Multiple strategies to prevent oxidative stress in Arabidopsis plants lacking the malate valve enzyme NADP-malate dehydrogenase.

Abstract

The nuclear-encoded chloroplast NADP-dependent malate dehydrogenase (NADP-MDH) is a key enzyme controlling the malate valve, to allow the indirect export of reducing equivalents. Arabidopsis thaliana (L.) Heynh. T-DNA insertion mutants of NADP-MDH were used to assess the role of the light-activated NADP-MDH in a typical C(3) plant. Surprisingly, even when exposed to high-light conditions in short days, nadp-mdh knockout mutants were phenotypically indistinguishable from the wild type. The photosynthetic performance and typical antioxidative systems, such as the Beck-Halliwell-Asada pathway, were barely affected in the mutants in response to high-light treatment. The reactive oxygen species levels remained low, indicating the apparent absence of oxidative stress, in the mutants. Further analysis revealed a novel combination of compensatory mechanisms in order to maintain redox homeostasis in the nadp-mdh plants under high-light conditions, particularly an increase in the NTRC/2-Cys peroxiredoxin (Prx) system in chloroplasts. There were indications of adjustments in extra-chloroplastic components of photorespiration and proline levels, which all could dissipate excess reducing equivalents, sustain photosynthesis, and prevent photoinhibition in nadp-mdh knockout plants. Such metabolic flexibility suggests that the malate valve acts in concert with other NADPH-consuming reactions to maintain a balanced redox state during photosynthesis under high-light stress in wild-type plants.

