003), Glc6-P dehydrogenase (Wenderoth et al., 1997), and
HCF164 (Motohashi and Hisabori, 2006), which is a
TRX-like protein located in the thylakoid membrane
required for the biogenesis of b6/f, were reduced by
TRXm. Recent data demonstrated that in Synechocystis
sp. PCC6803, PedR, a LuxR-type regulator, could be
inactivated by TRXm upon a shift to high light
(Horiuchi et al., 2010). Interestingly, ndhD2, one of the
genes encoding the NDHD subunit of the NDH complex in cyanobacteria, is negatively regulated by PedR.
In Arabidopsis trxm4 mutants, we could not detect any
change in the NDH amount (data not shown), excluding an effect of TRXm4 at the gene expression
level. Among the above-proposed targets for TRXm,
none appeared as an obvious mediator for NDH or
PGR complex regulation.
Recently, a few mutant or transgenic plants exhibiting a stimulated CEF have been described. All of them
lack Calvin cycle enzyme activity. Thus, in the Arabidopsis mutant lacking Fru-1,6-bisP aldolase, it was
suggested that CEF shifted from the NDH-dependent
pathway to the PGR-dependent pathway in response
to sink limitation of LEF (Gotoh et al., 2010). Jin et al.
(2008) obtained an antisense rice (Oryza sativa) line with
reduced Rubisco activase activity. In this line, the
enhancement of CEF combined with LEF depression
was involved in energy dissipation when CO2 availability became restricted. However, a mutant named
hcef1 affected in the Fru-1,6-bisphosphatase-encoding
gene (Livingston et al., 2010a) and a plant lacking
glyceraldehyde-3-phosphate dehydrogenase (Livingston
et al., 2010b) were demonstrated to display an enhanced NDH-dependent CEF. Both types of mutants
showed substantial increases in CEF linked to an increase in steady-state transthylakoid proton motive
force and subsequent activation of the energy-dependent
quenching of excess absorbed light energy response. In
hcef1, it was proposed that this activation aimed at
compensating a need for extra ATP that cannot be
fulfilled by alternative mechanisms. Interestingly, it
has been demonstrated that the four above-mentioned
enzymes of the Calvin cycle are regulated by TRX
(Montrichard et al., 2009). Thus, we could suggest that
TRXm4 specifically redox regulated any of them. If so,
however, an intriguing point remains concerning the
lack of a visible phenotype of trxm4. Indeed, plants
lacking the Calvin cycle enzyme activities displayed
striking phenotypes with inhibited growth (Livingston
et al., 2010a). It must be highlighted, however, that
in trxm4 leaves, Calvin enzymes are still present and
still functioning, and only their redox status may be
affected. Consequently, in order to clarify the mechanism underlying the redox regulation of the nonphotochemical PQ reduction pathways, the protein
target(s) of TRXm4 will have to be identified using, for
instance, strategies based on the overexpression of a
TRX active-site mutant (Rey et al., 2005).
MATERIALS AND METHODS
Plant Material and Growth Conditions
Arabidopsis (Arabidopsis thaliana) plants were grown in soil in a phytotron
with an 8-h photoperiod and a photon flux density of 150 mmol m22 s21. The
temperature regime was 22°C/16°C (day/night), and the relative humidity
was 55%. Arabidopsis T-DNA insertion mutant lines of plastidic TRX were
provided by the Salk Institute Genomic Analysis Laboratory (insertion lines
trxm3, N526367; trxm4-1, N532538; and trxm4-2, N523810). trxm1 (N653340),
trxf1 (N563799), trxx (N628906), and trxy2 (N528065) have been described
elsewhere (Laugier et al., 2012).
