Plant Cell Physiol.
40(5): 489-495 (1999)
JSPP © 1999
Organic Acid Metabolism in Aluminum-Phosphate Utilizing Cells of Carrot
(Daucus carota L.)
Eiji Takita, Hiroyuki Koyama' and Tetsuo Hara
Laboratory of Plant Cell Technology, Faculty of Agriculture, Gifu University, 1-1 Yanagido, Gifu, 501-1193 Japan
Carbohydrate metabolism in Al-phosphate utilizing
cells of carrot [designated as IPG, Koyama et al. (1992)
Plant Cell Physiol. 33: 171], which grow normally in Al-
phosphate medium accompanied by citrate excretion, was
investigated. The excretion of citrate was strongly related
to the availability of sucrose in medium, indicating that
citrate excretion was severely limited by sucrose in medium.
The ratio of the amount of carbon in the excreted citrate to
the consumed sucrose, was significantly higher in IPG cells
than in wild-type cells. When 50% of the sucrose in the
medium was consumed, the ratio was 0.6% for the IPG
cells and 0.2% the wild-type cells. Under these conditions,
IPG cells showed altered citrate synthesis metabolism,
which resulted in increased citrate production. Specific ac-
tivity of mitochondrial citrate synthase was higher in IPG
cells than in wild-type cells, whereas the activity of cyto-
solic NADP-specific isocitrate dehydrogenase was lower in
IPG cells than in wild-type cells.
Key words: Al-phosphate — Citrate synthesis — Citrate
synthase (EC 4.1.3.7) — Daucus carota — NADP spe-
cific isocitrate dehydrogenase (EC 1.1.1.42).
Aluminum toxicity and its related nutrient deficien-
cies are considered to be major environmental stresses that
limit plant growth on acid soils and as a result affect world
food production. One serious problem associated with Al
toxicity is the reduction in availability of phosphate from
soils due to the precipitation of aluminum-phosphate (Foy
1983). Since phosphate resources are nonrenewable, an
improvement of a plants capacity to utilize aluminum-
phosphate may increase crop productivity in such soils.
There are some strategies plants have adopted to in-
crease phosphate uptake from low P soils (Schachtman et
al. 1998). Since organic acids, such as citrate and malate,
can solubilize Pj from Al- and Fe-phosphate by chelating
the metals, the organic acids exudation from roots is
thought to be an effective P r stress rescue system used in
Abbreviations: Aco, aconitase; CS, citrate synthase; Cyt c
oxydase, cytochrome c oxydase; DTNB, 5'5-dithiobis (2-nitro-
benzoic acid); ICDH, isocitrate dehydrogenase; IPG, insoluble
phosphate grower; PEPC, phosphoenolpyruvate carboxylase;
TBS, tris buffered saline.
1
Corresponding author, e-mail, koyama@cc.gifu-u.ac.jp
489
higher plants (Johnson et al. 1994). In certain plant species,
organic acids are released from roots in response to P
starvation. Alfalfa has been found to release citrate (Lip-
ton et al. 1987) and rape (Hoffland et al. 1992) has been
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found to exude both citrate and malate from roots when
grown under Pj-stressed conditions. Lupin, which has a
high ability to utilize Ps from barely soluble phosphate
source, has been found to exude high amount of citrate
from the proteoid roots (Gardner et al. 1981, Johnson et
al. 1994). These plant species were also found to have
modified organic acid. Rape showed increased activities of
either CS or PEPC in roots during the excretion of organic
acids (Hoffland et al. 1990). Johnson et al. (1996) found on
increase in PEPC activity in proteoid roots of lupin, and
suggested that excretion of citrate was the result of en-
hanced nonphotosynthetic CO2 fixation. Transgenic plants
with modified organic acid metabolism have recently been
developed by introducing bacterial CS into tobacco and
papaya (de la Fuente et al. 1997). Since these transgenic
plants which carry an enhanced activity of CS showed an
enhanced ability to excrete citrate, modifications of organic
acid metabolism are thought to play a role in citrate ex-
cretion from plant roots. However, little is known about