Full text

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Journal of Experimental Botany, Vol. 63, No. 3, pp. 1445–1459, 2012
doi:10.1093/jxb/err386Advance Access publication 3 December, 2011
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER
Multiple strategies to prevent oxidative stress in Arabidopsis
plants lacking the malate valve enzyme NADP-malate
dehydrogenase
Inga Hebbelmann1,*,†, Jennifer Selinski1,†, Corinna Wehmeyer1, Tatjana Goss1, Ingo Voss1, Paula Mulo2,
Saijaliisa Kangasja ¨rvi2, Eva-Mari Aro2, Marie-Luise Oelze3, Karl-Josef Dietz3, Adriano Nunes-Nesi4,‡,
Phuc T. Do4, Alisdair R. Fernie4, Sai K. Talla5, Agepati S. Raghavendra1,5, Vera Linke1and Renate Scheibe1,§
1Department of Plant Physiology, FB5, University of Osnabrueck, D-49069 Osnabrueck, Germany
2Molecular Plant Biology, Department of Biochemistry and Food Chemistry, University of Turku, FIN-20014 Turku, Finland
3Biochemistry and Physiology of Plants, University of Bielefeld, D-33501 Bielefeld, Germany
4Max Planck Institute for Molecular Plant Physiology, Am Mu ¨hlenberg 1, D-14476 Potsdam-Golm, Germany
5Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India
* Present address: Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA.
yThese authors contributed equally to this work.
zPresent address: Max-Planck Partner Group, Departamento de Biologia Vegetal, Universidade Federal de Vic xosa, Vic xosa, Minas
Gerais, Brazil.
§To whom correspondence should be addressed. E-mail: scheibe@biologie.uni-osnabrueck.de
Received 12 May 2011; Revised 1 November 2011; Accepted 2 November 2011
Abstract
The nuclear-encoded chloroplast NADP-dependent malate dehydrogenase (NADP-MDH) is a key enzyme controlling
the malate valve, to allow the indirect export of reducing equivalents. Arabidopsis thaliana (L.) Heynh. T-DNA
insertion mutants of NADP-MDH were used to assess the role of the light-activated NADP-MDH in a typical C3plant.
Surprisingly, even when exposed to high-light conditions in short days, nadp-mdh knockout mutants were
phenotypically indistinguishable from the wild type. The photosynthetic performance and typical antioxidative
systems, such as the Beck–Halliwell–Asada pathway, were barely affected in the mutants in response to high-light
treatment. The reactive oxygen species levels remained low, indicating the apparent absence of oxidative stress, in
the mutants. Further analysis revealed a novel combination of compensatory mechanisms in order to maintain redox
homeostasis in the nadp-mdh plants under high-light conditions, particularly an increase in the NTRC/2-Cys
peroxiredoxin (Prx) system in chloroplasts. There were indications of adjustments in extra-chloroplastic components
of photorespiration and proline levels, which all could dissipate excess reducing equivalents, sustain photosynthe-
sis, and prevent photoinhibition in nadp-mdh knockout plants. Such metabolic flexibility suggests that the malate
valve acts in concert with other NADPH-consuming reactions to maintain a balanced redox state during
photosynthesis under high-light stress in wild-type plants.
Key words: Malate valve, NADP-malate dehydrogenase, oxidative stress, poising mechanisms, redox homeostasis.
Introduction
Malate dehydrogenases catalyse the reversible conversion of
oxaloacetate to malate using either NAD/H or NADP/H as
oxidant/reductant, respectively. Additionally, these enzymes
can indirectly function as a pacemaker of the transport of
Abbreviations: AAN, aminoacetonitrile; AOX, alternative oxidase; APX, ascorbate peroxidase; DHAR, dehydroascorbate reductase; GDC, glycine decarboxylase; GHA,
glycine hydroxamate; GL, growth light; GR, glutathione reductase; HL, high light; MDAR, monodehydroascorbate reductase; NADP-MDH, NADP-dependent malate
dehydrogenase; NPQ, non-photochemical quenching; NTRC, chloroplast NADPH-thioredoxin reductase; Prx, peroxiredoxin; qP, photochemical quenching; SHAM,
salicylhydroxamic acid.
ª 2011 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-
nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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reducing equivalents between subcellular compartments, in
cooperation with the membrane-bound dicarboxylate trans-
porters. NAD-dependent isoforms of MDH are present in
mitochondria, peroxisomes, cytosol, and plastids. Chloro-
plasts additionally possess an NADP-malate dehydrogenase
(NADP-MDH) with distinct regulatory properties. This
nuclear-encoded NADP-MDH is the key enzyme of the
malate valve (Scheibe, 2004). NADP-MDH converts oxalo-
acetate to malate using NADPH, facilitating the regenera-
tion of the electron acceptor NADP+in the chloroplasts,
particularly when CO2assimilation is restricted.
The malate valve is suggested to balance the ATP/
NADPH ratio of the chloroplast as required by changing
metabolic demands (Scheibe, 2004). Export of reducing
equivalents, however, needs to be well controlled in order
to avoid any imbalance or depletion of chloroplast energy
carriers. The reductive activation of NADP-MDH is inhi-
bited when the NADP/NADP(H) ratio is high (Scheibe and
Jacquot, 1983; Faske et al., 1995). The activation of NADP-
MDH, and consequently the rate of malate export from
the chloroplast, is high, only when there is a shortage of
NADP+. The activation state of NADP-MDH changes
within seconds to minutes, due to this post-translational
regulatory mechanism. Thus, the enzyme appears to play an
important role in the short-term adjustment of the stromal
NADP(H) redox state in response to changing environmen-
tal conditions, so as to ensure the maintenance of redox
homeostasis (Scheibe et al., 2005).
Knockout mutants of Arabidopsis thaliana (L.) Heynh.
(nadp-mdh) deficient in NADP-MDH activity were used to
assess the role of the light-activated NADP-MDH in C3
plants. Molecular and biochemical analyses of these plants
grown in or transferred to challenging high-light (HL)
conditions were conducted to understand potential compen-
satory mechanisms that counteract redox imbalances and
oxidative stress. Amongst these systems are the NTRC/2-
Cys peroxiredoxin (Prx) system in chloroplasts (Serrato
et al., 2004; Pe ´rez-Ruiz et al., 2006), other antioxidant en-
zymes and low molecular weight antioxidants in different
cellular compartments (Foyer and Noctor, 2009), photores-
piration (Wingler et al., 2000; Igamberdiev et al., 2001), and
even the mitochondrial alternative oxidase (AOX) pathway
(Yoshida et al., 2007; Strodtko ¨tter et al., 2009). The detailed
analysis presented here revealed that the nadp-mdh mutants
employed a combination of multiple strategies to counteract
the oxidative stress, and protect the chloroplasts from
photoinhibition under HL.
Materials and methods
Growth of plant material
Wild-type (WT) and transgenic Arabidopsis thaliana (L.) Heynh.
(ecotype Columbia) plants were cultivated in a growth chamber in
soil under short-day conditions with a 7.5 h daily light period,
a light intensity of 50 lmol quanta m?2s?1, and a temperature of
20 ?C. These conditions are defined as growth light (GL). To apply
stress conditions, plants were exposed to HL (750 lmol quanta
m?2s?1) for 7 h or for alternative periods as indicated. To analyse
plant growth in early stages, single seeds were placed in pots in
soil, and plants were grown under short-day conditions at a light
intensity of 150 lmol quanta m?2s?1for 5 weeks. Then the fresh
and dry weight of the above-ground biomass was determined in
100 seedlings of each genotype.
For growth analyses, single seeds were planted in Petri dishes on
agar. The nutrient medium described by Wilson et al. (1990) was
used with some modifications. The medium contained 2.5 mM
KCl, 1.25 mM KH2PO4, 1 mM MgSO4, 2 mM CaCl2, 80 lM Fe-
EDTA, 24 lM H3BO3, 4 lM MnCl2, 0.2 lM CuSO4, 0.4 lM
ZnSO4, 0.6 lM Na2MoO4, and 0.8% agar pro analysi (Carl Roth,
Karlsruhe, Germany). The pH was adjusted to 5.7 with KOH, and
the medium was supplemented with 1.8 mM nitrate. For uniform
germination, the plates were initially incubated for 2 d in the dark
at 4 ?C. Seedlings were grown under sterile conditions. After 4 weeks
of growth under short-day conditions at 150 lmol quanta m?2s?1,
the fresh weight of the total biomass was determined in 100 seedlings
of each genotype.
Screen for nadp-mdh mutants
The Arabidopsis nadp-mdh mutant lines At5g58330::tDNA-50
(Salk_012655) and At5g58330::tDNA-119 (Salk_063444) were
obtained from the Arabidopsis Biological Resource Centre (http://
www.arabidopsis.org/abrc). Homozygous knockout plants were
identified by PCR for T-DNA insertion within the gene region
of At5g58330. Genomic DNA was isolated from plant tissues
by standard methods. The sequence information for the gene- and
T-DNA-specific primers was taken from the Salk Institute (http://
signal.salk.edu). The positions of the T-DNA insertion were
confirmed by sequencing the PCR products.
Extraction of total RNA and northern blot analysis
For northern blot analysis, total RNA was isolated from frozen
leaf material by using the Purescript RNA-extraction kit (Gentra
Systems, Minneapolis, MN, USA). For RNA gel-blot hybrid-
ization, 10 lg of total RNA were denatured and separated on
a 1.0% (w/v) agarose–2.5% (v/v) formaldehyde gel, transferred, and
