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From Endoplasmic Reticulum to Mitochondria: Absence
of the Arabidopsis ATP Antiporter ER-ANT1
Perturbs Photorespiration
W
Christiane Hoffmann,a Bartolome Plocharski,a Ilka Haferkamp,a Michaela Leroch,a Ralph Ewald,b Hermann Bauwe,b
Jan Riemer,c Johannes M. Herrmann,d and H. Ekkehard Neuhausa,1
a Department
of Plant Physiology, University of Kaiserslautern, D-67663 Kaiserslautern, Germany
of Plant Physiology, University of Rostock, D-18059 Rostock, Germany
c Department of Cell Biochemistry, University of Kaiserslautern, D-67663 Kaiserslautern, Germany
d Department of Cell Biology, University of Kaiserslautern, D-67663 Kaiserslautern, Germany
b Department
The carrier Endoplasmic Reticulum Adenylate Transporter1 (ER-ANT1) resides in the endoplasmic reticulum (ER) membrane
and acts as an ATP/ADP antiporter. Mutant plants lacking ER-ANT1 exhibit a dwarf phenotype and their seeds contain
reduced protein and lipid contents. In this study, we describe a further surprising metabolic peculiarity of the er-ant1 mutants.
Interestingly, Gly levels in leaves are immensely enhanced (263) when compared with that of wild-type plants. Gly
accumulation is caused by significantly decreased mitochondrial glycine decarboxylase (GDC) activity. Reduced GDC activity
in mutant plants was attributed to oxidative posttranslational protein modification induced by elevated levels of reactive
oxygen species (ROS). GDC activity is crucial for photorespiration; accordingly, morphological and physiological defects in
er-ant1 plants were nearly completely abolished by application of high environmental CO2 concentrations. The latter
observation demonstrates that the absence of ER-ANT1 activity mainly affects photorespiration (maybe solely GDC), whereas
basic cellular metabolism remains largely unchanged. Since ER-ANT1 homologs are restricted to higher plants, it is tempting
to speculate that this carrier fulfils a plant-specific function directly or indirectly controlling cellular ROS production. The
observation that ER-ANT1 activity is associated with cellular ROS levels reveals an unexpected and critical physiological
connection between the ER and other organelles in plants.
INTRODUCTION
Eukaryotic cells, particularly those of plants, are characterized
by a high number of intracellular compartments. Specific solute
carriers and channel proteins in the surrounding membranes are
required for communication and metabolic embedding of the
respective organelles. Because nucleotides fulfill multiple and
important biological functions and because they are involved in
almost all metabolic processes, sufficient nucleotide import into
and export out of cells and organelles has to be guaranteed.
Nucleotides represent basic molecules for DNA and RNA synthesis, they serve as cofactors in many enzymatic reactions,
are crucial elements involved in intra- and extracellular signal
transduction, and are precursors of phytohormone production
(Buchanan et al., 2000; Roux and Steinebrunner, 2007; Rieder
and Neuhaus, 2011). Moreover, ATP is by far the most important
nucleotide as it constitutes the main energy currency for cellular
metabolism and is required in many biosynthetic pathways.
The size and charge of nucleotides renders free permeation
of membranes impossible and necessitates specific transport
1 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: H. Ekkehard Neuhaus
([email protected]).
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.113.113605
systems. So far, three different families of nucleotide transporting proteins have been identified on the molecular level: the
nucleotide transporter (NTT) family, VNUT-type carriers (vesicular nucleotide transporters), and the mitochondrial carrier family
(MCF) (Haferkamp et al., 2011).
NTT proteins are restricted to plant plastids as well as to
certain intracellular parasites or endosymbionts with highly impaired metabolic capacities. Most NTT-type transporters catalyze ATP uptake in exchange with ADP (plus phosphate) and
hence are essentially involved in energy provision to the plastid
or intracellular organism (Neuhaus et al., 1997; Emes and
Neuhaus, 1998; Trentmann et al., 2008). Bacteria of the orders
Chlamydiales and Rickettsiales (and diatom algae) possess additional, different NTTs with different specificities and transport
modes. These NTTs mediate, for example, NAD supply or
proton-driven net uptake of purine and pyrimidine nucleotides
and compensate for the missing nucleotide and cofactor synthesis (Haferkamp et al., 2004)
Mammalian VNUT-type carriers catalyze ATP import into vesicles destined for fusion with the plasma membrane and hence are
involved in ATP exocytosis. Extracellular ATP acts as an essential
and highly specific signal molecule stimulating various purinergic
responses (Sawada et al., 2008).
The MCF group is composed of multiple phylogenetically and
structurally related carriers. MCF proteins mainly act in an antiport manner and mediate the transport of diverse substrates,
such as nucleotides, organic acids, amino acids, phosphate, or
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diverse cofactors. They are generally located in the inner mitochondrial membrane. However, a few MCF members reside in
other organelles, like plastids, peroxisomes, or the endoplasmic
reticulum (ER).
The MCF of Arabidopsis thaliana comprises 58 carriers, and
;50% of these proteins have been biochemically and/or physiologically characterized (Picault et al., 2004; Palmieri et al.,
2011; Haferkamp and Schmitz-Esser, 2012). Phylogenetic investigations revealed that functionally related MCF proteins
from different organisms generally form a specific cluster. Accordingly, the affiliation to a certain subgroup allows postulation
of the transport properties of biochemically uncharacterized
MCF members. Nucleotide transporting MCF-type carriers are
subdivided into different subclusters, like Mg-ATP/Pi carriers,
adenylate carriers specific for CoA, phospho-adenosine phosphate or phospho-adenosine phosphor sulfate, peroxisomal
adenine nucleotide carriers (Linka et al., 2008) and NAD exchangers, or plastidial brittle proteins involved in ADP-Glc/
adenine nucleotide exchange or unidirectional adenylate export
(Leroch et al., 2005; Kirchberger et al., 2007). A prominent MCF
subcluster of nucleotide transport proteins is composed of mitochondrial ADP/ATP carriers (AACs) from mammals, yeast, and
plants. AACs catalyze ADP import in exchange with ATP and
hence connect ATP synthesis in the matrix with ATP consuming
reactions outside the mitochondrion (Aquila et al., 1987;
Haferkamp et al., 2011). Two further but more distantly related
plant specific carriers are also affiliated to the AAC subcluster:
the recently identified plasma membrane–located adenylate carrier plasma membrane-located adenylate transporter1 (PM-ANT1)
as well as the ER-located carrier ER-ANT1.
PM-ANT1 catalyzes ATP export out of the plant cell and is
involved in signaling processes required for controlled anther
development (Rieder and Neuhaus, 2011), whereas ER-ANT1 is
proposed to mediate energy provision to the ER of Arabidopsis
(Leroch et al., 2008). ER-ANT1 was shown to act as an ATP/ADP
antiporter when heterologously expressed in Escherichia coli
and to exhibit a submillimolar affinity for both substrates.
Arabidopsis ER-ANT1 loss-of-function mutants contain substantially decreased protein and lipid levels in seeds and exhibit
a dwarf phenotype (Leroch et al., 2008). The metabolic changes
are in line with the essential role of the ER in protein and storage
lipid accumulation. The reported er-ant1 mutant phenotype includes stunted growth, reduced chlorophyll contents, and an
altered protein/lipid ratio in seeds.
