Download The Mitochondrial External NADPH

Plant Cell Physiol. 49(2): 251–263 (2008)
doi:10.1093/pcp/pcn001, available online at www.pcp.oxfordjournals.org
ß The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
The Mitochondrial External NADPH Dehydrogenase Modulates
the Leaf NADPH/NADP+ Ratio in Transgenic Nicotiana sylvestris
Yun-Jun Liu 1, Fredrik E. B. Norberg 1, Anna Szilágyi 2, Rosine De Paepe 3, Hans-Erik Åkerlund
and Allan G. Rasmusson 1, *
2
1
Lund University, Department of Cell and Organism Biology, Sölvegatan 35B, SE-22362, Lund, Sweden
Department of Biochemistry, Center of Chemistry and Chemical Engineering, Lund University, POB 124, SE-22100 Lund, Sweden
3
Institut de Biotechnologie des Plantes, UMR CNRS 8618, Universite´ Paris Sud, Batiment 630, 91405 Orsay Cedex, France
2
NADP-dependent malate DH; NBT, nitro blue tetrazolium;
NP-GAPDH, non-phosphorylating glyceraldehyde-3-phosphate
DH; ROS, reactive oxygen species; PTOX, plastoquinol terminal
oxidase; RT–PCR, reverse transcription–PCR; UQ, ubiquinone;
WT, wild type.
Plant mitochondria contain alternative external
NAD(P)H dehydrogenases, which oxidize cytosolic NADH
or NADPH and reduce ubiquinone without inherent linkage
to proton pumping and ATP production. In potato, St-NDB1
is an external Ca2+-dependent NADPH dehydrogenase. The
physiological function of this enzyme was investigated in
homozygous Nicotiana sylvestris lines overexpressing St-ndb1
and co-suppressing St-ndb1 and an N. sylvestris ndb1. In leaf
mitochondria isolated from the overexpressor lines, higher
activity of alternative oxidase (AOX) was detected. However,
the AOX induction was substantially weaker than in the
complex I-deficient CMSII mutant, previously shown to
contain elevated amounts of NAD(P)H dehydrogenases
and AOX. An aox1b and an aox2 gene were up-regulated
in CMSII, but only aox1b showed a response, albeit smaller,
in the transgenic lines, indicating differences in AOX
activation between the genotypes. As in CMSII, the increase
of AOX in the overexpressing lines was not due to a general
oxidative stress. The lines overexpressing St-ndb1 had
consistently lowered leaf NADPH/NADP+ ratios in the
light and variably decreased levels in darkness, but unchanged
NADH/NAD+ ratios. CMSII instead had similar NADPH/
NADP+ and lower NADH/NAD+ ratios than the wild type.
These results demonstrate that St-NDB1 is able to modulate
the cellular balance of NADPH and NADP+ at least in the
day and that reduction of NADP(H) and NAD(H) is
independently controlled. Similar growth rates, chloroplast
malate dehydrogenase activation and xanthophyll ratios
indicate that the change in reduction does not communicate
to the chloroplast, and that the cell tolerates significant
changes in NADP(H) reduction without deleterious effects.
Introduction
The mitochondrial electron transport chain (mETC)
contains protein complexes that transfer electrons from
respiratory intermediates to O2. Matrix NADH is oxidized
by complex I. The electrons are donated to ubiquinone
(UQ), and successively to complex III, cytochrome c and
complex IV, where O2 is reduced. This process generates an
electrochemical proton gradient that drives ATP synthesis.
However, in the mETC of plant mitochondria the protonpumping complexes are bypassed by non-proton pumping
alternative enzymes that do not directly contribute to ATP
synthesis. The energy bypasses of the mETC include
alternative oxidase (AOX) (Vanlerberghe and McIntosh
1997) and at least four biochemically discernible types of
alternative dehydrogenases (DHs) that oxidize NADH or
NADPH (Rasmusson et al. 2004).
AOX has been studied intensely as compared with the
NAD(P)H DHs. There are at least two aox gene subfamilies
in higher plants, aox1 and aox2 (Considine et al. 2002,
Karpova et al. 2002). In several species, aox1 genes are
responsive to many stress treatments, whereas aox2 genes
have been shown to be affected by relatively few conditions,
for example treatments with low pH, ammonium and
paraquat (Vanlerberghe and McIntosh 1997, Clifton et al.
2005, Escobar et al. 2006). It has been suggested that AOX
has a physiological function in decreasing the reduction
level of mETC components (Purvis and Shewfelt 1993).
Over-reduction of the mETC may otherwise generate
reactive oxygen species (ROS), which can damage cellular
constituents if not recognized by cellular defense signaling
(Møller 2001, Foyer and Noctor 2005, Møller et al. 2007).
Consistently, overproduction and antisense suppression of
AOX in Nicotiana tabacum cells decreased and increased
Keywords: Alternative oxidase — Chloroplast — Electron
transport chain — Mitochondria — NADPH — Transgenic
plants.
Abbreviations: AOX, alternative oxidase; CaMV, cauliflower
mosaic virus; DAB, 3,30 -diaminobenzidine; DcQ, decylubiquinone;
DH, dehydrogenase; EST, expressed sequence tag; G-6-P DH,
glucose-6-phosphate DH; GR, glutathione reductase; MDA,
malondialdehyde; mETC, mitochondrial electron transport chain;
NADP-ICDH, NADP-dependent isocitrate DH; NADP-MDH,
*Corresponding author: E-mail, [email protected]; Fax, +46-46-2224113.
251
252
Mitochondrial regulation of cell NADPH reduction
ROS formation, respectively (Maxwell et al. 1999).
However, overexpressing or suppressing aox1a in
Arabidopsis plants did not affect ROS formation under
normal growth conditions (Umbach et al. 2005).
The inner membrane of plant mitochondria contains
alternative NAD(P)H DHs that are located internally,
oxidizing matrix NAD(P)H, and externally, oxidizing
cytosolic NAD(P)H. In potato, two NAD(P)H DH genes,
St-nda1 and St-ndb1, were identified as encoding internal
and external enzymes, respectively (Rasmusson et al. 1999,
Rasmusson and Agius 2001). St-NDB1 was shown to be a
Ca2+-dependent external NADPH DH (Michalecka et al.
2004). Arabidopsis contains seven NAD(P)H DH genes,
four of which belong to the ndb gene family (Michalecka
et al. 2003). At-NDB1 and At-NDB2 were shown to
target green fluorescent protein (GFP) to mitochondria
(Michalecka et al. 2003), and during in vitro mitochondrial
import At-NDB1, At-NDB2 and At-NDB4 become located
on the outside of the inner membrane (Elhafez et al. 2006).
When expressed as fusion proteins in Escherichia coli,
At-NDB1 is a Ca2+-dependent NADPH DH, whereas
At-NDB2 and At-NDB4 are Ca2+-stimulated and Ca2+independent NADH DHs, respectively (Geisler et al. 2007).
Thus, the set-up of external NAD(P)H DH in plants is
more complex than in other organisms, regarding both
homolog numbers and functional differentiation.
In several species, the expression of genes for AOXs
and alternative NAD(P)H DHs shows similar responses
to external effectors, including inhibitors, light, nutrients
and various stressors (Zhang et al. 1996, Svensson and
Rasmusson 2001, Clifton et al. 2005, Sieger et al. 2005,
Clifton et al. 2006, Elhafez et al. 2006, Escobar et al. 2006,
Rasmusson and Escobar 2007). Several mutants deficient in
mitochondrial components also jointly affect expression of
different alternative mETC enzymes (Karpova and Newton
1999, Brangeon et al. 2000, Karpova et al. 2002, Moore
et al. 2003, Ho et al. 2007). In particular, the Nicotiana
sylvestris CMSII mutant, which lacks the NAD7 subunit of
complex I (Gutierres et al. 1997), has increased AOX and
internal and external NAD(P)H DH activities (Sabar et al.
2000). Since alternative enzymes can mediate oxidation of
respiratory substrates without adenylic restriction, changes
in their abundance may control and balance levels of
cellular redox couples. However, very little direct evidence is
available regarding the maintenance of cellular NAD(P)H
levels or its physiological importance. Mitochondrial Ca2+dependent external NAD(P)H DHs can oxidize cytosolic
NAD(P)H and might thereby regulate the cytoplasmic
NAD(P)H pools (Rasmusson et al. 2004). It has also been
suggested that photosynthetic reductant may, via shuttles,
be oxidized by the Ca2+-dependent external NADPH DH
(Krömer 1995), which in potato is encoded by St-ndb1
(Michalecka et al. 2004).
In this study, homozygous transgenic N. sylvestris
plants expressing St-ndb1 were used to investigate its
primary physiological function. Our results demonstrate
that the overexpression of St-ndb1 leads to a modest
increase in levels of AOX. However, specific changes in the
cellular NADPH/NADP+ ratio demonstrate that St-NDB1
is active in the leaf and is able to decrease the balance
between NADPH and NADP. This change is apparently
not mediated to the chloroplast redox system.
