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Plant Physiology 132:2205-2217 (2003) © 2003 American Society of Plant Biologists Modulation of Citrate Metabolism Alters Aluminum Tolerance in Yeast and Transgenic Canola Overexpressing a Mitochondrial Citrate Synthase1Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9 (V.M.A., U.B., G.J.T.); and Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, Texas, 78229–3900 (M.T.M., L.M.-H.)
Aluminum (Al) toxicity is a major constraint for crop production in acid soils, although crop cultivars vary in their tolerance to Al. We have investigated the potential role of citrate in mediating Al tolerance in Al-sensitive yeast (Saccharomyces cerevisiae; MMYO11) and canola (Brassica napus cv Westar). Yeast disruption mutants defective in genes encoding tricarboxylic acid cycle enzymes, both upstream (citrate synthase [CS]) and downstream (aconitase [ACO] and isocitrate dehydrogenase [IDH]) of citrate, showed altered levels of Al tolerance. A triple mutant of CS (  cit123) showed lower levels
of citrate accumulation and reduced Al tolerance, whereas
aco1- and idh12-deficient mutants showed
higher accumulation of citrate and increased levels of Al tolerance.
Overexpression of a mitochondrial CS (CIT1) in MMYO11 resulted in a
2- to 3-fold increase in citrate levels, and the transformants showed enhanced
Al tolerance. A gene for Arabidopsis mitochondrial CS was overexpressed in
canola using an Agrobacterium tumefaciens-mediated system. Increased
levels of CS gene expression and enhanced CS activity were observed in
transgenic lines compared with the wild type. Root growth experiments revealed
that transgenic lines have enhanced levels of Al tolerance. The transgenic
lines showed enhanced levels of cellular shoot citrate and a 2-fold increase
in citrate exudation when exposed to 150 µM Al. Our work with
yeast and transgenic canola clearly suggest that modulation of different
enzymes involved in citrate synthesis and turnover (malate dehydrogenase,
CS, ACO, and IDH) could be considered as potential targets
of gene manipulation to understand the role of citrate metabolism in mediating
Al tolerance.
Aluminum toxicity is one of the major factors limiting crop productivity in acid soils. The root apex is considered the primary site of Al-induced injury, and inhibition of root elongation is one of the most visible symptoms of Al toxicity. Although most plants are sensitive to Al, several crop species exhibit genetic variation in their ability to tolerate Al. One possible mechanism of Al tolerance is the chelation of Al by organic anions within root cells or in the rhizosphere (Taylor, 1991  ). Exudation of a variety of low Mr
organic anions such as citrate, malate, or oxalate has been reported in
several crop species upon exposure to Al
(Miyasaka et al., 1991;
Basu et al., 1994;
Pellet et al., 1995;
Larsen et al., 1998). Although
a rapid release of organic anions (malate2-) was observed in near
isogenic, Al-tolerant wheat (Triticum aestivum) lines
(Delhaize et al., 1993),
several lines of evidence suggest that a lag phase may also exist between
exposure of roots to Al and excretion of organic anions. Citrate exudation
from roots of rye was observed only after 120 h of exposure
(Li et al., 2000). Similarly,
an Al-induced, de novo synthesis of malate leading to enhanced malate efflux
24 h after exposure was demonstrated in Al-tolerant wheat cv Katepwa
(Basu et al., 1994). Delayed
exudation of organic anions could possibly be due to alteration of cellular
metabolism leading to biosynthesis and release of anions.
Among the various organic acids, citrate is most commonly cited to be
involved in ameliorating the toxic effects of Al. Accumulation and/or efflux
of citrate can be enhanced by increased citrate production or by reduced
citrate catabolism (Neumann et al.,
2000
CS is a key enzyme involved in condensation of oxaloacetate (OAA) and
acetyl CoA to produce citrate. This biochemical reaction plays an important
role in the Krebs cycle, in
This study was conducted to investigate the role of citrate metabolism in
mediating Al tolerance in both yeast and plant model systems. Yeast
(Saccharomyces cerevisiae) is an excellent system for studies on
metal toxicity and resistance (MacDiarmid
and Gardner, 1998
In addition, we also tested the hypothesis that increased synthesis and
accumulation of citrate can mediate Al tolerance in plants using a transgenic
approach. We used an important oilseed crop, canola (Brassica napus
cv Westar), because the cultivars of this species are sensitive to acid soils
and Al toxicity (Clune and Copeland,
1999
Effect of Al on Growth of Wild-Type (WT) Yeast and Induction of Genes Involved in Citrate Metabolism A progressive reduction in growth was observed in WT yeast when exposed to increasing concentrations of Al, with a 50% inhibition at 400 µM Al (Fig. 2A). Northern analysis of WT yeast exposed to Al (0–400 µM) showed that the transcript level of CIT1 increased by 40% to 50% from 100 to 300 µM Al, and no significant increase in transcript abundance was observed at 400 µM Al. Transcript abundance for ACO1 increased by 430% to 477% of control at 100 to 400 µM Al, whereas the IDH1 transcript level was down-regulated to 60% of control by increasing concentrations of Al (Fig. 2, B and C).
