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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 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 (Lipton et al. 1987) and rape (Hoffland et al. 1992) has been 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 enhanced 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 excretion 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). 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 Alphosphate 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 activity of mitochondrial citrate synthase was higher in IPG cells than in wild-type cells, whereas the activity of cytosolic 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 specific isocitrate dehydrogenase (EC 1.1.1.42). Aluminum toxicity and its related nutrient deficiencies 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 aluminumphosphate may increase crop productivity in such soils. There are some strategies plants have adopted to increase 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 We have previously selected a number of Al-phosphate 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 excreted 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 stoichiometrically to forming non-phytotoxic compounds, namely Alcitrate. 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 Abbreviations: Aco, aconitase; CS, citrate synthase; Cyt c oxydase, cytochrome c oxydase; DTNB, 5'5-dithiobis (2-nitrobenzoic acid); ICDH, isocitrate dehydrogenase; IPG, insoluble phosphate grower; PEPC, phosphoenolpyruvate carboxylase; TBS, tris buffered saline. 1 Corresponding author, e-mail, [email protected] 489 490 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 Alphosphate 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 defined. Therefore, in the present study we have examined the activities of enzymes involved in citrate production, including 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 dehydrogenase 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 commercially 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 nitrogen 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 seedlings that were grown under darkness for 5 d after germination. In vitro enzyme assay—Citrate synthase was assayed spectrophotometrically according to Srere (1967) by monitoring reduction of Acetyl-CoA in the presence of DTNB at A412. PEPC was assayed by the method of Vidal and Gadal (1983). NADPICDH 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) insoluble 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 precipitate 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 mitochondrial lysate. To assess the purity of preparations the activities of mitochondria fumarase (Fum), a marker enzyme for mitochondria, 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 mitochondria was assayed using the method of Gardestrom and Edwards (1983). NAD-ICDH activity, a marker enzyme of TCA cycle, was assayed by the method of Cox (1967). Quantification of citrate, glucose in medium—Cells were removed 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 measured using a cycling reaction reagent (Kato et al. 1973). Glucose was measured colorimetrically using Glucose B-test Wako quantification 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 mitochondrial 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 initially 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 supplied to the cells is first converted to glucose and fructose Citrate production in AJ-phosphate utilizing cells within 2 d after inoculation (Ueda et al. 1974). Since uptake 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 transferred to the root exudate from metabolite significantly increased as organic acids were being released from the proteoid root (Johnson et al. 1996). Therefore, we estimated the amounts of carbon metabolism correlated with citrate excretion in both IPG cells and WT cells. By measuring the amount of citrate exuded and the amount of glucose remaining in the medium, we calculated the ratio of carbon present in the excreted citrate to the carbon consumed from free sugars in the medium. As shown in Fig. 2, we found a higher ratio in IPG cells to WT cells throughout the experimental period. The ratio ranged from CO £ o in. — 10 CO CO •"- 5 - 5 - 10 £ 2 E 0' 601 aim O) c - B 1.5 E • TX 1 T3 1.0 2 40 - o E £ CO 0.5 20 2 2 o CO X CD Cit ite • • u_ 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 modified in such a way as to allow an increase carbon allocation to citrate production. In vitro activity of enzymes relevant to citrate synthesis—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 acetyl-CoA and oxalacetate, in IPG cells was 30 to 100% higher than WT cells (Fig. 3B). The specific activity of CS in IPG cells was about 0.18 ^mol (mg protein)~' min" 1 unit, whereas that in the WT cells was lower than 0.13 unit. By contrast, the activity of Aco, which catalyzes the conversion of citrate to isocitrate, was similar in both cell types (Fig. 3C). This increased activity of CS in IPG cells may be one reason why this line produces more citrate than WT cells. Citrate metabolism is also affected by the activity of enzymes involved in TCA cycle in mitochondria, namely fumarase and NAD-ICDH. Under the given conditions, the activity of enzymes were similar between cell types (Table 1). The specific activities of fumarase and NADICDH of both cell types are about 0.08 and 0.08 unit, respectively. The activity of Cyt c oxidase, a mitochondrial inner membrane marker, was also similar between cell types (Table 1). The specific activity of Cyt c oxidase of both cell types was about 0.14 unit. This suggests that these parts of the TCA cycle are similar in both cell types, and 15 " A CD CO 491 0 n i u 0* 5 10 Days 15 Fig. 1 Changes in citrate and sucrose concentration in Al-phosphate medium during the growth of IPG and WT cells. Both IPG and WT cells were grown for 16 d in Al-phosphate medium containing 2 mM of anhydrous Al-phosphate as a sole phosphorus source at an initial pH of 5.6. A: cell suspension was harvested aseptically at designated time and weighed (IPG, • ; WT, o). B: concentrations of citrate (closed symbols) and sucrose (open symbols) in the medium containing IPG (•, • ) or WT cells (A, A). Means ±SD are indicated (n=3). 1 2 3 Consumed sucrose (mmole flask"1 ) Fig. 2 Loss of C by citrate exudation in IPG (•) and WT (o) cells grown in the medium containing Al-phosphate as a sole phosphorous source. Carbon ratio was designated as the ratio of the amount of carbon involved in excreted citrate to that in consumed free sugar in the medium. The values were calculated from the results shown in Fig. IB. 492 Citrate production in Al-phosphate utilizing cells 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-phosphate 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 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 contrast, a large difference was observed in NADP-ICDH activities between the two cell types (Fig. 3D). The specific 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 culture 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 enzyme 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-enzymes localized in cytosol, plastids and possibly in mitochondria 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 approximately 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 Cell line Activity of enzymes (^mol (mg protein) ' min ') NAD-ICDH Fumarase Cyt c oxydase 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 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 nonimmuno-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 (Breidenbach and Beevers 1967). However, a recent study of glyoxysome development in carrot suspension cells indicates 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 immunochemichal approaches. Using crude soluble proteins obtained from either IPG cells or etiolated seedlings, we performed an immunochemichal analysis using anti-serum raised against carrot mitochondrial CS. Although we found significant differences between immunotitration curves for each of the extracts (Fig. 4), when we performed western 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 extract. 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 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 dehydrogenase; 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) suggesting that most of the activity of this enzyme was localized 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 immunochemical approach mentioned above. Discussion In some plant species, citrate is excreted by plant roots following an induction period suggesting that some metabolic 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 Enzyme CS Fumarase NADP-ICDH ADH Activity (jimol (mg protein) ' min ') Crude extract (A) 10,000 xgppt (B) 0.19±0.02 0.09±0.01 0.11 ±0.02 0.52 ±0.04 0.46±0.07 0.27 ±0.02 0.03+0.00 0.11±0.01 B/A 2.4 2.9 0.3 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 Cytosol PM citrate 1 Aco Isocttrsto i (NADP-ICDH) 2-oxoglutarate t imino acid synthesis 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 overexpression of CS due to the introduction of a bacterial CS gene increased citrate excretion with the increased CS activities 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 concentration 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-phosphatetolerant 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 appears 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 (Schachtman et al. 1998). The results of the present study strongly suggest that the IPG cells have modified in their carbohydrate metabolism at least two steps (CS and NADPICDH). It would be interesting to determine whether these characteristics are caused by independent or coupled mutation. As determined in the present study (Fig. 2), the carbon loss by citrate excretion in IPG cells was within a few percent (<l%). This is why IPG cells can grow in Al-phosphate 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 nitrogen 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 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 specific 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). 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