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The Critical Role of Arabidopsis Electron-TransferFlavoprotein:Ubiquinone Oxidoreductase duringDark-Induced Starvation W
Kimitsune Ishizaki,a Tony R. Larson,b Nicolas Schauer,c Alisdair R. Fernie,c Ian A. Graham,b
and Christopher J. Leavera,1
a Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdomb Department of Biology, Centre for Novel Agricultural Products, University of York, Heslington, York YO10 5YW,
United Kingdomc Max-Planck-Institut fur Molekulare Pflanzenphysiologie, 14476 Golm, Germany
In mammals, electron-transfer flavoprotein:ubiquinone oxidoreductase (ETFQO) and electron-transfer flavoprotein (ETF) are
functionally associated, and ETF accepts electrons from at least nine mitochondrial matrix flavoprotein dehydrogenases
and transfers them to ubiquinone in the inner mitochondrial membrane. In addition, the mammalian ETF/ETFQO system
plays a key role in b-oxidation of fatty acids and catabolism of amino acids and choline. By contrast, nothing is known of the
function of ETF and ETFQO in plants. Sequence analysis of the unique Arabidopsis thaliana homologue of ETFQO revealed
high similarity to the mammalian ETFQO protein. Moreover, green fluorescent protein cellular localization experiments
suggested a mitochondrial location for this protein. RNA gel blot analysis revealed that Arabidopsis ETFQO transcripts
accumulated in long-term dark-treated leaves. Analysis of three independent insertional mutants of Arabidopsis ETFQO
revealed a dramatic reduction in their ability to withstand extended darkness, resulting in senescence and death within 10 d
after transfer, whereas wild-type plants remained viable for at least 15 d. Metabolite profiling of dark-treated leaves of the
wild type and mutants revealed a dramatic decline in sugar levels. In contrast with the wild type, the mutants demonstrated
a significant accumulation of several amino acids, an intermediate of Leu catabolism, and, strikingly, high-level accu-
mulation of phytanoyl-CoA. These data demonstrate the involvement of a mitochondrial protein, ETFQO, in the catabolism
of Leu and potentially of other amino acids in higher plants and also imply a novel role for this protein in the chlorophyll
degradation pathway activated during dark-induced senescence and sugar starvation.
INTRODUCTION
In mammals, the nuclear-encoded mitochondrial protein, elec-
tron-transfer flavoprotein:ubiquinone oxidoreductase (ETFQO),
which is associated with the inner mitochondrial membrane,
accepts electrons from the electron-transfer flavoprotein (ETF)
localized in the mitochondrial matrix and reduces ubiquinone
(Ruzicka and Beinert, 1977; Beckmann and Frerman, 1985,
1987). ETF is the physiological electron acceptor for at least
nine mitochondrial matrix flavoprotein dehydrogenases; hence,
the ETF/ETFQO system can be thought of as a branch of the
electron transport system with multiple input sites from seven
acyl-CoA dehydrogenases and two N-methyl dehydrogenases
(Frerman, 1988; Frerman and Goodman, 2001). These dehydro-
genases include four chain length–specific acyl-CoA dehydro-
genases involved in straight-chain fatty acid b-oxidation:
isovaleryl CoA dehydrogenase, 2-methyl branched-chain acyl-
CoA dehydrogenase, and glutaryl-CoA dehydrogenase, which
play a role in the oxidation of amino acids; and sarcosine and
dimethylglycine dehydrogenases, which are involved in choline
metabolism (Frerman, 1988; Frerman and Goodman, 2001). The
mammalian mitochondrial proteins, ETF and ETFQO, are essen-
tial for the catabolism of fatty acids, several amino acids, and
choline and are important in supplying mitochondria with re-
spiratory substrates auxiliary to those derived from sucrose. In
humans, mutations in either ETF or ETFQO results in the fatal
genetic disease type II Glutaric acidemia (multiple acyl-CoA
dehydrogenase dysfunctional disease) (Frerman and Goodman,
2001).
In the higher plantArabidopsis thaliana, botha- andb-subunits
of ETF (the corresponding genes are At1g50940 and At5g43430,
respectively) were identified in mitochondria by the use of gel-
based or liquid chromatography tandem mass spectrometry
mitochondrial proteomic analysis (Heazlewood et al., 2004).
However, the ETFQO protein has not been identified in plant
mitochondria, although a unique homologue of the ETFQO gene
(At2g43400) has been identified in the Arabidopsis genome
(Arabidopsis Genome Initiative, 2000).
One of the functions of the ETF/ETFQO system in mammals is
to allow respiration of substrates other than glucose. In plants,
sucrose availability can be restricted during extended darkness
1 To whom correspondence should be addressed. E-mail [email protected]; fax 44-01865-275144.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Christpher J.Leaver ([email protected]).W Online version contains Web-only data.Article, publication date, and citation information can be found atwww.plantcell.org/cgi/doi/10.1105/tpc.105.035162.
The Plant Cell, Vol. 17, 2587–2600, September 2005, www.plantcell.org ª 2005 American Society of Plant Biologists
(Graham et al., 1992); however, plants have apparently evolved
a range of metabolic responses that allow them to survive for
extended periods during dark-induced sucrose starvation. Such
metabolic responses lead to the mobilization of alternate cellular
components, which compensate for decreases in primary re-
spiratory substrate and maintain important biochemical pro-
cesses, thus allowing survival in adverse conditions (Aubert et al.,
1996). Genome-wide responses to sucrose starvation have been
recently highlighted by several microarray experiments, where it
was shown that a range of genes involved in metabolic pro-
cesses, such as lipid metabolism, chlorophyll degradation, pro-
tein degradation, and amino acid catabolism, are upregulated,
whereas genes encoding components of pathways such as
photosynthesis, carbon fixation, ribosome biogenesis, amino
acid biosynthesis, glycolysis, and many others are downregu-
lated (Contento et al., 2004; Lin and Wu, 2004; Thimm et al.,
2004; Buchanan-Wollaston et al., 2005).
In this study, we have investigated the role of ETFQO in
Arabidopsis during sucrose starvation induced by extended dark
treatment and have focused on the characterization of Arabi-
dopsis T-DNA knockouts of the gene encoding ETFQO at the
molecular, physiological, and metabolite levels. We have con-
firmed the mitochondrial location of the protein by means of
green fluorescent protein (GFP) localization experiments and
have performed expression analysis of ETFQO in Arabidopsis
seedlings following transfer to extended dark periods. Finally, we
have studied the consequences of these gene mutations on plant
viability following periods of extended darkness (up to 15 d) in
addition to studying their impact on cellular metabolism under
these conditions. The data are discussed in the context of
current models of mitochondrial metabolism in plant tissues.
