Journal of Experimental Botany, Vol. 64, No. 12, pp. 3605–3614, 2013
doi:10.1093/jxb/ert194 Advance Access publication 12 July, 2013
Research paper
Analysis of knockout mutants suggests that Arabidopsis
NADP-MALIC ENZYME2 does not play an essential
role in responses to oxidative stress of intracellular or
extracellular origin
Shengchun Li1,*, Amna Mhamdi1,*, Cyndie Clement2,3,4, Yves Jolivet2,3,4 and Graham Noctor1,†
1
Institut de Biologie des Plantes, Université de Paris sud, UMR CNRS 8618, 91405 Orsay cedex, France
Université de Lorraine, UMR1137 EEF, F-54500 Vandoeuvre-lès-Nancy, Cedex, France
3
INRA, UMR1137 EEF, F-54280 Champenoux, France
4
IFR110 EFABA, F-54500 Vandoeuvre-lès-Nancy, Cedex, France
2
* These authors contributed equally to this work.
Author for correspondence: [email protected]
† Received 8 April 2013; Revised 20 May 2013; Accepted 28 May 2013
Abstract
NADPH is a pivotal molecule in oxidative stress, during which it is potentially produced by several cytosolic NADPlinked dehydrogenases. This study investigated the response and functional importance of the major leaf cytosolic
NADP-malic enzyme in Arabidopsis (NADP-ME2) during oxidative stress. Data from both microarray and targeted
quantitative PCR analyses showed that NADP-ME2 transcripts accumulated in response to ozone or in mutants
undergoing intracellular oxidative stress. To test the functional importance of this response, loss-of-function nadpme2 mutants were obtained and the effects of oxidative stress of intracellular and extracellular origin were tested.
Despite much decreased leaf NADP-ME activity, nadp-me2 showed a wild-type phenotype when exposed to ozone.
Introduction of the nadp-me2 mutations into the catalase-deficient cat2 background did not alter growth inhibition
or lesions triggered by intracellular oxidative stress. Similarly, loss of NADP-ME2 function had little effect on cat2triggered changes in glutathione or NADPH. While single nadp-me2 mutations produced slight effects on basal resistance to one type of bacteria, they did not affect resistance induced by the cat2 mutation. Taken together, the results
suggest that, although NADP-ME2 induction is part of the response to oxidative stress, the enzyme is not an essential
determinant of the outcome of such stress.
Key words: Glutathione, H2O2, NADP(H), ozone, photorespiration, redox homeostasis.
Introduction
NADP(H) is a key player both in assimilatory metabolism
and in cellular redox homeostasis. In the chloroplast in the
light, ferredoxin-NADP+ reductase generates the reduced
form, NADPH, which then mainly powers the reduction of
1,3-bis-phosphoglycerate in the reaction catalysed by the stromal NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In the dark, or in non-photosynthetic
tissues, enzymes such as glucose-6-phosphate dehydrogenase (G6PDH) play important roles in converting plastidial
NADP+ to NADPH (Anderson and Duggan, 1976; Von
Schaewen et al., 1995; Wakao and Benning, 2005). While
attention has been paid to the roles of NADP-linked enzymes
in other highly redox-active organelles, such as the mitochondria and peroxisomes (Møller and Rasmusson, 1998; Meyer
Abbreviations: CAT, catalase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; G6PDH, glucose-6-phosphate dehydrogenase; GR, glutathione reductase;
GSH, glutathione; GSSG, glutathione disulphide; ICDH, isocitrate dehydrogenase; ME, malic enzyme.
© The Author [2013]. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved.
For permissions, please email: [email protected]
3606 | Li et al.
et al., 2011), less is known about the production and turnover of NADP(H) in the cytosol. As many signalling processes
occur in the cytosol, and because cytosolic and nuclear redox
states are likely to be closely linked, identifying the factors determining NADP(H) turnover in this compartment
remains key to understanding plant stress responses (Foyer
and Noctor, 2009). In particular, an important gap in our
knowledge is uncertainty over which enzymes are the most
important in producing reducing power to meet the increased
demand during stress (Valderrama et al., 2006, Dizengremel
et al., 2009). As well as being required to maintain the pools of
antioxidants such as ascorbate and glutathione in the reduced
form (Noctor, 2006), NADPH is considered to be the main
source of electrons for respiratory burst oxidase homologues,
also known as NADPH oxidases, which among other things
play important roles in biotic stress (Torres et al., 2006).