UV-cross-linked to a nylon membrane (Hybond-N, Amersham
Biosciences, UK). Pre-hybridization and hybridization were per-
formed at 65 ?C in Church buffer medium [0.25 M sodium
phosphate buffer, pH 7.2, 1 mM EDTA, 7% (w/v) SDS, and 1%
bovine serum albumin (BSA)]. Hybridization was performed
with an [a-32P]dCTP-labelled NADP-MDH cDNA-specific probe
(Ready-To-Go DNA-labelling beads, Amersham Biosciences,
UK). Membranes were washed twice for 15 min at 65 ?C in
washing buffer [40 mM sodium phosphate buffer, pH 7.2, 1 mM
EDTA, 0.5% (w/v) SDS, and 0.5% (w/v) BSA], then for 10 min
at room temperature in washing buffer containing 1% (w/v) SDS.
Finally, membranes were exposed to a Phosphor-Imager (GE
Healthcare, Freiburg, Germany).
RT-PCR analysis
Non-competitive reverse transcription-PCR (RT-PCR) was per-
formed essentially as described by Ahn (2002). cDNA was
synthesized from 5 lg of total RNA using oligo(dT) as primer
according to the manufacturer’s instructions (Fermentas Rever-
tAid? First Strand cDNA Synthesis Kit, Fermentas GmbH,
St. Leon-Rot, Germany). For a 25 ll PCR, 1 ll of cDNA was used
as template. The PCR settings were: first cycle at 95 ?C for 5 min,
then for the optimized number of cycles for each gene product
1 min at 95 ?C, 1 min at 47–67 ?C, and 1 min at 72?C, and a final
extension at 72 ?C for 5 min. Oligonucleotides that were used for
the detection of the transcripts are listed in Supplementary Table S1
available at JXB online. The intensity of each band after electro-
phoresis was determined with the Quantity One software (BioRad,
Munich, Germany).
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Microarray analysis
Arabidopsis thaliana 24k oligonucleotide arrays (MWG Biotech;
http://www.mwg-biotech.com; ArrayExpress database accession
no. A-ATMX-2; http://www.ebi.ac.uk/arrayexpress) were used to
study changes in nuclear gene expression. Leaf samples from the
WT and nadp-mdh knockout plants grown under standard con-
ditions and treated for 7 h with HL (750 lmol quanta m?2s?1)
were collected. Following total RNA isolation (Piippo et al., 2006),
cDNA synthesis, sample labelling, array hybridization and scan-
ning, as well as spot intensity quantification were performed as in
Kangasja ¨rvi et al. (2008). The expression data were normalized
and analysed by using the tool R/BioConductor limma (Smyth
et al., 2003).
Western blot analysis and immunodecoration
Equal amounts of soluble protein (50 lg per lane) were loaded on
12% discontinuous SDS–polyacrylamide gels using a vertical
minigel system (Mini-Protean II, BioRad). The gel was blotted
onto a nitrocellulose membrane. Immunodecoration was per-
formed essentially as described in Graeve et al. (1994). For the
detection, polyclonal antisera against NADP-MDH from pea
leaves (1:3000), against the 2-Cys Prx BAS1 from barley (to detect
2-Cys PrxA and B; Baier and Dietz, 1997) (1:5000), against the
chloroplast-localized NADPH-thioredoxin reductase C (NTRC;
Pe ´rez-Ruiz et al. 2006) (1:2000), and against the P-protein of
glycine decarboxylase (GDC) (1:3000; Hermann Bauwe, Rostock
University) were used. For the detection of the second antibody
linked to horseradish peroxidase (1:20 000), luminol was used as
the substrate as recommended by the supplier (GE Healthcare).
The steady-state levels of the ascorbate peroxidase isoforms
tAPX, sAPX, pAPX, and the cytosolic APXs (Asada 1999; Chew
et al. 2003; Narendra et al., 2006) were analyzed using the anti-
APX antibody from Agrisera (http://www.agrisera.com/en/artiklar/
apx-ascorbate-peroxidase-.html) as described in Kangasja ¨rvi et al.
(2008) using 10 lg of total leaf protein and immunodetection with
Arabidopsis anti-APX antibody (Kangasja ¨rvi et al., 2008) using
a Phototope?-Star Detection Kit (New England Biolabs, Beverly,
MA, USA; http://www.neb.com/).
Enzyme measurements in crude extracts
Leaves were cut from the plant, immediately transferred and
pulverized in liquid nitrogen, and stored until use at –80 ?C. For
extraction, buffers as required for the various assays were added to
aliquot portions of the powder. The total activity of NADP-MDH
was determined after exhaustive reduction with reduced dithio-
threitol (DTT) and corrected for unspecific NAD-MDH activity as
described by Scheibe and Stitt (1988). The total capacities of
catalase and APX were determined in extracts as in Del Longo
et al. (1993). The enzyme extractions and measurements of the
activities of NADP-dependent glyceraldehyde-3-P dehydrogenase
(NADP-GAPDH) were performed as in Baalmann et al. (1995), of
non-phosphorylating GAPDH (NP-GAPDH) as in Rius et al.
(2006), and of glycerol-3-P dehydrogenase (G3PDH) as in Shen
et al. (2006).
Chlorophyll (Chl) fluorescence measurements
A portable photosynthesis system LI-6400XT (LI-COR Biosciences,
Lincoln, NE, USA) was used to measure leaf Chl fluorescence in
800 lmol quanta m?2s?1actinic light (Table 2), while for measure-
ments in 50 lmol quanta m?2s?1the closed FluorCam FC 800-C
(Photon Systems Instruments, Brno, Czech Republic) was used. The
intensity of the saturating pulse was 5000 lmol quanta m?2s?1with
a duration of 800 ms. The dark adaption time was 20 min.
Maximum quantum yield in dark-adapted leaves (Fv/Fm), the
quantum yield of photosystem II [PSII; (UII)] and the quenching
coefficients photochemical quenching (qP) and non-photochemical
quenching (NPQ) were calculated according to Schreiber et al.
(1986), Walker (1988), and Genty et al. (1989).
Photosynthetic and respiratory performance of mesophyll
protoplasts
Mesophyll protoplasts were isolated from leaves of 11- to 12-week-old
WT and nadp-mdh plants that had been maintained under the
growth conditions as described above. Leaf sections without a midrib
were freed of the lower epidermis mechanically with a forceps and
subjected to enzymatic digestion with 1% (w/v) Cellulase Onozuka
R-10 and 0.4% (w/v) Macerozyme R-10 (Yakult Pharmaceutical
Industry, Tokyo, Japan) as described (Riazunnisa et al., 2007).
Rates of respiratory O2uptake in the dark and of photosyn-
thetic O2evolution in the light by mesophyll protoplasts of WT
and nadp-mdh mutants were monitored at 25 ?C using a Clark-type
O2electrode (Model DW2, Hansatech Ltd, King’s Lynn, UK). The
reaction medium for both photosynthesis and respiration determi-
nation was 1 ml containing 0.65 M sorbitol, 1 mM CaCl2, 1 mM
MgCl2, and 1 mM NaHCO3in 10 mM HEPES-KOH, pH 7.5, and
protoplasts equivalent to 10 lg Chl (Riazunnisa et al., 2007).
Illumination with 750 lmol quanta m?2s?1was provided by
a 35 mm slide projector (halogen lamp: Xenophot 24 V:150 W).
The inhibitors [SHAM, salicylhydroxamic acid (SHAM), glycine
hydroxamate (GHA), and aminoacetonitrile (AAN), all from
Sigma-Aldrich Co., St Louis, MO, USA] were added to the reaction
medium containing mesophyll protoplasts to obtain the required
final concentration, and the protoplasts were pre-incubated in
darkness at 25 ?C for 5 min before switching on the light. The
normal level of O2 in air-equilibrated reaction medium was
410 nmol ml?1. The reaction medium in the O2electrode chamber
was bubbled with N2, resulting in a marked decrease of O2 to
85 nmol ml?1.
Metabolite analysis
Starch content in leaves was determined according to Batz et al.
(1995). Malate was determined enzymatically with glutamate-
oxaloacetate transaminase (GOT; Sigma-Aldrich Co.) in a coupling
reaction as described in a protocol of R-Biopharm GmbH
(Darmstadt, Germany). Specifically, 100 mg of Arabidopsis leaves
were frozen in N2, ground to powder, resuspended in 1 ml of H2O,
and incubated at 95 ?C for 8 min. The supernatant was used for
the measurement in buffer containing 100 mM glycylglycine,
100 mM glutamate, 1 mM NAD+, and GOT (1 U ml?1). The
reaction was started by adding 1 U ml?1NAD-MDH (Sigma-
Aldrich Co.). For proline determination, Arabidopsis leaves
(500 mg) were frozen in N2, ground to powder, and 0.5 ml of 3%
sulphosalicylic acid was added. After mixing and centrifugation,
0.5 ml of the supernatant was transferred to a new reaction tube,
and proline concentrations were measured colorimetrically using
the ninhydrin method (Bates et al., 1973). Global metabolite
analysis was performed by gas chromatography–mass spectrome-
try (GC-MS) as described by Lisec et al. (2006).
Glutathione determination
The amounts of reduced and oxidized glutathione (GSH and
GSSG, respectively) were determined using the Total Glutathione
Detection Kit (Enzo Life Sciences, Lo ¨rrach, Germany). For each
genotype and treatment, Arabidopsis leaves from five plants were
frozen in N2and then ground to powder, and 50 mg of the tissue
was used to prepare the extract. For this, 1 ml of ice-cold 5%
metaphosphoric acid was added to the powder and vortexed for
30 s. After centrifugation, 50 ll aliquots of supernatant each were
used for total glutathione and oxidized glutathione, respectively.
For the latter, samples were incubated with 2-vinylpyridine at
room temperature for 1 h prior to the assay. The absorbance was
recorded at 405 nm using a plate reader (SPECTRAmax Plus 348,
Molecular Devices, Sunnyvale, CA, USA) at 1 min intervals over
NADP-malate dehydrogenase knockout induces multiple strategies | 1447