RESULTS
Arabidopsis ETFQO Is a Unique Homologue of Human
ETFQO and Is Localized in Mitochondria
We have identified a homologue of ETFQO in the Arabidopsis
genome, designated as ETFQO (At2g43400), that encodes
a protein with 70% similarity to human ETFQO (accession
number Q16134). The mammalian ETFQO is a unique integral
membrane protein containing one equivalent of flavin adenine
dinucleotide (FAD) and a [4Fe-4S]2þ1þ cluster (Goodman et al.,
1994), characteristics shared with the Arabidopsis ETFQO. The
N-terminal sequence of the Arabidopsis ETFQO protein has
characteristics consistent with that of a mitochondrial transit
peptide and is predicted to target ETFQO to the mitochondrion
by the three targeting prediction programs Predotar (http://
genoplante-info.infobiogen.fr/predotar/), MitoProt II (Claros and
Vincens, 1996), and PSORT (Nakai and Horton, 1999), consistent
with the location of mammalian ETFQO in mitochondria. Align-
ment of the amino acid sequence of Arabidopsis ETFQO
to homologues from bacteria to human shows a high over-
all similarity, including a putative FAD binding domain and
[4Fe-4S]2þ1þ cluster domain (see Supplemental Figure 1 online).
Taken together, the above observations give strong support to
our claim that Arabidopsis ETFQO is a functional homologue of
ETFQO from other organisms.
In order to confirm the in vivo subcellular localization of
Arabidopsis ETFQO, a full-length cDNA fragment was fused to
the cDNA of GFP (Figure 1A). The cDNA fusion construct was
subcloned downstream of the cauliflower mosaic virus (CaMV)
35S RNA promoter and transformed into Arabidopsis. Five-day-
old seedlings of stable transformants were incubated with
MitoTracker Orange for specific staining of mitochondria. In-
spection of the seedlings transformed with the Pro35S:ETFQO:
GFP construct by confocal microscopy revealed that the GFP
fluorescence within root cells was restricted to numerous spher-
ical or elongated bodies, which correspond well with mitochon-
drial morphology (Figures 1B to 1D). The colocalization of the
MitoTracker Orange dye and the GFP signal in root cells
confirmed mitochondrial targeting of the ETFQO:GFP fusion
protein (Figures 1B to 1D). These data clearly demonstrate the
mitochondrial targeting of the ETFQO:GFP fusion protein in vivo
and thus establish the mitochondrial localization of the ETFQO in
Arabidopsis.
ETFQO Is Induced under Sucrose-Starved Conditions
The ETFQO gene is represented on the Affymetrix ATH1 Ge-
nome Array, and analysis of microarray data stored in the
GENEVESTIGATOR database (Zimmermann et al., 2004) re-
vealed that ETFQO transcripts are expressed constitutively at a
low level in all plant tissues and developmental states, including
leaf senescence (Buchanan-Wollaston et al., 2005); however, in-
triguingly, it was highly expressed in dark-induced senescent
leaves (Lin and Wu, 2004), senescent cell cultures (Buchanan-
Wollaston et al., 2005), and sucrose-starved cell cultures
(Contento et al., 2004). RNA gel blot analysis confirmed that
ETFQO expression was not significantly induced during normal
developmental senescence (data not shown).
To further investigate regulation of ETFQO, transcript levels
were measured in 4-week-old Arabidopsis leaves following
transfer to darkness by RNA gel blot analysis. During dark
adaptation, the transcripts from a light-regulated photosynthetic
gene, Cab4, gradually declined (Figure 2) (Fujiki et al., 2001),
whereas the ETFQO transcripts accumulated to very high levels
(Figure 2). As a positive control, the transcript levels of the
isovaleryl-CoA dehydrogenase gene (IVD) and the E1a subunit of
branched-chain a-keto dehydrogenase gene (BCKDH), which
have previously been shown to be induced by sucrose starvation
(Daschner et al., 2001; Fujiki et al., 2001), were analyzed. The IVD
and BCKDH transcripts also accumulated in the dark-adapted
leaves (Figure 2), suggesting that expression of the ETFQO gene
is also induced by sucrose starvation following prolonged
darkness.
Isolation of T-DNA InsertionMutants ofArabidopsis ETFQO
To investigate the in vivo functions of the ETFQO protein, three
independent Arabidopsis lines containing T-DNA or dissociation
(Ds) transposon element insertional mutations of ETFQO were
isolated. Segregation of the encoded resistance markers of all
three insertion elements were in good agreement with the 3:1
(resistant:susceptible) ratio, suggesting insertion at a single Men-
delian locus. Homozygous lines for each mutant were confirmed
2588 The Plant Cell
by genomic PCR and designated etfqo-1, etfqo-2, and etfqo-3,
respectively (Figure 3A), and the respective sites of insertion of
these mutants were confirmed by sequencing PCR products
amplified from each mutant.
As described above, ETFQO contains a putative [4Fe-4S]2þ1þ
cluster domain near its C terminus that is likely to be required for
electron flow from flavin to ubiquinone. In all three mutant lines,
the insertional element is positioned upstream of the putative
[4Fe-4S]2þ1þ cluster, and it would be anticipated that this would
severely compromise its electron transfer capacity and so result
in a complete loss of functional protein. RT-PCR using primer
pairs designated to span the T-DNA insertion site of each mutant
locus was used to investigate ETFQO transcription. The Arabi-
dopsis b-tubulin gene was used as a control to demonstrate the
integrity of the RNA preparation. ETFQO mRNAs were detected
in the wild type (Columbia-0 [Col-0] and Landsberg erecta
[Ler-0]) using the primer sets L1/R1 and L2/R2. In etfqo-1,
ETFQO transcripts were detected using the L2/R2 primer set;
however, no amplification products were observed for the trans-
cripts using the L1/R1 primer set. In etfqo-2 and etfqo-3, ampli-
fication products were not detected using either the L1/R1 or
L2/R2 primer set (Figure 3B). These results confirmed that trans-
cripts spanning the T-DNA insertion site, and downstream of
the T-DNA insertion, are absent in all these mutant lines.
Phenotype of etfqoMutant Lines
After the molecular identity of the T-DNA insertional mutants was
established, they were grown in soil under long-day conditions
alongside the corresponding wild-type controls. Under these
conditions, there was no visible phenotype in the mutants during
the early stage of vegetative growth; however, in the reproduc-
tive stage, etfqo-1 and etfqo-2 plants often produced shorter
siliques with a lower number of seeds than the corresponding
wild type, whereas etfqo-3 had siliques with length and number
of seeds similar to the wild type (see Supplemental Table 1
online). This difference might be due to the fact that the etfqo-3
mutant is in a different wild-type strain background (Ler-0) from
Figure 1. Mitochondrial Localization of an ETFQO:eGFP Fusion Protein in Arabidopsis Seedlings.
(A) The construct carrying the gene encoding ETFQO:eGFP fusion protein. The ETFQO cDNA encoding the full-length coding sequence except stop
codon (ETFQO, yellow box), which includes a sequence for the predicted mitochondrial target peptide (red box), is cloned upstream of the eGFP
reading frame (eGFP, green box). Expression of the resulting fusion protein is controlled by the CaMV 35S promoter (blue box) and the NOS terminator
(gray box).
(B) to (D) Images taken from Arabidopsis root cells expressing the ETFQO:eGFP fusion protein. (B), (C), and (D) are GFP signal (green), MitoTracker
Orange signal (blue), and merged image of (C) and (D), respectively.
Figure 2. Accumulation of Transcripts from the ETFQO in Dark-Treated
Arabidopsis Leaves.
