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Plant Physiol. (1998) 117: 473-481
Metabolic Bypass of the Tricarboxylic Acid Cycle during Lipid
Mobilization in Germinating Oilseeds1
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Biosynthesis of sucrose from triacylglycerol requires the bypass of the CO2-evolving reactions of the tricarboxylic acid (TCA) cycle. The regulation of the TCA cycle bypass during lipid mobilization was examined. Lipid mobilization in Brassica napus was initiated shortly after imbibition of the seed and proceeded until 2 d postimbibition, as measured by in vivo [1-14C]acetate feeding to whole seedlings. The activity of NAD+-isocitrate dehydrogenase (a decarboxylative enzyme) was not detected until 2 d postimbibition. RNA-blot analysis of B. napus seedlings demonstrated that the mRNA for NAD+-isocitrate dehydrogenase was present in dry seeds and that its level increased through the 4 d of the experiment. This suggested that NAD+-isocitrate dehydrogenase activity was regulated by posttranscriptional mechanisms during early seedling development but was controlled by mRNA level after the 2nd or 3rd d. The activity of fumarase (a component of the nonbypassed section of the TCA cycle) was low but detectable in B. napus seedlings at 12 h postimbibition, coincident with germination, and increased for the next 4 d. RNA-blot analysis suggested that fumarase activity was regulated primarily by the level of its mRNA during germination and early seedling development. It is concluded that posttranscriptional regulation of NAD+-isocitrate dehydrogenase activity is one mechanism of restricting carbon flux through the decarboxylative section of the TCA cycle during lipid mobilization in germinating oilseeds.
Upon germination of oilseeds,
storage triacylglycerols are mobilized by conversion to carbohydrates
for transport to the root and shoot axes of the developing seedling.
Suc, the major carbohydrate transport form, is used as a substrate for
biosynthesis and is respired for energy. Lipid mobilization in
postgerminative oilseeds requires the metabolic coordination of four
subcellular compartments: the oil body, the glyoxysome, the
mitochondrion, and the cytosol (Trelease and Doman, 1984 Flux through the TCA cycle in plant tissues can vary,
depending on the metabolic requirements of the tissue. For example, a
reduction in the activity of the TCA cycle in the light compared with
its activity in the dark has been documented (Gemel and Randall, 1992 The proposed TCA cycle bypass was first demonstrated by in vivo
labeling studies in castor bean endosperm (Canvin and Beevers, 1961 We are interested in the developmental regulation of mitochondrial
function. Understanding the switch in the role of the mitochondrion in
lipid mobilization to its oxidative function during photosynthesis will
further our knowledge of mitochondrial biogenesis and mitochondrial development. To this end, we have re-examined and extended the observations of earlier workers (Canvin and Beevers, 1961 Plant Material and Chemicals
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
). The
elucidation of this complex interaction was dependent on the discovery
of the glyoxylate cycle (Kornberg and Krebs, 1957). Fatty acids are
cleaved by lipases from their glycerol backbone in the oil body and,
after being transported to the glyoxysome, are degraded by
-oxidation to acetyl-CoA. The glyoxylate cycle ultimately catalyzes
the condensation of two of these acetyl-CoA molecules to form
succinate, which is then transported to the mitochondrion and
metabolized by a partial TCA cycle. Since the complete TCA cycle
catalyzes two decarboxylative reactions, the quantitative conversion of
lipid to Suc can occur only if certain TCA cycle reactions are
bypassed. During gluconeogenesis, only the TCA cycle activities of
succinate dehydrogenase, fumarase, and malate dehydrogenase are
required. The activities of the TCA cycle decarboxylative enzymes
NAD+-IDH and 
-ketoglutarate dehydrogenase are
avoided.