the genetic basis of the modified organic acid metabolism
in plant. Although a number of mutants from Arabidopsis
thaliana showing a high ability to excrete citrate have been
isolated, a relationship between organic acid release and
their metabolism has not been fully elucidated (Larsen et
al. 1998).
We have previously selected a number of Al-phos-
phate utilizing cell lines from carrot (Koyama et al. 1988),
tobacco (Ojima et al. 1989) and alfalfa (Takita et al. 1997).
Since these selected cell lines could also utilize insoluble
ferrous phosphate as a phosphorous source, they were
designated as IPG (insoluble phosphate grower). During
growth in Al-phosphate medium, these IPG cell lines ex-
creted a large amount of citrate into the medium (Koyama
et al. 1988). A previous study suggested, the citric acid
chelated the Al in Al phosphate with 1 to 1 stoichiometri-
cally to forming non-phytotoxic compounds, namely Al-
citrate. This results in the solubilization of P; allowing the
IPG cells to grow a normal rate in Al-phosphate medium
(Koyama et al. 1990). The addition of citrate at a level
which was similar to that released from IPG cells allowed
wild type cells to grow at a normal level in the presence of
Al-phosphate. This observation suggested that enhanced
490 Citrate production in Al-phosphate utilizing cells
citrate excretion is an important characteristics of the IPG
cells (Koyama et al. 1988, 1990). We consider the IPG cells
to be genetically different from other cells, since the Al-
phosphate tolerant phenotype appears to be very stable and
it can be introduced into plants after somatic hybridization
(Koyama et al. 1995). However, the mechanisms involved
in this enhanced citrate excretion have not yet been de-
fined.
Therefore, in the present study we have examined the
activities of enzymes involved in citrate production, in-
cluding PEPC, CS, Aco and NADP-ICDH. The specific
activity of mitochondrial citrate synthase involved in IPG
cells was higher than that in the wild-type cells, whereas the
activity of cytosolic NADP-specific isocitrate dehydro-
genase was lower in IPG cells than in wild-type cells.
Materials and Methods
Plant materials and culture conditions—Suspension cultures
of carrot were obtained from calli induced from hypocotyls as
described previously (Koyama et al. 1988). Stock cell suspensions
were subcultured weekly and maintained in R2 liquid medium
(Ohira et al. 1973), containing either NaH 2 PO 4 (wild-type: WT) or
aluminum phosphate (IPG) as a sole phosphorous source, on a
rotary shaker (120 rpm) at 25°C as described previously (Koyama
et al. 1992). The IPG cells used in the present study were identical
to the cells used in our previous work (Koyama et al. 1988, 1990).
For experiments to estimate the growth response, citrate excretion
and enzyme activities, the cells (400 mg each) were cultured in 30
ml of medium containing either NaH2PO4 (2 mM) or commer-
cially available anhydrous aluminum phosphate (2 mM: Kanto
Chemical, Tokyo, Japan). Etiolated seedlings were grown under
darkness for 5 d after germination at 25°C.
Preparation of extracts—Cells were collected by vacuum
filtration on a Miracloth (Calbiochem-Novabiochem, La Jolla,
CA, U.S.A.) and washed thoroughly with distilled water. Cells (1
g) were packed in a polypropylene pouch, frozen in liquid nitro-
gen and kept in a deep freezer (—80°C) until use. Frozen cells
were disrupted by a Polytron PT-3000 (Kinematica, Luzern,
Switzerland) at 20,000 rpm for 1 min in extraction buffer [50 mM
HEPES-NaOH (pH 7.6), 5 M glycerol, 0.5% (v/v) Triton X-100,
1 mM dithiothreitol], and centrifuged at 28,000 xg for 10 min.
The supernatant was desalted using a Bio-Gel P-6 (Bio-Rad)
column prior to determination of enzyme activity. Extracts from
etiolated seedlings were obtained by the same protocol using see-
dlings that were grown under darkness for 5 d after germina-
tion.
In vitro enzyme assay—Citrate synthase was assayed spec-
trophotometrically according to Srere (1967) by monitoring re-
duction of Acetyl-CoA in the presence of DTNB at A412. PEPC