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a 10 min period. For analysis of the data, the software SoftMax
Pro 5.3 (Molecular Devices) was used.
Reactive oxygen species (ROS) determinations
H2O2 was quantified in leaf extracts prepared immediately after
exposure to HL for 5 h, as described by Liu et al. (2010). The
extract was diluted accordingly and then used for H2O2determina-
tion with an Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit
(Molecular Probes, Eugene, OR, USA). The values were obtained
from three samples, each from two plants, and the standard error
was calculated. The data were analysed by analysis of variance
(ANOVA), and means were compared using a Student’s t-test.
Chl and protein determination
To determine leaf Chl a and b contents, extraction, photometric mea-
surements, and calculation were performed as described in Sims and
Gamon (2002). The protein content in the soluble leaf extracts was
estimated according to Bradford (1976), with BSA as the standard.
Results
Verification of NADP-MDH gene knockout in two
independent lines of A. thaliana
Two independent nadp-mdh-T-DNA insertion lines were
identified in Arabidopsis. The lines At5g58330::tDNA-50
and At5g58330::tDNA-119 harbour a T-DNA insertion at
positions 54 200 and 55 324, respectively, of chromosome 5
(Fig. 1A). The combination of the NADP-MDH-specific
primers 119LP and 119RP for line 119 allowed for ampli-
fication of the expected PCR product (1028 bp) on genomic
DNA from the WT, but not on DNA from homozygous
nadp-mdh knockout plants from line 119 (Supplementary
Fig. S1A at JXB online). For line 50, the gene-specific
primers 121 and 84 were used and the corresponding PCR
product had a size of 1320 bp (Supplementary Fig. S1B).
PCR products were amplified in reactions containing the
T-DNA left border primer LBa1 and one gene-specific
primer on DNA from both homozygous nadp-mdh mutant
lines, but not on DNA from WT plants (Supplementary
Fig. S1A, B). The PCR result and the sequencing of the
PCR products confirmed that in both cases homozygous
T-DNA plants had been obtained.
Both homozygous lines lacked the NADP-MDH tran-
script as indicated by northern blot analysis (Supplementary
Fig. S2A at JXB online) and RT-PCR (data not shown),
and were devoid of the NADP-MDH protein as demon-
strated by western blot analysis (Supplementary Fig. S2B).
NADP-MDH activity, when corrected for the unspecific
activity with NADPH oxidation derived from NAD-MDH
isoforms (Scheibe and Stitt, 1988), was also completely
absent (data not shown). The results validated both in-
sertion lines as full nadp-mdh knockout lines. Given that the
two insertion lines exhibited identical phenotypes, further
work was concentrated on line 50, but selected results were
also confirmed using line 119.
Phenotypic appearance of nadp-mdh plants
The phenotype of nadp-mdh plants under standard growth
conditions (GL: 50 lmol quanta m?2s?1, short-day) on soil
was indistinguishable from that of the WT (Fig. 1B).
Likewise, Chl a, Chl b, and protein contents as well as fresh
weight and water content were not significantly altered in
the nadp-mdh plants (Table 1).
Cultivation of WT plants under stress conditions such as
HL, low temperature, or fluctuating light stimulates expres-
sion of NADP-MDH, indicating the requirement for an
increased capacity of the malate valve (Becker et al., 2006).
In this study, nadp-mdh plants treated with either HL or low
temperature also revealed an unaltered phenotype com-
pared with the WT. The GL-acclimated nadp-mdh mutant
plants that were transferred to HL for 1 week (Fig. 1C), as
well as the plants that were cultivated under HL for the
complete growth period (Fig. 1D, E), developed a WT-like
phenotype. Even under the more variable climatic condi-
tions of a greenhouse, the potted mutants developed like the
WT (data not shown).
HL effects on ROS formation and antioxidative systems
Accumulation of NADPH in the nadp-mdh plants could
stimulate the Mehler reaction and concomitantly increase
ROS formation during HL conditions. In the mutants,
a decreased level of ROS was apparent compared with the
WT, in HL as well as GL (Fig. 2A). The reduced and
oxidized glutathione increased in both the WT and mutants
upon exposure to HL, but the redox state was unaffected by
the lack of chloroplast NADP-MDH (Fig. 2B). The protein
levels, transcript amounts, and activities of selected Beck–
Halliwell–Asada pathway enzymes were examined in WT
and nadp-mdh mutant plants. No difference could be detected
in either protein levels of the various APX isoforms (Fig. 2D)
or the total APX activity (Fig. 2E). Semi-quantitative RT-
PCR analysis revealed a strong up-regulation of sAPX
transcript and a slight up-regulation of tAPX, dehydroascor-
bate reductase (DHAR), monodehydroascorbate reductase
(MDAR), and glutathione reductase (GR) after HL treat-
ment (Fig. 2C; Supplementary Fig. S3 at JXB online).
To address the ascorbate-independent water–water cycle,
the expression of NTRC and the various chloroplast Prx
isoforms in leaves of WT and nadp-mdh plants was analysed,
after 7 h HL. NTRC transcript and protein levels were in-
creased in the nadp-mdh plants after 7 h of HL (Fig. 3A, B),
as were the transcript levels of chloroplast 2-Cys Prx
isoforms PrxIIE and PrxQ (Fig. 3A), whereby the increase
of the PrxA/B and PrxIIE transcripts was significant. In
contrast to transcript regulation, the amount of 2-Cys
PrxA/B protein (named BAS1; Baier and Dietz, 1997) was
unchanged in both genotypes after HL treatment (Fig. 3C).
Transcriptome profiling revealed an increase in some tran-
scripts, among the most prominent being a C3HC4 zinc
finger RING-type protein (At4g26400), a WRKY-family
(At4g31550), and a MYB-related (At4g01060) transcription
factor (Supplementary Fig. S3 at JXB online). These
transcription factors are also found to be increased in
EXECUTER mutants where signalling from the chloroplast
to the nucleus is affected (Lee et al., 2007).
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Photosynthetic performance and photorespiratory
components in WT and nadp-mdh plants under HL
conditions
Photosynthetic CO2assimilation rates as a function of light
intensities were quite similar in leaves of nadp-mdh and WT
plants (data not shown). Chl fluorescence was used to
monitor the redox state of PSII in WT and nadp-mdh plants
after growth in moderate light (GL), after growth in HL
(750 lmol quanta m?2s?1) for long-term acclimation, and
during a HL treatment in the measurement (;20–30 min)
after growth in GL for short-term acclimation. Under all
conditions, no significant differences in the efficiency of
Fig. 1. Genome insertion sites and phenotype of the two independent homozygous nadp-mdh (At5g58330) mutants. (A) Gene structure
of the two individual AtNADP-MDH-T-DNA insertion lines Salk 012655 (line 50) and Salk 063444 (line 119). The insertion in line 50 is
localized in the fifth intron, whereas in line 119 the insertion is localized in the first exon. The primers used for PCR analysis are marked as
arrows. (B) Phenotype of plants of line 50 grown for 8 weeks under standard growth conditions at 50 lmol quanta m?2s?1under
a short-day photoperiod. (C) Plants were grown for 8 weeks under standard growth conditions (50 lmol quanta m?2s?1) and then
transferred to HL (750 lmol quanta m?2s?1) for 7 d. (D and E) Plants were cultivated under HL (750 lmol quanta m?2s?1) during their
entire growth period.
NADP-malate dehydrogenase knockout induces multiple strategies | 1449