RNA was isolated from leaves harvested at indicated times after the
onset of dark treatment. As control, we examined the expression of the
IVD gene, that of BCKDH E1a subunit gene, and that of the chlorophyll
a/b binding protein gene (Cab). Equal loading was confirmed by compar-
ing ethidium bromide staining of rRNA bands.
Functional Analysis of ETFQO in Arabidopsis 2589
etfqo-1 and etfqo-2 (Col-0). From crosses using wild-type pollen
as the male parent and etfqo-1 and etfqo-2 as female parents,
shorter siliques with lower seed set were produced, whereas
crosses using pollen from etfqo-1 and etfqo-2 to create crosses
with wild-type plants as female parent produced normal si-
liques with seed number similar to wild-type siliques, suggest-
ing an effect of the ETFQO defect on the development of
female gametophyte cells (see Supplemental Table 2 online).
When 5-week-old etfqo mutants grown under short-day con-
ditions were transferred to dark conditions, a dramatic pheno-
type became apparent. All of the homozygous mutant lines
started to wilt and show signs of senescence after 10 d of
continuous darkness and were apparently dead after 15 d of
continuous darkness, whereas both wild-type ecotypes are still
alive after 15 d of continuous darkness and show only limited
signs of senescence (Figure 4A). To further investigate this
apparent accelerated senescence in the etfqo mutants, we
measured two parameters related to the function of chloroplasts,
chlorophyll content and photochemical efficiency (maximum
variable fluorescence/maximum yield of fluorescence [Fv/Fm]),
as diagnostics for leaf senescence (Oh et al., 1996). During
extended dark conditions, chlorophyll content declined more
rapidly in etfqo mutants than in the wild type (Figure 4B), and
the decline of chlorophyll content was accompanied by a de-
crease in the photochemical efficiency of photosystem II (PSII)
(Fv/Fm) (Figure 4C). This result indicates that the process of se-
nescence is more rapid in etfqo mutants than wild-type plants
during extended dark conditions.
In order to confirm that the phenotypes of the mutant plants
were caused by mutations in ETFQO, a CaMV 35S promoter–
driven ETFQO open reading frame (ORF) was introduced into the
etfqo-1 mutant (designated as etfqo-1/OE line) and shown to
completely reverse the partial sterility and prevent the acceler-
ated senescence phenotype observed in the etfqo-1 mutant line
(Figure 4A; see Supplemental Table 1 online).
Leu Catabolism Is Blocked in etfqoMutants
In mammals, defects in ETFQO result in a functional deficiency of
mitochondrial flavoprotein dehydrogenases and accumulation of
their substrates (Frerman and Goodman, 2001). To gain further
insight into the role of the ETFQO protein inArabidopsis, we used
the method of Larson and Graham (2001) to analyze changes in
acyl-CoAs in the wild type and etfqo mutants during the
extended dark treatment. The results shown in Figure 5 demon-
strate a dramatic increase in the amount of five-carbon acyl-CoA
in the etfqo-1 and etfqo-2 mutants compared with the wild type
during the extended dark period. There were four candidates in
Arabidopsis for the five-carbon acyl-CoA compounds: isovaleryl-
CoA (3-methyl-butyryl-CoA), 2-methyl-butyryl-CoA, valeryl-CoA,
and acetoacetyl-CoA. However, the retention time of synthetic
acetoacetyl-etheno-CoA was earlier than the five-carbon acyl-
CoA peak (data not shown), eliminating acetoacetyl-CoA as a
candidate for the peak. Furthermore, given the improved reso-
lution among peaks in the short-chain acyl-CoA region by HPLC
using the Hypercarb porous graphitic carbon column, we could
successfully discriminate this five-carbon acyl-CoA peak from
valeryl-CoA and 2-methyl-butyryl-CoA, and identified this peak
as isovaleryl-CoA (Figure 5B). Isovaleryl-CoA is an intermediate
of the Leu catabolic pathway, being subsequently dehydrogen-
ated to 3-methylcrotonyl-CoA by isovaleryl-CoA dehydroge-
nase. In animals, ETF is the physiological electron acceptor of
isovaleryl-CoA dehydrogenase (Ikeda and Tanaka, 1983). These
results suggest a similar interaction of isovaleryl-CoA dehydro-
genase, ETF, and ETFQO in Arabidopsis.
Involvement of ETFQO in Phytol Degradation
Acyl-CoA profiles from both etfqo-1 and etfqo-2 mutants from
the 7- and 10-d extended dark treatments also showed dramat-
ically increased content of a compound that had the same mass
as the straight-chain eicosanoyl (20:0)-CoA, but which had
a different retention time from the 20:0-CoA peak in the standard
mixture. The retention time of this unknown acyl-CoA peak from
the etfqo-1 mutant was identical to synthetic phytanoyl-CoA
(Figure 6A), and both shared the same molecular mass and
diagnostic acyl-CoA–specific fragmentation pattern when ana-
lyzed by liquid chromatography–mass spectrometry (LC-MS)
(data not shown). Unfortunately, LC-MS fragmentation did not
provide any information on the branching pattern of the acyl
chain and so could not distinguish 20:0-CoA from phytanoyl-
CoA. Therefore, in order to further confirm the identity of this
unknown peak, it was purified in bulk, transmethylated, and
analyzed by gas chromatography–mass spectrometry (GC-MS).
The mass spectrum of the transmethylated peak was distin-
guishable from that of eicosanoic acid methyl ester but matched
Figure 3. Identification of etfqo-1, etfqo-2, and etfqo-3 Mutants.
(A) Genomic structure of ETFQO loci. Arrows represent positions of
primers used for genotype and RT-PCR analyses of wild-type and
mutant lines; closed boxes indicate exons. In etfqo-1 and etfqo-2, the
T-DNA is inserted in exon 4 and exon 12, respectively. In etfqo-3,
Ds transposon element is inserted in exon 1.
(B) RT-PCR analysis on total RNA from the wild type, Col-0, Ler-0, and
the mutant lines etfqo-1, etfqo-2, and etfqo-3, with primer sets indicated
on the left and the positions of each primer in ETFQO genomic loci
represented in (A).
2590 The Plant Cell
well with that of phytanic acid methyl ester, which displayed an
electron-impact fragmentation spectrum expected for methyl-
branched acyl chains (Figure 6B). From these results, the accu-
mulated unknown peak near 20:0 acyl-CoA was unequivocally
identified as phytanoyl-CoA. During extended dark treatment,
etfqo-1 and etfqo-2 mutants contained dramatically increased
concentrations of phytanoyl-CoA relative to the wild type (Fig-
ures 4B and 6A). However, analysis of leaf lipid fatty acids by GC
showed no measurable increase in phytanic acid during the ex-
tended dark treatment (data not shown). In humans, phytanoyl-
CoA is a known breakdown product of phytol, the hydrophobic
tail of chlorophylls a and b. That said, relatively little is currently
known about the exact mechanism of phytol or chlorophyll deg-
radation in plants (Matile et al., 1999; Hortensteiner, 2004);
however, our data suggest that Arabidopsis ETFQO is involved
in phytol degradation.