was assayed by the method of Vidal and Gadal (1983). NADP-
ICDH was assayed according to the method of Chen et al. (1988),
and Aco was measured using the method of Cooper and Beevers
(1969). Protein was quantified colorimetrically using the method
of Bradford (1976).
Partial purification of mitochondria—Twenty grams of fresh
cells were gently homogenized in 80 ml of 0.3 M mannitol solution
containing 10 mM of HEPES-KOH (pH 7.5), 1 mM EDTA, 0.1%
(w/v) bovine serum albumin (fatty acid free) and 0.6% (w/v) in-
soluble polyvinylpolypyrrolidone using a glass homogenizer. The
homogenate was then centrifuged at 2,500 x g for 10 min and the
supernatant was centrifuged at 10,000 x g for 10min. The pre-
cipitate fraction was used as mitochondria enriched fraction. The
precipitate fraction was treated with 20 mM of HEPES-KOH
buffer (pH7.5) containing 0.025% (w/v) bovine serum albumin
(fatty acid free) and 0.5% (v/v) Triton X-100 to obtain mito-
chondrial lysate. To assess the purity of preparations the activities
of mitochondria fumarase (Fum), a marker enzyme for mito-
chondria, and alcohol dehydrogenase (ADH), a marker enzyme
for cytosol were measured according to the methods of Cooper
and Beevers (1969), and Hart (1970), respectively. The activities
Cyt c oxidase activity, another marker enzyme for the mitochon-
dria was assayed using the method of Gardestrom and Edwards
(1983). NAD-ICDH activity, a marker enzyme of TCA cycle, was
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assayed by the method of Cox (1967).
Quantification of citrate, glucose in medium—Cells were re-
moved from the cultured medium by vacuum nitration. Spent
media was collected and then soluble proteins were eliminated by
centrifugation with 80% ethanol at 20,000 x g for 15 min. In order
to dissociate citrate from Al-citrate compounds, samples were
acidified to a pH below 2.0 by using 1 M HC1, and then passed
through a cation exchange column filled with Dowex 5OW-X8
(H + -form, Bio-Rad). Citrate concentrations in the eluate were
measured following the enzyme cycling method according to
Hampp et al. (1984). In this method, citrate is first converted to
NADH by citrate lyase (Sigma), and then, the NADH was meas-
ured using a cycling reaction reagent (Kato et al. 1973). Glucose
was measured colorimetrically using Glucose B-test Wako quan-
tification kit (Wako Pure Chemical, Osaka, Japan). One-milliliter
aliquots of medium were incubated with 100 fil of 8.5 M HC1 at
100°C for 2.5 h to hydrolyze sucrose in the media. The amount of
glucose was then determined and used as an indication of the
amount of sucrose that was present.
Immunotitration for citrate synthase in cells and etiolated
seedlings—Extracts obtained from cells and etiolated seedlings
containing each 0.02 unit of citrate synthase were incubated with
various amounts of rabbit anti-serum raised against carrot mito-
chondrial CS in TBS buffer (pH 7.2) containing 100//g per ml of
bovine serum albumin at 37°C for 30 min. Mixtures were then
kept at 4°C overnight, and centrifuged for 30 min at 15,000xg.
CS activity in supernatant was then measured using method of
Srere (1967).
Results
Carbon economy of citrate excretion from IPG cells—
In this cell culture system, growth rate was directly related
to the ability of cells to utilize Al-phosphate (Koyama et
al. 1990), and the availability of soluble phosphate was
directly related to the ability of cells to excrete citrate
(Koyama et al. 1990). Thus in the IPG cell line, the growth
rate in Al-phosphate medium primarily depends on the
capacity of these cells to excrete citrate. It is possible that
this excreted citrate may originate from carbohydrates ini-
tially supplied in the culture medium. To investigate this we
examined the relationship between the amount of citrate
excreted and the amount of carbohydrate consumed from
the medium.
In cultured cells, a large proportion of sucrose sup-
plied to the cells is first converted to glucose and fructose
 Citrate production in AJ-phosphate utilizing cells 491