There are at least four major types of cytosolic NADPlinked dehydrogenase that can oxidize various carbon substrates to generate reducing power in the form of NADPH.
As well as cytosolic G6PDH, the major form of leaf NADPlinked isocitrate dehydrogenase (cICDH) is found in this compartment (Hodges et al., 2003; Mhamdi et al., 2010a). Plants
also have a cytosolic non-phosphorylating NADP-GAPDH,
in addition to the classical NAD-linked enzyme (Kelly and
Gibbs, 1973; Rius et al., 2006). A fourth type of cytosolic
NAPDH-producing dehydrogenase is NADP-linked malic
enzyme (NADP-ME), which oxidatively decarboxylates
malate to pyruvate (Gerrard Wheeler et al., 2005, 2008, 2009).
While NAD- and NADP-dependent malic enzymes have long
been known to play key roles in C4 photosynthetic metabolism (Furbank and Foyer, 1988; Dever et al., 1995; Langdale,
2011; Maier et al., 2011), the roles of NADP-ME in C3 plants
are much less clear.
One possible function of C3-type NADP-ME is in plant
defence (Casati et al., 1999). Roles for NADP-ME in stress
responses have received support from studies that have shown
that the enzyme is upregulated by various environmental
challenges, including pathogen attack (Voll et al., 2012, and
references therein). However, such data can only provide correlative evidence that any given enzyme activity is important,
and the redox network may be composed of a complex matrix
of functionally interacting components that show complete
or partial redundancy. In olive plants subject to salt stress, for
example, multiple NADPH-generating activities are induced
in concert (Valderrama et al., 2006). Within this complex
redox network, knockout mutants are useful tools to identify
the most important players or to establish functional redundancy. Studies using this approach suggest that cICDH plays a
non-replaceable role in response to biotic and oxidative stress.
Loss-of-function icdh mutations in Arabidopsis cause activation of pathogenesis-related responses and bacterial resistance,
and alter the glutathione redox state during oxidative stress
(Mhamdi et al., 2010a; Dghim et al., 2013). In a similar vein,
a recent study reported altered responses to fungal infection
in Arabidopsis nadp-me2 knockout mutants (Voll et al., 2012).
However, it remains unclear whether NADP-ME2 plays an
important role in influencing the outcomes of oxidative stress,
a key factor in unfavourable environmental conditions.
The aim of the present study was to address this specific
question. To establish whether or not the major Arabidopsis
leaf malic enzyme (NADP-ME2) plays a major role during
biotic and oxidative stress, knockout mutants were subjected
to challenge with three bacterial strains as well as to two
independent types of stress that are triggered by increased
cellular oxidation: ozone exposure and a catalase-deficient
genetic background (cat2). The data showed that, while the
NADP-ME2 gene is induced both by exposure to ozone and
in cat2, loss of its function had only slight effects on resistance to bacteria and had little effect on the cellular redox
state or phenotypes determined by oxidative stress. Thus,
while induction of NADP-ME2 is part of the oxidative stress
responses, the enzyme does not appear to be an indispensable
player in these conditions.
Materials and methods
Plant material and growth conditions
All genotypes were in the Arabidopsis Columbia (Col-0) ecotype.
The cat2 mutant was cat2-2 (Queval et al., 2009), while two independent T-DNA lines for NADP-ME2 were obtained from the Salk
collection (SALK_073818 and SALK_020607). Double cat2 nadpme2 mutants were produced by crossing. After verification of the
double heterozygotes in F1 plants by PCR, double homozygotes
were identified similarly in the F2 generation (Supplementary Fig.
S1 at JXB online) and allowed to produce F3 seeds, which were
used for experiments. All seeds were sown on soil, incubated for 2
d in the dark at 4 °C, and then transferred to a controlled-environment growth chamber with long days (16 h light/8 h dark) and an
irradiance of 200 μmol m–2 s–1 at the leaf level, 20/18 °C, and 65%
humidity. Plants were supplied with nutrient solution twice weekly.