Cellular (Fig. 3A) and
extracellular (Fig. 3B) citrate
contents were estimated in different yeast mutants with altered citrate
metabolism. These include strains with all possible combinations of single,
double, and triple mutations in three genes, CIT1, CIT2, and
CIT3, encoding distinct CS isozymes. Two of the isozymes (CS1 and
CS3) are mitochondrial, whereas the other isozyme (CS2) functions in the
peroxisome. In addition, strains lacking the next two Krebs cycle isozymes
were also tested. The
No significant reduction in citrate efflux from cells of single mutants of
CS was observed, whereas citrate exuded from double and triple mutants of CS
was reduced 2-fold compared with the WT yeast (7.7 ± 0.9 nmol
109 cells-1 mL-1). Citrate released from
cells of
When yeast cells were challenged with Al, the single mutants
(
The gene encoding the major mitochondrial isoform of CS, CIT1, was overexpressed alone and with mitochondrial MDH gene, MDH1, in WT yeast. MDH is the enzyme involved in synthesizing OAA from malate. We therefore postulated that overexpression of MDH might supply additional OAA for citrate synthesis. Northern analysis of yeast transformants overexpressing CIT1 (MMYO11/CIT1) and CIT1 + MDH1 (MMYO11/CIT1 + MDH1) showed similar levels of increase in CIT1 transcript (Fig. 5A). When challenged with 500 µM Al, the transformants MMYO11/CIT1 and MMYO11/CIT1 + MDH1 showed significantly better growth (20% and 30%, respectively) compared with the WT and vector controls (Fig. 5B). Also, a significant increase (2.5- to 3-fold) in both cellular and extracellular citrate levels was observed in MMYO11/CIT1 and MMYO11/CIT1 + MDH1 transformants relative to the WT (Fig. 6, A and B). Interestingly, citrate levels were not higher with simultaneous overexpression of CIT1/MDH1 as in MMYO11/CIT1 + MDH1 compared with MMYO11/CIT1.
Transformation of canola cv Westar with pACS121-Hm (Fig. 7A) using an Agrobacterium tumefaciens-mediated system yielded 12 independent transgenic lines. The presence of the transgene, At-mtCS, was confirmed in four of these lines using genomic PCR analysis (primer positions used in genomic PCR are indicated in Fig. 7A). Amplification of an expected approximately 0.7-kb fragment was observed in transgenic lines and positive control when amplified with cauliflower mosaic virus (CaMV)-F and AtCS-R primers, but not in the WT and negative controls. When amplified with CaMV-F and HPTII-R primers, two bands of expected sizes (approximately 0.3 and 2.8 kb) were observed in transgenic lines and the positive control. Transgenic lines CS1 and CS12 were fertile and were raised to obtain homozygous seeds for further studies (Fig. 7B).
Transgenic lines were analyzed for enhanced levels of the CS transcript (Fig. 8A) and presence of the HPT II transcript by northern analysis. The transgenic lines showed up to a 2-fold increase in accumulation of the mt-CS transcript (approximately 1.6 kb) compared with the WT. The At-mtCS probe hybridizes to the mt-CS of both Arabidopsis and canola. Due to the short exposure time used (2 h), the endogenous CS transcript in the WT was only visible as a faint band, whereas the band intensity was stronger in CS1 and CS1 for the same exposure time. When the same membrane was stripped and reprobed with HPT II, the presence of HPT II transcript (1.8 kb) was observed only in transgenic lines (data not shown). The enhanced level of CS expression was also confirmed in the roots and shoots of T2 plants and several of T2 progenies, with At-mtCS probe in T1 and homozygous T2 generation (data not shown).