We performed an extended physiological examination of the
er-ant1 knockout plants and identified multiple phenotypical
alterations that are tightly associated to photorespiration.
The observation that lack of an ER carrier affects the photorespiratory pathway was unexpected. The cellular organization
of photorespiration and the proteins involved are well investigated (Tolbert, 1997; Foyer et al., 2009; Bauwe et al., 2010;
Bauwe et al., 2012). According to our current understanding,
photorespiration essentially requires the physiological interaction of three different organelles, namely, chloroplasts,
mitochondria, and peroxisomes (Foyer et al., 2009; Bauwe et al.,
2010), and 25 different carrier proteins have been described to
shuttle the required substrates, cofactors, and products between these organelles (Eisenhut et al., 2013a). However, in
animals and yeast, a structural junction and physiological association of ER and mitochondria have been described (Kornmann
et al., 2009; Kornmann and Walter, 2010; Michel and Kornmann,
2012). Thus, our current study provides important evidence for a
physiological (reactive oxygen species [ROS]–mediated) interplay
between the ER and mitochondria in plants.
RESULTS
ER-ANT1 Loss-of-Function Plants Accumulate Gly in
a Light-Dependent Manner
ER-ANT1 was shown to be capable of ATP/ADP exchange and
to reside in the membrane of the ER (Leroch et al., 2008). The
fact that direct homologs are missing in yeasts and animals
suggests that ER-ANT1 fulfills a plant-specific function and that
basic energy provision to the ER is generally catalyzed by
a different carrier type or system. To gain deeper insights into
the particular role of ER-ANT1, we determined primary metabolites like selected phosphorylated intermediates representing
key intermediates of Suc and starch synthesis, of glycolysis, and
of photosynthesis. In addition, we quantified Suc, Glc, Fru, and
starch as end products of photosynthesis, carboxylates as indicators for mitochondrial activities, and amino acids as C/N
integrators (see Supplemental Table 1, Supplemental Methods 1
and Supplemental References 1 online). From all of these 24
individual intermediates, Gly levels of mutant plants showed by
far the highest alterations. Wild-type plants contain ;1.2 µmol
g21 fresh weight (FW) Gly, whereas er-ant1 mutants exhibited
26-fold higher Gly contents (Figure 1A; see Supplemental Table
1 online).
Quantification of metabolite concentrations in leaves harvested at different time points allowed the investigation of diurnal changes in Gly content. During the course of the day, Gly
levels of wild-type plants ranged from ;0.25 µmol g21 FW at the
end of the night phase to ;1.2 µmol g21 FW after 8 h of illumination (Figure 1B). At the end of the dark, er-ant1 lines exhibited a Gly concentration of ;2 µmol g21 FW, which increased
to more than 28 µmol g21 FW during 8 h of photosynthesis
(Figure 1B). Accordingly, differences in Gly contents of wild-type
and mutant plants become more pronounced during the light
phase and hence are apparently associated to light-dependent
processes. The decline in Gly contents of er-ant1-2 mutants in
the dark (more precisely, the substantial difference between the
maximal and the minimal level) demonstrates that mutant plants
are in principle capable of Gly degradation.
Absence of ER-ANT1 Affects Photorespiration
Because Gly represents a key metabolite associated with photorespiration (Douce et al., 2001), we tested whether this lightdependent pathway is affected in er-ant1 plants. Physiological
and morphological defects of many photorespiration mutants
can be compensated by elevating the environmental CO2 concentration to ;1 to 2% (Somerville, 2001; Voll et al., 2006).
Therefore, we compared growth of er-ant1 and wild-type plants
under ambient (380 ppm, control) or permanently high CO2
Interaction of Endoplasmic Reticulum and Photorespiration
Figure 1. Determination of Gly Contents in Arabidopsis Wild-Type and
er-ant1 Plants.
Gly contents in 4-week-old wild-type and er-ant1 knockout plants were
determined by HPLC. Plants were grown under 2% CO2 and a light/dark
cycle of 10/14 h for 3 weeks and subsequently shifted to ambient air.
Samples were taken after 5 d of adaptation. Shown are mean values of
three individual replicates, 6 SE. Asterisks indicate the significance level
between wild-type and er-ant1 knockout plants according to Student’s t
test (***P < 0.001).
(A) Gly levels of wild-type and er-ant1 knockout plants at the end of the
light period.
(B) Time course of Gly accumulation during illumination.
conditions (2%; Timm and Bauwe, 2013). Indeed, development
and habitus of mutants were identical to the wild type in high
CO2 and accordingly suppression of photorespiration compensated the dwarf phenotype of er-ant1 lines (cf. Figure 2A, 380
ppm, and Figure 2B, high CO2).
To further pinpoint the postulated photorespiratory phenotype
of er-ant1 lines, we measured CO2 assimilation, the CO2 compensation points, and photosynthetic quantum efficiencies.
These quantitative parameters of photosynthetic gas exchange
are inherently codetermined by photorespiratory metabolism
(Bauwe et al., 2010; Timm and Bauwe, 2013). All gas-exchange
measurements were performed with plants permanently grown
in presence of elevated environmental CO2 (2%). Transfer to
moderate CO2 concentrations (380 ppm, ambient condition)
resulted in substantially lower CO2 fixation capacities of the two
er-ant1 lines (3.1 and 4.0 µmol CO2 m22 s21) when compared
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with that of wild-type plants (7.6 µmol m22 s21; Figure 3A).
However, exposure to high external CO2 (2.000 ppm) revealed
comparable CO2 fixation rates of mutant (9.8 µmol m22 s21) and
wild-type plants (11.5 µmol m22 s21; Figure 3B). This finding
demonstrates that particularly under ambient conditions er-ant1
mutants are impaired in CO2 assimilation.
The CO2 compensation point represents the external CO2
concentration at which photosynthetic CO2 fixation equals respiratory CO2 release. It was determined for each plant line by
stepwise increases of the external CO2. In the wild-type control,
the compensation point was reached at a CO2 concentration of
;60 ppm (Figure 3C), which corresponds to values previously
reported for Arabidopsis (Eisenhut et al., 2013b). Similar to other
photorespiratory mutants (Timm et al., 2012), the er-ant1 plants
required significantly more CO2 (;90 ppm) to reach the equilibrium between photosynthetic CO2 fixation and (photo)respiratory CO2 release.
In a further study, we investigated whether transfer from high
external CO2 to ambient conditions affects maximum photosynthetic quantum efficiency of photosystem II (PSII) (given as
the Fv/Fm ratio) of the different plant lines. Prolonged exposure
to environmental CO2 concentrations (380 ppm) reduced Fv/Fm
of the mutant lines, whereas wild-type plants showed no corresponding alteration. Moreover, Fv/Fm of er-ant1 plants apparently decreased with increasing exposure time to low CO2 (1
d compared with 5 d; Figure 3D). These data show that extended exposure to photorespiratory conditions results in a reduced photosynthetic efficiency of er-ant1 mutants probably
caused by increasing damage of the photosynthetic machinery.
This is also in line with the fact that er-ant1 plants permanently
grown in ambient CO2 show reduced chlorophyll contents when
compared with wild-type plants (Figure 2A; Leroch et al., 2008).
Figure 2. Typical Growth Phenotype of Wild-Type and er-ant1 Knockout
Plants.
Wild-type and er-ant1 plants were grown on soil for 4 weeks under different CO2 conditions at a day/night cycle of 10/14 h.