Following snap freezing in liquid nitrogen, samples were stored at
–80 °C until analysis. All data are means ±standard error (SE) of at
least three independent samples from different plants.
Ozone treatment
Ozone treatments were performed in phytotron chambers constantly
ventilated with charcoal-filtered air. The treatment (350 ppb) was
begun following a 7-d acclimation period. Ozone was produced from
pure O2 with two ozone generators (OZ500, Fischer, Bonn, German;
CMG3-3; Innovatec II, Rheinbach, Germany) and injected directly
along with the filtered air entering the chambers. Control plants
were exposed to ambient filtered air. A set of automated systems
and analysers (O341M; Environment S.A. Paris, France) were used
to monitor the concentrations and the length of ozone exposure.
Fumigation started 1 h after the beginning of the photoperiod (16 h
light/8 h dark). Ozone concentration was maintained at 350 ± 10 ppb
of ozone for 7 h (8 h into the photoperiod). Two 7 h fumigations
were performed on consecutive days. Prior to sampling, plants were
allowed to recover overnight following the second fumigation.
Quantification of lesions
The percentage of total rosette area displaying lesions during ozone
stress or in the cat2 background was quantified by imaging affected
areas of at least ten plants of each type using IQmaterials software.
Transcript analysis
Whole rosettes of four replicate plants per treatment were harvested
and frozen immediately in liquid nitrogen. Total RNA extraction
and quantitative reverse transcriptase (RT)-PCR were conducted as
described by Queval et al. (2007). The primer sequences are shown in
NADP-malic enzyme and oxidative stress | 3607
7
6
5
4
9
8
Pst-avrRpm1
7
6
5
4
9
8
Psm-ES4326
(1)
7
6
5
4
9
8
* *
Psm-ES4326
(2)
7
6
5
nadp-me2-1
nadp-me2-2
4
Col-0
Of the four NADP-ME genes found in Arabidopsis,
NADP-ME4 encodes a plastidial enzyme while the other
three enzymes are predicted to be localized in the cytosol
(Gerrard Wheeler et al., 2005). Of these three, NADP-ME2
encodes the major enzyme in leaf tissues (Gerrard Wheeler
et al., 2005; Voll et al., 2012). Consistent with this,
Genevestigator analysis (Hruz et al., 2008) showed that
NADP-ME2 was the most highly expressed in most tissues
and also during stress conditions (Supplementary Fig. S2A,
B, at JXB online). RT-PCR analysis in the growth conditions used in this study showed that, while NADP-ME2 transcripts were readily detected, NADP-ME1 and NADP-ME3
were expressed at very low levels in leaves (Supplementary
Fig. S2C). Knockout mutants for NADP-ME2 show less
than 10% wild-type extractable leaf activity (Voll et al.,
2012). The absence of detectable transcripts was confirmed
in the nadp-me2 mutants by RT-PCR (Supplementary Fig.
S3 at JXB online).
The knockout mutants were used to analyse the potential
role of NADP-ME2 in response to biotic and oxidative stress.
First, the impact on the response to bacterial challenge was
analysed. Two experiments, each based on quadruplicate
samples, were performed for three bacterial strains. Control
samples were taken immediately following bacterial inoculation (0 h post-inoculation; Fig.1, left panels) and bacterial
proliferation was analysed in samples taken 24 or 48 h later
(Fig. 1, right panels). For avirulent and virulent Pst, no significant difference between Col-0 and the nadp-me2 mutant lines
was observed in either experiment. For Psm, one experiment
revealed no significant difference, while the second showed
slightly but significantly increased bacterial growth in the
nadp-me2 mutants (Fig. 1).
Pst-DC3000
nadp-me2-1
Results
8
nadp-me2-2
Enzyme and metabolite assays
Soluble protein and extractable NADP-malic enzyme activity was
measured as described by Dghim et al. (2013). Pyridine nucleotides,
ascorbate, and glutathione were assayed spectrophotometrically
using a plate-reader protocol as described in detail by Queval and
Noctor (2007).