Results
Overexpression and co-suppression of Ndb1 in homozygous
lines
Hemizygous transgenic N. sylvestris plants containing
St-ndb1 driven by the cauliflower mosaic virus (CaMV)
35S promoter have previously been obtained (Michalecka
et al. 2004). To study the physiological role of St-ndb1,
hemizygous plants were selfed and homozygous plants
were selected (see Materials and Methods). To investigate
expression of the St-ndb1 gene and its homologous genes in
N. sylvestris, a weak PCR product was amplified from the
wild type (WT) using primers for St-ndb1. The 412 bp
fragment showed 92% nucleotide identity to St-ndb1 and
was designated as N. sylvestris ndb1 cDNA. We investigated
the transcript levels for St-ndb1 and the Ns-ndb1 by realtime reverse transcriptase–PCR (RT–PCR), using the gene
for the 28.5 kDa subunit of tobacco complex I (SchmidtBleek et al. 1997) as the control gene (Fig. 1A). Three
transgenic lines, S2, S6 and S17, had high transcript levels
for St-ndb1, whereas line S8 had a very low RNA level
compared with the WT and the vector control line E35.
Further, line S8 had much lower Ns-ndb1 transcript
levels than the WT and E35, whereas S2, S6 and S17 were
similar to the WT (Fig. 1A). By competitive RT–PCR with
primers against fully conserved regions, the ratio of St-ndb1
to Ns-ndb1 transcripts was at least 5 for lines S2, S6 and
S17 in three independent determinations. In line S8, only
an St-ndb1-derived product could be reproducibly detected
(results not shown). These results show that St-ndb1 was
strongly expressed in lines S2, S6 and S17 but not in line S8,
where St-ndb1 and Ns-ndb1 were co-suppressed.
Mitochondria were isolated from mature leaves of
flowering 10-week-old plants. Protein levels for St-NDB1
were investigated by Western blotting using antibodies
specific for the EF-hand domain (Michalecka et al. 2004).
A single band was detected in mitochondria from lines S2,
S6 and S17, whereas mitochondria from S8, the WT and
E35 displayed no signal (Fig. 1B). The activity of external
NAD(P)H DH was measured in the isolated mitochondria
using the UQ analog decylubiquinone (DcQ). Lines S2,
S6 and S17 had higher external NADPH-DcQ oxidation
activity than the WT and E35, whereas S8 had a very low
Mitochondrial regulation of cell NADPH reduction
A
Ns-28.5kD
St-ndb1
Ns-ndb1
120
Specific activity (%)
Transcript abundance (%)
A
80
40
300
200
100
0
0
WT
S2
WT
B
S6
S2
S6
S8
S8
253
S17
S17
E35
B
E35
WT
S2
S6
S8
S17
S35
WT
S2
S6
S8
S17
E35
WT
S2
S6
S8
S17
E35
AOX
St-NDB1
NAD9
76 kD
C
C
Activity (%)
300
log(2) AOX/NAD9
External NADPH DH
External NADH DH
200
100
1.5
1.0
0.5
0
0
WT
S2
S6
S8
S17
E35
Fig. 1 Expression of ndb1 in transgenic N. sylvestris.
(A) Transcript levels for St-ndb1 and Ns-ndb1. Total RNA was
prepared from leaves of 4-week-old homozygous transgenic plants,
WT and the vector control line E35. Transcripts of St-ndb1,
Ns-ndb1 and the 28.5 kDa subunit gene of tobacco complex I were
analyzed by real-time RT–PCR. Data are shown as the mean SD
for two separate RNA preparations. (B) Western analysis of
leaf mitochondria from transgenic N. sylvestris plants.
Mitochondria were purified as described in Materials and
Methods, and analyzed for St-NDB1 by Western blotting. The
76 kDa subunit of complex I was used as control. (C) NAD(P)H
oxidation activity in leaf mitochondria using DcQ as electron
acceptor. Activities are given as the percentage of the activity in
the WT in each experiment. Control activities were for external
NADPH DH 212.6 nmol NADPH min1 mg1 protein, and for
external NADH DH 329.6 nmol NADH min1 mg1 protein. Error
bars denote SD for three independent mitochondrial
preparations.
activity. External NADH-DcQ oxidation activity was
similar in all lines (Fig. 1C). Thus, the results on the transcript levels of St-ndb1 and Ns-ndb1, the protein levels of
St-NDB1 and the activity levels of external NAD(P)H DH
demonstrated that St-ndb1 was overexpressed in lines S2, S6
and S17, whereas both St-ndb1 and Ns-ndb1 were strongly
suppressed in line S8. These lines were used for the further
experiments.
Fig. 2 Analysis of AOX in mitochondria purified from leaves of
transgenic N. sylvestris plants. (A) AOX activity was measured
using O2 as electron acceptor with 100 mM NADH and 100 mM
NADPH as substrates. Activities are given as a percentage of the
activity in the WT in each experiment. Control activity was
14.6 nmol NAD(P)H min1 mg1 protein. S6 and S17 are
significantly different from the WT and E35 in t-tests at P50.05.
(B) Western analysis of mitochondrial protein. AOX was detected
with monoclonal antibodies against the Sauromatum guttatum
protein. The NAD9 subunit of complex I was used as control.
(C) Relative levels of AOX protein in mitochondria. To correct
for loading variations, the AOX/NAD9 signal ratios are shown,
using a log(2) scale with WT set to unity. As a group, the
overexpressor plants (S2, S6 and S17) are significantly different
from controls (WT and E35) in a t-test at P50.01. In (A) and (C),
error bars denote SE for three independent mitochondrial
preparations.
Secondary effects on expression of Aox genes
The purified mitochondria were used to determine
the enzymatic capacity of AOX. The AOX activity was
1.8- to 2.6-fold higher in mitochondria from S2, S6 and S17
than the WT, whereas AOX activity in mitochondria
from S8 and E35 was similar to that in the WT (Fig. 2).
The AOX activity was correlated to the activity of external
NADPH DH as revealed by regression (R240.85) using a
linear or exponential equation, however based on a
restricted variation. Mitochondria from the overexpressing
254
Mitochondrial regulation of cell NADPH reduction
A
S2
S6
S8
S17
AOX
RBCL
log(2) AOX/RBCL
B
300
CMS
II
250
Transcript level (%)
WT
200
150
100
5
50
4
0
3
2
1
0
WT
S2
S6
S8
S17
CMS
II
Fig. 3 Western analysis of AOX in total leaf protein extracts.
(A) Total protein was extracted from leaves of 6-week-old plants,
except for CMSII (9 weeks old) 7 h into the light period. A 40 mg
aliquot of total protein extract (20 mg for CMSII) plus 0.2%
2-mercaptoethanol was loaded in each lane. As internal control,
the 55 kDa Rubisco large subunit (RBCL) was stained with
Coomassie blue after SDS–PAGE. (B) Relative level of AOX protein
in leaf extracts. Ratios of AOX signal to RBCL signal are shown on
a log(2) scale with WT set to unity. Error bars denote SE for six
independent experiments.
lines (S2, S6 and S17) also contained elevated amounts
of AOX protein as compared with the WT, E35 and S8
(Fig. 2). To examine whether AOX was also affected in
leaves of plants in vegetative growth phase, total leaf
protein was extracted from 6-week-old plants and Western
blotted. We also analyzed the complex I CMSII mutant,
which has elevated AOX protein levels (Sabar et al. 2000),
to test if the two mitochondrial perturbations would
have similarities in AOX induction. Since the CMSII
mutant grows more slowly, it was harvested at an age of
9 weeks when the size is equal to that of a 6-week-old WT.
CMSII had an AOX protein signal strongly elevated
as compared with the WT, whereas in the transgenic
lines only moderate elevations over the WT was seen
(Fig. 3). Also, a relationship to ndb1 expression was
not visible, and a substantial variation between analyzed
plants excluded statistical support for the changes in the
transgenic lines. Upon Western blotting of gels run in the
absence of reductant, AOX migrated almost exclusively
as the reduced monomer form in all extracts (results not
shown).
To investigate further the AOX expression, we
analyzed transcripts. There are two aox gene families
WT
S17
S2
S6
S8
CMSII
Ns28.5kD
Stndb1
Nsndb1
Nsaox1b
Nsaox2
NsPTOX
Fig. 4 Transcript levels for alternative oxidase genes in
N. sylvestris leaves. Samples were taken from the second, third
and fourth fully developed leaf of 6-week-old plants (9-week-old
plants for CMSII) 7 h into the light period. Data are shown as
a percentage of the WT SE for three separate RNA preparations,
except for St-ndb1, which is shown as a percentage of maximum.
For aox1b, S17 and CMSII are significantly different from the WT
in t-tests at P50.05.
in dicot plants, aox1 and aox2, and most species investigated contain more than one aox gene of each type
(Considine et al. 2002). Real-time PCR primers were
designed against N. sylvestris expressed sequence tags
(ESTs) closely homologous to the N. tabacum aox1b
(Whelan et al. 1996). Also, an N. sylvestris aox2 cDNA
fragment was isolated by RT–PCR using primers designed
against an N. tabacum EST, the translation of which was
highly similar to that of the N. tabacum AOX2 (Whelan
et al. 1996). RNA was extracted from 6-week-old transgenic
plants and the transcript levels of aox1b and aox2 were
assayed by real-time RT–PCR. For Ns-aox1b, a small
increase was seen in the overexpressing lines S2, S6 and S17
over the WT and S8. Ns-aox2 was not affected in any of the
transgenic lines (Fig. 4). We also measured the transcript
levels of aox1b and aox2 in 9-week-old CMSII plants. The
results showed that for both genes the transcript level was
substantially increased in CMSII as compared with the
other genotypes (Fig. 4).
