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The Plant Cell, Vol. 25: 4195–4208, October 2013, www.plantcell.org ã 2013 American Society of Plant Biologists. All rights reserved.
The Importance of Cardiolipin Synthase for Mitochondrial
Ultrastructure, Respiratory Function, Plant Development, and
Stress Responses in Arabidopsis
W
Bernard Pineau,a,1 Mickaël Bourge,b Jessica Marion,c Caroline Mauve,a Francoise Gilard,a Lilly Maneta-Peyret,d
Patrick Moreau,d Béatrice Satiat-Jeunemaître,b,c Spencer C. Brown,b,c Rosine De Paepe,a and Antoine Danona,e,2
a Institut
de Biologie des Plantes, Saclay Plant Science, Université de Paris-Sud XI, Centre National de la Recherche Scientifique,
Unité Mixte de Recherche 8618, 91405 Orsay cedex, France
b Imagif, Centre National de la Recherche Scientifique, Institut Fédératif de Recherche 87/Fédération de Recherche du CNRS 3115,
91198 Gif-sur-Yvette cedex, France
c Institut des Sciences du Végétal, Centre National de la Recherche Scientifique, Unité Propre de Recherche 2355, 91198 Gif-surYvette cedex, France
d Laboratoire de Biogenèse Membranaire, Centre National de la Recherche Scientifique, Université Bordeaux Segalen, Unité Mixte de
Recherche 5200, 33076 Bordeaux, France
e Institut de Biologie Physico-Chimique, Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, Centre National de la
Recherche Scientifique, Université Pierre et Marie Curie, FRE3354, 75005 Paris, France
ORCID ID: 0000-0001-6311-6076 (A.D.).
Cardiolipin (CL) is the signature phospholipid of the mitochondrial inner membrane. In animals and yeast (Saccharomyces
cerevisiae), CL depletion affects the stability of respiratory supercomplexes and is thus crucial to the energy metabolism of
obligate aerobes. In eukaryotes, the last step of CL synthesis is catalyzed by CARDIOLIPIN SYNTHASE (CLS), encoded by
a single-copy gene. Here, we characterize a cls mutant in Arabidopsis thaliana, which is devoid of CL. In contrast to yeast cls,
where development is little affected, Arabidopsis cls seedlings are slow developing under short-day conditions in vitro and die if
they are transferred to long-day (LD) conditions. However, when transferred to soil under LD conditions under low light, cls plants
can reach the flowering stage, but they are not fertile. The cls mitochondria display abnormal ultrastructure and reduced content
of respiratory complex I/complex III supercomplexes. The marked accumulation of tricarboxylic acid cycle derivatives and amino
acids demonstrates mitochondrial dysfunction. Mitochondrial and chloroplastic antioxidant transcripts are overexpressed in cls
leaves, and cls protoplasts are more sensitive to programmed cell death effectors, UV light, and heat shock. Our results show
that CLS is crucial for correct mitochondrial function and development in Arabidopsis under both optimal and stress conditions.
INTRODUCTION
Mitochondrial internal membranes have a specific lipid composition involved in specific protein–protein and protein–lipid interactions that are essential for mitochondrial function (Schlame
et al., 2000). Cardiolipins (CLs) are dimeric anionic phospholipids localized in bacterial membranes and in mitochondrial internal membranes of eukaryotes (Bligny and Douce, 1980). They
are composed of four acyl chains, carry two phosphate groups,
and can adopt different structures. In mitochondria, CLs are involved in membrane curvature and cristae formation (Mileykovskaya
and Dowhan, 2009). In all eukaryotes examined so far, the last
step of CL synthesis is catalyzed by cardiolipin synthase (CLS)
from phosphatidyl-cytidine monophosphate (CMP) and phosphatidylglycerol (PG; Tamai and Greenberg, 1990; Schlame and
1 Deceased.
2 Address
correspondence to [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.plantcell.org) is: Antoine Danon, antoine.
[email protected]
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.113.118018
Hostetler, 1997). The CLS gene is present in single copy in yeast
(Saccharomyces cerevisiae; Chang et al., 1998), Arabidopsis
thaliana (Katayama et al., 2004), and mammals (Nowicki et al.,
2005). In yeast, cls1 disrupted (crd1) mutants are viable, although
they display reduced oxidative phosphorylation activities and
mitochondrial membrane potential (Jiang et al., 1997; Zhong
et al., 2004). CLs have been shown to bind with high affinity to
a large portion of the proteins of the inner mitochondrial membrane, such as complexes I, III, IV, and V, the carrier family
(ADP–ATP carrier, phosphate carrier, uncoupling protein), and
two peripheral membrane proteins (cytochrome c and creatine
kinase) in yeast and mammals (Sharpley et al., 2006). CL was
shown to be required for the formation and stabilization of respiratory chain supercomplexes, which have been proposed to
be essential for proper functioning of the mitochondrial electron
transport chain in the human respirasome and for supramolecular organization of ATP synthase (Acehan et al., 2011). CLs are
thus crucial for physiological ATP production. Moreover, it was
shown that CLs are implicated in the mitochondrial transport
machinery in yeast (Jiang et al., 2000) by interacting with translocases of the outer mitochondrial membranes (Gebert et al.,
2009, 2011). In human tissues, decreased CL content, alterations
in its acyl chain composition, and/or peroxidation have been
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associated with mitochondrial dysfunction in different pathological conditions, such as cardioskeletal myopathy Barth syndrome (Vreken et al., 2000; Gu et al., 2002; Scherer and Schmitz,
2011). Peroxidized CLs have also been reported to play a crucial
role in the animal apoptotic process (Ott et al., 2007).
Much less information is available on CL function in plants, as
the characteristics of plant cls mutants and of CL-lacking plants
are unclear. Although stable mitochondrial supercomplexes containing complex I (CI) and dimeric complex III have been described
in several species (Eubel et al., 2003, 2004) and a stable respirasome-like complex has been described in spinach (Spinacia
oleracea) green leaf mitochondria (Krause and Durner, 2004),
their association with CL in the plant kingdom is unclear. Here,
we characterize three mutants disrupted in the Arabidopsis CLS
single-copy gene, named cls mutants. In vitro, germinating seedlings
of cls mutants are able to develop under low-illumination conditions
only, and all three have a slow-growing phenotype. When
transferred to soil, they can eventually reach the flowering stage,
but they are not fertile. We show that the cls mutant is fully
devoid of CL, confirming that CLS activity is essential for CL
biosynthesis in plants as in other eukaryotes. The cls mitochondria
have an abnormal ultrastructure and reduced content in respiratory CI and supercomplex I/III (CI/CIII), showing alteration in
mitochondrial electron transport. Amino acid accumulation occurs in the mutants, which is indicative of extensive metabolic
perturbations. Antioxidant systems and programmed cell death
are also exacerbated in cls, indicating a general perturbation of
stress responses. Our results show that CL is necessary for correct
mitochondrial function and development in Arabidopsis, especially
under stress conditions.