9
Col-0
Pathogen tests
Three strains of Pseudomonas syringae were used in this study. To
test resistance to an avirulent bacterium, P. syringae pv. tomato
(Pst) strain DC3000 avrRpm1 was used. Resistance to a virulent
bacterium was tested using Pst strain DC3000, while P. syringae pv.
maculicola ES4326 (Psm) was employed as a second, less virulent
strain than P. syringae DC3000. Bacteria were selected on medium
containing 50 mg l–1 of rifampicin (Psm) or 100 mg l–1 of rifampicin
and 25 mg l–1 of kanamycin (Pst). Using a 1 ml syringe with no needle, the central leaves of five to seven plants of each genotype were
inoculated with bacteria. Leaf discs were taken for analysis either
immediately after inoculation (0 h, control for possible differences in
bacterial entry) and 24 or 48 h later to quantify bacterial proliferation in planta.
Ozone is an important pollutant whose stressful effects on
plants involve initial oxidation in the apoplast followed by
adjustments in intracellular metabolism, notably involving
respiratory pathways (Kangasjärvi et al., 2005; Dizengremel
et al., 2009). Exposure of Arabidopsis to ozone caused visible lesions to appear on the leaves, but there was no significant difference in the extent of lesions between Col-0 and
the nadp-me2 mutant (Fig. 2A, B). Total extractable leaf
Colony forming units (log 10 cm-2 leaf area)
Supplementary Table S1 at JXB online. Microarray data were analysed in a dataset of which the other features were described previously by Mhamdi et al. (2010b).
Fig. 1. Effect of nadp-me2 mutations on challenge with three
different bacterial strains. Shaded bars, Col-0; filled bars,
nadp-me2 mutants. Left, leaves sampled at 0 h post-inoculation.
Right, leaves sampled at 24 (Pst-DC3000) or 48 (others) h postinoculation. *Significant difference from Col-0 at P <0.05. For
Pst-DC3000 and Pst-avrRpm1, each graph shows the results of
one of two repeat experiments in which no significant difference
was observed. For Psm-ES4326, the two individual experiments
gave slightly different results, and so both are shown.
3608 | Li et al.
A
Control
Control
Col-0
nadp-me2-1
C
5
*
0
0,4
0,2
0,0
Col-0 + O3
10
*
Col-0
15
Transcript abundance
rel. to ACTIN2
nd
0,6
*
Col-0
Col-0 + O3
NADP-ME activity
nkat.g-1 FW
nd
nadp-me2-1
nadp-me2-1 + O3
0
Col-0
Col-0 + O3
Lesion area (%)
3
nadp-me2-1
D
20
6
+ Ozone
Col-0
nadp-me2-1
nadp-me2-1 + O3
B
+ Ozone
Fig. 2. Ozone responses of NADP-ME in Col-0 and nadp-me2-1 plants. (A) Representative photographs of plants before and
after ozone treatments. (B) Quantification of ozone-induced leaf damage. (C) Extractable NADP-ME enzyme activities in Col-0 and
nadp-me2-1 plants before and after ozone treatment. (D) NADP-ME2 transcript abundance in Col-0 plants. *Significant difference from
Col-0 at P <0.05.
NADP-ME activity was enhanced by ozone exposure in Col0, while remaining at very low levels in nadp-me2 (Fig. 2C).
Quantitative (q)RT-PCR analysis showed that the ozoneinduced increase in activity in Col-0 was accompanied by
enhanced NADP-ME2 transcript abundance (Fig. 2D).
Given its induction by ozone, the response of NADP-ME2
was examined under conditions where oxidative stress is initiated by intracellular H2O2 production. This was done by
exploiting the Arabidopsis cat2 mutant, a stress-mimic model
that is a useful system for uncovering the functions of enzymes
involved in producing reductant during oxidative stress
(Mhamdi et al., 2010a,b,c). In microarray analyses of cat2,
NADP-ME2 showed some induction, although the increase
was not statistically significant at the P <0.05 level compared
with the Col-0 control (Fig. 3, left). However, a statistically
significant induction of NADP-ME2 was observed in a double cat2 gr1 mutant, which showed exacerbated intracellular oxidative stress caused by additional loss of glutathione
reductase 1 activity (Fig. 3, left). A more sensitive quantification of responses in the single cat2 mutant using qRT-PCR
showed that NADP-ME2 was significantly induced, to more
than twofold the wild-type values (Fig. 3, right).