within 2 d after inoculation (Ueda et al. 1974). Since up-
take rates of glucose and fructose are similar in suspension
cultured cells (Amino and Tazawa 1988), we were able to
determine the availability of carbon in the medium by
measuring of both free glucose and sucrose-constructing
glucose. When free sugar in medium was exhausted, both
the increase of citrate excretion and the rate of cell growth
sharply decreased (Fig. 1). Under the given conditions, this
suggests that citrate excretion is accompanied by changes in
carbon metabolism. In lupin, the ratio of carbon trans-
ferred to the root exudate from metabolite significantly
increased as organic acids were being released from the
0.3-0.8% in IPG cells and 0.05-0.2% in WT cells. This
strongly suggests that the metabolism in IPG cells is mod-
ified in such a way as to allow an increase carbon allocation
to citrate production.
In vitro activity of enzymes relevant to citrate syn-
thesis—The TCA cycle may play a primary role in citrate
production. In the TCA cycle the citrate synthesis is
mediated by both CS and Aco. To investigate the role that
modified metabolism might play in the ability of IPG cells
to excrete higher amounts of citrate than WT cells, the
activities of these enzymes were measured in both cell types
(Fig. 3). The activity of CS, which produce citrate from

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proteoid root (Johnson et al. 1996). Therefore, we esti- acetyl-CoA and oxalacetate, in IPG cells was 30 to 100%
mated the amounts of carbon metabolism correlated with higher than WT cells (Fig. 3B). The specific activity of CS
citrate excretion in both IPG cells and WT cells. By meas- in IPG cells was about 0.18 ^mol (mg protein)~' min" 1
uring the amount of citrate exuded and the amount of unit, whereas that in the WT cells was lower than 0.13 unit.
glucose remaining in the medium, we calculated the ratio By contrast, the activity of Aco, which catalyzes the con-
of carbon present in the excreted citrate to the carbon version of citrate to isocitrate, was similar in both cell types
consumed from free sugars in the medium. As shown in (Fig. 3C). This increased activity of CS in IPG cells may be
Fig. 2, we found a higher ratio in IPG cells to WT cells one reason why this line produces more citrate than WT
throughout the experimental period. The ratio ranged from cells.
Citrate metabolism is also affected by the activity of
15 " A enzymes involved in TCA cycle in mitochondria, namely
CD
CO fumarase and NAD-ICDH. Under the given conditions,
CO
the activity of enzymes were similar between cell types
£
o in. (Table 1). The specific activities of fumarase and NAD-
— 10 - ICDH of both cell types are about 0.08 and 0.08 unit, re-
CO
CO
spectively. The activity of Cyt c oxidase, a mitochondrial
•"-
inner membrane marker, was also similar between cell
5 - 5 - types (Table 1). The specific activity of Cyt c oxidase of
10 both cell types was about 0.14 unit. This suggests that these
£ parts of the TCA cycle are similar in both cell types, and
u_
• •
0'
2 B - 1.5
E 601
E
O) TX • 1
c T3
aim