Metabolite Contents of ETFQOMutants during the
Extended Dark Condition
Further characterization of the accelerated senescence pheno-
type in etfqo mutants was performed using a nonbiased GC-MS
metabolic profiling protocol allowing the simultaneous determi-
nation of >100 compounds (Roessner et al., 2001) As was ex-
pected, the extended dark treatment led to a rapid decline in
sucrose and other sugar contents in both the wild type and
etfqo-1 (Figure 7). Most free amino acids increased significantly
in both the wild type and etfqo-1, including, Leu, Isle, Val, Asn,
Arg, Trp, and Phe, suggesting that protein degradation was
elevated under these conditions (Figure 7). The significantly
elevated levels of several amino acids, especially Leu and Trp, in
the etfqo mutants suggest involvement of ETFQO in their deg-
radation pathways. Analysis of tricarboxy acid (TCA) cycle
intermediates showed a dramatic increase in succinate, fuma-
rate, and malate in etfqo-1 at 7 and 10 d dark treatment (see
Supplemental Figure 2 online). While these accumulations are
striking, the exact mechanism underlying this phenomenon
cannot be elucidated from the results in this study. There are
two possible explanations for the progressive accumulation of
these metabolites. First, it is conceivable that the TCA cycle is
progressively upregulated in the mutant, during the course of the
extended darkness, in an attempt to compensate for the reduced
availability of respiratory substrate. Secondly, the accumulation
of TCA cycle intermediates may be a consequence of a general
downregulation of biosynthesis that would be anticipated un-
der conditions of carbon starvation. While we favor the second
Figure 4. Phenotype of etfqo Mutants in Extended Dark Treatment.
(A) Photos of 4-week, short-day grown Arabidopsis plants after further
growth for 0 and 10 d in extended darkness. The plant designated as
etfqo-1/OE is the etfqo-1 line transformed with the CaMV 35S promoter–
driven ETFQO ORF. The leaves of etfqo mutants, etfqo-1, etfqo-2, and
etfqo-3,wereyellowedanddehydrated following10dofgrowth indarkness
compared with the wild-type control, Col-0 and Ler-0, and etfqo-1/OE.
(B) Chlorophyll (line graph) and phytanoyl-CoA (bar graph) content of 9th
or 10th leaves of 4-week, short-day grown Arabidopsis plants of wild
type (Col-0), etfqo-1, and etfqo-2 after further growth for 0, 3, 7, and 10 d
in extended dark treatment. Values are means 6 SD for six independent
samplings. FW, fresh weight.
(C) Fv/Fm, maximum quantum yield of PSII electron transport (maximum
variable fluorescence/maximum yield of fluorescence) of 9th or 10th
leaves of 4-week, short-day grown Arabidopsis plant of the wild type
(Col-0), etfqo-1, and etfqo-2 after further growth for 0, 3, 7, and 10 d in
extended darkness. Values are means 6 SD for six independent
samplings.
Functional Analysis of ETFQO in Arabidopsis 2591
Figure 5. Accumulation of Isovaleryl-CoA in etfqo-1 and etfqo-2 Mutants in Extended Dark Treatment.
(A) Acyl-CoA profiles of the wild type (Col-0), etfqo-1, and etfqo-2 during the extended dark treatment. Values are means 6 SE for six samples of 10 mg
each, derivatized to their acyl-etheno-CoA esters, separated by HPLC, and detected fluorometrically. FW, fresh weight.
2592 The Plant Cell
hypothesis, further experimentation, including direct measure-
ments of fluxes within the constituent pathways, will ultimately be
required in order to fully comprehend these data. Interestingly,
Glu and pyroglutamic acid concentrations declined in etfqo-1 at
7 to 10 d, whereas they increased in the wild type (Figure 7; see
Supplemental Figure 2 online). It has been suggested, in sucrose
starvation experiments, that amino acid catabolism is activated
and supplies respiratory substrates to the TCA cycle (Contento
et al., 2004); however, in etfqo-1, one or more amino acid
catabolic pathways appear to be blocked. One possible conse-
quence of this block could be an overactivation of Glu catabolism
in order to compensate for the shortfall of respiratory substrate.
Furthermore, accumulation of uracil and ribose in etfqo-1 in the
10-d dark treatment suggests that RNA degradation was acti-
vated in etfqo mutants at this stage, and this is most probably
associated with the process of cell death (data not shown). The
above observations were all made by analyzing data on the basis
of specific metabolites; however, interesting features of the data
also emerge when the entire metabolite profiles are considered.
While the majority of the metabolite levels, in the etfqo-1 mutant,
exhibit monophasic behavior with respect to time in continuous
darkness, a subset of metabolites, such as Gln, Gly, oxyproline,
Pro, mannose and trehalose, exhibit biphasic behavior. It is
tempting to speculate that monophasic responses are observed
for the metabolites more intimately related to the reactions
catalyzed by ETFQO, while the biphasic responses are probably
secondary effects arising from the extreme stress that the etfqo
mutants are under following longer periods of darkness. How-
ever, further experimentation will be required to determine
whether this is indeed the case. Future work should also include
characterization of mannose and trehalose metabolism in these
lines, since both of these carbohydrates increase following
extensive periods in continuous darkness despite initially de-
creasing. It is important to note that although the data presented
here were solely determined from etfqo-1, essentially the same
results were obtained from analysis of a second mutant (etfqo-2).
DISCUSSION
The essential roles in central metabolism of mammalian ETF and
ETFQO have been well established; however, we have no
knowledge of the role of their homologues in higher plants. ETF
and ETFQO homologues were identified in the Arabidopsis
genome and also in the genomes and EST databases of many
other plant species, including, rice (Oryza sativa), tomato (Lyco-
persicon esculentum), barley (Hordeum vulgare), and wheat
(Triticum aestivum) (Plant Genome Database; http://
www.plantgdb.org/). This suggests that ETF and ETFQO are
widely expressed in higher plants and potentially play an impor-
tant role in plant metabolism. In this article, we describe an
investigation of the functional importance of ETFQO in Arabi-
dopsis and its role during dark-induced senescence.
In mammals, the reducing equivalents from various flavoprotein
dehydrogenases are transferred via ETF to ETFQO, which is
localized in the inner mitochondrial membrane, and the accepted
electrons are further passed from ETFQO into the electron trans-
port chain via coenzyme Q to the CoQH2-cytochrome c oxidore-
ductase (Complex III) (Parker and Engel, 2000). The deduced
amino acid sequence of Arabidopsis ETFQO showed high overall
similarity to that of human ETFQO, including its two putative
functional domains, the FAD binding domain, and the [4Fe-4S]2þ1þ
cluster domain (see Supplemental Figure 1 online) and a mito-
chondrial targeting peptide at the N terminus. We confirmed the
mitochondrial localization ofArabidopsis ETFQO by colocalization
of MitoTracker Orange with a fusion to GFP (Figure 1). In Arabi-
dopsis, a- and b-subunits of ETF have been found in mitochondria
(Heazlewood et al., 2004), and their amino acid sequences also
show high similarity to the both subunits of the human ETF (data
not shown). Hence, it is quite feasible that the basic function of
ETFQO in electron transfer to coenzyme Q in the mitochondrial
respiratory chain is conserved between plants and animals.