Tobacco (Nicotiana tabacum) plants were grown in soil under controlled
conditions in a growth chamber (14-h photoperiod at 25°C followed by a 10-h
night at 20°C) and watered with a complete nutrient solution as described
previously by Rumeau et al. (2005). Tobacco TRXf and TRXm coding sequences (GenBank accession nos. HQ338526 and HQ338525, respectively)
were amplified by PCR (Sanz-Barrio et al., 2011) and cloned into the pL3
chloroplast transformation vector under the plastid psbA gene promoter
(Farran et al., 2008). The resulting expression vectors were bombarded into in
vitro-grown tobacco (cv Petit Havana) leaves as described previously (Daniell,
1997). Homoplasmic T1 plants from the o/exTRXf and o/exTRXm transplastomic lines were used for further analysis.
Characterization of the Insertion Mutant Lines
Homozygous T-DNA insertion lines were verified by PCR analysis
with the Phire Plant Direct PCR Kit (Finnzymes) using gene-specific
primers: for N526367, O 3 (59-ATGCGTTCGTGATTCTCGAG-39) and O 4
(59-TTGTGTAACTTCAGCTGCTG-39); for N532538 and N523810, O13 (59-
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Courteille et al.
ACCAAATCTGTCTGATTCAGA-39) and O14 (59-TACTCGACCAAGAATCTTTCT-39). ACTIN2 was used as a positive control with the following
gene-specific primers: 59-AAAATGGCCGATGGTGAGGATAT-39 and 59CAATACCGGTTGTACGACCACT-39.
For RT-PCR analysis, total RNA was extracted from leaves using the
RNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s instructions.
RT was performed using total RNA (500 ng), SuperScript III Reverse Transcriptase (Invitrogen), and an oligo(dT)20 primer. RT-PCR was performed
using the same gene-specific primers as described above except for N526367,
which used O5 (59-CAAATCACTCTTCAATGGCG-39) and O6 (59-GTACCCATGATTGACTCTCG-39). ACTIN2 was used as a positive control for each
RT-PCR with the same primers as above.
In Vivo Chlorophyll Fluorescence Measurements
Chlorophyll florescence was measured at room temperature on attached
leaves using a PAM-2000 modulated fluorometer (Walz) as described previously (Rumeau et al., 2005). Briefly, postillumination chlorophyll fluorescence
rises were recorded under low nonactinic light following a 5-min actinic illumination (250 mmol photons m22 s21).
Chlorophyll fluorescence parameters were measured using a PAM-2000
modulated fluorometer (Walz). A saturating pulse of white light was applied on the leaf, and measurements were recorded during an actinic light
illumination (25–2,500 mmol photons m22 s21). The PSII photochemical efficiency during actinic illumination was calculated as DF/Fm9, where DF is
the steady-state chlorophyll fluorescence level and Fm9 is the maximal level.
NPQ reflects the dissipation of absorbed light energy from PSII as heat. NPQ
was calculated as (Fm/Fm9) 2 1, where Fm is the maximal fluorescence level
in the dark.
Thermoluminescence Measurements
The luminescence emitted by leaf discs (diameter of 12 mm) was measured
with a custom-made apparatus as described previously (Ducruet, 2003). The
sample was maintained at 0°C for 30 s in the dark, then it was illuminated for
20 s with FR light (more than 715 nm) at this temperature. FR light, produced
by a Schott KL1500LCD light source and a RG-715 Schott filter, was passed
onto the sample with one arm of a four-arm light guide (Walz). The fluence
rate of the FR light, measured with a Li-Cor Li185B/Li200SB radiometer, was
about 50 W m22. Immediately after switching off the FR light, the temperature
was increased from 0°C to 70°C at a rate of 0.5°C s21. The luminescence
emission by the sample was measured during heating by a photomultiplier
tube, protected by a red filter, via one arm of the light guide. The FR lightinduced thermoluminescence band peaking at around 40°C is the AG band,
which reflects a heat-induced electron flow from the stroma to the PQ pool
(Ducruet, 2003; Ducruet et al., 2005; Havaux et al., 2005).
centrifugation (5 min, 20,000g) and resuspended in a minimum volume of icecold extraction buffer (20 mM HEPES, pH 7.4, 0.5 M NaCl, 30 mM imidazole,
1 mM phenylmethylsulfonyl fluoride, and 5 mM DTT) supplemented with
Protease Inhibitor Cocktail (1 mL per 20 g of bacteria) and DNase (5 mg mL21).