;
Hanning and Heldt, 1993). The TCA cycle is regulated by the redox state
of the pyridine nucleotide pool (Oliver and McIntosh, 1995). NADH
competitively inhibits the activities of
NAD+-IDH, -ketoglutarate dehydrogenase, and
pyruvate dehydrogenase (although technically not a component of the TCA
cycle, the pyruvate dehydrogenase complex is the entry point of
glycolytically derived pyruvate into the TCA cycle). Furthermore,
NAD+-IDH is noncompetitively inhibited by NADPH
(McIntosh and Oliver, 1992). The regulation of the TCA cycle in plants
differs from its regulation in animals in that none of the plant
enzymes appears to be controlled by ratios of adenine nucleotides, e.g.
the ratio of ATP/ADP or of acetyl-CoA/CoA (Voet and Voet, 1990; Oliver
and McIntosh, 1995).
).
Radiolabeled acetate was shown to be converted into carbohydrate with
an experimental efficiency of 70% for the methyl carbon of acetate and
30% for the carboxyl carbon of acetate. The efficiency of conversion
was lower for the carboxyl carbon because it is this carbon that is
lost from succinate at the reaction of PEP carboxykinase, the only
decarboxylative step of gluconeogenesis. In later work, germinating
castor bean mitochondria were shown to rapidly oxidize succinate and
malate plus glutamate, whereas the TCA cycle intermediates in the
decarboxylative portion of the TCA cycle (isocitrate through succinate)
were only slowly oxidized (Millhouse et al., 1983). A more recent
report addressed the regulation of this bypass at the enzymatic level
in cucumber (Cucumis sativus) seedlings (Hill et al., 1992).
In this study several physiological and enzymological measurements of
postgerminative cucumber seedlings were conducted and it was concluded
that fumarase and NAD+-IDH activities are
regulated differently. Their data are consistent with a reduction in
carbon flux through the decarboxylative reactions of the TCA cycle
during lipid mobilization in cucumber cotyledons. Furthermore, the
authors compared fumarase and NAD+-IDH activities
in both the light and the dark in postgerminative seedlings and
determined that, whereas fumarase activity was unaffected, NAD+-IDH activity was significantly reduced in
the dark. These data suggest that new carbon fixation by photosynthesis
is required for full TCA cycle activity. Recently, it was suggested
that the TCA cycle may be limited postgerminatively by transcriptional control of mitochondrial pyruvate dehydrogenase (Grof et al., 1995).
; Millhouse et
al., 1983; Hill et al., 1992, 1994) using a biochemical and molecular
approach with Brassica napus, Arabidopsis, and cucumber. We
show that during the period of maximum gluconeogenesis the presence of
NAD+-IDH was not detectable, either catalytically
or immunologically, whereas fumarase activity was easily accounted for.
We will present evidence that the posttranscriptional regulation of
NAD+-IDH prevents its activity during
gluconeogenesis in germinating B. napus.
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
In Vivo Labeling
Each 10-mL flask contained 0.2 g of cotyledons (after seed coats were removed) or whole seedlings in 1 mL of 10 mM Mes, pH 5.2, and 0.5 µmol of sodium [1-14C]acetate (1 Ci/mol). Evolved 14CO2 was collected on a paper wick wet with 20 µL of 5 M KOH. After 30 min of incubation at 30°C in the light, the wick was removed and the reaction was stopped by boiling the tissue in 70% ethanol. After cooling on ice, the tissue was homogenized with a Ten-Broeck homogenizer (Thomas Scientific) and applied to a Dowex-1 formate column (pre-equilibrated in 1 M sodium formate and then washed in water) and then to a Dowex-50 column (pre-equilibrated in 0.1 M HCl and washed in water). The nonpolar eluent containing total carbohydrates was subjected to liquid-scintillation counting. To control for the efficiency of carbohydrate extraction, one sample of each time was spiked with 0.1 µCi of [U-14C]Glc (1.2 Ci/mol) after the tissue was cooled.Lipid Extraction