RESULTS
Morphological Characterization of the cls Phenotype
in Arabidopsis
A single gene encoding CLS, named CLS, was identified in the
ecotype Columbia (Col-0) nuclear genome (At4g04870; Katayama
et al., 2004). The SALK_49835, SALK_4984, and SALK_2263 lines
from the Salk Institute (Alonso et al., 2003) were identified as
containing independent T-DNA insertions within this gene. We
confirmed the localization of the predicted T-DNA insertions in
the CLS gene by PCR using different primer combinations
(Figures 1A to 1C). Homozygous cls mutants were easily recognized when these lines were germinated in vitro on gelose
medium under continuous light and very low intensity (30 mmol
m22 s21). Indeed, a few days after germination, approximately
one-quarter of the seedlings of the three lines exhibited a strong
dwarf phenotype accompanied by particularly short and thick
roots (Figures 2A to 2C). No CLS mRNA could be detected in
these seedlings (Figure 1D), which were therefore named cls1
(SALK_49835), cls2 (SALK_4984), and cls3 (SALK_2263). The
mutants were able to develop under higher light intensity,
;80 mmol m22 s21, but under short-day (SD) conditions only,
and they displayed a pale green phenotype (Figures 2D and 2E).
When transferred to long-day (LD) conditions, most cls plantlets
started to bleach and die (Figure 2F; see Supplemental Figure 1
Figure 1. Characterization of T-DNA Insertions in the Arabidopsis CLS
Gene.
(A) Positions of the T-DNA insertions for the three cls mutants we
identified.
(B) PCR products of genomic DNA extracted from Col-0 as well as
SALK_49835 (cls1), SALK_4984 (cls2), and SALK_2263 (cls3) homozygous mutant seedlings using primers surrounding the T-DNA insertions
(cls.5 and cls.3).
(C) PCR products of genomic DNA extracted from SALK_49835 (cls1),
SALK_4984 (cls2), and SALK_2263 (cls3) mutant seedlings using primers
surrounding the T-DNA insertions (cls.5 and cls.3) and T-DNA primer LBb1.
(D) Estimation of transcript levels of CLS was done by RT-PCR using
cDNA obtained from Col-0 and SALK_49835 (cls1), SALK_4984 (cls2),
and SALK_2263 (cls3) mutant seedlings and ACTIN2 (ACT2) as the reference gene. For each experiment, roughly 50 seedlings were used for
DNA or RNA extraction.
online). Taken together, these observations indicate a higher
susceptibility of cls seedlings to light quantity. When transferred
to soil in LD conditions under low light (<50 mmol m22 s21), only
a few cls plantlets were able to survive and initiate the transition
from vegetative to reproductive growth. cls rosette plants developed inflorescence stems in synchrony with the wild type;
Arabidopsis Cardiolipin Synthase Mutant
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40%, while phosphatidic acid decreased by 12% (Figure 3B). As
HPTLC analyses were first specifically designed to detect the
very-low-abundance CL, we could not in this case discriminate
phosphatidylinositol and phosphatidylserine, on the one hand,
and PE and PG, on the other hand (Figure 3B).
The cls Mutation Affects Mitochondrial Structure
and Function
Disruption of the CLS gene was expected to have marked consequences on mitochondrial populations in plants as in the yeast
crd1 mutant. Col-0 and cls1 tissues were stained with the Nernst
potential-sensitive markers MitoTracker Red and 3,3'-dihexyloxacarbocyanine iodide [DiOC6(5)].
These two probes were used in parallel not only to explore
complementary spectral domains but also to appreciate nonspecific
Figure 2. Phenotypes of cls Mutant Plants.
(A) to (C) Seedlings of SALK_49835 (cls1; [A]) SALK_4984 (cls2; [B]) and
SALK_2263 (cls3; [C]) lines grown in vitro under continuous light and
under a very low light intensity of 30 mmol m22 s21.
(D) Seedlings of the SALK_49835 line grown for 4 weeks under SD
(80 mmol m22 s21) conditions.
(E) and (F) cls1 plantlets grown for 3 weeks under SD (E) or transferred to
LD (80 mmol m22 s21) conditions for 1 additional week (F).
(G) Adult Col-0 and cls1 plants transferred to soil in LD conditions and
grown under low illumination (;50 mmol m22 s21). Insets show siliques
(arrows) of Col-0 and the cls1 mutant.
however, the size of leaves and inflorescence stems was severely
reduced (Figure 2G; see Supplemental Figure 1 online). Moreover, cls adult plants produced inordinately small siliques (arrowed
in insets of Figure 2G) containing only a few and irregularly-sized
seeds that were not able to germinate. Similar results were
obtained with all three SALK lines and provide evidence that the
drastic phenotypes we observed come from the CLS mutation.
In further analyses, we used the SALK_49835 line as a source of
cls mutant plants, referred to as cls1.
Absence of CL and Membrane Lipidic Changes in cls1
The cls1 mutant was expected to be devoid of CLs. Therefore,
we quantified CL content in Col-0 and cls1 by high performance
thin layer chromatography (HPTLC; see Methods). While the
concentration of CL in Col-0 reached 34 6 12 ng mg21 fresh
weight, no trace of CL could be detected in cls1 seedling lipids
(Figure 3A). In addition, we examined the content of some of the
main membrane lipids. The content of total polar lipids was similar
between Col-0 and cls1, but phosphatidylethanolamine (PE) + PG
increased by ;19% in the mutant and phosphatidylcholine by
Figure 3. Lipid Quantification of cls1 Mutant Plants.
Seedlings were grown in vitro in continuous light at very low intensity
(30 mmol m22 s21) for 3 weeks and transferred to soil for 2 additional weeks.