CS enzyme activity was measured in the WT and transgenic lines (Fig. 8B). Transgenic lines CS1 and CS12 showed a significant (2.5- to 4-fold) increase in CS enzyme activity (1.8 ± 0.005 and 3.2 ± 0.1 µmol CoA used min-1 mg-1 protein respectively) compared with the WT (0.8 ± 0.003 µmol CoA used min-1 mg-1 protein).
When exposed to toxic concentrations of Al, transgenic lines CS1 and CS12 showed better performance in the presence of Al (Fig. 9A). At 50 µM, a stimulatory effect of Al on root elongation was observed in CS1 and CS12 lines, whereas a growth reduction of 10% was observed in WT. At higher concentrations of Al, root elongation was greater in transgenic lines than in the WT. At 100 µM Al, no significant change in root elongation was observed in the transgenic lines compared with their controls (0 µM Al), whereas the WT showed a reduction of 9% in relative root growth. Larger differences in root elongation were observed at 150 and 200 µM Al. At 150 µM Al, root growth was reduced by 50% in WT, whereas CS1 and CS12 lines showed a reduction of 17% and 20%, respectively. At 200 µM Al, the transgenic lines showed a root growth inhibition of 65% to 70% their controls (0 µM Al), whereas the root growth of WT was reduced by 85% compared with its control.
Because the transgenic lines had the Atmt-CS expressed under control of the CaMV promoter, we expected higher levels of cellular citrate content and citrate exudation from roots of CS1 and CS12 under control conditions. But the amount of citrate (shoots and root exudates) in these lines was not significantly different from that of the WT at 0 µM Al (Fig. 9, B–D). The root citrate level was not different between WT and transgenic lines at 150 µM Al, although a significant difference was observed between WT and CS12 at 0 µM Al (Fig. 9B). However, when exposed to 150 µM Al, the citrate content of shoots was significantly increased (1.7- to 1.9-fold) in CS1 and CS12 compared with the WT (3.5 ± 0.7 µmol mg-1 protein; Fig. 9C). The amount of citrate exuded from the roots of CS1 (1.7 ± 0.2 µmol g -1 dry weight) and CS12 (1.2 ± 0.2 µmol g -1 dry weight) was 1.7- to 2.4-fold higher than WT (0.7 ± 0.2 µmol g -1 dry weight; Fig. 9D). These results indicate that overexpression of mitochondrial CS in canola results in an Al-induced increase in exudation of citrate levels and Al tolerance in the transgenic lines.
Several lines of evidence suggest that Al has a strong effect on the TCA cycle and glycolytic pathway. Modulation in activities of several enzymes involved in synthesis and catabolism of citrate has been reported in yeast and plants (Hoffland et al., 1992 ;
Neumann et al., 2000;
Zatta et al., 2000;
Neumann and Martinoia, 2002).
In our studies, exposure of yeast, MMYO11 to Al resulted in induction of
CIT1 and ACO1 and down-regulation of IDH1
transcript levels (Fig. 2, B and
C). Metabolic engineering of biochemical pathways involved in
organic acid metabolism would provide us an opportunity to understand the role
of these pathways in Al tolerance.
Genetic manipulation is relatively easy in yeast due to the availability of
mutants, ease of handling, and short experimental periods. Our data from yeast
mutants with altered citrate metabolism (defective in TCA and glyoxylate
cycles genes) revealed that accumulation of citrate in cellular and
extracellular pools can mediate Al tolerance. When measuring metabolite levels
in yeast, it is important to measure both cellular and extracellular levels,
because these metabolites are likely to be transported out of their site of
synthesis and exuded out of cells. Citrate is efficiently transported out of
mitochondria by tricarboxylate carrier proteins
(Barbier-Brygoo et al., 2000
In the present study, cellular and extracellular citrate levels were not
significantly different in single mutants (
Increased accumulation of citrate can also be mediated by reduced activity
of enzymes involved in citrate turnover
(Massonneau et al., 2001 Results from our yeast overexpression studies demonstrated that cellular and extracellular citrate levels can be enhanced in yeast by overexpressing a mitochondrial CIT1 either alone or with a mitochondrial MDH1. The transformants MMYO11/CIT1 and MMYO11/CIT1 + MDH1 showed a 2- to 3-fold increase in citrate content and reduced Al sensitivity compared with WT yeast. MMYO11/CIT1 + MDH1 was expected to synthesize and exude more citrate than MMYO11/CIT1 (we postulated reduced substrate limitations). However, citrate contents in both transformants were not significantly different. The difference in sensitivity of MMYO11/CIT1 + MDH1 compared with MMYO11/CIT1 again suggests that there could be other metabolites (perhaps malate) that attributed for the enhanced Al tolerance.