Tissue was ground in liquid N2 to a fine powder and extracted with CHCl3:methanol (v/v, 2:1) at a ratio of 0.5 g tissue to 10 mL of solvent. After 2 h of extraction, particulate matter was removed by filtration. The filtrate was back-extracted with 2.5 mL of 1% NaCl and the organic phase was delivered to a tared vial and dried under an N2 stream at room temperature to a constant weight.Isolation of Mitochondria
Mitochondria were prepared from B. napus developmentally staged seedlings, Arabidopsis mature leaves, etiolated pea seedlings, and green cucumber seedlings by a modification of the method of Hill et al. (1992). Chilled tissue was homogenized in 3 volumes of the following buffer: 0.3 M sorbitol, 10 mM KH2PO4, 2 mM EDTA, 2 mM MgCl2, 2 mM Gly, 1% PVP-40, 1% defatted BSA, 30 mM
ascorbate, 25 mM sodium PPi, 14 mM
-mercaptoethanol, 1% polyvinylpolypyrrolidone, 1 mM
benzamidine, and 0.1 mM PMSF, pH 7.6. This homogenate was filtered and mitochondria were collected by differential
centrifugation, washed, and purified on a Percoll gradient. The
mitochondria were washed in BSA-free medium, and the final pellet
was suspended in a minimal volume of 20 mM Mops
and 5 mM -mercaptoethanol, pH 7.5, and frozen. The
mitochondria were subjected to two freeze-thaw cycles and centrifuged
at 12,000g for 15 min, and the supernatant was collected for
enzyme assays, protein assays, and immunoblot analysis.
Enzyme and Protein Assays
NAD+-IDH activity was assayed as previously described (McIntosh and Oliver, 1992). The isocitrate- and
enzyme-dependent reduction of NAD+ was monitored
by the increase in A340 of a 1-mL
reaction. The reaction consisted of 20 mM Mops (pH
7.5), 5 mM MgSO4, 5 mM
-mercaptoethanol, 1 mM NAD+, and
10 mM isocitrate. Fumarase activity was assayed by
following the increase in A240 of a 1-mL
reaction containing 20 mM Mops (pH 7.5) and 10 mM malate (Behal and Oliver, 1997). Protein concentration was determined by the Bio-Rad Bradford protein assay reagent using ovalbumin as a standard.
cDNA Clones Used
Three cDNA clones were used in this study: idhI (accession no. U81993, Behal and Oliver, 1998), idhII
(accession no. U81994, Behal and Oliver, 1998), and fum
(accession no. U82201, Behal and Oliver, 1997).
Antibody Preparation
To obtain Arabidopsis IDH I protein for antibody preparation, IDH I was overexpressed in Escherichia coli. The cDNA for the mature IDH I protein was amplified by PCR using appropriate primers and cloned into the expression vector pET-24c (Novagen, Inc., Madison, WI). The construct was checked by sequencing to confirm that idhI had been cloned as a translational fusion with an N-terminal T7-tag. pET-IDH I was transformed into competent BL21(DE3) E. coli cells. SDS-PAGE-purified protein from isopropylthio--galactoside-induced cells was submitted to HTI
Bio-Products, Inc. (Ramona, CA) for antibody production in rabbits.
Resulting antiserum was enriched for IDH-specific IgGs by purification
on a column matrix of immobilized IDH I following the manufacturer's
instructions (AminoLink, Pierce). Purified anti-IDH was used at a
dilution of 1:20 for all B. napus immunoblots but detected
the recombinant protein and the native Arabidopsis protein at a higher
dilution (1:2000). Antibodies against fumarase (Behal and Oliver,
1997) and IDH II (Behal and Oliver, 1998) were produced from cDNA
clones by similar protocols.
Immunoblot Analysis
SDS-PAGE on 12.5% acrylamide gels was performed as described by Laemmli (1970). Proteins were transferred to a nitrocellulose membrane
in modified Towbin buffer (25 mM Tris, 192 mM
Gly, and 10% methanol, pH 8.3) (Towbin et al., 1979). Secondary
antibody was goat anti-rabbit alkaline phosphatase conjugate using
5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium as the
colorigenic substrates.