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dark-adapted PSII (Fv/Fm), qP, NPQ, and flux through PSII
(UII) were apparent in the mutants as compared with the
WT (Table 2). Also P700 absorption in leaves under GL as
well as HL conditions was not significantly altered between
the WT and mutant (data not shown). These observations
indicate that neither a higher reduction state of the primary
electron acceptor QAin PSII, nor a higher rate of cyclic
electron transport, nor photoinhibition occurred in the
mutants under HL.
Excess photosynthetic reductant could be consumed
during the reactions of photorespiration, depending on the
levels of CO2and O2. Gas exchange experiments on leaves
indicated a possibly increased photorespiratory component
in nadp-mdh mutants (data not shown). Therefore, an effect
of HL on photorespiratory activity was further investigated
in protoplasts of nadp-mdh knockout and WT plants. The
photosynthetic rates of mesophyll protoplasts from WT
leaves were not significantly changed at low O2compared
with ambient O2 when assayed at 1 mM bicarbonate in
the assay medium, while in the mutants photosynthesis was
strongly inhibited at low O2 compared with normal air
(Fig. 4A). Further, the transcript levels of GDC1 and GDC2
(P-protein) were higher in the mutant than in the WT after
7 h of HL treatment (Fig. 4B), while the immunoblot indi-
cated only a slight increase or no change in P-protein in the
mutant (Fig. 4C). Other activities related to photorespira-
tion, namely catalase and hydroxypyruvate reductase, were
not affected in whole leaf extracts following 7 h of HL
treatment, either in the WT or in the mutant plants (data
not shown).
Effect of lacking a malate valve on mitochondrial
activities and metabolite levels upon HL treatment
The AOX1A transcript level increased to a similar extent in
both genotypes when transferred from GL to HL (Fig. 5B).
However, the inhibitory effect of SHAM on photosynthesis of
protoplasts was more pronounced in mutants than in the WT
(Fig. 5A), suggesting an important role for mitochondrial
AOX in compensating the loss of chloroplast NADPH-MDH.
This was true both at low O2and in normal air (Fig. 5A).
Metabolite profiling using GC-MS revealed differences in
relative metabolite contents between WT and nadp-mdh plants
under GL (the full data set is presented in Supplementary
Table S2 at JXB online). However, the relative differences in
metabolite contents were pronounced following a 7 h HL
treatment. The most significant metabolic differences between
the WT and mutants are presented in Fig. 6 and include
increases in aspartate, proline (>2-fold), and succinate, along
with decreases in glutamine, 5-oxoproline, malate, and,
tentatively, ascorbate (Fig. 6). The levels of sucrose and
starch were unaltered between mutant and WT plants (Fig. 6,
Table 1). Starch levels decreased to very low levels during the
following dark phase in both genotypes (Table 1).
The decreased malate level in the mutants after 7 h of HL
was confirmed by an enzymatic determination yielding
18.0 lmol g?1fresh weight in the WT and 15.0 lmol g?1
fresh weight in the mutant (Fig. 7A). Intriguingly, there was
a 2.1-fold increase in proline in the mutants following the
exposure to HL, while it increased only 1.5-fold in the WT
(Fig. 7B).
Enzyme activities of alternative shuttle systems
Indirect transfer of reducing equivalents from the chloro-
plast to the cytosol and subsequently into mitochondria
might alternatively be mediated by oxidoreductases other
than MDH, in conjunction with appropriate transporters.
The enzyme activities of NADP-GAPDH of the Calvin
cycle, cytosolic NP-GAPDH, and mitochondrial NAD-
dependent G3PDH, thought to be involved in a mitochon-
drial shuttle for reducing equivalents (Shen et al., 2006)
were therefore determined. The activity of only NADP-
GAPDH was increased in the nadp-mdh mutants (Table 3).
Effect of lacking NADP-MDH on early seedling growth
Although there was no difference in biomass between the
WT and mutants at the mature stage (Fig. 1), in the early
stages of growth the mutants had a clear advantage, either
when directly cultivated as single plantlets on soil, or on
agar under sterile conditions with minimal medium contain-
ing 1.8 mM nitrate (Fig. 8). The mutant seedlings had
a significantly increased biomass after either 4 weeks of
growth on agar or 5 weeks of growth on soil, when grown
under 150 lmol quanta m?2s?1.
Discussion
Flexibility in redox metabolism prevents phenotypic
alterations in nadp-mdh plants
Experimental evidence and theoretical considerations sug-
gest that the malate valve can counteract over-reduction of
the photosynthetic electron transport chain (Scheibe, 2004).
Therefore, it was surprising to observe the WT-like per-
formance of nadp-mdh plants even when cultivated under
HL conditions, for example with respect to photosynthetic
performance and development (Table 1; Fig. 1). Obviously,
these mutants do not use excess reducing equivalents in the
Calvin cycle for CO2fixation and for biomass production as
Table 1. Leaf characteristics of WT and nadp-mdh Arabidopsis
plants
Arabidopsis plants were grown for 11 weeks under GL (50 lmol
quanta m?2s?1), then the leaves were analyzed.
WTnadp-mdh
Specific fresh weight (mg cm?2)
Water content (%)
Chl a (lg cm?2)
Chl b (lg cm?2)
Protein (lg cm?2)
Starch as glucose units (lmol mg?1Chl)
18.0961.14
92.4960.31
5.2161.34
2.1060.55
157.3066.89
27.3560.37a
17.9361.10
92.1160.41
5.5661.66
2.260.59
157.18615.20
27.4962.20a
aAfter 1 d of HL treatment.
1450 | Hebbelmann et al.