E. coli cells were lysed by three freezing/thawing cycles. The lysate was
centrifuged at 20,000g for 30 min. The recombinant protein was purified by
FPLC using a nickel-affinity column and desalted by gel filtration (Zeba Spin
Column; Thermo Scientific). To observe the purity of the recombinant protein,
a sample of each fraction obtained after FPLC was loaded in a SDS-PAGE
apparatus and Coomassie Brilliant Blue stained. The activity of reduced
TRXm4 (200 mM) was tested by spectrometry assay (optical density at 650 nm)
in the presence of 150 mM HEPES (pH 7.0), 2 mM EDTA, 10 mg mL21 insulin,
and 6 mM DTT. Recombinant TRXm4 was reduced and oxidized with 10 mM
reduced DTT (Sigma) and 10 mM oxidized DTT (Sigma), respectively, for 30
min at room temperature. Then, the protein solutions were desalted using
Zeba Desalt Spin Columns (Thermo Scientific) before addition to the chloroplast preparations. Elution buffer contained 50 mM HEPES (NaOH), pH 7.6,
and 20% glycerol.
In Vitro Assay of PQ Reduction by the Cyclic
Electron Pathways
PQ reduction activity was measured in ruptured chloroplasts prepared as
described by Munekage et al. (2002, 2004). Briefly, leaves of Arabidopsis and
tobacco were homogenized in a medium containing 0.4 M sorbitol, 20 mM
Tricine/KOH (pH 8.4), 10 mM EDTA, 10 mM NaHCO3, and 0.15% (w/v) bovine
serum albumin. After centrifugation for 90 s at 1,500g, the pellet was gently
resuspended in 0.4 M sorbitol, 20 mM HEPES/KOH (pH 7.6), 2.5 mM EDTA (pH
8), 5 mM MgCl2, 10 mM NaHCO3, and 0.15% (w/v) bovine serum albumin. Intact
chloroplasts were purified by centrifugation on a preformed Percoll gradient
(mix of 100% Percoll and 23 the suspension medium described above centrifuged at 38,700g for 55 min). After centrifugation for 10 min at 13,300g, chloroplasts were osmotically lysed in 50 mM HEPES/NaOH (pH 8.0), 7 mM MgCl2,
1 mM MnCl2, 2 mM EDTA, 30 mM KCl, and 0.25 mM KH2PO4 according to
Munekage et al. (2002). The reduction of the primary electron-accepting plastoquinone of PSII and PQ was measured with ruptured chloroplasts diluted in
the lysis medium (pH 8.0) at 1.5 mg chlorophyll mL21 under weak illumination (1.0 mmol photons m22 s21) with electron donors 5 mM ferredoxin and
250 mM NADPH. Chlorophyll fluorescence was measured with a PAM-2000
fluorometer (Walz). AA (2 mM), recombinant TRXm4 (200 mM), and commercial TRX E. coli (200 mM; Sigma) were added before the measurement.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. In vivo detection of the NDH-dependent electron
flow by chlorophyll fluorescence measurements in Arabidopsis leaves
infiltrated with NEM.
Redox State of P700
Changes in the redox state of the reaction center P700 of PSI were monitored
via leaf absorbance changes at around 820 nm. A Walz PAM-101 system
connected to a dual-wavelength P700 unit (ED-P700DW; Walz) was used in the
reflection mode. P700 oxidation or rereduction was measured with FR light
(more than 715 nm) or after switching off the FR light, respectively.
The P700 oxidation ratio was measured as described by Okegawa et al.
(2010) and was calculated as DA/DAmax, where DAmax is the maximal P700+
level induced by a 50-ms pulse of intense white light under FR light illumination and DA is the steady-state P700+ level during illumination with white
light (photon flux density of 100 or 1,000 mmol m22 s21).