RNA Analysis
Total RNA was extracted from developmentally staged B. napus and cucumber seedlings as previously described for Aspergillus nidulans (Timberlake, 1986) with one
modification: dry seed and d 1 and 2 postimbibition RNA was
differentially precipitated with 2-butoxyethanol to remove interfering
polysaccharides, as described by Schultz et al. (1994). RNA was
electrophoresed in formaldehyde gels and blotted to nylon as summarized
by Sambrook et al. (1989). Loading was standardized with rRNA.
Polysomes were extracted from 1-d-old postimbibition B. napus seedlings, according to the method of DeVries et al. (1988),
except that 250 µM -mercaptoethanol was added to the
extraction buffer. RNA was extracted from polysomes by
phenol-chloroform extraction (Timberlake, 1986).
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Time Course of Lipid Mobilization during B. napus Seedling Development
The relationship between gluconeogenesis and lipid mobilization in developing B. napus seedlings is demonstrated in Figure 1. Carbohydrate synthesis from fatty acids was measured as the ratio of 14CO2 released to [14C]carbohydrate synthesized when seedlings were fed [1-14C]acetate (Fig. 1, right y axis). When plant tissue is utilizing lipid as a carbon and energy source, the ratio of released 14CO2 to synthesized [14C]carbohydrate will be lower than when photosynthate is the sole carbon and energy source (Canvin and Beevers, 1961). Figure 1 shows that the ratio of
[1-14C]acetate incorporation into
14CO2 to
[14C]carbohydrate was 1.08 ± 0.09 for
light-grown and 1.48 ± 0.16 for dark-grown B. napus
seedlings at 12 h postimbibition. The difference between light-
and dark-grown seedlings was significant by a Student's t
test. By comparison, Canvin and Beevers (1961) measured a
14CO2 to
14C-carbohydrate ratio of 1:1 in 5-d-old castor
bean endosperm when it was labeled with
[1-14C]acetate. Although the metabolism of
endospermic seeds and nonendospermic (cotyledonary) seeds cannot be
directly compared, our measurement indicates that the fate of
[1-14C]acetate is primarily gluconeogenic in
B. napus seedlings at 12 h postimbibition.
|
). Various plants were used in this study.
B. napus was utilized for in vivo labeling experiments,
enzyme assays, immunoblots, and RNA blots. Arabidopsis has a smaller
and more completely described genome than B. napus and,
therefore, was used to clone the genes for
NAD+-IDH and fumarase. The enzyme assays,
immunoblots, and northern blots of cucumber were necessary to compare
our results with the previous data from this plant (Hill et al., 1992).
Activities of NAD+-IDH and Fumarase during Seedling Development in B. napus and Cucumber
As shown in Figure 1, gluconeogenesis extended from 12 h to 2 or 3 d postimbibition in developing B. napus seedlings. After this period, the in vivo labeling data suggested that the full TCA cycle was operational in these seedlings. The activities of fumarase and NAD+-IDH during this developmental period in B. napus and cucumber seedlings are shown in Figure 2. Enzyme activities are reported as total activity per seedling (Fig. 2). Because of the possibility that the efficiency of mitochondrial isolation changed during seedling growth, the data for fumarase and IDH were also reported on the basis of specific activity per milligram of mitochondrial protein (Fig. 2, A and B, insets) for B. napus. At all times measured, fumarase total and specific activity were 40- to 100-fold greater than the NAD+-IDH activity in both B. napus and cucumber (Fig. 2, note the difference in activity maxima on the y axes). Fumarase activity in B. napus (Fig. 2A) was low but detectable in seedlings at 12 h postimbibition and then increased rapidly in the light until reaching its maximum total activity at 4 d postimbibition. In the dark-grown seedlings, fumarase followed the same activity profile as in the light but its activity was reduced approximately 3-fold (Fig. 2A). The specific activity of fumarase (Fig. 2A, inset) reached a maximum at d 2 in the light-grown seedlings, 2 d earlier than the maximum total activity. This difference reflects the continuing expansion of the cotyledon concomitantly with general protein synthesis from d 2 to 4 postimbibition. Western analysis (data not presented) showed that fumarase protein level and enzyme activity increased in parallel from 12 h to 5 d postimbibition in the light and in the dark.