Enhanced levels of organic acid synthesis or efflux is often accompanied by
reduced growth under control conditions or requires specific culture
conditions for enhanced synthesis and exudation to occur
(Ryan and Delhaize, 2001
Changes in TCA cycle function have wide-ranging effects. Transcriptional
profiling using microarray analysis of responses to TCA cycle dysfunction
(McCammon et al., 2003
We also overexpressed a mitochondrial CS from Arabidopsis (At-mtCS) in
canola cv Westar. Between the two isoforms of CS in eukaryotes (glyoxysomal
and mitochondrial), modulating the mitochondrial isoform of CS might be a
better strategy to test whether enhanced synthesis and exudation of citrate
could be achieved by overexpressing CS. The mitochondria is the major site of
function for CS in eukaryotes. Allosteric inhibition of CS by ATP would be low
in mitochondria due to the low ATP to ADP ratio typically maintained in the
organelle. Moreover, compartmentation of TCA cycle enzymes within the
mitochondria not only ensures the availability of substrates but also physical
proximity of substrates required for proper functioning of CS. A number of
multi-enzyme complexes involving sequential enzymes termed
"metabolons" exist in the TCA cycle, and their interactions are
highly specific (Sumegi and Srere,
1984
In this study, transgenic canola overexpressing At-mtCS showed increased CS
expression at the transcript level and enhanced CS activity, leading to an
increase in levels of cellular (shoots) citrate and citrate exudation from
roots of transgenic lines relative to WT. Interestingly, the increase in shoot
citrate content and exudation of citrate were observed only upon exposure to
150 µM Al, even though At-mtCS was expressed under the control
of a constitutive promoter (CaMV). It is not clear why the root citrate level
in transgenic lines, CS1 and CS12 was not increased in the presence of Al. In
Al-tolerant cultivars of soybean (Glycine max), significant
difference was observed in the citrate levels of root tips and not in roots,
when exposed to Al (Silva et al.,
2001
It is important to note that our transgenic lines did not have any
difference in growth and development compared with WT. A reduction in
mitochondrial CS activity (up to 30% of the WT levels) in transgenic potato
resulted in a normal phenotype, but this reduction in CS activity was a
limiting factor during flowering because the transgenic potato plants had
deformed ovules (Landschütze et al.,
1995
The transgenic canola lines overexpressing mtCS showed reduced sensitivity
to Al. At 50 and 100 µM Al, root elongation rates were higher in
the transgenic lines compared with the control (0 µM Al). This
could be due to alleviation of H+ toxicity by Al as suggested by
Clune and Copeland (1999
Overexpression of a gene for CS from P. aeroginosa in cytoplasm of
tobacco and Papaya sp. resulted in enhanced levels of cellular
citrate and citrate exudation from roots of transgenic lines compared with the
WT, with a concomitant increase in Al tolerance
(de la Fuente et al., 1997
Several other studies have also suggested that an overexpression strategy
can be used to enhance the synthesis and exudation of organic anions. For
instance, transgenic alfalfa (Medicago sativa) overexpressing a
nodule-specific MDH showed enhanced exudation of organic anions from
their roots and enhanced Al tolerance
(Tesfaye et al., 2001 Despite a number of published studies on the topic, a holistic approach to understand citrate metabolism in relation to Al tolerance has not yet been undertaken. Our work with yeast and transgenic canola overexpressing At-mtCS has enabled us to explore the complexity of citrate metabolism. Our results clearly demonstrate that CS represents only a part of the complex system and that genetic manipulation of several enzymes involved in citrate metabolism (such as MDH, CS, ACO, and IDH) can be potentially used to increase citrate synthesis and its accumulation in cells (Fig. 1). Citrate levels could be modulated by up-regulation of MDH and CS by an overexpression strategy or by down-regulation of ACO and IDH using an antisense approach (perhaps, with an inducible promoter). These approaches might be adopted to generate a significant improvement in Al tolerance in plants. Because cellular metabolism involves an array of enzymes that are interrelated and function in coordination with each other, metabolic profiling of these transformants could help us to further understand the coordinated synthesis of other organic anions.