(A) Quantification of CL and total polar lipids in Col-0 and cls1 mutants.
FW, Fresh weight.
(B) Variation of lipid content (%) between cls1 and Col-0 seedlings.
PA, Phosphatidic acid; PC, phosphatidylcholine; PI, phosphatidylinositol;
PS, phosphatidylserine.
All measurements were done in duplicate on three independent samples.
Error bars represent SE.
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binding (to lipids and waxes), which tends to be higher with
DiOC6(5), even though it is a potential-sensitive dye, than with
MitoTracker Red (Bourdon et al., 2012). Using confocal microscopy,
we clearly identified active mitochondria in Col-0 leaf mesophyll
(Figure 4A) and epidermal (Figure 4B) cells. Both markers gave
comparable observations (the former fluoresces red and the
latter green), the organelles were punctate (1 µm or less), sometimes
fusiform, often mobile. Aggregation of mitochondria was occasionally observed in mesophyll cells (Figure 4A). In cls1, labeling
similar to Col-0 was observed in mesophyll (Figure 4D) and
epidermal (Figure 4E) cells; however, very small mitochondria
appeared, while mitochondrial aggregates were more frequent in
the mesophyll, and fivefold larger globular structures (considering their axes) were observed in labeled DiOC6(5) epidermal
cells (Figure 4E). Using 10-N-nonyl acridine orange (NAO), which
was initially described to specifically bind CLs in mitochondrial
membranes (Petit et al., 1992), similar classes of structures appeared in Col-0 as with the other dyes (Figure 4C). Unexpectedly,
mobile NAO+ mitochondria were also observed in cls1 epidermal
cells and stomata, which also contain large globular mitochondria.
Furthermore, in many cells of both genotypes (Col-0 and cls1),
NAO strongly labeled other membranes.
Similar observations were done in protoplasts isolated from
young in vitro–grown seedlings (Figures 4G to 4N). In Col-0 protoplasts, MitoTracker Red revealed typical small punctate mitochondria with high contrast (Figures 4G and 4H), while labeling
of both small punctate mitochondria and of large organelles
(arrows) was observed in cls1 protoplasts (Figures 4I and 4J).
Similarly, NAO labeled both punctate and large organelles in
Col-0 protoplasts (Figures 4K and 4L) and in cls1 protoplasts
(Figures 4M and 4N). Moreover, in addition to mitochondria, the
stain labeled a whole range of structures, such as cytoplasmic
membranes and the chloroplast periphery in both genotypes,
showing that the dye is not specific for CL in our experimental
conditions. This was confirmed using flow cytometry (Figure 4C),
gating on light scatter, and exclusion of propidium iodide in
order to target only intact protoplasts. In contradiction with any
putative binding of NAO to CLs, cls1 protoplasts were even
more fluorescent than Col-0 protoplasts after 5 min of staining
with 100 nM NAO. The capacity to label with NAO decreased if
the protoplasts had first been stored, but the same relationship
was observed (Figure 4C for protoplasts stored 2 or 4 h). If the
geometric mean of NAO fluorescence after 2 h of incubation is
set at 100 (arbitrary units) for intact wild-type protoplasts, the
cls1 population had a mean of 142 arbitrary units.
In summary, all fluorescent markers used, including NAO, outlined
the effects of the cls mutation on the size and three-dimensional
organization of the mitochondrial pool. The unspecificity of NAO for
CL binding is possibly due to the low concentration used (100 nM).
Similar unspecificity has already been reported in the yeast crd1
mutant (Gohil et al., 2005a; Mileykovskaya and Dowhan, 2009).
The cls Mutation Affects the Mitochondrial Ultrastructure
The lack of CL was previously shown to affect mitochondrial shape
and ultrastructure in the yeast crd1 mutant and in human Barth
cells (Acehan et al., 2007; Mileykovskaya and Dowhan, 2009). The
ultrastructure of Col-0 and cls1 leaf cells of plantlets maintained
in SD conditions was examined by transmission electron microscopy. Col-0 mitochondria showed the typical features of functional
mitochondria in plant cells (Frey and Mannella, 2000), consisting of
ovoid organelles, ranging in size from 0.5 to 1.2 µm (longer axis;
Figures 5A and 5B). The osmium/potassium ferrocyanide impregnation outlined the numerous cristae. The mitochondrial
population of cls1 leaf cells appeared far more heterogeneous in
size and aspect (Figures 5C to 5H). While a few cls1 mitochondria (roughly 2%) had size and shape similar to those of
Col-0 mitochondria (Figure 5C), most (roughly 70%) were oblong or asymmetrical and exhibited fewer cristae, reminiscent of
young, undifferentiated mitochondria (Figures 5E to 5G). In addition, giant mitochondria (roughly 28%) were observed, with
a 2- to 6-µm-long axis (Figures 5D and 5H). Giant mitochondria
might correspond to the large fluorescent structures shown in
Figure 4. Neither the shape, size, and thylakoid organization of
cls1 chloroplasts (Figure 5H) nor the endomembrane system
(endoplasmic reticulum, Golgi, vacuoles) appeared to be affected in the mutant leaf cells.
These results confirmed that the cls mutation induces drastic
changes in mitochondrial morphogenesis and ultrastructure.
Importance of CLs for the Assembly of Respiratory
Complexes of the Inner Mitochondrial Membrane
CLs have been shown to interact with different complexes of the
inner membrane in mammals and yeast (McAuley et al., 1999;
Zhong et al., 2004; Sharpley et al., 2006) and to stabilize respiratory chain supercomplexes (Pfeiffer et al., 2003; Gebert
et al., 2009). We compared the accumulation of respiratory CI
and CI/CIII in Col-0 and the cls1 mutant by blue native electrophoresis (Figure 6). In dodecyl maltoside–solubilized proteins
of Col-0 and cls1 leaf membrane extracts (Figure 6A), both in-gel
NADH/nitroblue tetrazolium (NBT) staining, which reveals NADH
dehydrogenase activity, and immune detection using an antibody directed against the NAD9 subunit of the CI peripheral arm
(Klodmann et al., 2010) showed that CI accumulation was lower
in cls1 than in Col-0. In contrast, the accumulation of complex
IV, revealed by the anti-COXII antibody, was unaffected (Figure 6A).