ETFQO Is Induced and Essential for Viability
in Sucrose-Starved Condition
Analysis of microarray data in public databases and RNA gel blot
analyses confirmed that ETFQO was constitutively expressed in
all tissues and stages of development in Arabidopsis plants and
was upregulated following transfer of mature plants to extended
periods of darkness over a period of 15 d (Figure 2). During
experimentally induced sucrose starvation, there is a marked
decrease in expression of many genes involved in carbohydrate
breakdown, glycolysis, the TCA cycle, mitochondrial electron
transport, and ATP synthesis (Buchanan-Wollaston et al., 2005).
This is associated with a decrease in respiratory energy metab-
olism and increased expression of a number of genes involved in
proteolysis, amino acid catabolism, and fatty acid degradation,
suggesting the induction of salvage mechanisms that release
amino acids and fatty acids from structural proteins and lipids as
an alternative source of respiratory substrate to maintain viability
of carbohydrate-starved cells (Contento et al., 2004; Lin and Wu,
2004; Thimm et al., 2004; Buchanan-Wollaston et al., 2005). The
increased expression ofETFQO in dark-adapted plants (Figure 2)
suggests a central role for ETFQO in supplying mitochondria with
an alternative source of respiratory substrates from proteolysis
and/or lipid degradation. The observation that the etfqo mutants
senesce and die before wild-type plants under extended dark
Figure 5. (continued).
(B) Identification of the increased short-chain acyl-CoA peak in etfqo-1. Short-chain acyl-CoAs were chemically synthesized from their respective fatty
acids, derivatized to their acyl-etheno-CoA esters, separated by HPLC on a Hypercarb porous graphitic carbon column, and detected by MS. The 5:0
acyl-etheno-CoA peaks were identified by their characteristic parent and daughter ion mass-to-charge (m/z) ratios of 876 and 452, respectively. Peaks were
aligned against added heptanoyl-etheno-CoA (7:0) internal standard (ISTD; m/z 904). Shown are the extracted ion chromatograms for m/z values of 876 and
904. i, Valeryl-etheno-CoA; ii, 2-methyl-butyryl-etheno-CoA; iii, isovaleryl (3-methyl butyryl)-etheno-CoA; iv, etfqo-1 extract at 10 d in extended darkness.
Functional Analysis of ETFQO in Arabidopsis 2593
Figure 6. Identification of Phytanoyl-CoA Peak.
(A) Comparison of phytanoyl-CoA peak in etfqo-1 with standards. HPLC profiles of acyl-etheno-CoA derivatives are shown for etfqo-1 extract at 10 d in
extended darkness (i), phytanoyl-CoA synthesized from phytanic acid by Pseudomonas acyl-CoA synthetase (ii), and the calibration standard mix (iii).
(B) Confirmation of phytanoyl-CoA identity by GC-MS. Collected fractions of the putative phytanoyl-CoA peak from HPLC runs of derivatized etfqo-1 extracts
at 10 d in extended darkness were subjected to alkaline transmethylation to form the methyl ester. Left panels show GC-MS chromatograms; right panels
show mass spectra for the corresponding peaks. i, eicosanoic acid methyl ester; ii, synthetic phytanic acid methyl ester; iii, transmethylated peak from etfqo-1.
2594 The Plant Cell
conditions (Figure 4A) taken together with their altered metabolic
profiles provides further evidence that ETFQO plays a key role in
supplying alternative respiratory substrates. However, it is pos-
sible that the accelerated senescence and early death of the
etfqo mutants (Figure 4) could be, at least in part, due to the
hyperaccumulation of toxic metabolites, since both branched-
chain amino acids and a-keto acids are known to be cytotoxic
compounds that can induce apoptosis in mammals (Eden and
Benvenisty, 1999; Ogier de Baulny and Saudubray, 2002). Thus,
our results suggest that ETFQO plays an essential role during
sucrose starvation in plants by facilitating the use of alternative
respiratory pathways either by supplying alternate substrates, by
promoting the metabolism of toxic products thereof, or both.
Contribution of ETFQO to the Reproductive Development
of Arabidopsis
The ecotype-specific reduced seed set and silique size of etfqo
mutants (see Supplemental Table 1 online) suggest an additional
role for ETFQO inArabidopsis. Numerous studies on cytoplasmic
male sterility have revealed the important role of the mitochon-
drion during pollen development (Hanson and Bentolila, 2004). In
Figure 7. Relative Levels of Amino Acids and Sugars in the Wild Type (Black Bars) and etfqo-1 Mutant (White Bars) during Extended Dark Conditions.
The y axis values represent metabolite level relative to the wild type. Data were normalized to mean response calculated for the 0-d dark-treated leaves
of the wild type (in case no response was detected at 0 d, normalization was done against 3-d dark-treated leaves of the wild type). Values presented are
mean 6 SE of determinations on six individual plants.
Functional Analysis of ETFQO in Arabidopsis 2595
this case, however, the reproductive phenotype was not asso-
ciated with pollen quality (see Supplemental Table 2 online).
Thus, the mitochondrion also appears to play an important role in
female gametophyte development as previously shown (Skinner
et al., 2001; Christensen et al., 2002).
Involvement of ETFQO in Leu Catabolism
In Arabidopsis, isovaleryl-CoA dehydrogenase is localized in
plant mitochondria (Daschner et al., 2001; Taylor et al., 2004),
and our metabolite profiling data (Figure 5) demonstrated
a marked accumulation of the isovaleryl-CoA dehydrogenase
substrate, isovaleryl-CoA, in etfqo mutants, suggesting dysfunc-
tion of isovaleryl-CoA dehydrogenase in these mutants. These
results further support the existence of the electron transfer
cascade from isovaleryl-CoA dehydrogenase to ETF, ETF to
ETFQO, and ETFQO to ubiquinone in the electron transport chain
of plant mitochondria as reported in mammals (Ikeda and
Tanaka, 1983), although the direct interaction of these four
components has yet to be confirmed.
The recent proteomic study on Arabidopsis mitochondria by
Taylor et al. (2004) revealed that the enzymes necessary for Leu
catabolism are all localized within plant mitochondria, confirming
the findings of 14C-Leu tracer experiments on isolated pea (Pisum
sativum) mitochondria (Anderson et al., 1998). In this study, our
demonstration of a marked accumulation of isovaleryl-CoA, as
a result of mutation in the mitochondrial protein ETFQO, provides
further support for the mitochondrial location of Leu catabolism in
Arabidopsis. By contrast, we did not detect an accumulation
of the intermediates of either Ile or Val catabolism, 2-methyl-
butyryl-CoA, and isobutyryl-CoA, respectively, in etfqo mutants
during the extended dark treatment, although it has been shown
in mammals that ETF/ETFQO is also involved in dehydrogenation
of 2-methyl-butyryl-CoA and isobutyryl-CoA (Ikeda et al., 1983).