|
). In their study the activity of
NAD+-IDH activity in the dark was much lower
relative to its activity in the light than what we observed.
Isolation of Arabidopsis NAD+-IDH and Fumarase cDNAs and Immunoblot Analysis of NAD-IDH
To obtain molecular tools for the analysis of fumarase and NAD+-IDH in developing seedlings, these genes were cloned from Arabidopsis (Behal and Oliver, 1997, 1998).
idhI and idhII are 86% identical at the amino
acid level and both possess most of the important residues of the
catalytic site, as determined from the crystal structure of the
NADP+-IDH protein from E. coli (Hurley
et al., 1990). The coding region of both of the Arabidopsis
idh genes is 1100 bp in length and encodes a protein with a
predicted molecular mass of 40 kD. When IDH I and IDH II were
overexpressed separately in E. coli, neither extract
displayed NAD+-dependent IDH activity, although
the native NADP+-dependent IDH activity was
easily detectable (data not shown). Experiments to denature and refold
the recombinant NAD+-IDH to obtain enzymatic
activity were unsuccessful.
).
Occurrence of NAD+-IDH Protein in Developing
Seedlings
-mercaptoethanol, and ascorbate, gave the
least amount of cross-reaction with nonspecific bands. B. napus seeds have high levels of phenolics in the seed coat and
contain 48% lipid. Proteins can be cross-linked by phenolic and
lipid-based mechanisms, and lipid peroxidation is a probable mechanism
for inactivation of mitochondrial proteins (Cohn et al., 1996). When
the antibody was immunoprecipitated against its antigen prior to
probing the developmental immunoblot, the 47-kD band representing
NAD+-IDH was preferentially eliminated (Fig.
4C). This further demonstrated that the
47-kD band in the B. napus mitochondrial extracts
represented NAD+-IDH. Antibody was also made
against IDH II produced using the pMAL overexpression system. This
antibody, anti-IDH II, also recognized a protein band at 47 kD in plant
mitochondrial extracts.
View larger version (41K):
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Figure 3.
Overexpression of recombinant Arabidopsis IDH I
protein in E. coli and immunoblot analysis of IDH I in
several plants. A to F, Electrophoresis on 12.5% (w/v) acrylamide
SDS-PAGE followed by transfer to nitrocellulose in modified Towbin
buffer. A, Coomassie blue stain of 10 µg of total protein of
recombinant pET-IDHI E. coli extract. B, Immunoblot
analysis of 50 ng of total protein of recombinant pET-IDHI E. coli extract, reacted with anti-IDH at 1:2000 dilution. C to F,
Immunoblot analysis of plant mitochondrial extracts with anti-IDH at
1:20 dilution. C, Arabidopsis leaf, 20 µg; D, cauliflower, 20 µg;
E, pea leaf, 100 µg; and F, B. napus seedling, 3 d
postimbibition, 40 µg.
View larger version (46K):
[in a new window]
Figure 4.
Immunoblot analysis of NAD+-IDH in
B. napus mitochondrial extracts during seedling development.
A to C, Forty micrograms of total protein from developmentally staged
mitochondrial extracts were loaded per lane and electrophoresed on
12.5% (w/v) acrylamide SDS-PAGE. Transfer and immunoblot development
were as described in Figure 3 and ``Materials and Methods''. A,
Light-grown tissues. B, Dark-grown tissues. C, Light-grown tissue,
probed with anti-IDH I that had been precipitated with recombinant
Arabidopsis IDH I prior to incubation with the blot for the purpose of
removing IDH-specific antibodies. Arrows indicate IDH band. DPI, Days
postimbibition.