Moreover, in proteins solubilized by digitonin, which maintains
the association of supercomplexes (Pineau et al., 2008), NAD9
was essentially incorporated into CI/CIII in Col-0, whereas such
supercomplexes where hardly detectable in cls1 (Figure 6B). The
level of CI/CIII in cls1 mitochondrial membranes also appeared
low after NADH/NBT staining (Figure 6B). The lack of accumulation
of CI and of CI/CIII indicates an alteration of the mitochondrial
electron transport and possibly of respirasome function (Krause
and Durner, 2004) in the cls1 mutant.
Changes in Antioxidant Gene Expression in cls
Alteration of mitochondrial electron transport in cls1 was expected to increase the probability of the formation of reactive
oxygen species (ROS) by respiratory complexes, essentially CI
and CIII (Møller, 2001). To test this hypothesis, transcript levels
of several genes previously reported to reflect cellular ROS
levels were quantified (Figure 7). First, transcript levels of AOX1
and NDB2, genes encoding alternative oxidase and external NAD
Arabidopsis Cardiolipin Synthase Mutant
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(P)H dehydrogenase, respectively, two mitochondrial respiratory
enzymes whose expression is induced under several stress
conditions (Yoshida and Noguchi, 2009), are markedly increased
in cls1 seedlings. Moreover, as overexpression of genes encoding
extramitochondrial antioxidant enzymes has been reported previously in mitochondrial CI mutants (Dutilleul et al., 2003b; Meyer
et al., 2009), several markers of cellular oxidative stress were
tested, such as Catalase1 (CAT1) to CAT3 for the peroxisome
catalase (Queval et al., 2007; Du et al., 2008) and Ascorbate
peroxidase1 (APX1) and APX2 for the cytosolic ascorbate peroxidase (Koussevitzky et al., 2008). Whereas no significant transcript changes were detected for the major CAT2 and APX1
genes, CAT1, CAT3, and APX2 transcript levels were markedly
increased in cls1. Among the genes previously reported to be
implicated in the chloroplastic response to ROS (op den Camp
et al., 2003; Kim et al., 2012), we tested distinctive markers that
are selectively induced either by superoxide/hydrogen peroxide,
FERRITIN1 (FER1), or by singlet oxygen, BONZAI1-ASSOCIATED
PROTEIN (BAP1). In cls1, transcript levels of BAP1 but not of
FER1 were strongly increased, suggesting singlet oxygen but
not superoxide/hydrogen peroxide overproduction in cls1 plastids.
Taken together, these results suggest that in cls1, oxidative stress
occurs, probably from both mitochondria (superoxide/hydrogen
peroxide) and plastids (singlet oxygen).
CLS Mutation Affects Tricarboxylic Acid–Derived
Metabolites and Amino Acid Levels
Figure 4. Confocal Microscopy of Col-0 and cls1 Cells and Flow
Cytometry of NAO Intensity in Protoplasts.
(A) to (F) Leaves. In Col-0 mesophyll (A) and epidermal (B) cells, the
Nernst potential-sensitive dyes MitoTracker Red (red) and DiOC6(5)
(green) revealed punctate (arrowhead) or fusiform mitochondria ;1 µm in
length; chloroplasts are coded blue. Occasional clusters of mitochondria
were also observed (arrows). In cls1 mesophyll (D) and epidermal (E)
cells, both very small punctate mitochondria (arrowhead) and large reticulate mitochondria (arrows) were labeled with MitoTracker Red, and
immobile globular structures were observed with DiOC6(5). The putatively potential-insensitive mitochondrial dye NAO (green) revealed
a similar range of mitochondrial forms in Col-0 mesophyll ([C]; chloroplasts coded red); in cls1, punctate mitochondria were observed in
epidermal cells ([F]; arrowhead), and large globular mitochondria ([F];
arrows) were present in stomata. The lipidic osteole was also labeled by
NAO. Bars = 5 µm.
(G) to (N) Protoplasts isolated from young in vitro–grown seedlings. In
Col-0 protoplasts, MitoTracker Red revealed typical small punctate
mitochondria with high contrast ([G] and [H]). Both small punctate mitochondria and large organelles (arrows) were labeled in cls1 protoplasts
([I] and [J]). Chloroplasts are coded in blue in (G) and (I), whereas (H) and
(J) are single-color micrographs. NAO (green) labeled tiny punctate
mitochondria and larger organelles in Col-0 protoplasts ([K] and [L]); in
cls1 protoplasts ([M] and [N]), both punctate mitochondria and a whole
Mitochondria play a crucial role in the plant nitrogen/carbon
balance (Fernie et al., 2004), and CI mutants have been shown to
accumulate tricarboxylic acid (TCA) cycle–derived metabolites
(Dutilleul et al., 2005; Meyer et al., 2009). Similarly, the balance
of primary metabolites, examined by gas chromatography–time
of flight–mass spectrometry (GC-TOF-MS; Figure 8), was markedly
altered in cls1 as compared with Col-0 plants. In addition to the
accumulation of soluble sugars in cls1 plants, most TCA cycle
intermediates were more abundant in the mutant than in Col-0.
Fumarate, the most abundant acid in Arabidopsis, synthesized in
part by a cytoplasmic fumarase (Pracharoenwattana et al., 2010), was
increased only marginally. In parallel to TCA cycle derivatives, most
amino acids were also more abundant in cls1 than in Col-0 plants,
except Glu and Gln, which were not significantly different, and
range of structures, such as cytoplasmic membranes and the chloroplast
periphery (arrow in [N]), were labeled by NAO. Bars = 5 µm.
(O) Flow cytometry of Col-0 and cls1 protoplasts to quantify NAO uptake. Histograms indicate the relative intensity of NAO charge in protoplasts after 5 min of staining with 100 nM NAO and counterstaining with
PI. The capacity to label with NAO was measured after storing the protoplasts at room temperature 2 h (left histogram) and 4 h (right histogram). The geometric mean of NAO fluorescence was determined for
Col-0 (shaded histogram) and cls1 protoplasts (unshaded histogram)
using analytical cytometry to exclude PI-permeant protoplasts. For each
histogram, the geometric mean was obtained and then reexpressed in
arbitrary units (au), assigning 100 to the mean of the wild type at 2 h.
Unstained protoplasts were <3 arbitrary units.