(A) Growth in ambient air (0.038% CO2). Col-0, Columbia-0.
(B) Growth in 2% CO2.
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Figure 3. Gas Exchange Measurements and Chlorophyll Fluorescence.
Wild-type and er-ant1 knockout mutants were grown under conditions of 2% CO2 in a light/dark cycle of 10/14 h for 4 weeks. CO2 assimilation rates
and compensation points were determined at a PFD of 1.000 µmol photons m22 s21 and at CO2 concentrations ranging from 0.038% to 0.2%. Data
represent mean values of at least four individual replicates per line, 6 SE. Asterisks indicate the significance level between wild-type and er-ant1
knockout plants according to Student’s t test (*P < 0.05, **P < 0.01, and ***P < 0.001).
(A) Photosynthetic net CO2 uptake rates at 0.038% CO2. Col-0, Columbia-0.
(B) Photosynthetic net CO2 uptake rates at 0.2% CO2.
(C) CO2 compensation points.
(D) Maximum quantum yield of PSII.
In summary, these observations demonstrate that er-ant1 mutants, in all important aspects, clearly resemble plants with defects
in photorespiration (Somerville, 2001). First, suppression of photorespiration by high CO2 cures the stunted growth phenotype.
Second, er-ant1 mutants display a distinctly higher CO2 compensation point than wild-type plants. Third, photorespiratory
conditions lead to reduced CO2 fixation in combination with
a lower PSII maximum quantum efficiency. This clear diagnosis of
a photorespiratory phenotype of the er-ant1 mutants provides
unexpected evidence for an interaction between ER metabolism
and the complex process of photorespiration.
To figure out whether the lack of ER-ANT1 is in fact causative for
the observed phenotype, we performed control experiments by
use of the rice (Oryza sativa) ortholog Os-ER-ANT1. For this, we
investigated the functional properties of the closest ER-ANT1
homolog from rice (see Supplemental Figure 1 and Supplemental
Table 2 online) by heterologous expression in E. coli and transport
measurements with intact recombinant cells. Import studies
demonstrated that Os-ER-ANT1 is able to transport ATP and ADP
(see Supplemental Figure 2 online), as also known for Arabidopsis
ER-ANT1 (Leroch et al., 2008). The rice carrier Os-ER-ANT1 exhibits apparent affinities (Km) for ATP and ADP of 8.9 and 12.7 µM,
respectively (see Supplemental Figures 2A and 2B online) and
hence possesses ;30-fold higher substrate affinities when compared with ER-ANT1 from Arabidopsis (Leroch et al., 2008). The
Os-ER-ANT1 gene has been introduced under control of a cauliflower mosaic virus promoter into the homozygous Arabidopsis
knockout line er-ant1-2 (see Supplemental Figures 3A to 3C, 4A,
and 4B online). Growth analyses confirmed that developmental
deficits of the er-ant1-2 line (under ambient CO2) were nearly
completely compensated for by integration of the orthologous rice
gene (see Supplemental Figures 4B and 4C online). These data,
together with the already conducted detailed molecular and
physiological characterization of the two independent er-ant1
mutant lines (Leroch et al., 2008), clearly demonstrate that absence of ER-ANT1 is the reason for the observed photorespiratory
phenotype.
er-ant1 Mutants Exhibit Highly Reduced Gly Decarboxylase
Activity but Only Slightly Reduced Abundance of Gly
Decarboxylase Components
With the aim to gain first insights into the interplay between ER and
photorespiration, we focused on the investigation of possible
Interaction of Endoplasmic Reticulum and Photorespiration
factors causing Gly accumulation in er-ant1 mutant plants (Figures
1A and 1B). An important enzymatic step during photorespiration
is the degradation of Gly to methylene tetrahydrofolate, CO2, and
NH3 via the mitochondrial enzyme Gly decarboxylase (GDC;
Douce et al., 2001; Bauwe et al., 2010). To test whether GDC
activity is affected in er-ant1 mutants, we performed enzyme activity assays with purified mitochondria isolated from the different
plant lines grown at ambient CO2. Mitochondria from the two
er-ant1 mutant lines showed ;40% GDC activity when compared
with that of wild-type plants (set to 100%; Figure 4A). Interestingly,
also under nonphotorespiratory growth conditions (2% CO2) GDC
activity in the er-ant1 lines was significantly reduced (36% in
er-ant1-1 and 39% in er-ant1-2, respectively; Figure 4B).
Functional GDC requires four different proteins, namely, P-,
T-, H-, and L-protein (Douce et al., 2001). Moreover, mitochondrial
Ser hydroxymethyltransferase (SHMT) is also important for adequate
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in vivo GDC activity under photorespiratory conditions because
it rapidly recycles methylene tetrahydrofolate to tetrahydrofolate
(Douce et al., 2001). To gain insight into the relative abundance of
these proteins associated with GDC function, we performed immunoblot analyses of leaf extracts. Immunodetection with a set of
specific antibodies raised against four of these five components
revealed that er-ant1 mutants show a reduced amount of the
P-protein, whereas all other investigated GDC component proteins
were present at about identical levels in wild-type plants and
er-ant1 mutants (Figures 4C and 4D). While we did not exactly
determine leaf P-protein levels, the reduced GDC activities (Figures 4A and 4B) correspond to the lower amounts of P-protein
at least to some degree.
To check whether the lowered levels of P-protein (Figure 4D)
might be causative for the decreased GDC activity (Figures 4A
and 4B), we generated er-ant1 plants overexpressing either the
Figure 4. GDC Activity and Immunodetection of Mitochondrial Protein Abundance.
(A) GDC activity in mitochondria isolated from wild-type and er-ant1 mutants grown in ambient air. Col-0, Columbia-0.
(B) GDC activity in mitochondria isolated from wild-type and er-ant1 knockout plants grown in 2% CO2. Mitochondria were isolated from green leaf
tissue, broken by sonication, and incubated in GDC assay buffer supplemented with [14C]-Gly as described in Methods. The 14CO2 released from
P-protein activity was trapped in 3 M KOH after 30 min, and the amount of radioactivity was quantified by scintillation counting. The data are means of
three individual replicates, 6 SE. Asterisks indicate the significance level between wild-type and er-ant1 knockout plants according to Student’s t test
(***P < 0.001).
(C) Coomassie blue–stained SDS-PAGE. Ten micrograms of protein of total leaf extracts from wild-type and the two er-ant1 knockout plant lines was
loaded.
(D) Immunoblots showing the protein abundance of GDC subunits and SHM.
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P-protein from Flaveria pringlei (PFP5) or from the cyanobacterium
Synechocystis sp PCC6803 (SLR0293) under control of a constitutive 35S promoter (see Supplemental Figures 5A to 5F online).
The corresponding genetic constructs (see Supplemental Figures
5A and 5B online) were introduced into the er-ant1 background
and led to substantial levels of corresponding mRNAs (see
Supplemental Figure 5C online). Immunoblot analysis confirmed
that the representative overexpressing mutant lines exhibited not
only detectable mRNA levels, but also increased levels of P-protein
(see Supplemental Figure 5D online). However, neither the mutant
line overexpressing the Flaveria gene nor the mutant line overexpressing the Synechocystis gene exhibited any rescue of the
dwarf phenotype (see Supplemental Figures 5E and 5F online).
er-ant1 Mutants Show Elevated Levels of ROS and
Increased ROS Defense Capacity
Previous analyses revealed that GDC activity is distinctly lower
in er-ant1 than in the wild type, both at high and ambient CO2. At
least in part this can be explained by the somewhat reduced
Figure 5. Histochemical Detection of ROS, Activity of ROS Scavenging Enzymes, and GSH Redox Status in Wild-Type and er-ant1 Plants.