Production of Recombinant TRXm4
The coding region of mature TRXm4 from Arabidopsis was amplified
by Phusion High-Fidelity PCR (Finnzymes). For that, the oligonucleotides
used were 59-GAGAAATTAACCATGGAGGCTCAGGACACCACT-39 and 59ATGGTGATGAGATCCCTCGACCAAGAATCTTTCT-39; this last one contains
a six-His tag. Then, the mature region was cloned in pQE60 vector between
BamHI and HindIII restriction sites. After transformation of this vector into
Escherichia coli BL21(DE3)pLysS, a carbenicillin/chloramphenicol-resistant
clone was cultured in 2 L of Luria-Bertani medium for 5 h at 37°C after
isopropylthio-b-galactoside (500 mM) induction. Cells were harvested by
Supplemental Figure S2. Molecular characterization of the Arabidopsis
trxm3 mutant.
Supplemental Figure S3. Molecular characterization of Arabidopsis
TRXm4-1 and TRXm4-2.
Supplemental Figure S4. Purification and characterization of recombinant
TRXm4.
Supplemental Figure S5. Characterization of the photosynthetic activity of
trxm4 mutants.
ACKNOWLEDGMENTS
We thank Prof. T. Shikanai and Dr. Y. Munekage for providing the seeds of
pgr5. We are very grateful to P. Henri for valuable technical assistance and to
the Groupe de Recherche Appliquée en Phytotechnologie (Commissariat à
l’Energie Atomique, Institut de Biologie Environnementale et Biotechnologie,
Service de Biologie Végétale et Microbiologie Environnementales) for technical
assistance with phytotrons.
Received September 14, 2012; accepted November 12, 2012; published
November 14, 2012.
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Redox Regulation of the NDH and PGR Pathways
LITERATURE CITED
Allen JF (2003) Cyclic, pseudocyclic and noncyclic photophosphorylation:
new links in the chain. Trends Plant Sci 8: 15–19
Apostol S, Szalai G, Sujbert L, Popova LP, Janda T (2006) Non-invasive
monitoring of the light-induced cyclic photosynthetic electron flow
during cold hardening in wheat leaves. Z Naturforsch C 61: 734–740
Baker NR, Harbinson J, Kramer DM (2007) Determining the limitations
and regulation of photosynthetic energy transduction in leaves. Plant
Cell Environ 30: 1107–1125
Battchikova N, Eisenhut M, Aro EM (2011) Cyanobacterial NDH-1 complexes:
novel insights and remaining puzzles. Biochim Biophys Acta 1807: 935–944
Benitez-Alfonso Y, Cilia M, San Roman A, Thomas C, Maule A, Hearn S,
Jackson D (2009) Control of Arabidopsis meristem development by
thioredoxin-dependent regulation of intercellular transport. Proc Natl
Acad Sci USA 106: 3615–3620
Breyton C, Nandha B, Johnson GN, Joliot P, Finazzi G (2006) Redox
modulation of cyclic electron flow around photosystem I in C3 plants.
Biochemistry 45: 13465–13475
Buchanan BB, Balmer Y (2005) Redox regulation: a broadening horizon.
Annu Rev Plant Biol 56: 187–220
Burrows PA, Sazanov LA, Svab Z, Maliga P, Nixon PJ (1998) Identification of
a functional respiratory complex in chloroplasts through analysis of tobacco
mutants containing disrupted plastid ndh genes. EMBO J 17: 868–876
Cardol P, Forti G, Finazzi G (2011) Regulation of electron transport in
microalgae. Biochim Biophys Acta 1807: 912–918
Clarke JE, Johnson GN (2001) In vivo temperature dependence of cyclic
and pseudocyclic electron transport in barley. Planta 212: 808–816
Collin V, Issakidis-Bourguet E, Marchand C, Hirasawa M, Lancelin JM,
Knaff DB, Miginiac-Maslow M (2003) The Arabidopsis plastidial thioredoxins: new functions and new insights into specificity. J Biol Chem
278: 23747–23752
Cornic G, Bukhov NG, Wiese C, Bligny R, Heber U (2000) Flexible coupling
between light-dependent electron and vectorial proton transport in illuminated leaves of C3 plants: role of photosystem I-dependent proton pumping.