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lack of CL on the initiation of PCD in plants was tested using
UV-C light and heat shock, two treatments known to induce
a mitochondria-dependent PCD in Arabidopsis (Danon et al.,
2004; Vacca et al., 2006; Gao et al., 2008). The quantification of
cell death reactions was achieved using protoplasts and Evans
Blue dye, which selectively stains dead cells (Wright et al., 2000;
Danon et al., 2005). Protoplasts from Col-0 and cls1 seedlings
were isolated, and the percentage of dead cells was calculated
2, 4, and 6 h after treatment. The frequency of dead cells increased in cls1 even after 2 h of treatment with UV-C light
(Figure 9A) or heat shock (Figure 9B), whereas there was no
significant increase in Col-0. For later time points, dead cells
increased both in Col-0 and cls1, but more rapidly and more intensively in the mutant. In the absence of treatments, the number
of dead Col-0 and cls1 cells remained almost constant throughout the experiment (Figure 9). Collectively, these results suggest
that, in response to different stimuli, the cls1 mutant develops an
exaggerated and accelerated PCD.
DISCUSSION
We have identified knockout mutants for the CLS gene in Arabidopsis, which has allowed us to evaluate the importance of CLs
for mitochondrial ultrastructure and function as well as for development and adjustment to light conditions and response to
Figure 5. Electron Micrographs of Col-0 and cls1 Leaf Mitochondria and
Plastids.
(A) and (B) Col-0 mitochondria (m) and plastid (pl).
(C) cls1 mitochondrion with similar size and internal structure to Col-0 mitochondria.
(D) Giant cls1 mitochondrion.
(E) to (G) Irregular cls1 mitochondria with poorly developed cristae.
(H) Giant cls1 mitochondrion and plastid.
The same magnification was used in all figures except in (H), with onethird less magnification. Bars = 500 nm.
Asp, which was decreased. In particular, levels of photorespiratory
Gly and Ser were markedly increased in the mutant, as was the
level of glycolate but not of glycerate, the end product of the
photorespiratory cycle. The glycolate:glycerate, Gly:Ser, and Ser:
glycerate ratios were higher in cls1 than in Col-0 leaves, suggesting impairment of the photorespiratory cycle and marked
limitation at the level of glycolate oxidase and Gly decarboxylase
activities (Douce et al., 2001). In contrast, integration of glycerate
into the Calvin cycle in the chloroplast appeared not to be limited.
Increased Sensitivity of cls Protoplasts to Programmed Cell
Death Effectors
CLs have been reported to mediate programmed cell death
(PCD) in animals (Schug and Gottlieb, 2009). The impact of the
Figure 6. Consequences of the Absence of CLS Expression for the
Composition of the Mitochondrial Respiratory Chain.
CI and CI/CIII were separated by blue native electrophoresis of protein
extracts, followed by either NADH/NBT staining or immunodetection
using antisera directed against the CI NAD9 subunit and the COXII
subunit of complex IV. Compared with Col-0 plants, the accumulation of
CI and CI/CIII is impaired in the cls1 mutant.
(A) Proteins from dodecyl maltoside (DDM)–solubilized membranes.
(B) Proteins from membranes solubilized by digitonin, which preserves
CI/CIII association (Pineau et al., 2008).
Arabidopsis Cardiolipin Synthase Mutant
Figure 7. Changes in Antioxidant Gene Expression Levels in the cls1
Mutant.
The accumulation of transcripts related to oxidative stress in mitochondria (AOX1a and NDB2), plastids (BAP1 and FER1), cytosols (APX1
and APX2), or catalases (CAT1, CAT2, and CAT3) was quantified by realtime PCR in Col-0 and cls1 seedlings. Results shown are mean relative
mRNA levels 6 SE from at least three independent samples. Asterisks
indicate significant differences between cls1 and Col-0 values.
PCD treatments. The cls1 mutants did not display a detectable
amount of CL (Figure 3A), which was expected since CLS is the
last step of CL synthesis in plants as in other eukaryotes and the
CLS gene is in single copy. CL deficiency induces changes in
the lipid homeostasis of whole plant cells, phosphatidylcholine,
PE, and PG being in higher amounts in the cls mutant than in
Col-0. Interestingly, PG is the direct precursor of CL, while PE
and CL have been reported to have overlapping functions and to
cooperate in maintaining mitochondrial morphology (Gohil et al.,
2005b; Joshi et al., 2012).
CLs are key molecules for plant mitochondrial structure and
function. Indeed, cls1 plants present some of the characteristics
of CL-deficient cells described in yeast and mammals, where the
lack of CL results in an aberrant morphology of mitochondrial
cristae (Mileykovskaya and Dowhan, 2009; Gonzalvez et al.,
2013) and the instability of respiratory chain supercomplexes
4201
(see Introduction). In the cls1 mutant, most mitochondria have
abnormal shape with few cristae (Figure 5), and the low abundance
of CI/CIII of the respiratory chain (Figure 6) strongly suggests that
mitochondrial electron transport is affected. Moreover, giant mitochondria are observed in cls1 cells (Figures 4 and 5), as has been
observed in other CL-deficient cells (Gonzalvez et al., 2013),
suggesting impairment of the plant mitochondrial fusion/fission
machinery (Westermann, 2010). Such a function of CL might be
supported by several nonexclusive mechanisms, including either intrinsic properties (Schlame, 2008; Furt and Moreau, 2009)
and/or properties related to its importance in mitochondrial ATP
production and membrane protein import (Herlan et al., 2004;
Joshi et al., 2009; Acehan et al., 2011; Gebert et al., 2011). Increased levels of TCA-derived organic acids (Figure 8) suggest
the blocking of glycolysis and the TCA cycle following impaired
respiration and mitochondrial protein import. Whatever the
mechanism(s) involved, these results demonstrate strong alterations
of mitochondrial biogenesis and activity in cls1 mutant plants.
However, the presence of at least partially functional mitochondria,
in particular giant mitochondria, is indicated by the accumulation of
membrane potential–sensitive dyes and NAO (Figure 4), showing
that this dye is definitely not specific for CL, as demonstrated
previously in yeast (Gohil et al., 2005a; Rodriguez et al., 2008;
Mileykovskaya and Dowhan, 2009).
While mitochondrial activity is sufficient to allow the development of the cls1 mutant under low light, although at a very
low rate, the mutant is not able to develop and then bleaches
under higher illumination conditions (either higher light intensity
or LD conditions). An analysis of several markers of oxidative stress
(Figure 7) suggests that light sensitivity can result, in last in
part, from ROS overproduction.