40 - 1.0 2
E o
X
£ CD
0.5
Cit ite

CO 20
2 2
o
CO n i 0
u 0* 5 10 15
Days
1 2 3
Fig. 1 Changes in citrate and sucrose concentration in Al-phos- Consumed sucrose
phate medium during the growth of IPG and WT cells. Both IPG (mmole flask"1 )
and WT cells were grown for 16 d in Al-phosphate medium
containing 2 mM of anhydrous Al-phosphate as a sole phospho- Fig. 2 Loss of C by citrate exudation in IPG (•) and WT (o)
rus source at an initial pH of 5.6. A: cell suspension was harvested cells grown in the medium containing Al-phosphate as a sole
aseptically at designated time and weighed (IPG, • ; WT, o). B: phosphorous source. Carbon ratio was designated as the ratio of
concentrations of citrate (closed symbols) and sucrose (open the amount of carbon involved in excreted citrate to that in con-
symbols) in the medium containing IPG (•, • ) or WT cells (A, sumed free sugar in the medium. The values were calculated from
A). Means ±SD are indicated (n=3). the results shown in Fig. IB.
492 Citrate production in Al-phosphate utilizing cells

involved in citrate production by supplying oxaloacetate to

the TCA cycle (Hoffland et al. 1990, Johnson et al. 1996),
which then used by CS in the formation of citrate. Citrate
can then be converted to 2-oxoglutarate by NADP-ICDH,
and utilized as a carbon source for amino acid synthesis in
the cytosol (Chen and Gadal 1990) (see Fig. 5): Activities of
these enzymes were also determined for both cell types.
Although a slight difference in PEPC activity was observed
between IPG and WT cells 7 d after inoculation, the
difference was not significant as a whole (Fig. 3A). In con-
trast, a large difference was observed in NADP-ICDH ac-
tivities between the two cell types (Fig. 3D). The specific

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2 4 6 8 10
Days
Fig. 3 Activity of enzymes relevant to citrate production. Cell
extracts were prepared from 1 g FW of cells grown in Al-phos-
phate medium at designated time points. Activities of PEPC (A),
CS (B), Aco (C) and NADP-ICDH (D) were measured for IPG (•)
and WT (o) cells, respectivery. Means±SD are indicated (n = 3).
mitochondrial content of each cell line is thought to be
identical.
Phoshoenolpyruvate carboxylase is another enzyme
activity of NADP-ICDH in IPG cells ranged from 0.15 unit
at day 5 to 0.09 unit at day 9. Wild type cells showed values
that were 30 to 150% higher than IPG cells throughout the
experimental period.
These results suggested that carbohydrate metabolism
in the IPG cells differed from that in WT cells both in the
direct step involved in citrate synthesis, in addition to the
steps involved in carbon flow from citrate to amino acid
synthesis. Soluble protein concentrations during the cul-
ture period were similar in both IPG and WT cells. The
protein levels measured were 12.0±1.3 and 12.2±2.9mg
(gFW)~ l , respectively. This means that difference in en-
zyme activities in IPG and WT cells were similar on a fresh
weight basis.
Intracellular localization of CS and NADP-ICDH in
IPG cells—In higher plants, iso-enzymes are localized in
different intracellular compartments and these enzymes
play different physiological roles. In higher plants, two
kinds of CS have been found. One is localized in the
mitochondria while the other is found in the glyoxysome
(Kato et al. 1995) and at least three NADP-ICDH iso-en-
zymes localized in cytosol, plastids and possibly in mito-
chondria are also known (Galvez et al. 1994, Rasmusson
and Moller 1990). To more fully understand the CS role, it
seems important to determine the intracellular location of
these enzymes in IPG cells.
In the preliminary experiments, we found that ap-
proximately 30% of the CS activity of etiolated seedlings
was inhibited by an inactivator specific for glyoxysomal
CS, called DTNB. Glyoxysomal enzymes, which are corn-

Table 1 Fumarase, NAD-ICDH and Cyt c oxidase activities in IPG and WT cells
Activity of enzymes (^mol (mg protein) ' min ')
Cell line
Fumarase Cyt c oxydase NAD-ICDH