Planta 210: 468–477
DalCorso G, Pesaresi P, Masiero S, Aseeva E, Schünemann D, Finazzi G,
Joliot P, Barbato R, Leister D (2008) A complex containing PGRL1 and
PGR5 is involved in the switch between linear and cyclic electron flow in
Arabidopsis. Cell 132: 273–285
Daniell H (1997) Transformation and foreign gene expression in plants by
microprojectile bombardment. Methods Mol Biol 62: 463–489
Ducruet JM (2003) Chlorophyll thermoluminescence of leaf discs: simple
instruments and progress in signal interpretation open the way to new
ecophysiological indicators. J Exp Bot 54: 2419–2430
Ducruet JM, Roman M, Havaux M, Janda T, Gallais A (2005) Cyclic electron flow
around PSI monitored by afterglow luminescence in leaves of maize inbred
lines (Zea mays L.): correlation with chilling tolerance. Planta 221: 567–579
Duek PD, Wolosiuk RA (2001) Rapeseed chloroplast thioredoxin-m:
modulation of the affinity for target proteins. Biochim Biophys Acta
1546: 299–311
Eberhard S, Finazzi G, Wollman FA (2008) The dynamics of photosynthesis. Annu Rev Genet 42: 463–515
Endo T, Shikanai T, Sato F, Asada K (1998) NAD(P)H dehydrogenasedependent, antimycine A-sensitive electron donation to plastoquinone
in tobacco chloroplasts. Plant Cell Physiol 39: 1226–1231
Fan DY, Nie Q, Hope AB, Hillier W, Pogson BJ, Chow WS (2007) Quantification of cyclic electron flow around photosystem I in spinach leaves
during photosynthetic induction. Photosynth Res 94: 347–357
Farran I, Río-Manterola F, Iñiguez M, Gárate S, Prieto J, Mingo-Castel
AM (2008) High-density seedling expression system for the production
of bioactive human cardiotrophin-1, a potential therapeutic cytokine, in
transgenic tobacco chloroplasts. Plant Biotechnol J 6: 516–527
Golding AJ, Johnson GN (2003) Down-regulation of linear and activation
of cyclic electron transport during drought. Planta 218: 107–114
Gotoh E, Matsumoto M, Ogawa K, Kobayashi Y, Tsuyama M (2010) A
qualitative analysis of the regulation of cyclic electron flow around
photosystem I from the post-illumination chlorophyll fluorescence
transient in Arabidopsis: a new platform for the in vivo investigation of
the chloroplast redox state. Photosynth Res 103: 111–123
Hald S, Nandha B, Gallois P, Johnson GN (2008) Feedback regulation of
photosynthetic electron transport by NADP(H) redox poise. Biochim
Biophys Acta 1777: 433–440
Hashimoto M, Endo T, Peltier G, Tasaka M, Shikanai T (2003) A nucleusencoded factor, CRR2, is essential for the expression of chloroplast ndhB
in Arabidopsis. Plant J 36: 541–549
Havaux M, Rumeau D, Ducruet JM (2005) Probing the FQR and NDH
activities involved in cyclic electron transport around photosystem I by
the ‘afterglow’ luminescence. Biochim Biophys Acta 1709: 203–213
Heber U, Egneus H, Hanck U, Jensen M, Koster S (1978) Regulation of
photosynthetic electron transport and photophosphorylation in intact
chloroplasts and leaves of Spinacia oleracea L. Planta 143: 41–49
Horiuchi M, Nakamura K, Kojima K, Nishiyama Y, Hatakeyama W,
Hisabori T, Hihara Y (2010) The PedR transcriptional regulator interacts
with thioredoxin to connect photosynthesis with gene expression in