Our data imply that the mitochondrial ETF/ETFQO is less involved
in Ile and Val catabolism in Arabidopsis than it is in Leu
catabolism. However, it is important to note that these amino
acids and several others accumulate in the etfqomutants relative
to the wild type (Figure 7). It is not clear why CoA intermediates for
Ile and Val were not detected. It is possible that they are not as
actively degraded as Leu; thus, intermediates are below the level
of detection. Alternatively, the CoA intermediates could be
actively and specifically deacylated to free fatty acids, but short
chain acyl-CoA thioesterases that would catalyze this process
have not been described in plants. Finally it is possible that the
breakdown of Val and Ile may not be dependent on the ETFQO,
and the dehydrogenation/oxidation step could be localized to the
peroxisomes, as is the case for straight-chain fatty acids in higher
plants. Such a hypothesis is consistent with the observations that
the peroxisomal short-chain acyl-CoA oxidase is able to oxidize
isobutyryl-CoA (Hayashi et al., 1999), and the demonstration that
several other enzymes involved in Ile and Val catabolism are
localized in peroxisomes in Arabidopsis (Lange et al., 2004;
Taylor et al., 2004). The mitochondrial isovaleryl-CoA dehydro-
genase does utilize 2-methyl-butyryl-CoA as a substrate in vitro;
however, the Km value of the enzyme for the isobutyryl-CoA is
;10 times larger than that for isovaleryl-CoA (Daschner et al.,
2001). Since the relative concentration of Val is only half that of
Leu, the large difference in Km values suggests that isovaleryl-
CoA dehydrogenase is not involved in Val oxidation in vivo. The
recent finding that the Arabidopsis mitochondrial branched-
chain aminotransferase (BCAT-1) is able to initiate degradation
of Leu, Ile, and Val (Schuster and Binder, 2005) along with
previous proteomic results (Taylor et al., 2004) and those of this
study provide an important foundation for understanding the
degradative pathways of branched-chain amino acids. Intrigu-
ingly, the etfqo mutants also displayed elevated accumulation of
aromatic amino acids, hinting that the ETF/ETFQO system may
also be involved in the metabolism of this family of amino acids.
The Involvement of ETFQO in the Phytol Catabolic
Pathway in Plants
Our observations show that there was a significant accumulation
of phytanoyl-CoA in etfqo mutants during extended dark treat-
ment (Figures 5 and 6), which parallels a decrease in chlorophyll
(Figure 4B). It is well known in humans as a breakdown in-
termediate of phytol, which is derived from chlorophyll in the
diet. In humans, phytol is oxidized to phytanic acid, subse-
quently converted into phytanoyl-CoA by a long-chain fatty acyl-
CoA synthetase, and further degraded in peroxisomes by the
a-oxidation system followed by b-oxidation in mitochondria
(Wierzbicki et al., 2002; Wanders et al., 2003). Disorders in
peroxisomal a-oxidation result in a syndrome called Refsum’s
disease, which results in a dramatic accumulation of phytanic
acid in plasma- and lipid-containing tissues (Wierzbicki et al.,
2002). In plants, phytol is derived from the first step of chlorophyll
degradation, which is catalyzed by chlorophyllase (EC 3.1.1.14)
(Tsuchiya et al., 1999); however, the latter enzymatic steps of
phytol degradation are poorly understood, especially in compar-
ison with the pathway in humans. Previous studies have sug-
gested that phytol degradation during leaf senescence occurs via
photooxidative conversion into various isoterpenoid compounds
(Rontani et al., 1996; Matile et al., 1999; Rontani and Aubert,
2005). This obviously cannot be the case for chlorophyll break-
down during the extended dark treatments in this study. There-
fore, increasing accumulation of phytanoyl-CoA in response to
the length of dark treatment in etfqo mutants suggests the
existence of an enzymatic pathway of phytol degradation in
Arabidopsis mitochondria. In the extended dark condition, chlo-
rophyll breakdown was greater in theetfqomutant than in the wild
type (Figure 4B), which could explain the dramatically increased
concentration of phytanoyl-CoA in etfqo mutants (Figure 4B).
However, it is also possible that ETFQO is directly involved in the
phytanoyl-CoA degradation pathway, and phytanoyl-CoA degra-
dation is blocked in etfqo mutants. In the analysis of leaf lipid
derived fatty acid content of etfqo mutants during the extended
dark treatment, no accumulation of phytanic acid, which is an up-
stream intermediate of phytanoyl-CoA, was observed (data not
shown), suggesting rapid conversion from phytol to phytanoyl-
CoA in Arabidopsis, at least under the conditions studied.
The Function of ETFQO in Mammals and Plants
In mammals, it has been shown that one of the main roles of
ETF/ETFQO is the reoxidation of four straight-chain acyl-CoA
2596 The Plant Cell
dehydrogenases that are essential for the first step of fatty acid
b-oxidation (Bartlett and Eaton, 2004). b-Oxidation of fatty
acids primarily occurs in the mitochondria in mammals, while
b-oxidation in higher plants occurs exclusively in peroxisomes
(Graham and Eastmond, 2002). Our observations are consis-
tent with a peroxisomal localization for b-oxidation in higher
plants in that straight-chain acyl-CoAs do not accumulate in
the etfqo mutants during the dark treatment (Figure 5). Fur-
thermore, measurement of the fatty acid content of etfqo
mutants and the wild type during germination indicated that
fatty acid breakdown was the same in etfqo mutants and the
wild type (data not shown). This result suggests that the
ETFQO is not involved in b-oxidation of straight-chain fatty
acids in Arabidopsis, supporting the postulate that b-oxidation
of straight-chain fatty acids is localized exclusively in peroxi-
somes in Arabidopsis.
In mammals, ETF and ETFQO are also involved in the catab-
olism of several amino acids via dehydrogenation of isovaleryl-
CoA dehydrogenase (Leu) catabolism, 2-methyl branched-chain
acyl-CoA dehydrogenase (Ile and Val), and glutaryl-CoA dehy-
drogenase (Lys, Hyl, and Trp) (Frerman and Goodman, 2001).
While the accumulation of isovaleryl-CoA in the etfqo mutants
showed the involvement of ETFQO in Leu catabolism via iso-
valeryl-CoA dehydrogenase as in mammals, the lack of accu-
mulation of 2-methyl-butyryl-CoA and isobutyryl-CoA does not
allow us to formally conclude a similar role for ETFQO in Ile and
Val catabolism. However, it should be noted that Ile and Val
accumulate dramatically in the etfqo mutants, and it is possible
that ETFQO is involved in catabolism of Ile and Val upstream of
the intermediates 2-methyl-butyryl-CoA and isobutyryl-CoA since
neither of these accumulates. The increase of Trp in GC-MS pro-
files of the etfqo mutants suggests involvement of ETFQO in Trp
catabolism as found in mammals. The accumulation of phytanoyl-
CoA and also amino acids in the etfqo mutants, as revealed by
GC-MS profiling, implies the involvement of ETFQO in the unique
metabolic processes that are not found in animals. As we dis-
cussed above, given the importance of the structure of the mam-
malian ETFQO to its basic functionality, the high amino acid
sequence conservation (70%) between mammalian and Arabi-
dopsis ETFQO suggests that the basic function of ETFQO as the
electron donor between ETF and ubiquinone is conserved. How-
ever, the specific cellular role will be dependent on the interacting
partners of the ETF. Thus, further understanding of these roles in
plants will require identification of the ETF interacting partners.
Conclusion
This study has revealed that ETFQO is essential for plants to
survive in sucrose-depleted conditions induced by extended
growth in the dark and is involved in the Leu catabolic pathway.