Yeast Strains and Media
Strains of yeast (Saccharomyces cerevisiae) used in this study are
listed in Table I. Most strains
harboring disruptions in different TCA cycle and glyoxylate cycle genes were
constructed previously (Przybyla-Zawislak
et al., 1999
To construct yeast overexpressing CIT1 and MDH1, strain
MMYO11 was transformed with Yep352-CIT1
(Kispal et al., 1989
While testing the growth response of different yeast strains to Al, a low
phosphate medium (LPP) was used (Schott
and Gardner, 1997
Total RNA was isolated from 5-mL yeast cultures grown with varying concentrations of Al (0–300 µM) for 20 h by an enzymatic method using the RNAeasy mini kit (Qiagen, Ontario, Canada). Northern-blot analysis was done using [32P]CTP-labeled CIT1, ACO1, and IDH1 probes.
Yeast cells grown in 3 mL of LPP medium for 20 h were pelleted and washed
three times with 3 mL of sterile water. The medium and the supernatant from
each wash were pooled as the extracellular fraction. The cells were
resuspended in 1 mL of sterile water, and cell density was determined by
estimating OD600. Cells were again pelleted, and the supernatant
was pooled into the extracellular fraction. Cells were finally suspended in 1
mL of 80% (v/v) ethanol in 15-mL screw-capped tubes and vortexed vigorously to
lyse the cells. The cell suspension was boiled at 80°C for 20 min with
frequent vortexing to ensure complete lysis of cells and denaturation of
proteins. The cell debris with denatured proteins was pelleted, the
supernatant was passed through a 0.45-µm filter, and the filtrate was used
in the estimation of citrate content in cells. To estimate citrate content in
the extracellular medium, the pooled supernatant was concentrated (from 13 to
1 mL) using an Evap-o-Vac (Cole-Parmer, Ontario, Canada) placed inside an
incubator at 50°C. Enzymatic analysis of citrate was conducted with 100
µL of either samples or standards. The reaction mix consisted of 100
mM Tris-Cl (pH 8.2), 0.2 mM ZnCl2, 3 units
mL-1 MDH, and 10 mM NADH. A stable initial
A340 was noted, the reaction was started by the addition
of citrate lyase (Sigma-Aldrich, St. Louis), and stable final
A340 was recorded
(Tompkins and Toffaletti,
1982
Agrobacterium tumefaciens strain EHA101 carrying a binary vector
pACS121-Hm was kindly provided by Dr. Hiroyuki Koyama (Gifu University, Japan)
and was used in the transformation of canola (Brassica napus cv
Westar). This binary vector had a full-length cDNA for At-mtCS (1.6 kb) under
the control of the CaMV promoter (Fig.
6A) and two selectable marker genes encoding kanamycin resistance
(NPT II) and hygromycin resistance (HPT II;
Koyama et al., 1999 Presumptive positive plants (T1) were transferred to soil (Metromix, Apache Seeds, Edmonton, Canada) and raised under controlled environment conditions (16 h of light and 300 µmol m-2 s-1 at 20°C/8 h of dark at 18°C). The presence of the transgene was tested using PCR on genomic DNA isolated from 12 independent lines. Among the four positive lines, two of these T1 plants were fertile. Thirty seeds from each of the T1 selfed-lines were surface-sterilized and germinated on seed germination medium with 150 µg mL-1 kanamycin. Seedlings (T2 plants), which remained green on antibiotic selection, were again tested by genomic PCR. Segregation ratio was determined based on number of green to bleached yellow seedlings. From each of the primary transformants (T1), 15 green T2 seedlings were transferred to soil and screened to identify homozygous lines. To select for homozygous lines in the T2 generation, about 20 to 30 seedlings from T2 progenies were screened by genomic PCR for the presence of transgene.