cyanobacteria. Biochem J 431: 135–140
Ifuku K, Endo T, Shikanai T, Aro EM (2011) Structure of the chloroplast
NADH dehydrogenase-like complex: nomenclature for nuclear-encoded
subunits. Plant Cell Physiol 52: 1560–1568
Issakidis-Bourguet E, Mouaheb N, Meyer Y, Miginiac-Maslow M (2001)
Heterologous complementation of yeast reveals a new putative function
for chloroplast m-type thioredoxin. Plant J 25: 127–135
Iwai M, Takizawa K, Tokutsu R, Okamuro A, Takahashi Y, Minagawa J
(2010) Isolation of the elusive supercomplex that drives cyclic electron
flow in photosynthesis. Nature 464: 1210–1213
Jin SH, Wang D, Zhu FY, Li XQ, Sun JW, Jiang DA (2008) Up-regulation of
cyclic electron flow and down-regulation of linear electron flow in
antisense-rca mutant rice. Photosynthetica 46: 506–510
Joët T, Cournac L, Peltier G, Havaux M (2002) Cyclic electron flow around photosystem I in C3 plants: in vivo control by the redox state of chloroplasts and
involvement of the NADH-dehydrogenase complex. Plant Physiol 128: 760–769
Johnson GN (2003) Thiol regulation of the thylakoid electron transport
chain: a missing link in the regulation of photosynthesis? Biochemistry
42: 3040–3044
Johnson GN (2005) Cyclic electron transport in C3 plants: fact or artefact? J
Exp Bot 56: 407–416
Johnson GN (2011) Reprint of: Physiology of PSI cyclic electron transport
in higher plants. Biochim Biophys Acta 1807: 906–911
Joliot P, Johnson GN (2011) Regulation of cyclic and linear electron flow in
higher plants. Proc Natl Acad Sci USA 108: 13317–13322
Joliot P, Joliot A (2005) Quantification of cyclic and linear flows in plants.
Proc Natl Acad Sci USA 102: 4913–4918
Joliot P, Joliot A (2006) Cyclic electron flow in C3 plants. Biochim Biophys
Acta 1757: 362–368
Josse EM, Alcaraz JP, Labouré AM, Kuntz M (2003) In vitro characterization of
a plastid terminal oxidase (PTOX). Eur J Biochem 270: 3787–3794
Kalituho L, Beran KC, Jahns P (2007) The transiently generated nonphotochemical quenching of excitation energy in Arabidopsis leaves is
modulated by zeaxanthin. Plant Physiol 143: 1861–1870
Kamruzzaman Munshi M, Kobayashi Y, Shikanai T (2005) Identification
of a novel protein, CRR7, required for the stabilization of the chloroplast
NAD(P)H dehydrogenase complex in Arabidopsis. Plant J 44: 1036–1044
Kofer W, Koop HU, Wanner G, Steinmüller K (1998) Mutagenesis of the
genes encoding subunits A, C, H, I, J and K of the plastid NAD(P)Hplastoquinone-oxidoreductase in tobacco by polyethylene glycolmediated plastome transformation. Mol Gen Genet 258: 166–173
Kohzuma K, Cruz JA, Akashi K, Hoshiyasu S, Munekage YN, Yokota A,
Kramer DM (2009) The long-term responses of the photosynthetic
proton circuit to drought. Plant Cell Environ 32: 209–219
Kramer DM, Avenson TJ, Edwards GE (2004) Dynamic flexibility in the
light reactions of photosynthesis governed by both electron and proton
transfer reactions. Trends Plant Sci 9: 349–357
Kramer DM, Evans JR (2011) The importance of energy balance in improving photosynthetic productivity. Plant Physiol 155: 70–78
Kuntz M (2004) Plastid terminal oxidase and its biological significance.