First, restricted mitochondrial electron transport is expected
to generate more superoxide/H2O2 (Møller, 2001), and the accumulation of mitochondrial and extramitochondrial antioxidant
transcripts was previously observed using inhibitors of the respiratory chain (Vidal et al., 2007; Umbach et al., 2012). Accordingly,
transcripts of AOX1 and NDB2, which are responsive to H2O2
(Wagner, 1995), accumulate in cls1 (Figure 7), as reported previously
in Nicotiana sylvestris (Sabar et al., 2000; Dutilleul et al., 2003b; Vidal
et al., 2007), maize (Zea mays; Karpova et al., 2002), and Arabidopsis CI mutants (Meyer et al., 2009). The accumulation of peroxisomal CAT1 and CAT3 mRNAs, which, unlike CAT2, are not
related to photorespiratory activity (Du et al., 2008; Mhamdi et al.,
2012), might also respond to H2O2 accumulation and favor acclimation of the cls1 mutant to mitochondrial impairment.
Second, the impairment of the photorespiratory pathway
originating from the absence of CL in cls mitochondria might
also have an impact on plastid functioning, as a deficiency in
photorespiration has been shown to interrupt the Calvin cycle
and to affect the stability of the photosystem II (Takahashi et al.,
2007). In cls plants, a photosynthetic impairment might generate
singlet oxygen and result in photooxidative stress, as reported
elsewhere (Apel and Hirt, 2004; Krieger-Liszkay, 2005). In cls1
seedlings, we found that mRNAs of the singlet oxygen–responsive
gene BAP1 (op den Camp et al., 2003; Kim et al., 2012) strongly
accumulate, which is consistent with the production of singlet
oxygen in plastids, as would be expected from a lack of stability
in photosystem II. Another possible but not exclusive explanation
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The Plant Cell
Figure 8. Leaf Metabolic Content Is Altered in the cls1 Mutant.
TCA-derived metabolites, amino acids, and total sugars are from GC-TOF-MS analyses. Results shown are mean metabolite contents according to
fresh weight (arbitrary units) 6 SE from three independent samples. Asterisks indicate significant differences between cls1 and Col-0 values. TCAfumarate indicates all TCA-derived organic acids except fumarate, by far the most abundant organic acid in Arabidopsis.
Arabidopsis Cardiolipin Synthase Mutant
Figure 9. Effects of UV-C Light and Heat Shock on the Extent of Cell
Death in cls1 and Col-0 Protoplasts.
(A) Protoplasts subjected to UV-C irradiation (10 kJ m22).
(B) Protoplasts subjected to heat shock (HS; 55°C, 10 min).
In both cases, the percentage of dead protoplasts was calculated 0, 2, 4,
and 6 h after treatment using Evans Blue (0.04%). Data represent
means 6 SE of four independent experiments, with a minimum of 100
protoplasts being counted per sample.
for singlet oxygen production in cls1 plastids comes from the
fact that mitochondria are able to dissipate redox equivalents
from the chloroplast to protect them from photoinhibition (Woodson
and Chory, 2008); therefore, the altered ultrastructure and respiratory function in cls1 could also explain the overproduction
of singlet oxygen in plastids. The expression of FER1, a plastidic
superoxide/H2O2-responsive gene (op den Camp et al., 2003;
Kim et al., 2012), was not affected in the mutant, suggesting that
only singlet oxygen, but not superoxide/H2O2, was produced in
cls1 plastids (Figure 7). Mitochondria were reported to have an
impact on proper chloroplast functioning in a N. sylvestris cytoplasmic male sterility II mutant lacking CI, which has low photosynthesis levels (Sabar et al., 2000; Dutilleul et al., 2003a) and
impaired adjustment to higher growth irradiance (Priault et al.,
2006).
Taken together, these results show that various putative sources
of ROS overproduction are present in cls1 following CL deficiency,
which would explain why cls1 seedlings are highly susceptible to
4203
high-illumination conditions. This is not the first time that
the crucial role of CL has been described, not only in the functioning of mitochondria but also in its relationship with other cell
compartments. In yeast, a new mitochondria–vacuole signaling
pathway was identified in a crd1 mutant lacking CL (Chen et al.,
2008).
Mitochondria play a key role in most types of PCD signaling
identified so far. In animals, CLs have been shown to be important regulators of PCD mainly because they anchor cytochrome c, which is released from mitochondria into the cytosol
during apoptosis (Garrido et al., 2006; Vacca et al., 2006). Peroxidation of CLs has been shown to be involved in the regulation
of cytochrome c release (Kagan et al., 2009). However, the impact of the lack of CLs on the onset of PCD is still controversial,
as it has been described as either enhancing (Choi et al., 2007;
Kagan et al., 2009) or inhibiting (Huang et al., 2008; Schug and
Gottlieb, 2009), depending on the system used. Mitochondria
are considered to be key players in the execution of PCD in
plants as well, and the release of cytochrome c during PCD has
been described in several situations (Reape et al., 2008). The
cytoplasmic male sterility II CI mutant has been shown to display
enhanced resistance to Tobacco mosaic virus in an incompatible interaction (Dutilleul et al., 2003b). We took advantage of
the cls1 mutant to test the impact of the lack of CLs on the onset
of mitochondria-dependent PCD induced by UV-C light (Danon
et al., 2004; Gao et al., 2008) and heat shock (Vacca et al., 2006,
2007). While untreated protoplasts from Col-0 and cls1 exhibited
the same death frequency, for both PCD treatments, cls1 protoplasts were much more sensitive than the wild type (Figure 9).
This could suggest that, as described for animals, the lack of CL
would facilitate the release of cytochrome c into the cytosol,
exacerbating the onset of PCD (Choi et al., 2007; Kagan et al.,
2009). It is also possible that ROS accumulation in the mutant,
indicated by the upregulation of ROS-responsive marker genes,
might have an impact on the onset of PCD in cls1 cells under
stress conditions. Indeed, mitochondrial ROS appear to be involved in the onset of PCD in both animals and plants (Fleury
et al., 2002; Rhoads et al., 2006), and more recently, the importance of chloroplastic ROS has also been demonstrated in
Arabidopsis PCD (Kim et al., 2012).