(A) and (B) DAB staining for detection of H2O2 accumulation (A) and NBT staining for determination of superoxide accumulation (B). Wild-type and
er-ant1 plants were grown in 2% CO2 for 4 weeks to ensure equal growth patterns. Prior to staining of the individual ROS species, plants were kept
under high CO2 conditions or shifted to ambient air for additional 5 d. The control solutions contained 10 mM ascorbate (DAB staining) or 10 units mL21
SOD (NBT staining). Col-0, Columbia-0.
(C) Total catalase and SOD activity in Arabidopsis leaf extracts from plants grown under different CO2 conditions.
(D) Levels of reduced and oxidized GSH and GSH redox status (ratio of GSH:GSSG). The data represent mean values of three individual replicates, 6 SE.
Asterisks indicate the significance level between wild-type and er-ant1 knockout plants according to Student’s t test (*P < 0.05, **P < 0.01, and ***P < 0.001).
Interaction of Endoplasmic Reticulum and Photorespiration
amount of P-protein, which is the actual Gly decarboxylating
subunit. Since GDC is known to be regulated by diverse factors,
however, we hypothesized that the reduced amount of P-protein
is one but probably not the exclusive and perhaps not even the
major reason for the very strongly elevated Gly levels and that
other processes might also interfere with GDC activity.
Because GDC is highly sensitive to attack by ROS (Taylor
et al., 2002; Palmieri et al., 2010), we investigated ROS production in er-ant1 mutant plants as another possible cause for
GDC inhibition. For this, plants were first grown for 4 weeks
under high CO2 (to assure similar development; see Figure 2B)
and subsequently cultivated for five additional days under either
high CO2 (2%) or control CO2 levels (380 ppm). Leaves from
wild-type and mutant plants were collected 4 h after onset
of light and the relative abundances of two prominent ROS
species, namely, H2O2 and the superoxide anion, monitored.
Staining with 3,39-diaminobenzidine (DAB) allowed the detection
of H2O2, and nitro-blue tetrazolium chloride (NBT) was used to
assess superoxide accumulation. Preincubation with ascorbate
or the superoxide-degrading enzyme superoxide dismutase
(SOD) confirmed that leaf staining was solely caused by H2O2
and superoxide, respectively (Figures 5A and 5B, control; see
Supplemental Figures 6A and 6B online for further independent
replicates of NBT-stained leaves). Comparison of the staining
intensity revealed that leaves of er-ant1 mutants contained
higher amounts of H2O2 and superoxide than those of wild-type
plants (Figures 5A and 5B; see Supplemental Figures 6A and 6B
online). Interestingly, enhanced accumulation of these two ROS
in mutant leaves not only became detectable after plant exposure to ambient CO2 but also occurred when plants were cultivated under elevated environmental CO2 (Figures 5A and 5B).
The increased H2O2 levels were directly confirmed via luminometric quantification. At ambient CO2, wild-type plants contained 200 nmol H2O2 g21 FW, but both er-ant1-1 and er-ant1-2
plants exhibited between 330 and 340 nmol H2O2 g21 FW (see
Supplemental Figure 6C online). When grown under high CO2,
wild-type plants contained nearly identical H2O2 levels as
present under ambient CO2, namely, 195 nmol H2O2 g21 FW.
Similar to the situation in ambient CO2, growth of both er-ant1
plant lines under high CO2 led again to increased H2O2 levels
when compared with the wild type, namely, 285 nmol g21 FW in
er-ant1-1 and 348 nmol g21 FW in er-ant1-2 (see Supplemental
Figure 6D online).
ROS generation is connected to various physiological processes in higher plants (Foyer and Noctor, 2005), and different
reactions contribute to the defense machinery directed against
these reactive compounds. Catalase and SOD are important
factors controlling cellular ROS levels, and plants contain multiple isoforms of these proteins (Willekens et al., 1994; Bowler
et al., 1989). Exposure to ambient CO2 results in significantly
enhanced total catalase and SOD activities in er-ant1 knockout
lines when compared with wild-type plants, whereas under the
nonphotorespiratory conditions of high environmental CO2,
corresponding enzyme activities of mutant plants only marginally exceeded activities in the wild type (Figure 5C). Quantitatively, photorespiration is the major ROS-producing pathway in
illuminated leaves (Foyer et al., 2009). It is hence likely that ROS
accumulation in mutant plants is higher under ambient than
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under elevated CO2 conditions. This difference cannot be seen
in the staining analysis (Figures 5A and 5B) but is probably
sufficient to significantly stimulate catalase and SOD activity.
GSH is a prevalent reducing equivalent in the cell and represents a further factor controlling cellular ROS state (Noctor et al.,
2011). Wild-type plants exposed to high CO2 contained GSH
concentrations of 201 nmol g21 FW, and identically treated
mutant lines showed slightly higher GSH levels (283 nmol g21
FW in er-ant1-1 and 260 nmol g21 FW in er-ant1-2; Figure 5D).
Increase of GSH in er-ant1 mutants is accompanied by moderately decreased oxidized glutathione GSSG levels when
compared with that of the wild type, resulting in a marginally
altered GSH pool (Figure 5D). However, when exposed to ambient conditions, mutant lines exhibit strongly increased GSH
levels (619 nmol g21 FW in er-ant1-1 and 418 nmol g21 FW in
er-ant1-2), whereas wild-type plants contain ;243 nmol GSH g21
FW (Figure 5D). Moreover, apart from GSH also GSSG concentrations of the er-ant1 lines were significantly increased (by
;2.8 and 4.2 nmol g21 FW) when compared with wild-type
values (Figure 5D). As a consequence, er-ant1 lines exhibit an
elevated pool of reduced GSH, particularly under ambient
conditions (;96.7% GSH in er-ant1-1 and 96.4% in er-ant1-2
plants, respectively, versus 93.7% GSH in wild-type plants;
Figure 5D).
GDC from er-ant1 Mutants Exhibits Oxidative Inhibition
Reactive oxygen and nitrogen species are important factors
causing oxidative alterations of the Cys thiol side chains in
proteins. Cys side chains can be oxidized to disulfides, different
sulfur oxoacids, mixed disulfides with other thiols, or become
nitrosylated. ROS inhibit plant GDCs (Taylor et al., 2002),
and posttranslational Cys modifications (S-glutathionylation/Snitrosylation) are involved in the oxidative downregulation of
GDCs (Palmieri et al., 2010).
As shown above, mutant plants lacking ER-ANT1 exhibit increased ROS and reduced GDC activity. We applied the reducing agent DTT to check for an oxidative GDC processing and
to examine whether the low GDC activity in er-ant1 mutants is
due to such protein modification (when compared with that of
the wild-type enzyme). Enriched mitochondria from wild-type
plants and of one representative mutant line (er-ant1-2) were
disrupted and treated with DTT. Application of DTT stimulated
wild-type GDC threefold from 0.52 nmol min21 mg21 protein to
;1.56 nmol min21 mg21 (Figure 6). Interestingly, the low GDC
activity of er-ant1-2 mutants was highly increased (to 1.94 nmol
min21 mg21 protein) by DTT and even slightly exceeded the
corresponding wild type (Figure 6). The extent of activity stimulation by DTT addition could be explained by an oxidative
modification of GDC in the er-ant1 mutants. This is consistent
with the observation that the reducing agent DTT reversed oxidation, which suggests that thiol-based modification accounts
for the highly reduced GDC activity in er-ant1 mutants.