Plastids contain a plastoquinol terminal oxidase
(PTOX), which is homologous to AOX and oxidizes
plastoquinol with O2 as acceptor, thus possibly preventing
the over-reduction of the plastoquinone pool (Joët et al.
2002). An N. sylvestris cDNA fragment for PTOX was
obtained by RT–PCR using the primers against an
N. tabacum EST. The transcript levels for PTOX were
assayed by real-time RT–PCR, but were found to be similar
in the different lines (Fig. 4).
Taken together, the results indicate that in the
vegetative state there is an increase in AOX expression
upon St-ndb1 overexpression, but the increase is modest
when compared with the strong increase in CMSII.
Mitochondrial regulation of cell NADPH reduction
A
Superoxide (relative level)
6
4
2
B
Hydrogen peroxide (relative level)
0
C
WT
6
S17
S2
S6
CMSII
*
*
2
WT
S17
S2
S6
S8
CMSII
Control
Plus KCN
50
40
30
*
*
20
10
0
WT
S17
S2
Ascorbate (µmol g-1 FW)
D
Overexpression of St-ndb1 decreased the leaf NADPH/
NADP+ ratio in the light
A crucial issue for physiological analysis of the
transgenic plants is whether NAD(P)H pools in plant cells
are affected by overexpression or co-suppression of the
external NADPH DH. The total leaf pyridine nucleotide
pools were assayed on 6-week-old plants in the light and
in darkness. Individually, leaf NAD+, NADH, NADP+
and NADPH contents in the transgenic plant were
not significantly different from that in the WT in leaves
sampled 4 h into the light period (Fig. 6A). There was
also no change in the NADH/NAD+ ratio between lines.
However, NADPH/NADP+ ratios in the overexpressing
lines decreased by 19–31%, as compared with the WT,
each line being significantly different from the WT
(Fig. 6B). No difference was seen between the WT and
the co-suppressed line S8. Under the same conditions,
the CMSII leaves contained all four nucleotides in higher
S8
4
0
MDA equivalents (nmol g-1 FW)
Expression of St-ndb1 did not affect the cellular levels
of ROS
Since NADPH is an electron donor for both NDB1
and ROS detoxification systems (Møller 2001), and cellular
ROS are known to affect aox gene expression (Clifton et al.
2006), we investigated ROS levels in the transgenic plants.
The levels of superoxide ðO
2 Þ and hydrogen peroxide
(H2O2) were detected by in situ staining of leaves with nitro
blue tetrazolium (NBT) and 3,30 -diaminobenzidine (DAB),
respectively. Both NBT and DAB staining were relatively
similar in all transgenic lines, though slightly decreased
NBT staining was observed in the overexpressing lines S6
and S17 and a somewhat increased DAB staining was seen
in S17 (Fig. 5). CMSII displayed a somewhat increased
staining with NBT but decreased DAB staining.
Oxidation of polyunsaturated fatty acids produces
malondialdehyde (MDA), the levels of which gives a general
measure of lipid peroxidation (Hodges et al. 1999).
The levels of MDA were relatively similar between different
lines, with only small decreases visible in S17 and S2
as compared with the WT. Also, none of the lines showed
an increase in MDA levels after the leaf disks had been
treated with KCN (Fig. 5C). This indicates that the basal
AOX level was sufficient for preventing ROS-induced
lipid peroxidation caused by KCN inhibition of the
cytochrome oxidase. Ascorbate, an important substrate
for H2O2 detoxification in plant cells (Noctor and Foyer
1998), was examined in leaves. The results showed that
the contents of both reduced and oxidized ascorbate
were similar in all genotypes (Fig. 5D). Taken together,
the results suggest that overexpression or co-suppression
of St-NDB1 did not cause oxidative stress in the plant
cells, although the relative magnitude of different ROS
markers was changed in some lines.
255
S8
Reduced
Total
4
3
*
2
1
0
WT
S17
S2
S6
S8
CMSII
Fig. 5 Oxidative stress markers in N. sylvestris leaves. Leaf disks
were sampled from the second fully expanded leaf of 7-week-old
plants (9-week-old CMSII plants) and analyzed as described
in Materials and Methods. (A) Relative levels of superoxide.
(B) Relative levels of hydrogen peroxide. (C) Levels of MDA
equivalents in leaf disks incubated in the presence or absence of
KCN. (D) Content of reduced and total ascorbate. Data are
shown SE for five (A and B) or three (C and D) independent
experiments. Asterisks denote a significant difference from the WT
in t-tests at P50.05.
A
Mitochondrial regulation of cell NADPH reduction
Nucleotide amount (nmol g−1 FW)
256
60
*
50
40
30
20
10
0
NADH
B
WT
S17
S2
S6
S8
CMSII
*
NADPH
NAD
WT
S17
S2
S6
S8
CMSII
1.4
Ratio
1.2
1
0.8
0.6
0.4
NADP
*
* * *
*
0.2
0
C
Nucleotide amount (nmol g−1 FW)
NADPH/NADP
NADP(H)/NAD(H)
60
50
WT
S17
S2
S6
S8
CMSII
*
40
30
20
10
0
NADH
D
NADH/NAD
NAD
NADPH
2.0
Ratio
NADP
WT
S17
S2
S6
S8
CMSII
1.6
1.2
*
0.8
*
0.4
*
0.0
NADPH/NADP
NADH/NAD
amounts than in the WT, and NAD+ in particular was
abundant in CMSII. The NADH/NAD+ ratio in CMSII
was decreased compared with the WT, but the NADPH/
NADP+ ratio was unchanged (Fig. 6B).
In leaves sampled in the dark period, the leaf pyridine
nucleotide amounts and NAD(P)H/NAD(P)+ ratios were
not consistently affected in the St-ndb1 transgenic lines.
The NADPH/NADP+ ratio was significantly decreased in
line S17, as compared with the WT, whereas S2 and S6
showed only slightly lower levels (Fig. 6D). CMSII had
a significantly higher NAD+ content and a lower
NADH/NAD+ ratio as compared with the WT (Fig. 6C,
D). Both in the light and in darkness, the NADP(H)/
NAD(H) ratio was similar in the WT and the transgenic
plants, but significantly decreased in CMSII plants.
The NADPH/NADP+ ratio was higher in all lines in the
night than in the day (Fig. 6), consistent with previous
investigations of diurnal changes in NADP(H) reduction
under optimum growth conditions (Raymond et al. 1987,
Rao et al. 1989). Total NADP(H) is higher in the day
than in the night (Fig. 6), consistent with the presence of
a light-regulated NAD kinase (Muto and Miyachi 1981).
NADP(H)/NAD(H)
Fig. 6 Pyridine nucleotide contents and ratios in N. sylvestris
plants. Samples were taken from the first fully expanded leaf of
9-week-old (CMSII) and 6-week-old (other lines) plants 4 h into
the light period (A, B) and 4 h into the dark period (C, D). Values
are means SE of three independent extracts. Asterisks denote
a significant difference from the WT in t-tests at P50.05.
Analysis of putative compensatory reactions in the cells
A primary change in the cytosolic NADPH/NADP+
ratio may be compensated in several ways. Chloroplast
reductant may be exported via the putative triose phosphate
shuttle (Kelly and Gibbs 1973). It has also been discussed
whether the external NADPH DHs may oxidize chloroplast-derived reductant and affect the stromal redox level
(Krömer 1995, Hoefnagel et al. 1998, Padmasree et al.
2002). This issue can now be addressed.
The transgenic plants grew normally, without any
differences in biomass accumulation visible between
transgenic lines and the WT under normal light conditions,
indicating that primary assimilation was unaffected.
Also under fluctuating light, which challenges the stabilization of the reduction level in chloroplast, biomass accumulation was unaffected (Fig. 7). To investigate further if
the change in the tissue NADPH/NADP+ ratio also
involved a modified chloroplast redox state, we investigated
NADP-dependent malate DH (NADP-MDH) activation.
Initial and maximum activities of NADP-MDH were
measured in protein extracts from plant leaves 7 h into
the light period. The results showed that both activities were
similar in all lines and in CMSII (Fig. 8A). NADP-MDH
activities were also examined on the leaves of dark-adapted
and high-light-illuminated plants. Light induced the
initial activity of NADP-MDH, whereas initial and maximum NADP-MDH activities were similar in all lines
(Fig. 8B). The amount of chlorophyll, on a fresh weight
basis, showed some differences between lines, but was not
correlated to the expression or suppression of St-ndb1
DW (%)
A 150
Root
Shoot
A
100
50
Activity (mmol min−1 mg−1 Chl)
Mitochondrial regulation of cell NADPH reduction
Initial
Maximum
4
3
2
1
0
WT
WT
S17
S2
S6
DW (%)
B 150
S8
S17
S2
S6
S8
CMSII
B
Activity (mmol min−1 mg−1 Chl)
0
257
Root
Shoot
100
50
5
Initial
Maximum
4
3
2
1
0
WT
S2 S6
Dark
S8
WT
S2 S6
Light
S8
0
S17
S2
S6
S8
C
FW)
WT
Chlorophyll (mg
g−1
Fig. 7 Dry weight (DW) of 7-week-old N. sylvestris plants. Plants
were grown in the greenhouse with a 16 h light period per day.