IPG 0.085 ±0.013. 0.130±0.011 O.O8O±O.OO5

WT 0.073 ±0.005 0.121 ±0.023 0.075 ±0.009

Both cell types were grown in Al-phosphate medium for 9 d. Cell extracts were prepared from 1 g FW
of cells. Means SD are shown (n=3).
 Citrate production in Al-phosphate utilizing cells 493

blotting analysis followed by SDS-PAGE, both IPG cells

and etiolated seedlings showed only one signal around
45 kDa (data not shown), whereas there is a significant
difference between immunotitration curves for each ex-
tract. As shown in Fig. 4, the activity of CS was completely
inhibited by the presence of 3 //I of anti-serum in IPG cells,
whereas the activity of CS in etiolated seedlings was only
inhibited about 70% by the addition of 5 jul of anti-serum.
These results strongly suggest that mitochondrial CS is
the dominant isoenzyme in IPG cells. On the other hand,
most of NADP-ICDH was thought to be localized in
the cytosol of IPG cells judging from the activity in the

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0 1 2 3 4 5
Amount of antiserum (ui)
Fig. 4 Immunotitration curves of mitochondrial citrate synthase
from IPG cells. CS (at 0.02 unit activity) was incubated with
various amounts of either antiserum (•) and nonimmuno-serum
(A). Samples were incubated at 37°C for 30 min, and then kept at
4°C over night. Samples were centrifuged for 30 min at 15,000xg
and CS activity remaining in the supernatant was determined.
Curves for the extracts from etiolated seedlings with nonimmu-
no-serum (•) and immunoserum (o) are also shown.
monly found in etiolated seedlings (Cooper and Beevers
1969, Schnarrenberger et al. 1971), are known to play a
role in fatty acid metabolism during germination (Brei-
denbach and Beevers 1967). However, a recent study of
glyoxysome development in carrot suspension cells indi-
cates that the glyoxysomal CS may affect total activity of
the CS found in IPG cell (Lee et al. 1998). According to
Kato et al. (1995) and la Congnata et al. (1996), there is
only a weak similarity (below 20%) between mitochondrial
and glyoxysomal CS isoenzymes in higher plants, and thus
these enzymes can be distinguished using immunochemi-
chal approaches. Using crude soluble proteins obtained
from either IPG cells or etiolated seedlings, we perform-
ed an immunochemichal analysis using anti-serum raised
against carrot mitochondrial CS. Although we found sig-
nificant differences between immunotitration curves for
each of the extracts (Fig. 4), when we performed western
mitochondria-enriched fraction. As shown in Table 2, the
mitochondrial matrix marker enzyme, fumarase was 2.9
times more concentrated in the mitochondria-enriched
fraction, whereas the cytosolic marker (alcohol dehydro-
genase; ADH) was reduced to less than 20% compared to
the crude extract. NADP-ICDH activity was also lower in
mitochondria-enriched fraction (0.3 times of cytosol) sug-
gesting that most of the activity of this enzyme was local-
ized in the cytosol. In contrast, the CS activity was 2.4
times higher in mitochondrial fraction than in the crude
extract (Table 2), supporting the results of the immuno-
chemical approach mentioned above.
Discussion
In some plant species, citrate is excreted by plant roots
following an induction period suggesting that some meta-
bolic changes in carbon metabolism may precede the citrate
excretion (Lipton et al. 1987, Ma et al. 1997, Miyasaka et
al. 1991). The results in the present experiments clearly
showed that carbohydrate metabolism involved in citrate
production is modified in IPG cells compared to WT cells,
suggesting that this may play an important role their ability
to excrete high levels of citrate. The IPG cells had a high
CS activity but a low NADP-ICDH activity, which could
result in an increased accumulation of citrate in the cells
(Fig. 5). Transgenic approaches have recently been taken to
examine carbohydrate metabolism in some plant species.