).
Postgerminative Expression of TCA Cycle Genes in B. napus
To examine whether the lack of immunodetectable NAD+-IDH protein until 2 d postimbibition in B. napus was due to a lack of its mRNA, RNA-blot analysis was undertaken. Total RNA was isolated from developmentally staged B. napus and cucumber seedlings and probed with the full-length idhI and fum cDNAs. Figure 5 shows the mRNA levels of idh and fum in B. napus and cucumber, from imbibed seeds to seeds at 4 d postimbibition when equivalent amounts of RNA for each time were hybridized with the indicated DNA. As shown in Figure 5A, the idh transcript was detectable in seeds and seedlings of B. napus allowed to imbibe in the light and in the dark. The level of IDH mRNA was similar for imbibed seeds and at 12 h postimbibition. It increased until at least d 3 postimbibition. The transcript level of this gene was reduced in the dark compared with the transcript level in the light in a manner that coincided with the enzyme activity profile for NAD+-IDH. Fumarase mRNA, on the other hand, was not detected until d 1 postimbibition (Fig. 5B). Fumarase enzymatic activity was also detected at this time and it appears that the regulation of this gene occurs primarily at the level of mRNA availability.
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Translational Analysis of idh in B. napus Seedlings
Since the idh mRNA was detectable at 1 d postimbibition in B. napus seedlings, whereas both protein and enzyme activity were not detected until d 2, we were interested in determining whether this transcript was actually being translated. We performed a polyribosomal analysis of the idh mRNA in 1-d postimbibition seedlings. At d 1 postimbibition, the B. napus seed was undergoing gluconeogenesis. At this developmental stage, NAD+-IDH activity and the NAD+-IDH protein were not detected, although the mRNA was present. Two possibilities arise: (a) translational control was preventing the message from being translated, (b) or the message was translated and the protein was rapidly degraded or modified in some way rendering it inactive and undetectable by immunoblot. An RNA-blot analysis of idh in the polysomal fractions is shown in Figure 6, and the spectrophotometer trace of the polysome fractionation on a Suc gradient is shown in Figure 6C. This figure demonstrates that the idh message was associated with polyribosomes at 1 d postimbibition. This is strong evidence that the NAD+-IDH protein was being made and that regulation of NAD+-IDH during gluconeogenesis was posttranslational.
|
). When
mitochondrial extracts from 1- and 7-d postimbibition B. napus seedlings were incubated with shrimp alkaline phosphatase (1 unit in 1 mL of 20 mM Tris, 10 MgCl2,
pH 8.0, for 15 min at room temperature), no change in IDH activity or
immunoreactivity was observed (data not shown). Also, as previously
mentioned, we determined that there were no soluble inhibitory factors
present in d-1 mitochondria that affected activity in d-7 mitochondria.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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The development of the TCA cycle during oilseed germination and seedling development was examined. In B. napus seedlings triacylglycerol metabolism started at imbibition and proceeded until about 3 d postimbibition; after this time the carbon and energy source for seedling growth switched to mainly photosynthetic reactions. Based on the in vivo labeling results, during the period of lipid degradation, metabolism began as gluconeogenic and then switched to being respiratory. This transition was accompanied by a rapid increase in the TCA cycle activities, including fumarase and NAD+-IDH (Fig. 2). Whereas gluconeogenesis predominated, the decarboxylative reactions of the TCA cycle were bypassed to provide quantitative conversion of lipid to Suc in the cotyledons of the developing seedling. The activity of fumarase, an essential TCA cycle for carbohydrate synthesis from lipid, was detected in B. napus seeds 12 h after imbibition. The activity of NAD+-IDH, a decarboxylative dehydrogenase in the section of the TCA cycle thought to be bypassed during carbohydrate synthesis, was not detected until 2 d postimbibition. Thus, during gluconeogenesis the decarboxylative reactions were bypassed by delaying the expression of NAD+-IDH (and possibly other enzymes).