The shoots and roots were dried at 908C for 20 h. Values are shown
as a percentage of total DW of the WT. Error bars denote the SE
for four plants. (A) Plants were grown at 200 mmol m2 s1 with
16 h of light per day. The total DW of the WT was 11.8 g. (B) Plants
were grown under conditions where light fluctuates between
200 and 5 mmol m2 s1 every 15 min during the 16 h light period.
The total DW of the WT was 5.4 g.
Chl b
Chl a
1.0
0.5
0.0
WT
D
160
% of total xanthophyll
(Fig. 8C). Also, the Chl a/b ratio in leaves did not show
any difference between lines. Chloroplast photosynthetic
electron flow can, under overexcitation conditions, cause
formation of a sufficiently high transthylakoid pH gradient
that drives the xanthophyll cycle conversion of violaxanthin
to antheraxanthin and zeaxanthin (Gilmore 1997).
Violaxanthin, antheraxanthin and zeaxanthin were examined in the overexpressing line S17 and the co-suppressed
line S8, but displayed similar contents in both lines
(Fig. 8D). Together, these results indicate that the
chloroplast redox level in leaves of transgenic plants was
not significantly affected by the modified ndb1 expression,
nor by the change in total NADPH/NADP+ ratio.
It has been suggested that the external NADPH
DH may re-oxidize NADPH formed by the cytosolic
non-phosphorylating glyceraldehyde-3-phosphate DH
(NP-GAPDH), NADP-dependent isocitrate DH (NADPICDH) or increase the pentose phosphate pathway flux
by decreasing NADPH inhibition of the cytosolic glucose6-phosphate DH (G-6-P DH) (Douce and Neuburger 1989,
S17
S2
S6
S8
Zeaxanthin
Antheraxanthin
Violaxanthin
120
80
40
0
S17
S8
Fig. 8 Chloroplast parameters in transgenic N. sylvestris plants.
(A and B) For initial and maximum activities of NADP-MDH, leaf
samples were taken from different sets of 6-week-old plants
(9-week-old plants for CMSII). Values are means SE of three
independent determinations. (A) Samples were taken from the
plants 7 h into the light period. (B) Plants 7 h into the light period
were dark adapted for 1 h (Dark), then treated at 700 mmol m2 s1
for 20 min (Light). For determination of chlorophylls (C) and
xanthophylls (D), leaf samples were taken from the second
fully developed leaf of 6-week-old plants. Data are shown SE
for 3–5 independent experiments.
258
Table 1
Mitochondrial regulation of cell NADPH reduction
Activities of NAD(P)H-metabolizing enzymes in extracts from N. sylvestris leaves
NP-GAPDH
G-6-P DH
NADP-ICDH
NAD-ICDH
NAD-MDH
NAD-GAPDH
NADP-GAPDH
GR
S8
S17
S2
S6
WT
50.4 4.5
39.8 6.1
20.7 3.9
0.24 0.04
23.7 1.3
5.99 0.37
6.87 0.53
0.23 0.02
55.4 3.9
38.5 6.0
19.9 3.1
0.27 0.04
25.8 0.3
6.95 0.48
7.22 0.31
0.25 0.06
57.3 3.6
43.7 1.9
25.8 4.0
ND
ND
ND
ND
ND
46.8 1.9
38.1 0.9
15.8 2.4
ND
ND
ND
ND
ND
52.4 2.9
38.4 2.6
17.3 1.3
ND
ND
ND
ND
ND
Leaves were sampled for extraction in the middle of the light period. Activities are given in nmol NADPH min1 mg1 protein for
NP-GAPDH, G-6-P DH and NADP-ICDH, and in mmol NAD(P)H min1 mg1 chlorophyll for other enzymes. Values are means SE
for 3–6 independent experiments.
ND, not determined.
Krömer 1995). Compensatory changes in these enzymes
may also affect the cellular NADPH/NADP+ ratio
(Krömer 1995). However, neither of these three enzyme
activities was affected in transgenic lines, as compared
with the WT (Table 1). Several other metabolic enzymes
were also found to be similar in leaves of the overexpressing
line S17 and the co-suppressing line S8 (Table 1).
These results indicate that the modified NADPH/NADP+
ratio did not provoke compensatory gene expression
changes for NADPH-providing cytosolic enzymes.
Discussion
The presence of mitochondrial external NADPH
oxidation in plants has been known for many years,
yet virtually nothing is known about its physiological
significance nor under which developmental and environmental conditions the process is active (Douce and
Neuburger 1989, Rasmusson et al. 2004). We have therefore
constructed homozygous transgenic N. sylvestris plants
modified for expression of St-ndb1. Transcript, protein
and activity analyses show that three lines stably overexpress St-ndb1 whereas one line co-suppresses St-ndb1
and a tobacco homolog, Ns-ndb1, extending previous
observations in hemizygous plants (Michalecka et al. 2004).
The potential effects of a modified NDB1 level may
occur upstream of the enzyme, by changing the levels of
NADP(H), or downstream via changes in electron donation
to the mETC. Downstream, an increased reduction of
the mETC may lead to ROS formation (Mittler et al. 2004).
In an Arabidopsis NP-GAPDH mutant, both NADPH
reduction and ROS levels are elevated (Rius et al. 2006),
indicating that changes in substrate availability to the
mETC may affect ROS production. However, leaves
of the N. sylvestris CMSII mutants are not subject to
oxidative stress, possibly due to an increase in antioxidant
systems (Dutilleul et al. 2003b, Vidal et al. 2007). In the
plants modified for St-ndb1, the content of ascorbate
and glutathione reductase (GR), both involved in ROS
detoxification (Noctor and Foyer 1998), as well as three
oxidative stress indicators were relatively similar to the
WT, with no genotype showing a consistent change in
several ROS markers (Fig. 5, Table 1). This suggests that
the overproduced NADPH DH does not cause an overreduction of the mETC nor compete for NADPH with
ROS-metabolizing enzymes, many of which use NADPH
as the primary reductant source via the GR reaction
(Mittler et al. 2004). The latter is consistent with a 5-fold
lower Km(NADPH) for cytosolic GR than for mitochondrial NADPH DH (Krömer and Heldt 1991, Stevens
et al. 2000).
ROS production due to mETC over-reduction has been
suggested to be ameliorated by AOX, and ROS is also
a signal for induction of AOX (Maxwell et al. 1999, Møller
2001, Rhoads and Subbaiah 2007). Thus, elevated AOX
levels may prevent ROS production upon overexpression
of an NADPH DH. Consistently, AOX protein and activity
were elevated in mitochondria from plants overexpressing
St-ndb1 (Figs. 2, 3), with correlated NADPH DH and
AOX activities over several lines. AOX and external
NADPH DH activities are jointly increased in the CMSII
mutant (Sabar et al. 2000) and by phosphate deficiency
(Sieger et al. 2005), but our data show that the overexpression of an NADPH DH alone is sufficient for
elevated AOX. It is however not known whether protein
levels and in vitro measured capacities will reflect the in vivo
activities of NAD(P)H DH and AOX (Priault et al. 2007,
Vidal et al. 2007).
Most dicot plant species have more than one gene
of the aox1 and aox2 type (Considine et al. 2002). In
N. tabacum, aox1a, aox1b and a partial deduced
peptide sequence of an aox2 have been described
(Vanlerberghe and McIntosh 1994, Whelan et al. 1995,
Whelan et al. 1996). In N. sylvestris, two aox1 transcripts
Mitochondrial regulation of cell NADPH reduction
were previously detected, one of which was specifically
induced in the CMSII mutant (Vidal et al. 2007), but
sequences were not determined. We cloned fragments of an
aox1b and an aox2. Both genes had increased transcript
levels in CMSII, but only aox1b was elevated in the
overexpression lines (Fig. 4). In conjunction with the
findings of Vidal et al. (2007), the results indicate that
there are at least three aox genes in N. sylvestris, and that
the different mitochondrial perturbations in the overexpressors and CMSII mediate different patterns of AOX
activation. This situation is similar to maize, where different
aox genes are induced in complex I-deficient NCS2 and
complex IV-deficient NCS6 mutants (Karpova et al. 2002).
A modified expression of St-ndb1 may instead cause an
effect upstream of the enzyme by modifying the cellular
homeostasis of its substrate NADPH. The, on average,
27% decrease in daytime leaf NADPH/NADP+ ratios in
the overexpressors (Fig. 6) shows that the enzyme is active
in the light in vivo. In darkness, smaller differences
were generally observed, but the NADPH/NADP+ ratio
was markedly decreased in line S17. The difference between
the overexpressing lines may be due to variations in
NADPH oxidation between lines, superseding the compensatory capacity of NADP-reducing enzymes. An Arabidopsis mutant lacking NADPH-producing NP-GAPDH even
had an increased NADPH/NADP+ ratio, possibly due to
the induction of cytosolic G-6-P DH, leading to overcompensation (Rius et al. 2006). Here, the unchanged
activities of NP-GAPDH, NADP-ICDH and G-6-P DH
(Table 1) exclude a compensation by expression, but the
enzyme levels already present are expected to compensate
allosterically for a modified NADPH DH activity.