To establish whether NADP-ME2 is functionally important in response to intracellular oxidative stress, the two
allelic nadp-me2 mutants were each crossed into the cat2
background, and the effects on phenotype and redox state
were examined. Oxidative stress in the cat2 mutant induces
visible phenotypes of decreased growth and characteristic lesions that appear on the leaves (Queval et al., 2007;
Chaouch et al., 2010). These responses are modulated by the
introduction of secondary mutations for several NADP(H)linked enzymes located in the cytosol or at the plasmalemma
(Mhamdi et al., 2010a,b; Chaouch et al., 2012). However, neither the decreased rosette size nor lesion spread observed in
cat2 was affected by the introduction of the secondary nadpme2 mutations (Fig. 4).
NADP-malic enzyme and oxidative stress | 3609
1
A
2
3
4
5
6
NADP-ME2
*
0.4
ACTIN2
0.2
0
Fig. 3. Response of NADP-ME2 transcript abundance to
intracellular oxidative stress. Left, microarray analysis of
transcripts in cat2 and cat2 gr1 plants. Right, qRT-PCR analysis
of NADP-ME2 transcripts in Col-0 and cat2 plants. *Significant
difference from Col-0 at P <0.05.
Col-0
cat2
nadp-me2-1
cat2 nadp-me2-1
nadp-me2-2
cat2 nadp-me2-2
C
600
12
400
8
200
4
0
nd
1 2 34 5 6
nd
nd
1 2 34 5 6
Lesion area
(%)
The potential impact of the nadp-me2 mutations on oxidative stress-induced changes in cellular redox state was
explored. In agreement with other studies in barley and
tobacco (Smith et al., 1984; Willekens et al., 1997), the
clearest and most reproducible biochemical effect of leaf
catalase deficiency in Arabidopsis was on glutathione. In
cat2, this key redox marker of intracellular redox state
becomes more oxidized and the disulphide form (GSSG)
typically accumulates to 10- to 20-fold higher levels than
in Col-0 (Queval et al., 2007; Fig. 5). In contrast to glutathione, changes in H2O2 are much less apparent or undetectable in cat2, reflecting the difficulty of quantifying this
reactive molecule and/or its rapid removal by reducing
enzymes in the absence of catalase-dependent dismutation (Chaouch et al., 2010; Han et al., 2013). In the Col-0
background, one of the nadp-me2 mutants had a decreased
content of glutathione (Fig. 5), but neither showed a significant change in glutathione redox state. In the cat2 oxidative stress background, neither allelic nadp-me2 mutation
affected the accumulation of GSSG, and the glutathione
redox state was similar in all three cat2 backgrounds, at
about 60% glutathione (Fig. 5). No significant effects of
any of the mutations on ascorbate or NAD(H) pools were
observed (Fig. 5). While neither single nadp-me2 mutant
showed a significant change in leaf NADP(H), the cat2
mutant showed a tendency towards an increase in the total
pool, and this was associated with significantly increased
NADPH compared with Col-0. A similar effect was
observed in cat2 nadp-me2-1 but was less evident in cat2
nadp-me2-2 (Fig. 5).
Oxidative stress in cat2 leads to induction of resistance to
virulent bacteria above basal levels, and metabolite profiling
suggests that this response shows many of the hallmarks of
induced resistance triggered by biotic challenge (Chaouch
et al., 2010, 2012). To analyse whether the nadp-me2 mutations affected this response, bacterial growth was analysed
in cat2 and the double mutants. While cat2 showed lower
growth of both Pst-DC3000 and Psm relative to Col-0, this
enhanced resistance was not affected by the secondary nadpme2 mutations (Fig. 6).
B
Rosette FW
(mg)
0
cat2 gr1
0.5
qRT-PCR
cat2
*
Rel. ACTIN2
1.0
0.6
Col-0
Microarray
cat2
Log2 transcript
(rel. Col-0)
1.5
0
Fig. 4. Characterization of cat2 nadp-me2 double mutants.
(A) NADP-ME2 transcript levels in Col-0 and single and
double mutants. Lanes: 1, Col-0; 2, cat2; 3, nadp-me2-1;
4, cat2 nadp-me2-1; 5, nadp-me2-2; 6, cat2 nadp-me2-2.