Taken together, our results show that CLs are essential for
plant development, mitochondrial ultrastructure, and the assembly of CI/CIII of the internal mitochondrial membrane. This
diversity of CL roles explains the striking consequences for plant
growth and morphology. The cls phenotype is much more accentuated in plants than in yeast, where development is slightly
affected only under nonfermentable conditions. This might be
attributable to the impaired functioning of mitochondria and altered metabolism leading to ROS production in mitochondria
but also to photooxidative stress in plastids, which would explain why cls plants are only viable under a low-light regime.
Even though the impact of CLs on the onset of PCD in Arabidopsis is clear, an analysis of the peroxidation of CLs and the
release of cytochrome c in cls would help us to understand
further the mechanisms involved. The Arabidopsis cls mutant,
therefore, constitutes an important model for the study of the
multiple roles of CL in mitochondrial biogenesis and function in
obligate aerobes.
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The Plant Cell
METHODS
Plant Material
Arabidopsis thaliana seeds of the SALK_49835, SALK_4984, and SALK_2263
lines carrying a T-DNA insertion in the At4g04870 gene were obtained from the
Nottingham Arabidopsis Stock Centre. Seeds were sterilized and sown under
aseptic conditions on agar plates containing Gamborg B5 salt and vitamins
(Duchefa) supplemented with 0.5% Suc. The plated seeds were kept at 4°C for
5 d and transferred to an illuminated, temperature-controlled growth chamber.
Plants were grown at 20°C under an 8-h-day/16-h-night photoperiod (short
days) of white light (;80 mmol m22 s21) if not indicated otherwise.
Lipid Extraction and Quantification
Lipids were extracted three times with 2 mL of chloroform:methanol (2:1, v/v)
at room temperature for 1 h and then washed three times with 0.9% (w/v)
NaCl. The upper phase was discarded, and the solvent was evaporated under
nitrogen. The lipids of the different extracts were dissolved in an adequate
volume of chloroform:methanol (2:1, v/v) to adjust all samples according to
their initial amounts of plant fresh weight. Lipids were sprayed (7-mm-wide
bands) onto HPTLC plates (60F254; Merck) with a Linomat IV (CAMAG).
Plates were developed in chloroform:methanol:acetic acid (65:25:5, v/v).
Lipids were identified by comigration with known standards and quantified by
densitometry (Macala et al., 1983) using a TLC Scanner 3 (CAMAG).
RNA Isolation and RT-PCR Analysis
Total RNA was prepared using the Trizol reagent (Invitrogen) following the
manufacturer’s instructions. RNA (1 µg) was treated with RQ1 RNase-Free
DNase (Promega) and reverse transcribed using random hexamers and
the SuperScript III first-strand kit (Invitrogen) following the manufacturer’s
recommendations. One one-hundredth of the cDNA solution, 200 nM of
each primer, and the LightCycler 480 SYBR Green I master mix (Roche
Applied Science) were mixed in a final volume of 20 mL. Real-time PCR
products were analyzed with the LightCycler 480 detection system.
Melting curves were used to verify the specificity of amplification products. Relative mRNA abundance of the selected genes was calculated
using Exor4 relative quantification software (Roche Applied Science),
based on the Delta-Deltacycle threshold method, using TIF4A as an internal reference for the normalization of transcript levels. Data were from
duplicates of three biological replicates. The expression levels of AOX1a
and NDB2 were determined using primers described by Ho et al. (2008).
APX1, APX2, CAT1, CAT2, and CAT3 mRNA levels were determined
using primers described by Mhamdi et al. (2010). BAP1 and FER1 expression levels were determined using primers described by op den Camp
et al. (2003). The expression levels of TIF4A were determined using TIF4A5 (59-AGTCTCCCTGGTTATCAACTTC-39) and TIF4A-3 (59-CGACTTCTTTTCTCCCTTCAC-39) primers. Equivalent efficiencies between the different
probes and our internal standard were observed during the PCRs.
Primers used to detect the expression levels of CLS by RT-PCR were
as follows: cls.5 (59-TTACGTGGCTCGGAGGATGAAG-39) and cls.3
(59-AGTATTGTACACCATAAGCTGC-39); those used for ACTIN2 were
act2.5 (59-ATTAAGGTCGTTGCACCAC-39) and act2.3 (59-CATTTTCTGTGAACGATTCCTG-39). cls.5 and cls.3 primers were also used for
the PCRs, involved in the characterization of T-DNA insertions in the
three different mutants, in combination with the T-DNA–specific primer
LBb1 (59-GCGTGGACCGCTTGCTGCAACT-39).
After blue native electrophoresis, the NADH dehydrogenase activity of CI
was revealed by incubation of the gel in the presence of 1 mM NBT and
0.2 mM NADH in 0.05 M 3-(N-Morpholino)propanesulfonic acid, 4Morpholinepropanesulfonic acid, pH 7.6, after washing for 20 min in 0.1 M
3-(N-Morpholino)propanesulfonic acid, 4-Morpholinepropanesulfonic acid,
pH 7.6. The cytochrome oxidase activity was revealed after washing the gel
in 50 mM sodium phosphate, pH 7.4, by incubation in the same buffer but
containing 0.22 M Suc, 0.5 mM bovine heart cytochrome c, 1.4 mM 3.39diaminobenzidine, and 24 units mL21 catalase (Sigma-Aldrich).
Protein Gel Blot Analyses
Gels were electroblotted onto nitrocellulose membranes for SDS-PAGE
and polyvinylidene difluoride for blue native electrophoresis, and immunodetection studies (Pineau et al., 2008) were performed using wheat
(Triticum aestivum) anti-NADH dehydrogenase subunit9 antibody at a 1:1000
dilution (a gift from J.M. Grienenberger, Institut de Biologie PhysicoChimique). Signals were visualized by enhanced chemiluminescence
according to the manufacturer’s instructions (Roche Diagnostics).
Confocal Microscopy of Col-0 and cls1 Leaf Cells and Protoplasts
with Mitochondrial Dyes
For imaging tissue, leaves were torn with tweezers in 5 µM dye solutions
containing 0.1% (w/v) Tween 20, first using potential-sensitive mitochondrial markers (Fricker et al., 2001): DiOC6(5) (Kodak) and MitoTracker
Red (Invitrogen). These were imaged with a confocal microscope (SP2
Leica) using a 403, numerical aperture 1.25 oil objective with a 488-nm
laser (8% power), measuring the emission at 505 to 540 nm, or for
MitoTracker Red, excitation at 543 nm and emission at 570 to 630 nm.