studied the
development of the E1-subunit of pyruvate dehydrogenase during cucumber seedling development. It has been shown that this subunit undergoes light-dependent inactivation by phosphorylation to
potentially limit the activity of the TCA cycle during photorespiration
(Gemel and Randall, 1992). During germination and early seedling
development of cucumber, the steady-state level of the E1 mRNA was
maximal 2 d postimbibition. The E1 protein was most active and
most abundant at d 4 and 5 postimbibition. The timing of maximal PDC
activity corresponded to photosynthetic development. The authors
postulated that the delay in PDC activity during seedling development
could prevent carbon flow through the full TCA cycle, until the
seedling is photosynthetically competent and not dependent on stored
cotyledonary reserves.
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FOOTNOTES |
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Received November 10, 1997;
accepted March 4, 1998.
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ABBREVIATIONS |
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Abbreviations: IDH, isocitrate dehydrogenase. TCA, tricarboxylic acid.
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LITERATURE CITED |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Behal RH, Oliver DJ (1997) Biochemical and molecular characterization of fumarase from plants. Cloning, sequencing, and expression of the Arabidopsis thaliana fumarase gene; purification and characterization of Pisum sativum fumarase enzyme. Arch Biochem Biophys 347: 65-74
Behal RH, Oliver DJ (1998) NAD+-dependent isocitrate dehydrogenase from Arabidopsis thaliana. Characterization of two closely related subunits. Plant Mol Biol 36: 691-698
Canvin DT,
Beevers H
(1961)
Sucrose synthesis from acetate in the germinating castor bean: kinetics and pathway.
J Biol Chem
236:
988-995
Carpenter WD, Beevers H (1958) Distribution and properties of isocitritase in plants. Plant Physiol 34: 403-409
Cohn JA, Tsai L, Friguet B, Szweda LI (1996) Chemical characterization of a protein-4-hydroxy-2-nonenal cross-link: immunochemical detection in mitochondria exposed to oxidative stress. Arch Biochem Biophys 328: 158-194 [CrossRef][ISI][Medline]
De Vries S, Hoge H, Bisseling T (1988) Isolation of total and polysomal RNA from plant tissues. In SB Gelvin, RA Schilperoort, DPS Verma, eds, Plant Molecular Biology Manual, Vol B6. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 1-13
Gemel J,
Randall DD
(1992)
Light regulation of leaf mitochondrial pyruvate dehydrogenase complex. Role of photorespiratory carbon metabolism.
Plant Physiol
100:
908-814
Grof CPL,
Winning BM,
Scaysbrook TP,
Hill SA,
Leaver CJ
(1995)
Mitochondrial pyruvate dehydrogenase: molecular cloning of the E1 subunit and expression analysis.
Plant Physiol
108:
1623-1629
[Abstract]
Hanning I, Heldt HW (1993) On the function of mitochondrial metabolism during photosynthesis in spinach (Spinacia oleracea L.) leaves. Partitioning between respiration and export of redox equivalents and precursors for nitrate assimilation products. Plant Physiol 103: 1147-1154 [Abstract]
Hill SA,
Bryce JA,
Leaver CJ
(1994)
Pyruvate metabolism in mitochondria from cucumber cotyledons during early seedling development.
J Exp Bot
45:
1489-1491
Hill SA,
Groff CPL,
Bryce JH,
Leaver CJ
(1992)
Regulation of mitochondrial function and biogenesis in cucumber (Cucumis sativus L.) cotyledons during early seedling growth.
Plant Physiol
99:
60-66
Hurley JH,
Dean AM,
Sohl JL,
Koshland DE Jr,
Stroud RM
(1990)
Regulation of an enzyme by phosphorylation at the active site.