(B) Representative photographs of plants. (C) Rosette fresh weight
(left) and H2O2-triggered lesions (right) in cat2 and the double
mutants. No significant differences in either rosette size or lesion
extent were observed (n≥10 plants). Genotypes are indicated by
the same numbers as in (A).
Discussion
Both detoxification and signalling processes linked to oxidative stress depend on NADPH production, but the relative importance of different NADPH-generating enzymes
remains unclear (Foyer and Noctor, 2009). Some conclusions
have been drawn based on the relative extractable activities
of NADP-generating enzymes (Valderrama et al., 2006;
Dizengremel et al., 2009). While such data may be useful
pointers, for several reasons it is not possible to reach definitive conclusions based solely on them. Measurable in vitro
enzyme activities are often a composite of several isoforms
3610 | Li et al.
8
Pst-DC3000
* * *
7
6
5
9
8
Psm-ES4326
* * *
cat2 nadp-me2-1
4
cat2 nadp-me2-1
Colony forming units (log 10 cm-2 leaf area)
9
7
6
5
cat2 nadp-me2-2
cat2
cat2 nadp-me2-2
cat2
4
Fig. 6. Impact of nadp-me mutations on cat2-induced resistance
to Pst-DC3000 and Psm-ES4326. Open bars, cat2; filled bars,
cat2 nadp-me double mutants. Left, leaves sampled at 0 hours
post-inoculation; right, leaves sampled at 24 (Pst-DC3000) or
48 (Psm-ES4326) h post-inoculation. *Significant difference from
Col-0 at P <0.05 (for both bacteria, growth was significantly lower
in cat2 backgrounds than in Col-0). Dotted lines indicate the Col-0
value.
Post-translational regulation, well known for the chloroplast
G6PDH (Anderson and Duggan, 1976; Von Schaewen et al.,
1995; Wenderoth et al., 1997), may also modify in vivo activities compared with those measured in vitro. For these reasons,
a genetic approach able to target specific isoforms can provide important information on the functional importance of
a specific enzyme. Using this strategy, evidence was reported
that cytosolic NADP-ICDH plays a non-redundant role in
oxidative stress responses and signalling (Mhamdi et al.,
2010a). The present genetically based study of NADP-ME2
allows the following conclusions to be drawn.
Cytosolic NADP-ME is induced by oxidative stress of
intracellular and extracellular origin
Fig. 5. Redox profiling of Col-0, nadp-me2, cat2, and cat2
nadp-me2 double mutants. Open bars, reduced forms; filled bars,
oxidized forms. Significant differences are indicated at P <0.05
by black symbols on the white bars (reduced forms) and white
symbols on the black bars (oxidized forms). *Comparison of
mutants with Col-0. +Comparison of double mutants with cat2.
located in different subcellular compartments. Secondly, such
assays are usually carried out at optimal substrate concentrations, whereas these may be less optimal or variable in vivo.
Previous work has established that NADP-ME2 is induced
by various stresses (Voll et al., 2012, and references therein).
The present work focused on inducibility by oxidative stress,
because this is a component common to many environmentally induced challenges. Both in response to ozone, whose
primary action is at the cell surface, and in the cat2 mutant,
where the initial oxidative trigger is peroxisomal, NADP-ME2
was significantly induced, by several fold. In the case of
ozone, this induction at the transcript level appeared to be of
significance for enzyme capacity, because it was accompanied
NADP-malic enzyme and oxidative stress | 3611
by an increase in extractable NADP-ME activity, an effect
that was not observed in the nadp-me2 knockout (Fig. 2).
Thus, increased NADP-ME activity seems to be part of the
oxidative stress responses, and this increase is linked to induction of NADP-ME2 at the transcriptional level.
A search of available microarray datasets for the cat2
mutant showed that NADP-ME2 was the only ME-encoding
gene that was significantly induced. Indeed, of all the candidate NADPH-generating enzymes in the cytosol, NADP-ME2
showed the clearest response. Despite this, it seems that the
responses of Arabidopsis to oxidative stress are little affected
by the loss of its function.
NADP-ME does not play an irreplaceable role in
responses to oxidative stress
Ectopic expression of NADP-ME2 has been reported to
enhance stress tolerance (Laporte et al., 2002; Liu et al., 2007).