For genomic PCR and Southern analyses, genomic DNA was isolated from shoots or roots of canola using Qiagen DNeasy mini kit. For genomic PCR analysis, genomic DNA was used as template DNA, and the sequences of primers used for amplification were as follows: 35S-F, 5'-CCACTGACGTAAGGGATGACG-3'; AtCS-R, 5'-AAGCCTCCAG ACTGGGCAGTA-3'; and HPT II-R 5'-GCCATCGGTCCAGACGGCC-3'. RNA from shoots and roots were isolated using the Qiagen RNAeasy mini kit, separated by electrophoresis on agarose formaldehyde denaturing gels, and transferred to nitrocellulose membranes (Genescreen, NEN Research Products, Perkin Elmer Life Sciences Inc., Boston). Membranes were hybridized at 42°C overnight and washed under standard stringent conditions recommended by Genescreen. Hybridization probes were radioactively labeled with [32P]CTP using Oligolabelling kit (Amersham Biosciences, Uppsala).
Roots and shoots were collected, frozen in liquid nitrogen, and stored at
-70°C until used for enzyme assays. Approximately 1 g of tissue was ground
in liquid nitrogen and homogenized with 2 mL of ice-cold extraction buffer (50
mM HEPES, 0.5% [w/v] Triton-X, 1 mM EDTA, 1
mM iodoacetamine, and 10% [w/v] glycerol). The extract was
centrifuged for 10 min at 4°C, and the supernatant was collected and
desalted by passing through PD-10 columns (Bio-Rad Laboratories, Hercules, CA)
equilibrated with 10 mM HEPES and eluted with 2 mL of 10
mM HEPES. The reaction mix for CS enzyme assay consisted of 1
mM 5,5'-dithio-bis(2-nitrobenzoic acid) in Tris-Cl (pH 8.1),
10 mM acetyl CoA, and the enzyme. The reaction was started by the
addition of 10 mM OAA, and increase in absorbance due to
deacetylation of acetyl CoA was measured at 412 nm
(Srere et al., 1963
WT and transgenic lines (T2 homozygous) were tested for their
sensitivity to Al by the root growth elongation assay described by Basu et al.
(2001
Shoot and root tissues (approximately 0.2 g) of WT and transgenic lines
were ground with liquid nitrogen, homogenized with 1 mL of 80% (v/v) ethanol,
and vortexed thoroughly. The samples were centrifuged for 2 min at 13,000 rpm,
and 100 µL of the supernatant was taken for protein estimation. The
remaining portion of the samples was then vortexed and boiled at 80°C for
15 min. Samples were centrifuged at 13,000 rpm for 5 min, the supernatant was
collected and passed through 0.45-µM filters, and 100 µL was
used in citrate assay as described above
(Delhaize et al., 1993
Seeds of WT and transgenic lines were surface-sterilized and plated on seed
germination medium. Two-day-old seedlings (13–15 seedlings per vessel,
plated on a 14' mesh) were transferred to 75 mL of sterile FNS in
magenta vessels (Basu et al.,
1994
All experiments included three to 10 independent replicates, and statistical analyses were performed using Sigmastat statistical analysis package (v1.0, Jandel Scientific, Chicago). Single- or two-factor ANOVA was performed, and a P value below 0.05 was considered statistically significant based on Student Newman-Keul's test or Dunnett's test. Experiments were repeated three to six times to ensure reproducibility of results.
Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes.
We thank Dr. Hiroyuki Koyama (Gifu University, Japan) for providing with the construct (At-mtCS) for plant transformation. Received March 19, 2003; returned for revision April 21, 2003; accepted May 12, 2003.
1 This work was supported by the Natural Sciences and Engineering Research Council of Canada, by the Department of Biological Sciences, University of Alberta (Edmonton, Alberta, Canada), and by the National Institutes of Health (grant no. AG17477 to L.M.-H.).  * Corresponding author; e-mail mv{at}ualberta.ca; fax 780–492–9234.
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TOP
ABSTRACT
RESULTS
) and down-regulation of genes
involved in citrate catabolism (
) would enhance citrate synthesis and/or
its accumulation in cells.
-oxidation of fatty acids, and also in
photo-respiratory glycolate pathways. As an important intermediate in various
biochemical reactions, such as amino acid synthesis and fatty acid synthesis,
citrate biosynthesis and catabolism are strictly regulated
(Ryan and Delhaize, 2001