For example, a decrease in the cytosolic NADPH/
NADP+ ratio of 27% is predicted to have a notable
allosteric effect on G-6-P DH, isoforms of which have
similar magnitudes of Km(NADP+) and Ki(NADPH) in
Arabidopsis (Wakao and Benning 2005). Also the decreased
NADPH DH activity in the co-suppressor line S8 (Fig. 6)
may have been compensated by other NADPH-utilizing
enzymes under the conditions analyzed. Pathway flux
changes without noticeable changes in metabolite levels
have previously been reported (Fernie et al. 2005). The
unchanged NADPH/NADP+ ratio in CMSII indicates that
a strong up-regulation of AOX content is not sufficient to
lower cellular NADP(H) reduction, consistent with previously reported data, though for somewhat different
conditions (Dutilleul et al. 2005). In summary, these results
demonstrate that St-NDB1 can significantly modulate the
NADP(H) reduction homeostasis of the cell. Thus, the
St-NDB1 must be active in the leaf at least in the light, and
allosteric agents (e.g. Ca2+) must be present in sufficient
concentrations to activate the NADPH DH activity. This
is consistent with the kinetically derived prediction of
259
NADPH being a preferred respiratory substrate in pea
leaves in the light (Krömer and Heldt 1991).
In leaf cells, cytosol, mitochondria and chloroplast
have total NADP(H) concentrations of 0.3, 0.3 and 0.4 mM
(Noctor et al. 2006, Noctor et al. 2007), and their volumes
constitute 5, 1 and 8% of potato mesophyll cells, respectively (Leidreiter et al. 1995). Thus, leaf cell cytosol and
chloroplast contain 30 and 64% of the total NADP(H),
respectively, and constitute the major NADP(H) pools.
St-NDB1 oxidizes cytosolic NADPH, so the cytosolic
NADPH/NADP+ ratio should be more affected than
other cellular pools. In barley protoplasts, the chloroplast
and extrachloroplastic (mainly cytosolic) NADPH/NADP+
ratios are differently responsive to short-term treatments
with low CO2 and light (Igamberdiev et al. 2001b),
indicating a restricted communication between the pools.
The average 27% decrease in total NADPH/NADP+ ratio
in the overexpressor lines (Fig. 6) would at maximum, i.e. if
the plastid pool is unaffected, constitute a decrease in the
cytosolic NADPH/NADP+ ratio of 43%.
In pea leaf protoplasts, the cytosolic NADH/NAD+
ratio increased in the light and at low CO2, but NADPH/
NADP+ was relatively unchanged. However, the mitochondrial reduction level of both NAD(H) and NADP(H)
increased in the light (Igamberdiev and Gardeström 2003).
In our study, the overexpressing lines had a decreased
NADPH/NADP+ ratio without affecting the NADH/
NAD+ ratio, whereas CMSII showed the reverse pattern
(Fig. 6). This demonstrates that the reduction levels of
the major pools of NAD(H) and NADP(H) in the leaf
are independently controlled with little communication.
A communication between NADP(H) and NAD(H) inside
mitochondria (Igamberdiev and Gardeström 2003) may
still take place, since the redox transfer between the
mitochondria and the cytosol may be restricted.
A change in the cytosolic NADPH/NADP+ ratio may
affect the transfer of redox equivalents between the
chloroplast and the cytosol. Redox equivalents have been
suggested to be exported from the chloroplast via the
malate valve (forming NADH in the cytosol) or the
triose phosphate/3-phosphoglycerate shuttle. In the latter,
either NADH and ATP are formed by cytosolic phosphorylating GAPDH, or NADPH is formed by cytosolic
NP-GAPDH (Kelly and Gibbs 1973, Krömer 1995,
Hoefnagel et al. 1998, Scheibe 2004). In pea leaves in the
light, the external mitochondrial NADPH DHs were
calculated to oxidize NADPH at faster rates than NADH
(Krömer and Heldt 1991), suggesting that chloroplast
reductant that is exported via the NADPH-linked triose
phosphate/3-phosphoglycerate shuttle may be oxidized by
the mETC (Krömer 1995). We therefore compared the
transgenic lines for several chloroplast redox markers.
NADP-MDH activation responds to the chloroplast
260
Mitochondrial regulation of cell NADPH reduction
redox level by thiol–disulfide interchange (Scheibe 2004),
and is increased in CMSII as compared with the WT after
light treatment due to increased stroma reduction (Dutilleul
et al. 2003a, Priault et al. 2006). PTOX is stress induced and
was suggested to modulate the plastoquinone redox state
(Joët et al. 2002, Rizhsky et al. 2002, Kong et al. 2003),
though it may have a developmental role in Arabidopsis
(Rosso et al. 2006). Xanthophylls respond to excess photon
energy by formation of zeaxanthin, which dissipates the
energy as heat (Gilmore 1997). Additionally, transient
changes in photosynthetic conditions, such as fluctuating
light, must be compensated by alternative redox and energy
sinks in the chloroplast or via redox export shuttles
(Krömer 1995, Scheibe et al. 2004). The similarity between
the transgenic lines regarding the chloroplast redox parameters (Figs. 4, 8) as well as growth under fluctuating
and normal light (Fig. 7) suggests that chloroplast redox
state and function were unaffected by the change in the
cellular NADPH/NADP+ ratio. This indicates that the
suggested redox export via the triose phosphate shuttle
and oxidation via the external NADPH DH (Krömer 1995)
is not involved in chloroplast redox stabilization under
the conditions studied. Instead, the results suggest that
N. sylvestris can tolerate a significant change in cellular
NADP(H) reduction level without deleterious effects on
photosynthesis and growth. This stands in contrast to
the postulated NADP(H) involvement in redox shuttling
and to the number of respiratory proteins that have been
shown by inhibitors or transgenics to affect photosynthesis
and growth (Krömer 1995, Hoefnagel et al. 1998,
Padmasree et al. 2002, Raghavendra and Padmasree 2003,
Sweetlove et al. 2006, Nunes-Nesi et al. 2007).
Materials and Methods
Plant growth and homozygote selection
Seeds from selfed hemizygous transgenic N. sylvestris line T
(Gutierres et al. 1997) transformed with the St-ndb1 gene driven
by the CaMV 35S promoter (Michalecka et al. 2004) were sown
on half-strength MS medium containing 50 mg l1 kanamycin and
germinated under 16 h of light (60 mmol m2 s1) per day at 238C.
Kanamycin-resistant seedlings of each line were transferred to soil
in pots and grown in a greenhouse with 16 h of light
(200 mmol m2 s1) per day at 238C. All hemizygous lines used as
starting material were single chromosome insertions, as determined
by segregation analysis (Michalecka et al. 2004), upon crossing
with the WT (1 : 1) and by selfing (3 : 1). Plants from each
hemizygous line were self-crossed, and plants showing a segregation of 1 : 1 upon crossing with the WT and 1 : 0 upon self-crossing
were determined as homozygotes.
For experiments, and unless otherwise specified, seeds were
sown on soil in pots and grown in a greenhouse with 16 h of
supplemental light per day at 200 mmol m2 s1 at 238C. Plants
were watered every 2 d and fertilized after 5 weeks with 0.6 g of
complete nutrient mix per plant. Unless otherwise specified, plants
were analyzed after 6 weeks when they had approximately
12 leaves. Plants used for mitochondrial isolation were grown in
a climate chamber under similar conditions. The homozygous
transgenic lines did not display any visual phenotype.
Transcript analysis
Total leaf RNA isolation, cDNA synthesis and real-time
RT–PCR were carried out as previously described (Svensson et al.
2002). The real-time RT–PCR primers for St-ndb1 and the
28.5 kDa subunit gene were used as described (Michalecka et al.
2004). To isolate the N. sylvestris ndb1 cDNA fragment,
the primers for St-ndb1 were used for RT–PCR. A 412 bp length
fragment of the Ns-ndb1 (accession No. EL738644) was obtained.
Real-time RT–PCR primers for Ns-ndb1 were designed as 50 -TTT
GCTGCTGAGCTACACG-30 and 50 -TGATCTCCAGATTGGA
CAAG-30 . Competitive RT–PCR for St-ndb1 and Ns-ndb1 was
carried out with primers 50 -TGGAATTTGCTGCTGAGCTAC-30
and 50 -CCAACTCCAGTTGACCATACAA-30 using 538C annealing but otherwise as described (Michalecka et al. 2004). The
product was restricted with SpeI and BstUI, which specifically cut
St-ndb1 and Ns-ndb1, respectively, and bands were quantified by
agarose gel imaging (Svensson and Rasmusson 2001).
The real-time RT–PCR primers for Ns-aox1b were designed
as 50 -GCGTCTTCTTCAACGCCTAC-30 and 50 -CTCGTCAGCC
CTAACAACCA-30 on the basis of two overlapping N. sylvestris
ESTs (BP752608 and BP748729) that are 98% identical to the
N. tabacum aox1b (Whelan et al. 1996). By Blast searching
GenBank, we found an N. tabacum EST (EB683363) with 83%
nucleotide identity to Arabidopsis thaliana aox2 (AB003176).