Page 7

evidenced by the identical CO2assimilation rates of leaves,
similar photosynthesis rates in isolated protoplasts (Fig. 4A),
and unaltered levels of the photosynthetic products, sucrose
and starch (Fig. 6, Table 1). An inhibitory effect on the
growth rate was detected in antisense tobacco plants
expressing <10% of WT amounts of NADP-MDH when
grown under natural light conditions in a greenhouse
(Faske et al., 1997). Overexpression of NADP-MDH
Fig. 2. Quantification of ROS and the Beck–Halliwell–Asada antioxidant system. (A) Quantitation of ROS in extracts using Amplex Red.
Asterisks indicate that the differences (P < 0.05) between the WT and nadp-mdh mutants are statistically significant as determined by the
t-test. (B) Contents of GSH and GSSG in WT and nadp-mdh plants. Values are presented as the mean 6SD of six individual
determinations per genotype. (C) Semi-quantitative RT-PCR for transcript analysis of Beck–Halliwell–Asada pathway enzymes. RNA was
isolated from WT and mutant plants maintained in GL or transferred to HL for 7 h, transcribed in cDNA, and amplified by PCR at the
linear amplification rate using the primers listed in Supplementary Table S1 at JXB online. The following transcripts were analysed: sAPX
(At4g08390; chloroplast/mitochondria), tAPX (At1g77490; chloroplast), stromal DHAR (At5g16710; chloroplast), MDAR 6 (At1g63940;
chloroplast/mitochondria), GR 2 (At3g54660; chloroplast/mitochondria), and CuZnSOD 2 (At2g28190; chloroplast/apoplast). The result is
representative for two independent experiments. (D) Western blot of an SDS–gel with crude extracts from leaves of WT and nadp-mdh
plants, and immunodecoration with antiserum against APX isoforms. (E) Total APX activity
NADP-malate dehydrogenase knockout induces multiple strategies | 1451

Page 8

stimulated tobacco plant development until the pot size
became limiting (Faske et al., 1997).
In this study, the photosynthetic electron transport was
also broadly unaffected by altered NADPH+H+oxidation
Fig. 3. Transcript and protein levels of the NTRC and chloroplast Prx system. (A) Densitometric analysis of RT-PCR for NTRC, 2-Cys
PrxA/B, PrxQ, and PrxIIE in leaves of WT and nadp-mdh knockout plants after 7 h of HL treatment. Ubiquitin (UBQ) was used as the
reference transcript. (B and C) Protein amounts of NTRC and 2-Cys Prx. Western blot and immunodetection using antiserum against
NTRC and 2-Cys Prx were performed with extracts from WT and nadp-mdh knockout plants after 7 h of HL treatment. In the lower part,
the Coomassie-stained band of the RubisCO large subunit (LSU) is shown as a loading control. Asterisks indicate that the differences
(P < 0.05) between WT and nadp-mdh mutants are statistically significant as determined by the t-test.
Table 2. Photosynthetic parameters of WT and nadp-mdh Arabidopsis plants under GL and HL conditions
Experimenta
50/50750/50 50/750
WT nadp-mdh WTnadp-mdh WTnadp-mdh
Fv/Fv
qP
NPQ
UII
0.8460.00
0.8060.04
0.2660.04
0.6460.01
0.8460.00
0.8260.01
0.1960.04
0.6660.01
0.7560.03
0.8660.04
0.2360.05
0.6160.01
0.7460.03
0.8860.02
0.2760.14
0.6260.04
0.8160.01
0.3260.02
1.9760.14
0.1760.02
0.8160.01
0.3160.03
1.8460.13
0.1660.01
aEach set of data in the three experiments was generated with a different combination of light intensities during pre-treatment and measurement
for WT and mutant plants, e.g. 50/50: pre-treatment of the plants at 50 lmol quanta m?2s?1, measurement at 50 lmol quanta m?2s?1.
1452 | Hebbelmann et al.