Plant 218: 896–899
Laugier E, Tarrago L, Courteille A, Innocenti G, Eymery F, Rumeau D,
Issakidis-Bourguet E, Rey P (2012) Involvement of thioredoxin y2 in the
preservation of leaf methionine sulfoxide reductase capacity and growth
under high light. Plant Cell Environ (in press)
Lemaire SD, Michelet L, Zaffagnini M, Massot V, Issakidis-Bourguet E
(2007) Thioredoxins in chloroplasts. Curr Genet 51: 343–365
Lindahl M, Mata-Cabana A, Kieselbach T (2011) The disulfide proteome
and other reactive cysteine proteomes: analysis and functional significance. Antioxid Redox Signal 14: 2581–2642
Plant Physiol. Vol. 161, 2013
519
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2013 American Society of Plant Biologists. All rights reserved.
Courteille et al.
Lintala M, Allahverdiyeva Y, Kangasjärvi S, Lehtimäki N, Keränen M,
Rintamäki E, Aro EM, Mulo P (2009) Comparative analysis of leaf-type
ferredoxin-NADP oxidoreductase isoforms in Arabidopsis thaliana.
Plant J 57: 1103–1115
Livingston AK, Cruz JA, Kohzuma K, Dhingra A, Kramer DM (2010a) An
Arabidopsis mutant with high cyclic electron flow around photosystem I
(hcef) involving the NADPH dehydrogenase complex. Plant Cell 22:
221–233
Livingston AK, Kanazawa A, Cruz JA, Kramer DM (2010b) Regulation of
cyclic electron flow in C3 plants: differential effects of limiting photosynthesis at ribulose-1,5-bisphosphate carboxylase/oxygenase and
glyceraldehyde-3-phosphate dehydrogenase. Plant Cell Environ 33:
1779–1788
Meyer Y, Buchanan BB, Vignols F, Reichheld JP (2009) Thioredoxins and
glutaredoxins: unifying elements in redox biology. Annu Rev Genet 43:
335–367
Miranda T, Ducruet JM (1995) Characterization of the chlorophyll thermoluminescence afterglow in dark-adapted or far-red-illuminated plant
leaves. Plant Physiol Biochem 33: 689–699
Montrichard F, Alkhalfioui F, Yano H, Vensel WH, Hurkman WJ,
Buchanan BB (2009) Thioredoxin targets in plants: the first 30 years. J
Proteomics 72: 452–474
Motohashi K, Hisabori T (2006) HCF164 receives reducing equivalents
from stromal thioredoxin across the thylakoid membrane and mediates
reduction of target proteins in the thylakoid lumen. J Biol Chem 281:
35039–35047
Munekage Y, Hashimoto M, Miyake C, Tomizawa KI, Endo T, Tasaka M,
Shikanai T (2004) Cyclic electron flow around photosystem I is essential
for photosynthesis. Nature 429: 579–582
Munekage Y, Hojo M, Meurer J, Endo T, Tasaka M, Shikanai T (2002)
PGR5 is involved in cyclic electron flow around photosystem I and is
essential for photoprotection in Arabidopsis. Cell 110: 361–371
Nandha B, Finazzi G, Joliot P, Hald S, Johnson GN (2007) The role of
PGR5 in the redox poising of photosynthetic electron transport. Biochim
Biophys Acta 1767: 1252–1259
Okegawa Y, Kagawa Y, Kobayashi Y, Shikanai T (2008) Characterization
of factors affecting the activity of photosystem I cyclic electron transport
in chloroplasts. Plant Cell Physiol 49: 825–834
Okegawa Y, Kobayashi Y, Shikanai T (2010) Physiological links among
alternative electron transport pathways that reduce and oxidize plastoquinone in Arabidopsis. Plant J 63: 453–468
Peeva VN, Tóth SZ, Cornic G, Ducruet JM (2012) Thermoluminescence
and P700 redox kinetics as complementary tools to investigate the
cyclic/chlororespiratory electron pathways in stress conditions in barley
leaves. Physiol Plant 144: 83–97