The deduced protein sequence was 97% identical to the deduced
amino acid sequence of the N. tabacum AOX2 protein (Whelan
et al. 1996). Based on the EST sequence, primers for cloning
N. sylvestris aox2 were designed as 50 -GCAACCCAAATGGTAT
GAGAG-30 and 50 -CCACTATGCAGGAACAGAACA-30 . A
516 bp N. sylvestris aox2 fragment (accession No. EL738645) was
obtained which is 99% identical to EB683363. The real-time RT–
PCR primers for Ns-aox2 were designed as 50 -CAACCCAAATG
GTATGAGAGG-30 and 50 -GTCAATTGCAATGGCAGGA-30 .
Attempts to clone a fragment for a homolog to Nt-aox1a
(Vanlerberghe and McIntosh 1994) were unsuccessful, by designing
PCR primers against the sequence itself, as well as against
conserved regions (Whelan et al. 1996).
An N. tabacum EST (EB425680) had 80% nucleotide
identity with A. thaliana cDNA for PTOX (AJ004881). The
deduced protein sequence was 75% identical with the A. thaliana
PTOX protein, confirming the identity of the N. tabacum EST.
A 549 bp N. sylvestris cDNA fragment (accession No. EL738646)
with 97% nucleotide identity to EB425680 was obtained by using
the primers 50 -TGAAGAGGAAGTGGTTGTGG-30 and 50 -AAT
TCCTCTCCTTGCTCCTTG-30 , designed according to the
N. tabacum sequence. Real-time RT–PCR primers for Ns-PTOX
were designed as 50 -GAGCTTTGGTTGGTGGAGAA-30 and
50 -TCTTGGGCTCAAAGCATACA-30 .
Mitochondrial isolation
Six-week-old plants growing in a greenhouse were transferred
to a climate chamber and grown for another 4 weeks. After initial
flower bud formation, the shoot apex was pruned and the time of
mitochondrial isolation corresponded to the time of repeated
flowering. A 50 g aliquot of leaf material was used to purify
mitochondria, as previously described (Michalecka et al. 2004).
Mitochondrial regulation of cell NADPH reduction
NAD(P)H oxidation measurement
Oxidation of NAD(P)H was measured in mitochondria as
previously described (Svensson and Rasmusson 2001). AOX
activity was measured as previously described (Escobar et al.
2006), except that the reaction was started by addition of 100 mM
NADH and 100 mM NADPH.
Western blotting
A 0.1 g aliquot of leaves was ground in liquid nitrogen and
total leaf proteins were extracted in 1 ml of 2 SDS–PAGE sample
buffer without 2-mercaptoethanol. Protein concentration was
determined using the bicinchoninic acid reagent according to the
manufacturer’s instructions (Sigma, St Louis, MO, USA)
A 5 mg aliquot of mitochondrial protein or 40 mg of total
leaf protein with or without 0.2% 2-mercaptoethanol was resolved
by one-dimensional SDS–PAGE on 10% gels, and proteins
were wet-blotted to polyvinylidene difluoride membranes with
transfer buffer [25 mM Tris, 10 mM glycine and 10% (v/v)
methanol, pH 8.3]. St-NDB1 was immunodetected with antisera
against the St-NDB1-EFD peptide, which specifically detects
the potato homolog (Michalecka et al. 2004). AOX was detected
with monoclonal antibodies against the Sauromatum guttatum
protein (Elthon et al. 1989). For complex I, the 76 kDa subunit
was detected by antibodies against the 78 kDa subunit of the
Neurospora crassa enzyme as previously described (Rasmusson
et al. 1998), whereas the NAD9 subunit antiserum was made
against the wheat homolog (Lamattina et al. 1993). Antibody
recognition was visualized using horseradish peroxidaseconjugated secondary antibodies (Duval et al. 2002). Autoradiography films of Western blots were quantified by densitometry
with Kodak 1D 3.5 software.
ROS and ascorbate measurements
MDA equivalents were measured according to Hodges et al.
(1999) and Umbach et al. (2005). Before measurement, leaf disks
were incubated for 6 h at 50 mmol m2 s1 light intensity in 15 mM
TES, pH 7.0 (control) or the same buffer plus 5 mM KCN, which
inhibits cytochrome oxidase in N. sylvestris leaves (Vidal et al.
2007). In situ superoxide was estimated using the NBT staining
method and H2O2 using the DAB staining method (Dutilleul et al.
2003b). For visual quantification (Amirsadeghi et al. 2006), stained
leaf disks were number coded and scored visually by 3–4 persons,
for which a mean was calculated before averaging between the
experiments. Ascorbate was extracted and measured as described
(Queval and Noctor 2007) except that the A265 was recorded with a
spectrophotometer and the concentration was calculated using the
extinction coefficient of 12.6 mM1 cm1.
Chlorophyll and xanthophyll measurement
Leaf samples were ground in liquid nitrogen and extracted in
80% (v/v) acetone. Chlorophyll concentration was determined
spectrophotometrically (Arnon 1949), and xanthophylls were
determined by reverse phase-HPLC essentially as described
(Thayer and Björkman 1990).
Determination of pyridine nucleotide level
Analyses were carried out in separate experiments for plants
in the light and in darkness. Pyridine nucleotides were extracted
as described (Hajirezaei et al. 2002) with some modifications: 1 g of
leaves were homogenized in liquid nitrogen and the powder
was divided into two aliquots that were weighed for correction
of the relative amounts. The pyridine nucleotides were assayed
261
by enzyme cycling in microtiter plates at 308C as previously
described (Gibon et al. 2004). Extracts were stored at 808C and
analyzed within 24 h for NAD(P)H and within 3 d for NAD(P)+.
Recovery of pyridine nucleotides added to the homogenate was
between 85 and 145%.
Enzyme activity measurement
NADP-MDH was extracted and assayed as previously
described (Dutilleul et al. 2003a). NP-GAPDH, G-6-P DH and
NADP-ICDH were extracted as described by Rius et al. (2006) and
the enzyme extract was treated as previously described (Wessel
and Flügge 1984) for protein concentration determination.
The activities of NP-GAPDH and G-6-P DH were measured as
described by Rius et al. (2006) except that the temperature was
258C. NAD-ICDH, NAD-MDH, NAD-GAPDH, NADPGAPDH and GR were extracted as previously described
(Gibon et al. 2004). NADP-ICDH, NAD-ICDH and NADMDH activity was measured as described (Igamberdiev et al.
2001a). NAD-GAPDH and NADP-GAPDH were assayed spectrophotometrically using the reaction mix described by Gibon et al.
(2004). GR was assayed as described (Foyer et al. 1995).
Funding
The Swedish Research Council; Carl Tesdorpfs
Stiftelse; Carl Tryggers Stiftelse.
Acknowledgments
The authors are grateful to Drs. T.E. Elthon, T. Friedrich
and J.-M. Grienenberger for antibodies.
References
Amirsadeghi, S., Robson, C.A., McDonald, A.E. and Vanlerberghe, G.C.
(2006) Changes in plant mitochondrial electron transport alter cellular
levels of reactive oxygen species and susceptibility to cell death signaling
molecules. Plant Cell Physiol. 47: 1509–1519.
Arnon, D.I. (1949) Copper enzymes in isolated chloroplasts: polyphenol
oxidase in Beta vulgaris. Plant Physiol. 24: 1–15.
Brangeon, J., Sabar, M., Gutierres, S., Combettes, B., Bove, J., Gendy, C.,
Chetrit, P., des Francs-Small, C.C., Pla, M., Vedel, F. and De Paepe, R.
(2000) Defective splicing of the first nad4 intron is associated with lack of
several complex I subunits in the Nicotiana sylvestris NMS1 nuclear
mutant. Plant J. 21: 269–280.
Clifton, R., Lister, R., Parker, K.L., Sappl, P.G., Elhafez, D., Millar, A.H.,
Day, D.A. and Whelan, J. (2005) Stress-induced co-expression
of alternative respiratory chain components in Arabidopsis thaliana.
Plant Mol. Biol. 58: 193–212.
Clifton, R., Millar, A.H. and Whelan, J. (2006) Alternative oxidases in
Arabidopsis: a comparative analysis of differential expression in the gene
family provides new insights into function of non-phosphorylating
bypasses. Biochim. Biophys. Acta 1757: 730–741.
Considine, M.J., Holtzapffel, R.C., Day, D.A., Whelan, J. and Millar, A.H.
(2002) Molecular distinction between alternative oxidase from monocots
and dicots. Plant Physiol. 129: 949–953.
Douce, R. and Neuburger, M. (1989) The uniqueness of plant mitochondria. Annu. Rev. Plant Physiol. Plant Mol. Biol. 40: 371–414.
Dutilleul, C., Driscoll, S., Cornic, G., De Paepe, R., Foyer, C.H. and
Noctor, G. (2003a) Functional mitochondrial complex I is required
by tobacco leaves for optimal photosynthetic performance in photorespiratory conditions and during transients. Plant Physiol. 131: 264–275.
262
Mitochondrial regulation of cell NADPH reduction
Dutilleul, C., Garmier, M., Noctor, G., Mathieu, C., Chétrit, P.,
Foyer, C.H. and De Paepe, R. (2003b) Leaf mitochondria modulate
whole cell redox homeostasis, set antioxidant capacity and determine
stress resistance through altered signaling and diurnal regulation.
Plant Cell 15: 1212–1226.
Dutilleul, C., Lelarge, C., Prioul, J.L., De Paepe, R., Foyer, C.H. and
Noctor, G. (2005) Mitochondria-driven changes in leaf NAD status exert
a crucial influence on the control of nitrate assimilation and the integration of carbon and nitrogen metabolism. Plant Physiol. 139: 64–78.