The study also shows that phytanoyl-CoA accumulates when
ETFQO is disrupted, thus implicating the mitochondrial ETF/
ETFQO in the degradation of the phytol tail of chlorophyll. The
phytol tail represents ;50% of the carbon in chlorophylls, the
other 50% belongs to the porphyrin ring. On a global scale,
the annual cycle of synthesis and breakdown of chlorophyll is
massive, and more work is now needed to fully understand the
role of the mitochondrial ETFQO in this important process. The
identification of the ETFQO protein in plants clearly demon-
strates the existence and importance of a novel mitochondrial
electron transport complex in higher plants. In mammals, ETF is
associated with ETFQO and several flavoprotein dehydroge-
nases (Parker and Engel, 2000). A similar relationship is likely to
function in higher plants with at least some interacting partners
conferring plant-specific roles, and it is on these that future
studies need to focus.
METHODS
Plant Material and Dark Treatment
Arabidopsis thaliana seeds were surface-sterilized and imbibed for 4 d at
48C in the dark on 0.8% (w/v) agar plates containing half-strength
Murashige and Skoog (MS) media (Sigma-Aldrich; pH 5.7). Seeds were
subsequently germinated and grown at 228C under long-day conditions
(16 h light/8 h dark) with 150 mmol m�2 s�1 white light. For examination of
phenotype in the long-day treatment, seedlings were transferred to soil 7
to 10 d after germination and placed in a growth chamber at 228C under
long-day conditions (16 h light/8 h dark). For dark treatments, 7- to 10-d-
old seedlings were transferred to soil and then grown at 228C under short-
day conditions (8 h light/16 h dark) for 4 weeks. Following bolting, plants
were grown at 228C in the dark in the same growth cabinet. The fully
expanded, 7th to 12th, rosette leaves were harvested at intervals of 0,
3, 7, and 10 d from control and dark-grown plants for the subsequent
experiments.
RNA Gel Blot Analysis
Total RNA was isolated from light-grown control, from dark-treated
leaves, and from cell suspension culture using the TRIzol reagent
(Invitrogen). cDNA fragments for ETFQO, IVD, the E1a subunit ofBCKDH,
chlorophyll a/b binding protein (Cab4), and nitrate reductase (NR2), were
amplified from Arabidopsis cDNA by PCR. RNA was fractionated by
electrophoresis on 1% (w/v) agarose gels, blotted onto positively charged
nylon membrane, and hybridized at 658C with the appropriate cDNA
probes labeled with [a-32P]dCTP using the MegaPrime kit (Amarsham-
Pharmacia). The hybridized membrane was washed for 1 h in a solution
containing 23 SSC and 0.1% (w/v) SDS at 258C followed by two washes
with 0.13 SSC and 0.5% SDS at 558C for 1 h.
Isolation of T-DNA Insertion Mutants and
Genotype Characterization
The three mutant lines, SALK T-DNA line SALK_007870 (etfqo-1, back-
ground ecotype Col), Syngenta Arabidopsis insertion line SAIL_91_E03
(etfqo-2, background ecotype Col), and John Innes Centre Gene Trap line
GT_3_4705 (etfqo-3, background ecotype Ler) were obtained from the
Nottingham Arabidopsis Stock Centre (University of Nottingham). The
mutants were isolated according to published procedures: SIGnAL
(Alonso et al., 2003), Syngenta SAIL line (Sessions et al., 2002), and
John Innes Centre Gene Trap line (Sundaresan et al., 1995). Genotypes of
the different knockout mutant lines were analyzed by PCR using primers
specific for the ETFQO ORF (for etfqo-1, L1 59-AAGGTGGTACCGTG-
CTTCAG-39 and R1 59-CACCACCTTCAGGCACTGTC-39; for etfqo-2 and
etfqo-3, L2 59-AAAGCTATCTTCTTCTTCTTCACCA-39 and R2 59-GGT-
TGCAATCCCAACAACTT-39) and primers specific for the insertion
elements (for etfqo-1, LBa1 59-TGGTTCACGTAGTGGGCCATCG-39;
for etfqo-2, TMR1_LB1 59-GCCTTTTCAGAAATGGATAAATAGCCTTG-
CTTCC-39; for etfqo-3, Ds3-1 59-ACCCGACCGGATCGTATCGGT-39 and
Ds5-1 59-GAAACGGTCGGGAAACTAGCTCTAC-39).
Functional Analysis of ETFQO in Arabidopsis 2597
Complementation of etfqo-1with the CaMV 35S Promoter–Driven
ETFQO ORF
The full-length ORF including stop codon of ETFQO was amplified by RT-
PCR from wild-type Arabidopsis (ecotype Col-0) and subcloned into the
pH2GW7 vector (www.psb.ugent.be/gateway/) (Karimi et al., 2002) using
the Gateway recombination system (Invitrogen). Heterozygous etfqo-1
mutant plants were subsequently transformed by Agrobacterium tume-
faciens–mediated gene transfer utilizing the floral dip method (Clough and
Bent, 1998) because of the partial sterility exhibited by the homozygous
etfqo-1 plant. Positive transformants were selected on half-strength MS
medium containing 0.8% (w/v) agar and 30 mg/mL hygromycin. Homo-
zygous etfqo-1 lines containing the CaMV 35S promoter–driven ETFQO
ORF were characterized by genomic PCR using the primer set L1 and R1
described above in order to check the transformed ETFQO ORF and the
primer set L1 and RI1 (59-TTGTGCAGTGGATGGTTTTG-39) in order to
confirm that there was no amplification from a disrupted copy of ETFQO.
The primer RI1 was designed on the basis of the intron sequence
of ETFQO in order to eliminate amplification from the transformed
ETFQO ORF.
Analysis of ETFQOmRNA Expression by RT-PCR
Total RNA was isolated from rosette leaves of 3 d dark-treated plants
using TRIzol reagent. First-strand cDNA was synthesized from 10 mg of
total RNA with Superscript II Rnase H� reverse transcriptase (Invitrogen)
and oligo(dT) primer. PCR amplification of ETFQO cDNA-specific se-
quence was performed using primers specific for the ORF, L1 and R1 and
L2 and R2, described above. PCR amplification of the cDNA encoding the
b-tubulin of Arabidopsis (TUB9) with a forward primer (59-GATATC-
TGTTTCCGTACCTTGAAGC-39) and a reverse primer (59-ACCGACTG-
TAGCATCTTGATATTGC-39) served as a control.
Subcellular Localization Experiments
In order to generate CaMV 35S promoter–driven ETFQO:eGFP fusion
protein constructs, the 1899-bp coding region of ETFQO was amplified
by RT-PCR from wild-type Arabidopsis (ecotype Col-0) with primers
flanked with attB1 and attB2 sites. The resultant sequence was then
cloned into the entry vector pDONR201 by Gateway recombination
(Invitrogen). This cloned sequence was then transferred from the
pDONR201 vector to the pK7WFG2 vector (www.psb.ugent.be/
gateway/) (Karimi et al., 2002) by Gateway recombination (Invitrogen).