Peng L, Fukao Y, Fujiwara M, Takami T, Shikanai T (2009) Efficient operation of NAD(P)H dehydrogenase requires supercomplex formation
with photosystem I via minor LHCI in Arabidopsis. Plant Cell 21: 3623–
3640
Rey P, Cuiné S, Eymery F, Garin J, Court M, Jacquot JP, Rouhier N, Broin
M (2005) Analysis of the proteins targeted by CDSP32, a plastidic thioredoxin participating in oxidative stress responses. Plant J 41: 31–42
Rintamäki E, Martinsuo P, Pursiheimo S, Aro EM (2000) Cooperative
regulation of light-harvesting complex II phosphorylation via the plastoquinol and ferredoxin-thioredoxin system in chloroplasts. Proc Natl
Acad Sci USA 97: 11644–11649
Rochaix JD (2011) Regulation of photosynthetic electron transport. Biochim
Biophys Acta 1807: 375–383
Rumeau D, Bécuwe-Linka N, Beyly A, Louwagie M, Garin J, Peltier G
(2005) New subunits NDH-M, -N, and -O, encoded by nuclear genes, are
essential for plastid Ndh complex functioning in higher plants. Plant
Cell 17: 219–232
Sanz-Barrio R, Fernández-San Millán A, Carballeda J, Corral-Martínez P,
Seguí-Simarro JM, Farran I (2012) Chaperone-like properties of tobacco
plastid thioredoxins f and m. J Exp Bot 63: 365–379
Sanz-Barrio R, Millán AF, Corral-Martínez P, Seguí-Simarro JM, Farran I
(2011) Tobacco plastidial thioredoxins as modulators of recombinant
protein production in transgenic chloroplasts. Plant Biotechnol J 9: 639–
650
Schürmann P, Buchanan BB (2008) The ferredoxin/thioredoxin system of
oxygenic photosynthesis. Antioxid Redox Signal 10: 1235–1274
Shikanai T (2007) Cyclic electron transport around photosystem I: genetic
approaches. Annu Rev Plant Biol 58: 199–217
Shikanai T, Endo T, Hashimoto T, Yamada Y, Asada K, Yokota A (1998)
Directed disruption of the tobacco ndhB gene impairs cyclic electron
flow around photosystem I. Proc Natl Acad Sci USA 95: 9705–9709
Suorsa M, Järvi S, Grieco M, Nurmi M, Pietrzykowska M, Rantala M,
Kangasjärvi S, Paakkarinen V, Tikkanen M, Jansson S, et al (2012)
PROTON GRADIENT REGULATION5 is essential for proper acclimation of Arabidopsis photosystem I to naturally and artificially fluctuating
light conditions. Plant Cell 24: 2934–2948
Takahashi S, Milward SE, Fan DY, Chow WS, Badger MR (2009) How
does cyclic electron flow alleviate photoinhibition in Arabidopsis? Plant
Physiol 149: 1560–1567
Wenderoth I, Scheibe R, von Schaewen A (1997) Identification of the
cysteine residues involved in redox modification of plant plastidic
glucose-6-phosphate dehydrogenase. J Biol Chem 272: 26985–26990
Yamamoto H, Kato H, Shinzaki Y, Horiguchi S, Shikanai T, Hase T, Endo
T, Nishioka M, Makino A, Tomizawa K-i, et al (2006) Ferredoxin limits
cyclic electron flow around PSI (CEF-PSI) in higher plants: stimulation
of CEF-PSI enhances non-photochemical quenching of Chl fluorescence
in transplastomic tobacco. Plant Cell Physiol 47: 1355–1371
Yamamoto H, Peng L, Fukao Y, Shikanai T (2011) An Src homology 3
domain-like fold protein forms a ferredoxin binding site for the chloroplast NADH dehydrogenase-like complex in Arabidopsis. Plant Cell 23:
1480–1493
Zhang H, Whitelegge JP, Cramer WA (2001) Ferredoxin:NADP+ oxidoreductase is a subunit of the chloroplast cytochrome b6f complex. J Biol
Chem 276: 38159–38165
520
Plant Physiol. Vol. 161, 2013
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2013 American Society of Plant Biologists. All rights reserved.