However, such studies cannot provide information on genespecific functions. Using a specific loss-of-function approach,
NADP-ME2 was shown to be required for responses of
Arabidopsis to fungal infection (Voll et al., 2012). The present data on resistance to different bacteria also suggest
some role for the enzyme in biotic stress responses, although
statistically significant increases in pathogen growth were
observed in nadp-me2 compared with Col-0 only in one of
two experiments in which responses to Psm were analysed
(Fig. 1). A key point may be timing, as the effects on fungal responses were observed during the very early signalling
events that occur in the first few minutes after infection (Voll
et al., 2012). However, plants can be exposed to prolonged
oxidative stress in the natural environment, and this may be
a major determinant of plant performance and yield (Foyer
and Noctor, 2009). Such conditions can occur, for example,
during chronic exposure to ozone. Despite its induction by
ozone treatment, loss of NADP-ME2 function produced no
effect on the visible symptoms produced by exposure to elevated doses of this oxidizing pollutant.
The cat2 mutant is a useful system for evaluating potential
functions of other antioxidative enzymes. When CAT2 is functional, it prevents photorespiration-linked oxidative stress in
optimal conditions (Vandenabeele et al., 2004; Queval et al.,
2007). As the other two catalase genes (CAT1 and CAT3) are
not thought to be expressed at appreciable levels in photosynthetic cells (Mhamdi et al., 2010c), loss of CAT2 function
places a greater load on pathways that supply reductants to
peroxidases, and this is notably reflected in the shift of cellular
thiol-disulfide status towards an oxidized condition (Fig. 5).
This increased oxidative load provides a useful context for
evaluating the potential functions of reductant-generating
enzymes. For example, loss of glutathione reductase 1 function in gr1 knockout mutants has in itself no obvious effect on
plant phenotype, but it dramatically impacts on phenotypes
and redox state in the cat2 background (Mhamdi et al., 2010b).
With respect to NADPH-producing enzymes, loss of cytosolic
NADP-ICDH function also modulates responses in cat2:
both pathogenesis responses and glutathione oxidation are
reinforced in double cat2 icdh mutants (Mhamdi et al., 2010a).
The gr1 and icdh mutations also modulate ozone responses in
Arabidopsis (Dghim et al., 2013), showing that their impact
during oxidative stress is not limited to the cat2 background.
Another NADPH-linked enzyme, the NADPH oxidase
encoded by AtRbohF, also produces specific effects on cat2triggered redox and pathogenesis responses (Chaouch et al.,
2012). In contrast to these previous observations, and despite
its clear inducibility by oxidative stress, loss of NADP-ME2
function produced little or no effect on cat2 characteristics.
Introduction of this secondary mutation did not alter cat2
phenotypes (Fig. 4) or affect cat2-induced resistance to bacteria (Fig. 6). A previous study of nadp-me2 mutants reported
increases in the carbon substrate, malate, and decreases in the
carbon product, pyruvate (Voll et al., 2012), suggesting that the
enzyme contributes to respiratory pathways in vivo. However,
no data were presented on NADP(H) or related redox pools.
Similar to the lack of effect on cat2 phenotypes, introduction
of the secondary nadp-me2 mutations had little effect on redox
profiles. Although one double mutant showed a tendency
towards lower NADPH, in the other double mutant the status
of NADP(H) was very similar to cat2 (Fig. 5).
Accumulation of GSSG is a well-known response in catalase-deficient plants but also occurs in response to many
environmental stresses (Vanacker et al., 2000; Bick et al.,
2001; Gomez et al., 2004; Koornneef et al., 2008). Recent data
suggest that modulation of glutathione status may play an
important role in linking oxidative stress to downstream phytohormone-linked pathways (Han et al., 2013; Mhamdi et al.,
2013). Previous studies of the cat2 mutant strongly suggest
that a large part of the accumulated GSSG is found in compartments other than the cytosol, notably the chloroplast and
vacuole (Queval et al., 2011; Han et al., 2013). Nevertheless,
the extent of GSSG accumulation in cat2 is secondarily
affected by loss of function of other NADPH-linked enzymes
found in the cytosol, even though these mutations produce
much less obvious effects on NADP(H) contents measured in
whole leaf extracts (Mhamdi et al., 2010a,b). Thus, irrespective of the subcellular compartments in which GSSG most
strongly accumulates, glutathione status can be considered
a useful marker that provides a more sensitive indication of
changes in cytosolic redox processes than NADP(H) itself
in oxidative stress conditions. However, nadp-me2 mutations
produced little or no effect on the cat2-dependent modulation
of glutathione status. This contrasts with the effect of knocking out the cytosol-located GR1 activity, which dramatically
exacerbates cat2-triggered accumulation of GSSG (Mhamdi
et al., 2010b). In view of this observation, a requirement for
NADP-ME to contribute NADPH to maintain GR activity
would predict more severe accumulation of GSSG in cat2
nadp-me2 mutants than in cat2. This effect is indeed observed
in cat2 icdh mutants, implicating cICDH as a non-redundant
player in providing NADPH to GR (Mhamdi et al., 2010a).