Glutathionylation is an important posttranslational modification responsible for the reduction of GDC activity in plant mitochondria (Palmieri et al., 2010). Thus, to compare GDC
glutathionylation of wild-type and er-ant1 plants, we investigated
activity changes induced by deglutathionylation due to application
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Figure 6. Effects of Thiol-Modifying Agents on GDC Activity in Arabidopsis Wild-Type and er-ant1 Mitochondria.
Mitochondria from 6-week-old wild-type and er-ant1 knockout plants
were broken by sonication. Before starting the GDC assay, the samples
were either pretreated with 25 mM DTT for 1 h or with a regenerating
glutaredoxin system for 15 min. The activity of the P-protein was determined by trapping the released 14CO2 in KOH and subsequent scintillation counting. The data represent mean values of three individual
replicates, 6 SE. Asterisks indicate the significance level between wildtype and er-ant1 knockout plants according to Student’s t test (*P < 0.05,
**P < 0.01, and ***P < 0.001). Col-0, Columbia-0.
of the regenerating glutaredoxin system (Peltoniemi et al.,
2006; Bedhomme et al., 2012). GDC activity of wild-type mitochondria was approximately threefold stimulated (from 0.52
nmol min21 mg21 protein to 1.45 nmol min21 mg21 protein) by
glutaredoxin treatment (Figure 6). However, GDC from er-ant1-2
plants was hardly affected (reached 0.4 nmol min21 mg21 protein)
and corresponds to only 27% of the observed wild-type GDC
value (Figure 6). This finding implies that GDC from wild-type
plants is measurably downregulated by glutathionylation, whereas
the enzyme of er-ant1 plants is apparently inhibited by other
factors.
ROS Accumulation Is Not Caused by Impaired Protein
Synthesis in the ER
ER-ANT1 was shown to reside in the ER (Leroch et al., 2008), and
absence of this carrier results in increased ROS production and
finally in downregulation of GDC activity. To investigate whether
ROS production is due to basic malfunction of the ER, we analyzed
protein synthesis and folding in er-ant1 mutants. Accumulation of
unfolded proteins in the ER initiates the so-called unfolded protein
response (UPR), detectable by increased transcription of genes
encoding ER-located chaperones (Lai et al., 2007). However, we
obtained no indications for induction of UPR in er-ant1 plants. In
fact, as also previously documented (Leroch et al., 2008), the levels
of mRNAs coding typical ER-located chaperones, namely, BIP1/2,
BIP3, and calreticulin, were not enhanced but rather were even
lower in er-ant1 lines when compared with that of wild-type plants
(see Supplemental Figure 7 online).
To investigate whether er-ant1 mutants are defective in UPRassociated signal transfer from the ER to the nucleus, we applied
the toxin tunicamyin, known to induce UPR reactions by blocking
glycoprotein synthesis. After addition of tunicamycin, transcription
of BIP1/2, BIP3, and calreticulin was highly stimulated in the
wild type as well as in er-ant1 plants (see Supplemental Figure
7 online).
These results demonstrate that in er-ant1 mutants signal
transduction induced by the accumulation of unfolded proteins
in the ER lumen operates well and that basic metabolic processes in the ER apparently are not considerably altered. Conclusively, ROS production in er-ant1 plants is not caused by
drastically impaired ER function. Moreover, massive failures in
protein synthesis would severely affect plant growth and development. However, the dwarf phenotype of er-ant1 mutants
occurs solely under ambient CO2, whereas suppression of
photorespiration by elevated CO2 results in mutant plants morphologically resembling the wild type. This indicates that only
minor but important changes in the ER induce ROS production.
DISCUSSION
er-ant1 Mutants Exhibit a Photorespiratory Phenotype
Photorespiration occurs in cyanobacteria, algae, and at particularly high rates in most higher plant species (Foyer et al., 2009;
Bauwe et al., 2012). This metabolic process serves to recycle
2-phosphoglycolate, which is unavoidably produced by
ribulose-1,5-bis-phosphate carboxylase/oxygenase, into 3phosphoglycerate but also results in considerable rates of
CO2 release. Because photorespiration was long considered
a wasteful process leading to reduced plant growth and crop
yield, it is not surprising that several molecular-genetic strategies were pursued to possibly reduce photorespiratory CO2
losses (Kebeish et al., 2007; Maier et al., 2012). After its discovery in the 1950s, it turned out photorespiration is based
on a complex metabolic pathway spread over three cellular
compartments: chloroplasts, peroxisomes, and mitochondria
(Tolbert, 1997; Foyer et al., 2009).
In this study, we provide first evidence that a further organelle,
namely, the ER, is associated with photorespiration. We found
that Arabidopsis mutants lacking the ER-located MCF carrier
ANT1 (er-ant1 loss-of-function lines) exhibit a very distinct photorespiratory phenotype. First, these mutants accumulate high
amounts of Gly in the light. Second, CO2 compensation points are
elevated relative to wild-type plants, whereas CO2 fixation rates
and PSII quantum efficiency are reduced. These features collectively result in chlorotic leaves and strongly impaired growth.
Third, all these effects observed with the er-ant1 mutants in normal air can be more or less completely reverted to a wild-type-like
phenotype by plant growth at a high external CO2 concentration.
These symptoms are characteristic of mutants defective in photorespiratory metabolism (Somerville and Ogren, 1981; Eisenhut
et al., 2013b). The observation that er-ant1 plants exhibit altered
levels of some other metabolites than Gly (see Supplemental
Table 1 online) does not contradict this conclusion. It is generally
known that limitation of carbon flow on photorespiration also affect levels of metabolites not directly involved in this pathway
(Somerville, 2001; Timm et al., 2012; Eisenhut et al., 2013b).
Interaction of Endoplasmic Reticulum and Photorespiration
The Photorespiratory Phenotype of er-ant1 Mutants Is
Caused by Reduced GDC Activity
Investigation of the metabolite composition demonstrated that
er-ant1 mutants massively accumulate Gly under photorespiratory
conditions (Figures 1A and 1B). This metabolic peculiarity was
previously observed in Arabidopsis mutants lacking functional
GDC (Somerville and Ogren, 1982; Engel et al., 2007). GDC is an
essential component of the mitochondrial part of photorespiration
and catalyzes, together with SHMT, the formation of Ser from two
Gly molecules. Analysis of individual and combined T-DNA insertion mutants for the GDC’s P-protein verified that, apart from
photorespiration, the enzyme is also involved and indispensable
for basic plant metabolism (C1 metabolism; Engel et al., 2007).
Arabidopsis plants completely lacking the P-protein die, even
under nonphotorespiratory conditions (Engel et al., 2007). By
contrast, individual knockout lines of either of the two P-protein
genes in Arabidopsis grow well in ambient air and do not show any
visible difference to the wild type (Engel et al., 2007).