Page 9

capacity as an electron acceptor in nadp-mdh plants (Table 2).
Likewise, potato plants expressing minimal amounts of
NADP-MDH (<10% of the WT) are able to adjust photo-
synthesis and CO2assimilation for unaffected performance,
most probably by employing various energy-dissipating
cycles at PSI and PSII (Laisk et al., 2007). Since there is no
evidence for over-reduction at PSII even in the nadp-mdh
A. thaliana plants under HL conditions (Table 2), it is
expected that these mutants use compensatory strategies to
protect themselves from excess reductant in chloroplasts
and subsequent oxidative stress.
A set of diverse mechanisms can help to avoid de-
velopment of oxidative damage (Noctor and Foyer, 1998;
Niyogi, 2000; Scheibe et al., 2005; Hanke et al., 2009;
Scheibe and Dietz, 2011). The analysis of the mutants,
lacking nadp-mdh, revealed a novel combination of different
mechanisms to cope with excess reducing equivalents as
discussed below: (i) a stimulated NTRC/2-Cys Prx system;
(ii) adjustments in photorespiratory metabolism; and (iii)
possibly, proline biosynthesis.
Fig. 5. Components of the mitochondrial electron transport.
(A) Rates of photosynthetic O2evolution by protoplasts from WT
(grey bars) and nadp-mdh knockout mutants (white bars) at an
optimal bicarbonate concentration (1 mM) under either normal O2
(;410 nmol O2ml?1) (empty bars) or low O2(;85 nmol O2ml?1)
(dotted bars) without inhibitors and with SHAM (600 lM). (B)
Effects of HL treatment on AOX1A expression. Northern blot
analysis of WT and nadp-mdh knockout plants after 7 h under GL
(50 lmol quanta m?2s?1) and after 7 h under HL (750 lmol
quanta m?2s?1), respectively. Total RNA was extracted from
leaves. Ethidium bromide staining confirmed equal RNA loading.
The blot was hybridized with an AOX1A-specific probe. Asterisks
indicate that the differences (P < 0.05) between normal and low
oxygen are statistically significant as determined by the t-test.
Fig. 4. Effect of inhibition of photorespiration on photosynthesis in
protoplasts. (A) Rates of photosynthetic O2evolution by proto-
plasts from WT (grey bars) and nadp-mdh knockout mutants
(white bars) at an optimal bicarbonate concentration (1 mM) under
either normal O2(;410 nmol O2ml?1) (empty bars) or low O2
(;85 nmol O2ml?1) (dotted bars). (B) Densitometric analysis of
RT-PCR for GDC1/2 (P-protein) expression in leaves of WT and
nadp-mdh plants after 7 h of HL treatment. Ubiquitin (UBQ)
transcript was used for normalization. (C) Western blot and
immunodetection using antiserum against the P-protein of GDC
were performed with extracts from WT and nadp-mdh knockout
plants after 7 h of HL treatment. The lower part depicts
a Coomassie-stained gel showing the intensity of the band for
RubisCO large subunit (LSU). Data represent mean values (6SE)
from at least three independent experiments. Asterisks indicate
that the differences (P < 0.05) between normal and low oxygen
(in A) and also between WT and nadp-mdh (in B) are statistically
significant as determined by the t-test.
NADP-malate dehydrogenase knockout induces multiple strategies | 1453

Page 10

ROS production and the importance of the NTRC/Prx-
based antioxidative system
One might expect stimulated electron transfer to O2, and
thus increased ROS production and oxidative stress, in
plants lacking the malate valve. Glutathione (GSH) and
ascorbate are important to protect plants from oxidative
damage (Noctor and Foyer, 1998; Mullineaux and Rausch,
2005), through the Beck–Halliwell–Asada pathway (Foyer
and Halliwell, 1976) and in the GSH–glutaredoxin-type II
Prx pathway (Tripathi et al., 2009). However, there were no
marked changes in either glutathione (Fig. 2B) or ascorbate/
DHA (Fig. 6). Expression of various enzymes of the Beck–
Halliwell–Asada cycle in nadp-mdh plants was also unchanged,
even in HL (Fig. 2C–E). This suggests that the ROS-
scavenging systems, based on the ascorbate/glutathione and
Beck–Halliwell–Asada pathway, are unaffected in nadp-mdh
plants. However, the mutant plants showed increased levels
of the NTRC/Prx system.
Fig. 6. Normalized metabolite contents of WT and nadph-mdh plants after 7 h of HL treatment. Relative metabolite contents were
determined in leaf discs of 11-week-old WT plants (grey bars) and nadp-mdh mutants (white bars). Data were normalized with respect to
the mean response calculated for the WT. Values are presented as the mean 6SE of n¼6 per genotype. An asterisk indicates values that
were determined by the t-test to be significantly different (P < 0.05) from the WT.
Fig. 7. Malate and proline content. (A) Malate content in leaves of
WT (grey bars) and nadp-mdh mutants (white bars) under GL and
under 7 h HL, respectively. (B) Proline content in WT and nadp-
mdh mutants after 7 h under GL and under HL, respectively.
Asterisks indicate that the differences (P < 0.05) between WT and
nadp-mdh mutants are statistically significant as determined by
the t-test.
Table 3. Activities (mU mg?1protein) of oxidoreductases possibly
involved in redox shuttles
WTnadp-mdh
NADP-GAPDH
NP-GAPDH
NAD-G3PDH
167.63617.03
24.3862.29
7.4761.26
232.06616.70
22.5761.14
6.3862.16
1454 | Hebbelmann et al.