If, on the other hand, NADP-ME activity were essential to
provide reducing power for reactive oxygen species-producing
NADPH oxidases, cat2-triggered GSSG accumulation might
be expected to be weakened in cat2 nadp-me2 double mutants,
as observed in cat2 atrbohF (Chaouch et al., 2012). The present
results do not allow us to discount that NADP-ME is required
3612 | Li et al.
to generate NADPH for both antioxidant and pro-oxidant
processes, and that the similar glutathione status in cat2
and cat2 nadp-me2 lines is the result of opposing effects that
exactly cancel each other. Nevertheless, the simpler conclusion, based on the observations of Figs 4 and 5 taken together,
is that NADP-ME does not play an important, irreplaceable
role in producing NADPH for oxidative stress responses.
Concluding remarks
A recent report implicated NADP-ME2 in early events during
plant–pathogen interactions (Voll et al., 2012), but the present
data suggest that this enzyme is not functionally required for
longer-term redox homeostatic or phenotypic responses to
oxidative stress. This contrasts with the evident response of
the gene to oxidative stress at the transcriptional level. While
the slight effects on basal resistance to one bacterial strain
(Fig. 1) provide further evidence that NADP-ME2 plays a role
during biotic stress responses, such effects may not be redoxlinked: emerging evidence suggests that specific organic acids
could have a signalling role in stress responses (Finkemeier
et al., 2013). Given that NADP-ME2 encodes most of the leaf
NADP-linked ME activity, the apparent dispensability of its
expression could reflect biochemical redundancy due to the
presence in plant cells of other enzymes that can link malate
to pyruvate. Similarly, in terms of NADPH, several NADPlinked dehydrogenases may cooperate to generate reductant
for redox reactions during oxidative stress. Interestingly, the
dispensability of NADP-ME in such conditions contrasts with
our previous data for cICDH, even though the latter is much
less obviously induced by oxidative stress than NADP-ME2.
Together, these observations emphasize the importance of
establishing the impact of loss of function, as well as the danger of drawing conclusions on a given enzyme’s importance
from its activity or inducibility. Future studies will aim at
identifying the importance or redundancy of other NADPHgenerating enzymes in the response to oxidative stress.
Supplementary data
Supplementary data are available at JXB online.
Supplementary Fig. S1. Genotyping of cat2 nadp-me double mutants. Primer sequences are listed in Supplementary
Table S1.
Supplementary Fig. S2. Expression analysis of genes
encoding cytosolic NADP-ME in Arabidopsis.
Supplementary Fig. S3. Characterization of nadp-me2
mutants.
Supplementary Table 1. Oligonucleotide sequences used
in this study for genotyping (PCR) or analysis of transcript
abundance (RT-PCR, qRT-PCR).
Acknowledegments
We thank the Salk Institute Genomic Analysis Laboratory,
CA, USA, for providing the sequence-indexed Arabidopsis
T-DNA insertion mutants and the Nottingham Arabidopsis
Stock Centre, UK, for supply of seed stocks. This work
was partly funded by the French Agence Nationale de la
Recherche projects ‘Vulnoz’ project no. ANR-08-VULN-012
and ‘Cynthiol’ project no. ANR-12-BSV6-0011. The authors
thank Stéphane Martin for technical support. S.L. thanks the
Université de Paris Sud and Michel Dron (IBP, Orsay) for
help with funding towards his PhD studies.
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