Enzyme assays revealed that er-ant1 mutants exhibit only
;40% of wild-type GDC activity (Figure 4A). This residual GDC
activity is apparently sufficient to degrade Gly during the night
but insufficient to cope with the high photorespiratory carbon
flux during the light phase (Figure 1B). Inhibition of photorespiration by high CO2 results in normal growth and development of
the er-ant1 mutants, demonstrating that the remaining GDC
activity readily meets the demands of basic C1 metabolism.
Oxidative Modification Mainly Causes the Reduced GDC
Activity in er-ant1 Mutants
With the aim to unravel factors connecting ER metabolism and
photorespiration, we focused on the basis of GDC activity reduction. It is imaginable that limited presence of one or more
GDC-associated proteins in er-ant1 mutants causes the reduced in vivo activity. However, diverse subunits of the GDC
holoprotein as well as the interacting mitochondrial enzyme
SHMT exhibited similar abundance in wild-type and mutant
plants (Figures 4C and 4D). Also, the amount of P-protein was
slightly reduced (Figure 4D); hence, activity reduction might be
explained, if at all, only to a very limited extent by lowered
presence of the GDC holoprotein (Figure 6). Moreover, the observation that er-ant1 mutants overexpressing either the gene
coding for the P-protein from Flaveria or Synechocystis show
increased P-protein abundance but no rescue of the dwarf
phenotype (see Supplemental Figures 5A to 5F online) further
speaks against an influence of the decreased P-protein amount
on total GDC activity in er-ant1 plants.
Thus, apart from a slightly reduced abundance of the P-protein,
the vast majority of activity reduction was clearly attributed to
a substantial GDC inhibition due to oxidative posttranslational
modification. The GDC is highly susceptible to reactive oxygen
since ROS accelerate carbonylation or S-glutathionylation, processes known to downregulate GDC activity (Taylor et al., 2002,
2004; Palmieri et al., 2010; Lounifi et al., 2013). We believe that
this type of GDC modification is caused by increased oxidative
stress in er-ant1 plants as demonstrated by various indicators: (1)
elevated H2O2 and superoxide levels, (2) stimulated catalase and
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SOD activities, and (3) increased GSH levels (Figures 5A to 5D;
see Supplemental Figure 6 online). The latter observation is,
moreover, fully in line with the observation that oxidative stress
per se stimulates Glu-Cys ligase activity provoking increased
GSH levels (Hicks et al., 2007) since this enzyme is rate limiting
for GSH biosynthesis.
Carbonylation is an irreversible modification that targets proteins to degradation (Rao and Moller, 2011; Lounifi et al., 2013).
However, this type of modification is probably not responsible
for GDC activity reduction in er-ant1 mutant plants. Although
er-ant1 mutants harbored slightly lower amounts of the P-protein,
the other GDC proteins were present at wild-type levels (Figure
4D). Remarkably, the low GDC activity of er-ant1 mitochondria
could be fully restored by DTT treatment and then even exceeded
activities of the correspondingly treated GDC from wild-type mitochondria (Figure 6). This finding pointed to an oxidative modification of Cys residues.
Cys side chains can undergo different states of oxidation. For example, they are modified by S-thiolation (mainly S-glutathionylation)
or S-nitrosylation, form intra- or intermolecular protein disulfide
bridges, and are oxidized to sulfenic, sulfinic, and sulfonic
acids. By use of a regenerating glutaredoxin assay, suitable to
remove GSH residues from proteins (Bedhomme et al., 2012),
we investigated the S-glutathionylation status of GDC from
wild-type plants and er-ant1 mutants. Several glutathionylated
Cys residues have been identified in the GDC in vivo and were
shown to be responsible for reduced enzyme activity (Palmieri
et al., 2010). We found that GDC activity of the wild type was
partially reduced due to glutathionylation, but the corresponding
enzyme from mutant plants did not respond to the glutaredoxin
assay (Figure 6).
The latter observation does not fully exclude glutathionylation,
but we presently think that another type of oxidative alteration
is responsible for the inhibition of GDC in er-ant1 plants. DTT
can reduce several modified thioles (Scheibe and Stitt, 1988;
Kettenhofen et al., 2007), and GDC activity from er-ant1 plants
was totally restorable by application of reduced DTT (Figure 6).
Therefore, activity reduction of GDC in er-ant1 lines is most
likely caused by ROS induced oxidative alterations/damages,
like disulfide bond formation absent in wild-type mitochondria
(Figure 6). At this stage, it is important to mention that we cannot
rule out that this type of modification overrides the inhibitory
impact of a still possible S-glutathionylation in the GDC of mutant plants.
The reduced GDC activity is apparently causative for the
photorespiratory phenotype of er-ant1 mutants. It was shown to
result from oxidative protein modification and, likely to a smaller
degree, less P-protein. Oxidative modification of GDC is fully
consistent with an enhanced ROS production in er-ant1 mutants. Interestingly, er-ant1 mutants accumulated significantly
higher amounts of H2O2 and superoxide not only under ambient,
but also under high external CO2 concentrations when compared with wild-type plants (Figures 5A and 5B). However, other
physiological and morphological defects emerge exclusively/
mainly under photorespiratory conditions. These observations
indicate that the increased ROS level nearly exclusively affects
photorespiration, whereas other metabolic processes remain
rather unchanged or cope better with this situation.
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The Plant Cell
The Missing Link: What Connects ER-ANT1 to
ROS Formation?
ER-ANT1 is unambiguously affiliated to with the ATP/ADP carrier subgroup of the MCF. Moreover, it exhibits typical and essential protein motifs that are conserved among all AACs
(Leroch et al., 2008) and therefore was primarily suggested to
represent a fourth AAC isoform in Arabidopsis. In fact, biochemical analyses verified that both recombinant Arabidopsis
ER-ANT1 and the rice ER-ANT1 protein mediate counterexchange of ATP and ADP (Leroch et al., 2008; see Supplemental
Figures 2A and 2B online). Noteworthy, Arabidopsis ER-ANT1
and its rice homolog exhibit a marked structural difference
to the AACs from Arabidopsis as its amino acid sequence is
shorter because it lacks the mitochondrial targeting sequence
characteristic for latter carriers. This feature already points to
a different subcellular targeting and diverse methods clearly
localized the carrier in the ER (Leroch et al., 2008).
It appears quite unlikely that ER-ANT1 acts as the main ATP/
ADP carrier in this compartment. Of course ATP entry into the
ER is mandatory to enable various anabolic reactions within this
organelle (Clairmont et al., 1992; Pimpl et al., 2006). However,
the fact that ER-ANT1 homologs are restricted to the plant
kingdom but energy provision to the ER is required in all eukaryotes suggests that energy provision to this organelle is
mainly/generally mediated by a different mechanism (Leroch
et al., 2008; Haferkamp and Schmitz-Esser, 2012). This assumption is further supported by the observation that lack of
ER-ANT1 especially interferes with photorespiration but not
so markedly with basic cell metabolism. Thus, ER-ANT1 might
contribute only to a limited extend to the overall ATP supply into
the ER; moreover, it cannot be ruled out that ER-ANT1 catalyzes
the transport of an additional so far not identified substrate.
Regardless of its transport function, there is a clear metabolic
connection between ANT1 presence in the ER and ROS production; however, the mechanistic basis of this is still elusive.