Wild-type Arabidopsis Col-0 was subsequently transformed by
Agrobacterium-mediated gene transfer utilizing the floral dip method
(Clough and Bent, 1998). Positive transformants were selected on half-
strength MS medium containing 0.8% (w/v) agar and 50 mg/mL kana-
mycin. Following selection, transformants were transferred to soil and
seeds collected. The T2 generation was used for the observation of GFP
fluorescence. Five-day-old seedlings were harvested into a freshly made
staining solution of 200 nM CMTMRos (MitoTracker Orange; Molecular
Probes) in half-strength MS medium and allowed to stain for 15 min at
room temperature. After staining, the seedlings were submerged in a half-
strength MS solution for 10 min. The T2 seedlings were visualized by
confocal microscopy with a LSM510 confocal microscope (Zeiss) set to
measure an emission band of 475 to 525 nm for GFP (excitation 488,
emission 522) and an emission band of 565 to 615 nm for MitoTracker
Orange CMTMRos (excitation 551, emission 576). Excitation for GFP was
provided by the 458-nm line of a 25-mW argon laser and for MitoTracker
by a 543-nm HeNe laser. The software LSM examiner (Zeiss) was used for
postacquisition image processing. Several T2 generation lines for each
construct were investigated, and all showed the same fluorescence
pattern.
Measurement of Senescence Parameters
For chlorophyll content, 20 mg of leaf tissue were frozen and ground in
liquid nitrogen and extracted by heating in 1 mL of methanol at 658C for
20 min. After clarification by centrifugation, the chlorophyll concentration
per fresh weight of leaf tissue was calculated as described previously
(Porra et al., 1989). Photochemical efficiency was examined at the given
time points for the dark treatment, using attached leaves of the wild type
(Col-0) and etfqo mutants. The photochemical efficiency of PSII was de-
duced from the characteristics of chlorophyll fluorescence using a por-
table plant efficiency analyzer (Hansatech Instruments) (Oh et al., 1996).
The ratio of Fv to Fm, which corresponds to the potential quantum yield
of the photochemical reactions of PSII, was used as the measure of
the photochemical efficiency of PSII (Oh et al., 1996).
Acyl-CoA and Fatty Acid Profiling
Ten-milligram portions of leaf material were frozen in liquid nitrogen and
extracted for subsequent quantitative analysis of fluorescent acyl-
etheno-CoA derivatives by HPLC with a LUNA 150 3 2 mm C18(2) col-
umn (Phenomenex) using the gradient conditions described previously
(Larson and Graham, 2001; Larson et al., 2002). The lipid portion of the
acyl-CoA extracts was transmethylated for fatty acid analysis by GC with
flame ionization detection (Larson and Graham, 2001).
Commercially available phytanic acid (3,7,11,15-tetramethylhexade-
canoic acid) (Sigma-Aldrich) was used to synthesize phytanic acid methyl
ester by acidic transmethylation (Larson and Graham, 2001). Phytanoyl-
CoA, for use as an HPLC retention time standard, was enzymatically
synthesized from phytanic acid using Pseudomonas acyl-CoA synthe-
tase from Sigma-Aldrich (Taylor et al., 1990). Putative phytanoyl-CoA
peaks from HPLC runs of synthetic and leaf extracts were further
characterized by LC-MS to determine molecular weight and verify acyl-
CoA specific fragmentation patterns (Larson and Graham, 2001). Addi-
tional confirmation of phytanoyl-CoA identity was obtained by GC-MS
analysis of collected HPLC fractions transmethylated to their fatty acid
methyl esters. Putative phytanoyl-CoA fractions from 10-fold concen-
trated and derivatized extracts run on the HPLC were collected, dried
under vacuum, and transmethylated with 500 mL 0.1 M sodium meth-
oxide (Sigma-Aldrich) for 2 h at 858C. The formed methyl ester was
partitioned against 0.25 mL 0.9% (w/v) KCl and removed in 200 mL
hexane. The hexane phase was dried under vacuum and reconstituted in
20 mL fresh hexane, and a 2 mL aliquot was analyzed by GC-MS as
described previously (Tonon et al., 2005), with the exception that a ZB5
30 m 3 0.25 mm i.d. 3 0.25 mm film thickness capillary column (Pheno-
menex) was used.
Short branched-chain acyl-CoA esters were commercially unavailable,
and Pseudomonas acyl CoA synthetase was unable to catalyze the
formation of short branched-chain acyl-CoA esters from branched-chain
5-carbon acids (Sigma-Aldrich). Instead, these were synthesized by the
carbonyldiimidazole method (Kawaguchi et al., 1981). The acyl-CoA
products were derivatized to their etheno derivatives and characterized
by LC-MS. In order to increase the resolution between the C5 isomers,
a Hypercarb 100 3 3 mm porous graphitic carbon column (Thermo
Hypersil) was used in place of the LUNA 15032 mm C18(2) column, using
the same gradient program.
Extraction, Derivatization, and Analysis of Arabidopsis Leaf
Metabolites Using GC-MS
Metabolite analysis by GC-MS was performed by a method modified from
that described by Rossener-Tunai et al. (2003). Arabidopsis leaf tissue
(;180 mg) was homogenized using a mortar and pestle precooled with
liquid nitrogen and extracted in 1400mL of methanol, and 60mL of internal
standard (0.2 mg ribitol mL�1 water) was subsequently added as a
2598 The Plant Cell
quantification standard. The extraction, derivatization, standard addition,
and sample injection were exactly as described previously (Roessner-
Tunali et al., 2003). The GC-MS system and settings were exactly as
described by Roessner et al. (2001). Both chromatograms and mass
spectra were evaluated using the MASSLAB program (ThermoQuest),
and the resulting data were prepared and presented as described by
Roessner et al. (2001).
Accession Numbers
Sequence data from this article can be found in the GenBank/EMBL data
libraries under the following accession numbers: for Arabidopsis ETFQO,
At2g43400; IVD, At3g45300; BCKDH, At1g21400; Cab4, At3g47470; NR2,
At1g37130; TUB9, At4g20890; for rice OsETFQO, AAK39567; for human
HsETFQO, Q16134; for rice MmETFQO, Q921G7; for fly DmETFQO,
NP_610536; for worm CeETFQO, D88483; for yeast ScETFQO, Q08822;
for protobacteria LpETFQO, YP_095306.
ACKNOWLEDGMENTS
We thank Ian Moore and Ooi-kock Teh for assistance with the confocal
microscopy and Nicholas J. Kruger for help with the measure of the
photochemical efficiency of PSII. We also thank Lee J. Sweetlove for
invaluable suggestions. This work was supported in part by a grant from
the Biotechnology and Biological Science Research Council (to C.J.L.),
by a research fellowship of the Japan Society for the Promotion of
Science for Young Scientists (to K.I.), and by a grant from the German-
Israeli Cooperation Project, Deutsch-Israelisches-Projekt (to N.S. and
A.R.F.).
Received June 13, 2005; revised July 5, 2005; accepted July 5, 2005;
published July 29, 2005.
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2600 The Plant Cell
DOI 10.1105/tpc.105.035162; originally published online July 29, 2005; 2005;17;2587-2600Plant Cell
J. LeaverKimitsune Ishizaki, Tony R. Larson, Nicolas Schauer, Alisdair R. Fernie, Ian A. Graham and Christopher
during Dark-Induced Starvation Electron-Transfer Flavoprotein:Ubiquinone OxidoreductaseArabidopsisThe Critical Role of
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References /content/17/9/2587.full.html#ref-list-1
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