Science
249:
1012-1016
Kornberg HL, Krebs HA (1957) Synthesis of cell constituents from C2-units by a modified tricarboxylic acid cycle. Nature 179: 988-991 [CrossRef][Medline]
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacterophage T4. Nature 227: 680-685 [CrossRef][Medline]
LaPorte DC,
Thorsness PE,
Koshland DE Jr
(1985)
Compensatory phosphorylation of isocitrate dehydrogenase: a mechanism for adaptation to the intracellular environment.
J Biol Chem
260:
10563-10568
McIntosh CA,
Oliver DJ
(1992)
NAD+-linked isocitrate dehydrogenase. Isolation, purification and characterization of the protein from pea mitochondria.
Plant Physiol
100:
69-75
Millhouse J, Wiskich JT, Beevers H (1983) Metabolite oxidation and transport in mitochondria of endosperm from germinating castor bean. Aust J Plant Physiol 10: 167-177
Oliver DJ, McIntosh CA (1995) The Biochemistry of the Mitochondrial Matrix. In CS Levings III, IK Vasil, eds, The Molecular Biology of Plant Mitochondria. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 237-280
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Schultz DJ, Craig R, Cox-Foster DL, Mumma RO, Medford JI (1994) RNA isolation from recalcitrant tissue. Plant Mol Biol Rep 12: 310-316
Timberlake WE (1986) Isolation of stage- and cell-specific genes from fungi. In J. Bailey, ed, Biology and Molecular Biology of Plant-Pathogen Interactions. North Atlantic Treaty Organization-Advanced Study Institute Series, Vol H1. Springer-Verlag, Berlin, pp 343-357
Towbin H,
Staehelin T,
Gordon J
(1979)
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Proc Natl Acad Sci USA
76:
4350-4354
Trelease RN, Doman DC (1984) Mobilization of oil and wax reserves. In DR Murray, eds, Seed Physiology, Vol 2: Germination and Reserve Mobilization. Academic Press, Orlando, FL, pp 201-245
Voet D, Voet JG (1990) Biochemistry. John Wiley, New York
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 A. Elorza, H. Roschzttardtz, I. Gomez, A. Mouras, L. Holuigue, A. Araya, and X. Jordana A Nuclear Gene for the Iron-Sulfur Subunit of Mitochondrial Complex II is Specifically Expressed During Arabidopsis Seed Development and Germination Plant Cell Physiol., January 1, 2006; 47(1): 14 - 21. [Abstract] [Full Text] [PDF]
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G. R. Gray, A. R. Villarimo, C. L. Whitehead, and L. McIntosh Transgenic Tobacco (Nicotiana tabacum L.) Plants with Increased Expression Levels of Mitochondrial NADP+-dependent Isocitrate Dehydrogenase: Evidence Implicating this Enzyme in the Redox Activation of the Alternative Oxidase Plant Cell Physiol., October 15, 2004; 45(10): 1413 - 1425. [Abstract] [Full Text] [PDF]
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D. C. Logan, A. H. Millar, L. J. Sweetlove, S. A. Hill, and C. J. Leaver Mitochondrial Biogenesis during Germination in Maize Embryos Plant Physiology, February 1, 2001; 125(2): 662 - 672. [Abstract] [Full Text]
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,
Lipid content in light-grown seedlings; 
, lipid content in
dark-grown seedlings. In vivo labeling data (the ratio of
14CO2 released from
[1-14C]acetate to 14C incorporated into the
neutral fraction containing soluble sugars) on the right
y axis represent the means ± SD for
three replicates per data point. Analysis of in vivo labeling results
by a Student's t test showed that data for d 0.5 and 3 were significantly different between dark and light at P < 0.05. Days 1, 2, 4, and 5 were not significantly different between light and
dark at P < 0.05. 
, Labeling ratio for light-grown seedlings;
, labeling ratio for dark-grown seedlings.