It has frequently been reported that uncontrolled processes in
the ER, for example, accumulation of unfolded proteins, can
induce ROS production (Santos et al., 2009). However, er-ant1
plants do not show typical molecular symptoms associated with
a UPR because the expression of genes coding for ER-located
chaperones is not enhanced and also the UPR signal transduction pathway was proven to operate well (see Supplemental
Figure 7 online). Accordingly, enhanced ROS production in
er-ant1 mutants is apparently not associated with UPR-related
processes. We suggest that the ER of er-ant1 mutants exhibits
a subtle physiological impairment because main characteristic
ER functions, like protein synthesis and folding, proceed with no
visible alterations and because the majority of the observed
symptoms in mutants can be cured by permanent growth in
a high CO2 environment. The resulting ROS (H2O2 and superoxide) accumulating in er-ant1 mutants (Figures 5A and 5B; see
Supplemental Figures 6A and 6B online) most likely derive from
a modified mitochondrial metabolism, which is a result of impaired ER processes. This assumption is based on the general
properties of ROS because H2O2 is highly polar, and both H2O2
and superoxide are extremely reactive molecules, giving them
a limited capacity to diffuse across the cell (Apel and Hirt, 2004).
Interestingly, the proposed primary (and so far unresolved)
metabolic changes become detectable by the increased ROS
production occurring in mutants grown under ambient air but
also in mutants cultivated under high CO2 conditions (Figures 5A
and 5B; see Supplemental Figures 6A and 6B online). GDC is
suggested to be much more sensitive to oxidative stress than
other enzymes (Taylor et al., 2002); in fact, comparably slight
oxidative stress in er-ant1 plants grown under high CO2 (Figures
5B to 5D) already caused GDC inhibition (40% residual activity;
Figure 4B). However, excessive oxidative modification and activity impairment of GDC is solely problematic during photorespiration and not under nonphotorespiratory conditions (Figures 2A
and 2B). Therefore, we propose that er-ant1 plants exhibit a cumulative defect that becomes potentiated by high carbon flux
through photorespiration. In this context, er-ant1 plants resemble
typical photorespiratory mutants lacking one of the enzymes
critical for photorespiratory metabolism (Bauwe and Kolukisaoglu,
2003; Bauwe et al., 2010).
Conclusion
Our data indicate that the Arabidopsis carrier ER-ANT1 is required to prevent uncontrolled ROS generation and, thus, to
guarantee maintenance of photorespiration. Absence of ERANT1 causes essential but subtle and therefore so far unresolved physiological changes in the ER that lead to ROS
accumulation. The increased ROS level promotes oxidative
modification and inhibition of the enzyme GDC under both
photorespiratory and nonphotorespiratory conditions. However,
impaired GDC activity is only detrimental under photorespiratory
conditions that further potentiate defects in er-ant1 mutants.
In animal and fungal cells, the bidirectional communication
between ER and mitochondria has been observed for a long
time and is required for important processes like Ca2+ signaling,
lipid metabolism, intracellular energy provision, and even programmed cell death (Kim et al., 2006; de Brito and Scorrano
2010; Michel and Kornmann, 2012). Our studies shed unexpected light on the ER/mitochondria communication by a so
far unknown interaction of a plant-specific ER resident carrier
(Leroch et al., 2008) and a plant-specific metabolic pathway. In
the future, more elaborate analyses of the er-ant1 mutants are
required to completely unravel ER-ANT1–associated metabolic
alterations in the ER and the basis of the observed ROS
production.
METHODS
Plant Material and Growth Conditions
All studies were performed with Arabidopsis thaliana wild-type plants (ecotype
Columbia-0) and T-DNA insertion mutants for er-ant1-1 (Salk_043626) and
er-ant1-2 (Salk_023441) as described earlier (Leroch et al., 2008). Prior to
germination, seeds were incubated for 2 d in the dark at 4°C on standardized
ED73 soil (Weigel and Glazebrook, 2002). Plants were grown at 22°C and 120
µmol quanta m22 s21 in a 10-h-light/14-h-dark regime either at ambient CO2
levels (380 ppm CO2), or at high CO2 levels (20,000 ppm CO2; Timm and
Bauwe, 2013) in Percival plant climate chambers (CLF Plant Climatics). For
measurements under ambient conditions, plants were shifted to normal
air for 1 week prior to the experiment.
Interaction of Endoplasmic Reticulum and Photorespiration
RNA Isolation, Generation of cDNA, and Quantitative RT-PCR
Total RNA was prepared from Arabidopsis leaf tissue using the NucleoSpin
RNA Plant Kit (Macherey-Nagel). Quantitative RT-PCR was performed
as described (Leroch et al., 2005) using a MyIQ-Cycler (Bio-Rad) and IQ
SYBR Green Supermix (Bio-Rad), according to the manufacturer’s instructions. The gene At5g60390, encoding the elongation factor 1a, was
used for quantitative normalization. The respective primers are listed in
Supplemental Table 3 online.
11 of 14
(GFS-3000; Walz). All measurements were performed with 4-week-old
wild-type and er-ant1 plants grown under high CO2 conditions. Net CO2
assimilation rates were determined at ambient CO2 levels (380 ppm) or
elevated CO2 levels (2.000 ppm), a PFD of 1.000 µE, a leaf temperature of
25°C, 60% humidity, and an air flow of 750 µmol m22 s21. For the determination of CO2 compensation points, CO2 concentrations varied from
40 to 400 ppm. The maximum quantum yield of PSII (Fv/Fm) was quantified
according to Harbinson et al. (1989) before and after shifting the plants to
ambient air for 1 week (Timm et al., 2011).
Protein Isolation and Immunodetection of GDC Subunits
Isolation of Mitochondria and Determination of GDC Activity
For the isolation of total protein, 100 mg of leaf tissue was homogenized in
liquid nitrogen using a mortar and pestle and extracted in 200 mL of buffer
medium (50 mM HEPES-KOH, pH 7.6, 10 mM NaCl, 5 mM MgCl2, 100 mM
sorbitol, and 1 mM phenylmethylsulfonyl fluoride). The homogenate was
centrifuged (4°C, 10 min, 20,000g), and the total protein content was determined (Bradford, 1976). For immunological analysis, 10 mg of total leaf
protein was separated by SDS-PAGE and stained with Coomassie Brilliant
Blue R 250 (Laemmli, 1970) or blotted onto a polyvinylidenfluoride membrane
according to standard protocols. Immunoblots were decorated with primary
antibodies raised against SHM or the GDC proteins P, T, and H.
Isolation of a crude mitochondrial extract from 10 g of Arabidopsis leaves
was performed according to a standard protocol (Keech et al., 2005).
Mitochondria were washed twice in buffer medium (0.3 M Suc, 10 mM
TES, 2 mM EDTA, and 10 mM KH2PO4, pH 7.5), resuspended in 0.6 mL of
GDC assay medium (0.36 M Suc, 20 mM MOPS, pH 7.2, 8 mM KCl, 4 mM
NaH2PO4, 4 mM MgCl2, and 0.1% BSA), and broken by sonication for 3 3
30 s on ice. The total protein content was determined according to
Bradford (1976) after another centrifugation step (4°C, 5 min, 16,000g).