Table 2 CS and NADP-ICDH activity in mitochondria enriched fraction of IPG cells

Activity (jimol (mg protein) ' min ')
Enzyme B/A
Crude extract (A) 10,000 xgppt (B)
CS 0.19±0.02 0.46±0.07 2.4
Fumarase 0.09±0.01 0.27 ±0.02 2.9
NADP-ICDH 0.11 ±0.02 0.03+0.00 0.3
ADH 0.52 ±0.04 0.11±0.01 0.2
Fresh cells (20g) were gently ground with a glass homogenizer and centrifuged at 10,000xg for 10
min. Enzyme activities in ppt fraction were compared to those in cell extracts. Marker enzymes of
mitochondria matrix (fumarase) and cytosol (ADH) were also examined.
494 Citrate production in Al-phosphate utilizing cells
Fig. 5 Diagrammatic representation of carbon metabolisms relevant to the citrate excretion in carrot cells. Aco, aconitase; CS, citrate
synthase; NAD-ICDH, NAD specific isocitrate dehydrogenase; NADP-ICDH, NADP specific isocitrate dehydrogenase; OAA,
oxaloacetate; PEP, phosphoenolpyruvate; PEPC, phosphoenolpyruvate carboxylase; PM, plasma membrane.
For example, tobacco and papaya which showed an over-
expression of CS due to the introduction of a bacterial CS
gene increased citrate excretion with the increased CS ac-
tivities found in the transgenic lines (de la Fuente et al.
1997). A 5-fold increase citrate efflux was observed in
transgenic lines which had 4 fold higher CS activity as
compared with control plants. On the other hand, in
potato plants when NADP-ICDH activity was reduced by
the antisense inhibition caused an increase in citrate con-
centration in leaves was observed (Kruse et al. 1998). These
results are in agreement with our results presented in here.
Previous studies have shown that the Al-phosphate-
tolerant capacity of the IPG cells was very stable in IPG
cells (Koyama et al. 1990), suggesting that IPG traits were
produced as a result of genetical changes. However, it ap-
pears that this high tolerance of IPG cells to Al-phosphate
is caused by an increased citrate excretion and more
efficient P; uptake (Koyama et al. 1992), which are known
as Pi-starvation rescue systems in higher plants (Schacht-
man et al. 1998). The results of the present study strongly
suggest that the IPG cells have modified in their carbo-
hydrate metabolism at least two steps (CS and NADP-
ICDH). It would be interesting to determine whether these
characteristics are caused by independent or coupled mu-
tation.
As determined in the present study (Fig. 2), the carbon
loss by citrate excretion in IPG cells was within a few per-
cent (<l%). This is why IPG cells can grow in Al-phos-
phate medium as well as in P; sufficient medium (Koyama
et al. 1990). In addition, the increased citrate synthesis and
excretion did not affect the overall protein concentration in
IPG cells. However, a sufficient amount of carbon and ni-
trogen sources was given in our experiments. According
to Taylor (1991) carbon loss is thought to be a serious
problem associated with organic acid excretion, and recent
studies on N-nutrition suggested that the carbohydrate
metabolism (involving citrate synthesis) is directly affected
Cytosol PM
citrate
1 Aco
Isocttrsto
i (NADP-ICDH)
2-oxoglutarate
t
imino acid synthesis
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by nitrogen in the environment (Scheible et al. 1997). Thus,
it will be necessary to further examine citrate productivity
in our system under nutrient deficient conditions.
Citrate excretion from plant roots should require spe-
cific transport system in plasma membrane (Ryan et al.
1995). For malate excretion from wheat roots, an anion
channel seems to be involved and this channel appears to
be activated by aluminum (Ryan et al. 1997). Although
a similar carrier located in the plasma membrane with
citrate-transport activity has not been identified, such a
carrier system may affect citrate exudation of IPG cells.
Further analysis is required to determine this factor.
A part of this work was supported by a Grant-in-Aid for
Scientific Research from the Ministry of Education, Science and
Culture of Japan (for HK, No. 90234921). The authors wish to
thank Ms. Julie Stephens at the University of Alberta for editorial
comments regarding this manuscript.
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(Received August 27, 1998; Accepted February 21, 1999)