Page 11

Besides the Beck–Halliwell–Asada pathway, the NTRC/
Prx system quenches H2O2in the chloroplast (Ko ¨nig et al.,
2002; Serrato et al., 2004; Pe ´rez-Ruiz et al., 2006) and this
peroxide detoxification cycle was named the ascorbate-
independent or Prx-dependent water–water cycle (Dietz
et al., 2006). Recent studies using knock-down plants show
that the NTRC-dependent regeneration of 2-Cys Prx is
more important thanthe
(Pulido et al., 2010). In nadp-mdh mutants under HL, 2-Cys
Prx B and NTRC transcripts, as well as NTRC protein
increased compared with WT plants (Fig. 3A, B). Data
from Spinola et al. (2008) indicate that the NTRC system
plays a specific role in eliminating ROS in the dark, and this
point explains that stress treatment in the dark has more
effect on Arabidopsis ntrc knockout lines than on WT plants
(Pe ´rez-Riuz et al., 2006). In nadp-mdh plants too, the Prx-
dependent water–water cycle appears to function as an
alternative poising system that is increased to use up
NADPH when malate cannot be formed (Fig. 9). Transcript
profiling gave no additional clues as to which alternative
pathways might have been up-regulated (Supplementary
Fig. S3 at JXB online).
thioredoxin-dependent one
Adjustments in components of photorespiration,
mitochondria, and proline biosynthesis may all
compensate for the lack of malate valve
Despite being a major source of ROS, photorespiration is
crucial for maintaining the redox state in plant cells (Foyer
et al., 2009; Bauwe et al., 2010). Therefore, in nadp-mdh
plants, the photorespiratory pathway may provide an alter-
native mechanism to transport excess reducing equivalents
from the chloroplast to the mitochondrion, facilitating
electron transfer to O2via the cytochrome c oxidase path-
way or AOX (Fig. 9). Transcript profiling of mutant plants
lacking NTRC had increased transcript levels for photo-
respiratory genes such as those coding for catalase, P-protein,
and hydroxypyruvate reductase, and showed multiple signs
of metabolic imbalances (Lepisto ¨ et al., 2009). The marked
decrease in photosynthesis of protoplasts under low oxygen
(Fig. 4A), high expression of GDC (Figs. 4B, C), and a shift
in the glycine-to-serine ratio (Fig. 6) suggest altered patterns
in photorespiration of nadp-mdh mutants.
Inhibition of AOX or lack of the AOX1A isoform in
mitochondria of transgenic plants is known to cause over-
reduction of the photosynthetic electron transport chain in the
light (Padmasreee and Raghavendra, 1999; Yoshida et al., 2007;
Strodtko ¨tter et al., 2009). The increase in protein and activity of
AOX under HL and drought (Clifton et al., 2006; Giraud et al.,
2008) suggest that AOX plays an important role in the
consumption of excess reducing equivalents exported from the
chloroplasts in light. Although the malate valve-dependent
transport was disabled, the increase in AOX1A transcript levels
in nadp-mdh mutants was similar to that in WT plants after HL
treatment (Fig. 6C), indicating activation of alternative path-
ways for NADH re-oxidation in the mitochondria of both the
WT and mutants. Interestingly, transgenic tomato plants with
decreased mitochondrial NAD-MDH exhibited even en-
hanced photosynthetic performance (Nunes-Nesi et al.,
2005), indicating the redundancy of some of the oxidative
processes involved in optimizing photosynthesis. In double
mutants expressing neither mitochondrial MDH isoform,
photorespiration was increased (Tomaz et al., 2010).
Possible function of proline in nadp-mdh knockout
plants
The increased proline contents of nadp-mdh mutants in HL
(Figs 6, 7B) might represent a strategy of the plants to
increase stress tolerance, since proline functions as a compat-
ible solute in drought/salt stress (Hare et al., 1999) and
stabilizes the redox status of the cell (Bellinger and Larher,
1987; Hare et al., 1999; Szabados and Savoure ´, 2009). Further
experiments are needed to confirm that the accumulation of
proline in the nadp-mdh mutants under HL can help to
consume excess NADPH (Fig. 9).
Early seedling growth
A highly reproducible increase in biomass occurred in the
mutant plants during early development when grown on
soil, or on agar under sterile conditions with 1.8 mM nitrate
Fig. 8. Seedling growth on agar under sterile conditions and on
soil. (A) Sterilized seeds were placed on agar containing 1.8 mM
nitrate as an N source, and seedlings were grown for 5 weeks
under short-day conditions in sealed Petri dishes. The fresh weight
of 100 seedlings of each genotype with standard deviations is
given. (B) Seeds were grown in pots with soil, and the fresh weight
of the above-ground biomass was determined for 100 seedlings of
each genotype at 5 weeks of age. Asterisks indicate that the
differences (P < 0.05) between WT and nadp-mdh mutants are
statistically significant as determined by the t-test.
NADP-malate dehydrogenase knockout induces multiple strategies | 1455

Page 12

in minimal medium (Fig. 8). The compensatory responses in
the nadph-mdh plants seem to become established early to
achieve a metabolic pattern and altered redox state which
are even beneficial under certain conditions.
Conclusions
The nadp-mdh mutants employ a combination of mechanisms
to compensate for the lack of the malate valve function under
HL conditions. Higher capacity of the NTRC system, along
with increased expression of Prxs could facilitate balancing
the NADP/NADPH ratio in the chloroplasts and sustain
ROS scavenging. Adjustments in photorespiratory compo-
nents might also reflect the need to dissipate excess reducing
equivalents and prevent photoinhibition. Further experiments
are required to establish the role of proline as a protective
mechanism against oxidative stress in nadp-mdh mutants. It is
suggested that ROS may act as a ‘prime’ to trigger changes
initially in chloroplasts and then in other compartments of
plant cells, resulting in acclimation of nadp-mdh plants.
Supplementary data
Supplementary data are available at JXB online.
Figure S1. PCR analyses for proof of homozygous T-DNA
insertion and NADP-MDH gene knockout.
Figure S2. Northern blot (A) and western blot with
immunodecoration (B) to document the NADP-MDH
(At5g58330) gene knockout in the lines 119 (Salk_063444)
and 50 (Salk_012655).
Figure S3. Heatmap showing up- and down-regulated genes
in the HL-treated (7 h at 1000 lmol quanta m?2s?1) nadp-
mdh versus WT plants grown for 5 weeks under 100 lmol
quanta m?2s?1, 8/16 h light/dark rhythm. Results from three
independent experiments with | log2(expression ratio) | > 1
and P-value <0.06 are shown. The analysis was run in
R/BioConductor using topTable and heatmap.2 functions.
Table S1. Oligonucleotides used for RT-PCR.
Table S2. Relative metabolite content in WT and nadp-
mdh mutants grown under low light (GL) and exposed to
HL for 7 h.
Acknowledgements
This study was financially supported by the Deutsche
Forschungsgemeinschaft to V.L. and R.S. (EM166/1),
grants from the Department of Science and Technology-
DeutscherAkademischerAustauschdienst
A.S.R. and R.S.), the DAAD-Finland program (to R.S.
and E.-M.A.), and by a DFG-Mercator Visiting Professor-
ship to A.S.R. Funds were also received from the Academy
of Finland to E.-M.A. (118637, 8133293), P.M. (130075),
and S.K. (130595). The authors thank Dr Francisco Javier
Cejudo (Sevilla) for the gift of anti-NTRC serum as well as
probes for NTRC, and Dr Eevi Rintama ¨ki for the antibody
against Arabidopsis APXs. We thank Professor Hermann
Bauwe (Rostock) for probes and antiserum against GDC
(P-protein). We also thank Guiseppe Forlani (Ferrara,
Italy) for providing P5C, and Dietmar Funck (Konstanz,
Germany) for advice concerning proline metabolic enzyme
measurements. Thanks are due to Silke Walter for expert
technical assistance. We also thank Heike Wolf-Wibbel-
mann and Kirsten Ja ¨ger for cultivating the plants, and
(DAAD,to
Fig. 9. Overview of compensatory pathways which prevent over-reduction and overenergization of the chloroplast in nadp-mdh
knockout mutants. The pathways which are stimulated in mutants are all shown by red arrows, normal pathways in black, and
suppressed routes in grey. See text for further description.
1456 | Hebbelmann et al.

Page 13

Heike Schwiderski and Nicolas Ko ¨nig for their help with
the preparation of the manuscript.
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