Duval, F.D., Renard, M., Jaquinod, M., Biou, V., Montrichard, F. and
Macherel, D. (2002) Differential expression and functional analysis
of three calmodulin isoforms in germinating pea (Pisum sativum L.) seeds.
Plant J. 32: 481–493.
Elhafez, D., Murcha, M.W., Clifton, R., Soole, K.L., Day, D.A. and
Whelan, J. (2006) Characterization of mitochondrial alternative
NAD(P)H dehydrogenases in Arabidopsis: intraorganelle location
and expression. Plant Cell Physiol. 47: 43–54.
Elthon, T.E., Nickels, R.L. and Mcintosh, L. (1989) Monoclonal
antibodies to the alternative oxidase of higher plant mitochondria.
Plant Physiol. 89: 1311–1317.
Escobar, M.A., Geisler, D.A. and Rasmusson, A.G. (2006) Reorganization
of the alternative pathways of the Arabidopsis respiratory chain by
nitrogen supply: opposing effects of ammonium and nitrate. Plant J. 45:
775–788.
Fernie, A.R., Geigenberger, P. and Stitt, M. (2005) Flux an important,
but neglected, component of functional genomics. Curr. Opin. Plant Biol.
8: 174–182.
Foyer, C.H. and Noctor, G. (2005) Redox homeostasis and antioxidant
signaling: a metabolic interface between stress perception and physiological responses. Plant Cell 17: 1866–1875.
Foyer, C.H., Souriau, N., Perret, S., Lelandais, M., Kunert, K.J.,
Pruvost, C. and Jouanin, L. (1995) Overexpression of glutathione
reductase but not glutathione synthetase leads to increases in antioxidant
capacity and resistance to photoinhibition in poplar trees. Plant Physiol.
109: 1047–1057.
Geisler, D.A., Broselid, C., Hederstedt, L. and Rasmusson, A.G. (2007)
Ca2+-binding and Ca2+-independent respiratory NADH and
NADPH dehydrogenases of Arabidopsis thaliana. J. Biol. Chem. 282:
28455–28464.
Gibon, Y., Blaesing, O.E., Hannemann, J., Carillo, P., Hohne, M.,
Hendriks, J.H., Palacios, N., Cross, J., Selbig, J. and Stitt, M. (2004)
A robot-based platform to measure multiple enzyme activities in
Arabidopsis using a set of cycling assays: comparison of changes of
enzyme activities and transcript levels during diurnal cycles and in
prolonged darkness. Plant Cell 16: 3304–3325.
Gilmore, A.M. (1997) Mechanistic aspects of xanthophyll cycle-dependent
photoprotection in higher plant chloroplasts and leaves. Physiol. Plant.
99: 197–209.
Gutierres, S., Sabar, M., Lelandais, C., Chetrit, P., Diolez, P., Degand, H.,
Boutry, M., Vedel, F., de Kouchkovsky, Y. and De Paepe, R. (1997)
Lack of mitochondrial and nuclear-encoded subunits of complex I and
alteration of the respiratory chain in Nicotiana sylvestris mitochondrial
deletion mutants. Proc. Natl Acad. Sci. USA 94: 3436–3441.
Hajirezaei, M.R., Peisker, M., Tschiersch, H., Palatnik, J.F., Valle, E.M.,
Carrillo, N. and Sonnewald, U. (2002) Small changes in the activity
of chloroplastic NADP+-dependent ferredoxin oxidoreductase lead to
impaired plant growth and restrict photosynthetic activity of transgenic
tobacco plants. Plant J. 29: 281–293.
Ho, L.H., Giraud, E., Lister, R., Thirkettle-Watts, D., Low, J., Clifton, R.,
Howell, K.A., Carrie, C., Donald, T. and Whelan, J. (2007) Characterization of the regulatory and expression context of an alternative
oxidase gene provides insights into cyanide-insensitive respiration
during growth and development. Plant Physiol. 143: 1519–1533.
Hodges, D.M., DeLong, J.M., Forney, C.F. and Prange, R.K. (1999)
Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other
interfering compounds. Planta 207: 604–611.
Hoefnagel, M.H. N., Atkin, O.K. and Wiskich, J.T. (1998) Interdependence
between chloroplasts and mitochondria in the light and the dark.
Biochim. Biophys. Acta 1366: 235–255.
Igamberdiev, A.U., Bykova, N.V., Lea, P.J. and Gardeström, P. (2001a)
The role of photorespiration in redox and energy balance of photosynthetic plant cells: a study with a barley mutant deficient in glycine
decarboxylase. Physiol. Plant. 111: 427–438.
Igamberdiev, A.U. and Gardeström, P. (2003) Regulation of NAD- and
NADP-dependent isocitrate dehydrogenases by reduction of levels of
pyridine nucleotides in mitochondria and cytosol of pea leaves.
Biochim. Biophys. Acta 1606: 117–125.
Igamberdiev, A.U., Romanowska, E. and Gardeström, P. (2001b)
Photorespiratory flux and mitochondrial contribution to energy and
redox balance of barley leaf protoplasts in the light and during light–dark
transitions. J. Plant Physiol. 158: 1325–1332.
Joët, T., Genty, B., Josse, E.M., Kuntz, M., Cournac, L. and Peltier, G.
(2002) Involvement of a plastid terminal oxidase in plastoquinone
oxidation as evidenced by expression of the Arabidopsis thaliana enzyme
in tobacco. J. Biol. Chem. 277: 31623–31630.
Karpova, O.V., Kuzmin, E.V., Elthon, T.E. and Newton, K.J. (2002)
Differential expression of alternative oxidase genes in maize mitochondrial mutants. Plant Cell 14: 3271–3284.
Karpova, O.V. and Newton, K.J. (1999) A partially assembled complex I in
NAD4-deficient mitochondria of maize. Plant J. 17: 511–521.
Kelly, G.J. and Gibbs, M. (1973) A mechanism for the indirect transfer
of photosynthetically reduced nicotinamide adenine dinucleotide
phosphate from chloroplasts to the cytoplasm. Plant Physiol. 52:
674–676.
Kong, J., Gong, J.M., Zhang, Z.G., Zhang, J.S. and Chen, S.Y. (2003)
A new AOX homologous gene OsIM1 from rice (Oryza sativa L.) with an
alternative splicing mechanism under salt stress. Theor. Appl. Genet. 107:
326–331.
Krömer, S. and Heldt, H.W. (1991) Respiration of pea leaf mitochondria
and redox transfer between the mitochondrial and extramitochondrial
compartment. Biochim. Biophys. Acta 1057: 42–50.
Krömer, S. (1995) Respiration during photosynthesis. Annu. Rev. Plant
Physiol. Plant Mol. Biol. 46: 45–70.
Lamattina, L., Gonzalez, D., Gualberto, J. and Grienenberger, J.M. (1993)
Higher plant mitochondria encode an homolog of the nuclear-encoded
30-kDa subunit of bovine mitochondrial complex I. Eur. J. Biochem. 217:
831–838.
Leidreiter, K., Kruse, A., Heineke, D., Robinson, D.G. and Heldt, H.W.
(1995) Subcellular volumes and metabolite concentrations in potato
(Solanum tuberosum cv. Desirée) leaves. Bot. Acta 108: 439–444.
Maxwell, D.P., Wang, Y. and McIntosh, L. (1999) The alternative
oxidase lowers mitochondrial reactive oxygen production in plant cells.
Proc. Natl Acad. Sci. USA 96: 8271–8276.
Michalecka, A.M., Agius, S.C., Møller, I.M. and Rasmusson, A.G. (2004)
Identification of a mitochondrial external NADPH dehydrogenase by overexpression in transgenic Nicotiana sylvestris. Plant J. 37:
415–425.
Michalecka, A.M., Svensson, Å.S., Johansson, F.I., Agius, S.C.,
Johanson, U., Brennicke, A., Binder, S. and Rasmusson, A.G. (2003)
Arabidopsis genes encoding mitochondrial type II NAD(P)H
dehydrogenases have different evolutionary origin and show distinct
responses to light. Plant Physiol. 133: 642–652.
Mittler, R., Vanderauwera, S., Gollery, M. and Van Breusegem, F. (2004)
Reactive oxygen gene network of plants. Trends Plant Sci. 9: 490–498.
Moore, C.S., Cook-Johnson, R.J., Rudhe, C., Whelan, J., Day, D.A.,
Wiskich, J.T. and Soole, K.L. (2003) Identification of AtNDI1,
an internal non-phosphorylating NAD(P)H dehydrogenase in Arabidopsis mitochondria. Plant Physiol. 133: 1968–1978.
Muto, S. and Miyachi, S. (1981) Light-induced conversion of nicotinamide
adenine dinucleotide to nicotinamide adenine dinucleotide phosphate in
higher plant leaves. Plant Physiol. 68: 324–328.
Møller, I.M. (2001) Plant mitochondria and oxidative stress: electron
transport, NADPH turnover and metabolism of reactive oxygen species.
Annu. Rev. Plant Physiol. Plant Mol. Biol. 52: 561–591.
Møller, I.M., Jensen, P.E. and Hansson, A. (2007) Oxidative modifications to cellular components in plants. Annu. Rev. Plant Biol. 58:
459–481.