Chlorophyll was recorded simultaneously at 670 to 720 nm. Transmission
images were taken in differential interference contrast mode. Likewise,
leaves were torn in a 5 µM NAO solution containing 0.1% (w/v) Tween 20
prepared freshly from 2 mM NAO (ethanol stock of 10-nonyl acridine
orange bromide [Invitrogen Molecular Probes]) and imaged with 488-nm
excitation (as above). Protoplasts were imaged after 5 min of incubation in
100 nM solutions of these four dyes. The same material was also imaged
on a wide-field microscope (DMI6000; Leica) using a 633, numerical
aperture 1.40 oil objective and the filter blocks fluorescein isothiocyanate
or XF93 Red. Deconvolution was applied to the best Z-stacks.
Protoplast Preparation and Determination of Cell Death
Col-0 protoplasts were isolated from leaves of seedlings that were incubated for 15 h in the dark in the presence of the digestion medium:
Gamborg B5 medium salt and vitamins (Duchefa), 0.5 M mannitol, 1.2%
cellulose (Duchefa), and 0.8% macerozyme (Duchefa), pH 5.8. Protoplasts were then separated from cellular debris by filtration through
a 100-µm mesh filter (Milian), followed by flotation on a 0.4 M Suc solution
at 60g for 10 min. They were then collected and centrifuged at low speed
(60g) for 5 min and washed. Finally, the protoplasts were resuspended in
culture medium containing Murashige and Skoog medium (supplemented
with 0.4 M Suc and 0.4 M mannitol). The frequency of dead cells was
determined by light microscopy of protoplasts stained with Evans Blue
(Sigma-Aldrich) at a final concentration of 0.04% (w/v). Protoplasts were
exposed to 55°C for 10 min or to UV-C irradiation (254 nm) at a dose of
10 kJ m22 using UV Stratalinker 1800 (Stratagene).
Preparation of Crude Leaf Membrane Extracts, Blue Native
Electrophoresis, and In-Gel Activities
Measurement of Intracellular NAO Fluorescence of Protoplasts by
Flow Cytometry
The preparation of crude leaf membrane extracts and blue native electrophoresis were performed as described previously (Pineau et al., 2008).
Mesophyll protoplasts from Arabidopsis were suspended in B5-mannitol
buffer on a 24-well plate (3 3 10 5 mL21). For each point of kinetic
Arabidopsis Cardiolipin Synthase Mutant
measurements, NAO was diluted at 10 µM in B5-mannitol buffer from 1 M
stock solution in DMSO. Fifty microliters of protoplasts was taken from the
plate and gently added to a tube with 250 mL of B5-mannitol. Then, 3 mL of
10 µM NAO (final concentration of 100 nM) was added, and the samples
were incubated 5 min in darkness at room temperature. After the addition
of 1 µg mL21 propidium iodide (PI) to check cell integrity, samples were
analyzed by flow cytometry. Every kinetic point was prepared in duplicate
to confirm measurements.
Acquisitions were made on an ELITE cytometer (Beckman-Coulter)
using a 488-nm argon laser at 15 mW. Fluorescence emission of NAO was
collected through a 525-nm band-pass filter, and fluorescence emission
of PI was collected through a 610-nm band-pass filter. We checked for the
presence of chlorophyll by autofluorescence through a 685-nm bandpass filter. Dead or leaky protoplasts were excluded from the analyzed
population by gating out PI-positive objects.
4205
AUTHOR CONTRIBUTIONS
A.D. and R.D.P. supervised the study. S.C.B. and B.S.-J. were in charge
of the cell biology strategy. A.D. ordered single mutant plants and
performed their genetic characterization. B.P. performed blue native
PAGE and immunodetection experiments. S.C.B., A.D., and M.B. performed cytometry, light microscopy, and fluorochrome experiments. J.M.
performed the electron microscopy studies. P.M. and L.M.P. did lipid
quantifications. Metabolomic quantifications were performed by C.M.
A.D. performed transcriptomic analysis (RT–quantitative PCR) and PCD
studies. R.D.P., A.D., and S.C.B. wrote the article.
Received September 3, 2013; revised September 21, 2013; accepted
October 2, 2013; published October 22, 2013.
Electron Microscopy
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Accession Numbers
Sequence data from this article can be found in the GenBank/EMBL data
libraries under the following accession numbers: CLS, At4g04870; AOX1a,
At3g22370; NDB2, At4g05020; BAP1, At3g61190; FER1, At5g01600;
APX1, At1g07890; APX2, At3g09640; CAT1, At1g20630; CAT2, At4g35090;
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Supplemental Data
The following material is available in the online version of this article.
Supplemental Figure 1. Phenotype of the cls2 Mutant.
ACKNOWLEDGMENTS
This work was supported by the Centre National de la Recherche
Scientifique and the Université Paris-Sud 11. We used the facilities and
expertise of the Plate-forme Métabolisme Métabolome (Université Paris-Sud 11)
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Supplemental Data. Pineau et al. (2013). Plant Cell 10.1105/tpc.113.118018
A
Col0
cls2
B
Col0
cls2
Supplemental Figure 1. Phenotype of the cls2 mutant.
A. In vitro seedlings of the cls2 line (SALK4984) grown under LD at
80 μmol m-2 s-1. Bleaching homozygous cls2 plants are arrowed.
B. cls2 plant compared to Col0 plant at the adult stage in
greenhouse.
The Importance of Cardiolipin Synthase for Mitochondrial Ultrastructure, Respiratory Function,
Plant Development, and Stress Responses in Arabidopsis
Bernard Pineau, Mickaël Bourge, Jessica Marion, Caroline Mauve, Francoise Gilard, Lilly
Maneta-Peyret, Patrick Moreau, Béatrice Satiat-Jeunemaître, Spencer C. Brown, Rosine De Paepe and
Antoine Danon
Plant Cell 2013;25;4195-4208; originally published online October 22, 2013;
DOI 10.1105/tpc.113.118018
This information is current as of January 17, 2014
Supplemental Data
http://www.plantcell.org/content/suppl/2013/10/11/tpc.113.118018.DC1.html
References
This article cites 86 articles, 37 of which can be accessed free at:
http://www.plantcell.org/content/25/10/4195.full.html#ref-list-1
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