GDC activity of the supernatant was quantified by measuring the decarboxylation of [14C]-Gly (Somerville and Ogren, 1982). One hundred
micrograms of mitochondrial protein were incubated with 100 mL of GDC
assay buffer supplemented with 8 mM Gly (4 µCi mL21 [14C]-Gly) in 1.5mL reaction tubes. The effects of DTT were analyzed after incubation of
the mitochondrial protein with 25 mM DTT for 1 h on ice before starting the
assay. The effects of deglutathionylation were analyzed using a regenerative glutaredoxin system (Peltoniemi et al., 2006; Bedhomme et al.,
2012). Samples were incubated in GDC assay buffer containing 0.05 units
mL21 glutaredoxin, 0.02 units mL21 GSH reductase, 1 mM GSH, and
50 µM NADPH as final concentration (all compounds were provided by
Sigma-Aldrich) and incubated for 15 min at room temperature before
starting the assay. Released [14C]-CO2 was captured in small reaction
vessels containing 100 mL 3 M KOH. The reaction was stopped after
30 min by injecting 100 mL of 2 M HCl into the reaction mixture with
a syringe through the closed lid. The hole in the lid was subsequently
sealed with grease and the vials were stored for one night to allow total
absorption of radioactively labeled CO2. The trapped radioactivity was
quantified by scintillation counting (Perkin-Elmer Tricarb 2810 TR).
Quantification of Metabolites and Enzyme Activities of CAT and SOD
For the determination of metabolites and enzyme activities, Arabidopsis
leaf samples were harvested in the middle of the light phase and ground in
liquid nitrogen using mortar and pestle. Quantification of Gly by HPLC was
performed according to a routine method in our lab (Jung et al., 2009)
using 80% ethanol (v/v) as extraction medium. GSH was quantified from
neutralized HCl extracts according to the 5,59-dithiobis-C2-nitrobenzoic
acid-based cycling assay (Queval and Noctor, 2007) using a Tecan
Infinite-200 plate reader. For determination of CAT and SOD activities,
100 mg of frozen plant material was resuspended in 500 mL PBS (137 mM
NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4, and 1 mM
phenylmethylsulfonyl fluoride) and centrifuged to remove cell debris (4°C,
10 min, 20.000g), and total protein content of the supernatant was determined
(Bradford, 1976). CAT and SOD activity measurements were conducted
according to Jacobo-Velazquez et al. (2011). CAT activity was determined by
adding 5 to 15 mL of sample to CAT assay buffer medium (0.05 M KH2PO4, pH
7.4, and 0.01 M H2O2). The decrease in absorbance at 240 nm was monitored
with a Hitachi U-2000 spectrophotometer. SOD activity was determined with
a NBT-based assay using a Tecan Infinite-200 plate reader.
Detection of ROS
Histochemical stainings were performed as described (Lee et al., 2002;
Reiser et al., 2004) with the following modifications: To detect the accumulation of superoxide, leaves from wild-type and er-ant1 plants were
vacuum-infiltrated for 10 min with 0.1 mg mL21 NBT in 25 mM HEPES
buffer, pH 7.6. The control solution additionally contained 10 units mL21
SOD and 10 mM MnCl2. Leaf samples were left for 2 h at room temperature in the dark and were subsequently destained by incubation at
60°C in 95% ethanol (v/v) for 30 min.
The accumulation of H2O2 was visualized by vacuum infiltration of
leaves in a solution containing 0.1 mg mL21 DAB in 10 mM MES, pH 6.0,
and KOH. The control solution contained additionally 10 mM ascorbate.
After infiltration, the leaves were incubated for 16 h in the dark and destained by boiling in lactic acid:glycerol:ethanol (1:1:3) for 5 to 10 min.
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or GenBank/EMBL databases under the following accession
numbers: Arabidopsis ER-ANT1 (At5g17400), Arabidopsis AAC1 (At3g08580),
Arabidopsis AAC2 (At5g13490), Arabidopsis AAC3 (At4g28390), Arabidopsis
EF-1a (At1g07930), Arabidopsis BiP1 (At5g28540), Arabidopsis BiP2
(At5g42420), Arabidopsis BiP3 (At1g09080), Arabidopsis CRT1 (At1g56340),
Arabidopsis CRT2 (At1g09210), rice ER-ANT1 (Os11g0661300), putative rice
AAC1/2 (Os02g0718900), and putative rice AAC3 (Os05g0302700).
Supplemental Data
The following materials are available in the online version of the article.
Supplemental Figure 1. Alignment of the Predicted Amino Acid
Sequence of Rice ER-ANT1 and Related MCF Carriers from Arabidopsis and Rice.
Supplemental Figure 2. Heterologous Expression of Rice ER-ANT1 in
E. coli Cells and Substrate Dependency of Adenine Nucleotide Uptake.
Gas Exchange and Chlorophyll Fluorescence Measurements
Supplemental Figure 3. Molecular Characterization of 35S:Os-er-ant1
Plants by PCR.
Gas exchange measurements and fluorescence measurements were
performed with a gas exchange and chlorophyll fluorescence system
Supplemental Figure 4. Complementation of er-ant1-2 Knockout
Plants with the Closest Ortholog from Rice, Os-ER-ANT1.
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The Plant Cell
Supplemental Figure 5. Overexpression of pfp5 and slr0293 in
er-ant1 Knockout Plants.
Supplemental Figure 6. Further Evidence for Enhanced ROS Accumulation in Wild-Type and er-ant1 Knockout Plants by NBT Staining
and Determination of H2O2 in Leaf Extracts.
Supplemental Figure 7. Transcript Levels of ER Chaperone Genes in
Response to ER Stress.
Supplemental Table 1. Selected Metabolites of Wild-Type and
er-ant1 Knockout Plants under Ambient CO2 Conditions.
Supplemental Table 2. Comparison between the Amino Acid Sequences of Arabidopsis ER-ANT1 and Related MCF Carriers from
Arabidopsis and Rice.
Supplemental Table 3. Primers Used for Screening, Cloning, and
qRT-PCR Studies.
Supplemental Methods 1. Detailed Procedures for Supplemental Data.
Supplemental References 1. References for Supplemental Figures
and Methods.
ACKNOWLEDGMENTS
Work in the laboratories of H.E.N. was financially supported by the
Deutsche Forschungsgemeinschaft (Reinhard Koselleck-Grant). Work in
the lab of H.E.N., I.H., J.M.H., and J.R. was further supported by the
Landesschwerpunkt “Membrantransport” (http://www.uni-kl.de/rimb).
We thank Mohammad Hajirezaei, IPK Gatersleben, Germany, for quantifications of some metabolites.
AUTHOR CONTRIBUTIONS
C.H. conducted most experiments. B.P. and M.L. cloned the rice ER-ANT1
and conducted uptake experiments on E. coli. R.E. conducted immunoblot
experiments and established mitochondria purification protocols. I.H., J.R.,
J.M.H., and H.B. supported development of experimental strategies.
H.E.N. wrote the article.
Received May 13, 2013; revised June 21, 2013; accepted June 28, 2013;
published July 16, 2013.
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From Endoplasmic Reticulum to Mitochondria: Absence of the Arabidopsis ATP Antiporter
ER-ANT1 Perturbs Photorespiration
Christiane Hoffmann, Bartolome Plocharski, Ilka Haferkamp, Michaela Leroch, Ralph Ewald, Hermann
Bauwe, Jan Riemer, Johannes M. Herrmann and H. Ekkehard Neuhaus
Plant Cell; originally published online July 16, 2013;
DOI 10.1105/tpc.113.113605
This information is current as of June 17, 2017
Supplemental Data
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