Noctor, G., De Paepe, R. and Foyer, C.H. (2007) Mitochondrial redox
biology and homeostasis in plants. Trends Plant Sci. 12: 125–134.
Mitochondrial regulation of cell NADPH reduction
Noctor, G. and Foyer, C.H. (1998) Ascorbate and glutathione: keeping
active oxygen under control. Annu. Rev. Plant Physiol. Plant Mol. Biol.
49: 249–279.
Noctor, G., Queval, G. and Gakiere, B. (2006) NAD(P) synthesis and
pyridine nucleotide cycling in plants and their potential importance in
stress conditions. J. Exp. Bot. 57: 1603–1620.
Nunes-Nesi, A., Sweetlove, L.J. and Fernie, A.R. (2007) Operation and
function of the tricarboxylic acid cycle in the illuminated leaf. Physiol.
Plant. 129: 45–56.
Padmasree, K., Padmavathi, L. and Raghavendra, A.S. (2002) Essentiality
of mitochondrial oxidative metabolism for photosynthesis: optimization
of carbon assimilation and protection against photoinhibition. Crit. Rev.
Biochem. Mol. Biol. 37: 71–119.
Priault, P., Fresneau, C., Noctor, G., De Paepe, R., Cornic, G. and Streb, P.
(2006) The mitochondrial CMSII mutation of Nicotiana sylvestris impairs
adjustment of photosynthetic carbon assimilation to higher growth
irradiance. J. Exp. Bot. 57: 2075–2085.
Priault, P., Vidal, G., De Paepe, R. and Ribas-Carbo, M. (2007) Leaf agerelated changes in respiratory pathways are dependent on complex I
activity in Nicotiana sylvestris. Physiol. Plant. 129: 152–162.
Purvis, A.C. and Shewfelt, R.L. (1993) Does the alternative pathway
ameliorate chilling injury in sensitive plant tissues. Physiol. Plant. 88:
712–718.
Queval, G. and Noctor, G. (2007) A plate reader method for the
measurement of NAD, NADP, glutathione and ascorbate in tissue
extracts: application to redox profiling during Arabidopsis rosette
development. Anal. Biochem. 363: 58–69.
Raghavendra, A.S. and Padmasree, K. (2003) Beneficial interactions of
mitochondrial metabolism with photosynthetic carbon assimilation.
Trends Plant Sci. 8: 546–553.
Rao, I.M., Arulanantham, A.R. and Terry, N. (1989) Leaf phosphate
status, photosynthesis and carbon partitioning in sugar beet: II. Diurnal
changes in sugar phosphates, adenylates and nicotinamide nucleotides.
Plant Physiol. 90: 820–826.
Rasmusson, A.G. and Agius, S.C. (2001) Rotenone-insensitive NAD(P)H
dehydrogenases in plants: immunodetection and distribution of native
proteins in mitochondria. Plant Physiol. Biochem. 39: 1057–1066.
Rasmusson, A.G. and Escobar, M.A. (2007) Light and diurnal regulation of
plant respiratory gene expression. Physiol. Plant. 129: 57–67.
Rasmusson, A.G., Heiser, V., Irrgang, K.D., Brennicke, A. and
Grohmann, L. (1998) Molecular characterisation of the 76 kDa iron–
sulphur protein subunit of potato mitochondrial complex I. Plant Cell
Physiol. 39: 373–381.
Rasmusson, A.G., Soole, K.L. and Elthon, T.E. (2004) Alternative
NAD(P)H dehydrogenases of plant mitochondria. Annu. Rev. Plant
Biol. 55: 23–39.
Rasmusson, A.G., Svensson, A.S., Knoop, V., Grohmann, L. and
Brennicke, A. (1999) Homologues of yeast and bacterial rotenoneinsensitive NADH dehydrogenases in higher eukaryotes: two enzymes are
present in potato mitochondria. Plant J. 20: 79–87.
Raymond, P., Gidrol, X., Salon, C. and Pradet, A. (1987) Control involving
adenine and pyridine nucleotides. In The Biochemistry of Plants. Edited
by Davies, D.D. Vol. 11, pp. 129–176. Academic Press, Inc., San Diego.
Rhoads, D.M. and Subbaiah, C.C. (2007) Mitochondrial retrograde
regulation in plants. Mitochondrion 7: 177–194.
Rius, S.P., Casati, P., Iglesias, A.A. and Gomez-Casati, D.F. (2006)
Characterization of an Arabidopsis thaliana mutant lacking a cytosolic
non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase. Plant
Mol. Biol. 61: 945–957.
Rizhsky, L., Hallak-Herr, E., Van Breusegem, F., Rachmilevitch, S.,
Barr, J.E., Rodermel, S., Inze, D. and Mittler, R. (2002) Double antisense
plants lacking ascorbate peroxidase and catalase are less sensitive to
263
oxidative stress than single antisense plants lacking ascorbate peroxidase
or catalase. Plant J. 32: 329–342.
Rosso, D., Ivanov, A.G., Fu, A., et al. (2006) Immutans does not act as a
stress-induced safety valve in the protection of the photosynthetic
apparatus of Arabidopsis during steady-state photosynthesis. Plant
Physiol. 142: 574–585.
Sabar, M., De Paepe, R. and de Kouchkovsky, Y. (2000) Complex I
impairment, respiratory compensations and photosynthetic decrease in
nuclear and mitochondrial male sterile mutants of Nicotiana sylvestris.
Plant Physiol. 124: 1239–1249.
Scheibe, R. (2004) Malate valves to balance cellular energy supply. Physiol.
Plant. 120: 21–26.
Schmidt-Bleek, K., Heiser, V., Thieck, O., Brennicke, A. and Grohmann, L.
(1997) The 28.5-kDa iron sulfur protein of mitochondrial complex I is
encoded in the nucleus in plants. Mol. Gen. Genet. 253: 448–454.
Sieger, S.M., Kristensen, B.K., Robson, C.A., Amirsadeghi, S., Eng, E.W.,
Abdel-Mesih, A., Moller, I.M. and Vanlerberghe, G.C. (2005) The role
of alternative oxidase in modulating carbon use efficiency and
growth during macronutrient stress in tobacco cells. J. Exp. Bot. 56:
1499–1515.
Stevens, R.G., Creissen, G.P. and Mullineaux, P.M. (2000) Characterisation
of pea cytosolic glutathione reductase expressed in transgenic tobacco.
Planta 211: 537–545.
Svensson, Å.S., Johansson, F.I., Møller, I.M. and Rasmusson, A.G. (2002)
Cold stress decreases the capacity for respiratory NADH oxidation in
potato leaves. FEBS Lett. 517: 79–82.
Svensson, Å.S. and Rasmusson, A.G. (2001) Light-dependent gene
expression for proteins in the respiratory chain of potato leaves.
Plant J. 28: 73–82.
Sweetlove, L.J., Lytovchenko, A., Morgan, M., Nunes-Nesi, A.,
Taylor, N.L., Baxter, C.J., Eickmeier, I. and Fernie, A.R. (2006)
Mitochondrial uncoupling protein is required for efficient photosynthesis. Proc. Natl Acad. Sci. USA 103: 19587–19592.
Thayer, S.S. and Björkman, O. (1990) Leaf xanthophyll content and
composition in sun and shade determined by HPLC. Photosynth. Res. 23:
331–343.
Umbach, A.L., Fiorani, F. and Siedow, J.N. (2005) Characterization of
transformed Arabidopsis with altered alternative oxidase levels and
analysis of effects on reactive oxygen species in tissue. Plant Physiol. 139:
1806–1820.
Vanlerberghe, G.C. and McIntosh, L. (1994) Mitochondrial electron
transport regulation of nuclear gene expression. Studies with the
alternative oxidase gene of tobacco. Plant Physiol. 105: 867–874.
Vanlerberghe, G.C. and McIntosh, L. (1997) Alternative oxidase: from gene
to function. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48: 703–734.
Vidal, G., Ribas-Carbo, M., Garmier, M., Dubertret, G., Rasmusson, A.G.,
Mathieu, C., Foyer, C.H. and De Paepe, R. (2007) Lack of respiratory
chain complex I impairs alternative oxidase engagement and modulates
redox signaling during elicitor-induced cell death in tobacco. Plant Cell
19: 640–655.
Wakao, S. and Benning, C. (2005) Genome-wide analysis of glucose-6phosphate dehydrogenases in Arabidopsis. Plant J. 41: 243–256.
Wessel, D. and Flügge, U.I. (1984) A method for the quantitative recovery
of protein in dilute solution in the presence of detergents and lipids.
Anal. Biochem. 138: 141–143.
Whelan, J., Smith, M.K., Meijer, M., Yu, J.W., Badger, M.R., Price, G.D.
and Day, D.A. (1995) Cloning of an additional cDNA for the alternative
oxidase in tobacco. Plant Physiol. 107: 1469–1470.
Whelan, J., Millar, A.H. and Day, D.A. (1996) The alternative oxidase is
encoded in a multigene family in soybean. Planta 198: 197–201.
Zhang, Q.S., Mischis, L. and Wiskich, J.T. (1996) Respiratory responses of
pea and wheat seedlings to chloramphenicol treatment. Austr. J. Plant
Physiol. 23: 583–592.
(Received October 13, 2007; Accepted December 29, 2007)