JXB Advance Access originally published online on October 10, 2005
Journal of Experimental Botany 2005 56(421):2959-2969; doi:10.1093/jxb/eri293
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RESEARCH PAPER |
Phosphoenolpyruvate carboxykinase and its potential role in the catabolism of organic acids in the flesh of soft fruit during ripening
1Dipartimento di Scienze Agrarie e Ambientali, Università degli Studi di Perugia, Borgo XX Giugno 74, I-06121 Perugia, Italy
2Istituto di Biologia Agroambientale e Forestale—Consiglio Nazionale delle Ricerche, Viale Marconi 2, I-05010 Porano (TR), Italy
3Department of Animal and Plant Sciences, University of Sheffield, Sheffield, S10 2TN, UK
* To whom correspondence should be addressed. E-mail: rob.walker{at}talktalk.net; ffamiani{at}unipg.it
Received 13 April 2005; Accepted 18 August 2005
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Abstract |
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Previous studies of grapes and tomatoes have shown that the abundance of phosphoenolpyruvate carboxykinase (PEPCK) increases in their flesh at the start of ripening, and that this coincides with a decrease in its citrate and/or malate content. Thus, PEPCK might function in the catabolism of organic acid anions during the ripening of these fruits. In the present study, the abundance of PEPCK was determined in the flesh of blueberries, raspberries, red currants, and strawberries at different stages of their development. In addition, changes in the amounts of citrate, malate, soluble sugars, isocitrate lyase, NADP-malic enzyme, phosphoenolpyruvate carboxylase, and pyruvate, orthophosphate dikinase in the flesh were determined. PEPCK was not detected in strawberry flesh, in which there was no dissimilation of malate or citrate. In the flesh of the other fruits, the abundance of PEPCK increased during ripening to an amount that was similar to that in grapes and tomatoes. In the flesh of blueberries and red currants, PEPCK was most abundant when there was dissimilation of malate. In the flesh of raspberries, PEPCK was most abundant when there was dissimilation of malate and citrate. These results are consistent with PEPCK playing a role in the dissimilation of citrate and/or malate in the flesh of these fruits during ripening. However, PEPCK was also present in the flesh of blueberries, raspberries, and red currants when there was no dissimilation of malate or citrate, and this raises the possibility that PEPCK might have additional functions. Dissection of blueberries provided evidence that both PEPCK and phosphoenolpyruvate carboxylase were present in the same cells, and possible functions for this are discussed.
Key words: Blueberry, citrate, malate, phosphoenolpyruvate carboxykinase, raspberry, red currant, soft fruit, strawberry
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Introduction |
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At certain stages of development many fruits accumulate organic acids, such as citric, malic, quinic, and ascorbic acids, together with their anions (Ulrich, 1971
). These acids are a metabolically diverse group, for example, the anions of citric and malic acid, citrate and malate, are metabolized by the Krebs cycle and reactions that closely interface with it, whereas those of quinic and ascorbic acid are not (Ulrich, 1971). Citric, and to a lesser extent malic acid, together with their anions, accounts for the bulk of the organic acid content of blueberries, raspberries, currants, and strawberries (Whiting, 1958; Green, 1971). In blueberries (Kushman and Ballinger, 1967), black currants (Toldam-Andersen and Hansen, 1997), and probably raspberries (Perkins-Veazie and Nonnecke, 1992), but not strawberries (Moing et al., 2001), there is a decrease in their content of citric and/or malic acid during ripening. In some fruits, such as Hamlin orange, this decrease can be accounted for by dilution arising from expansion of the fruit (Ting and Vines, 1966). By contrast, in blueberries (Kushman and Ballinger, 1967) and black currants, dilution alone is not sufficient, and it appears that catabolism is also involved (Toldam-Andersen and Hansen, 1997). In soft fruit, little is known about the metabolic pathways involved in this catabolism (Manning, 1993), however, this has been studied in the flesh of grapes and tomatoes, and in these a number of fates for malate/citrate have been suggested. These are oxidation by the Krebs cycle, gluconeogenesis, fermentation reactions that produce ethanol, anthocyanin synthesis, and amino acid interconversions (Farineau and Laval-Martin, 1977; Ruffner, 1982; Famiani et al., 2000). However, uncertainty exists as to what proportion of malate/citrate is used by each of these processes. In the case of oxidation by the Krebs cycle, it should be noted that malate/citrate cannot be dissimilated by the cycle alone. This is because when malate (4 carbons) is fed directly into the cycle it is converted to oxaloacetate (4 carbons), acetyl CoA (2 carbons) is then condensed with it to form citrate (6 carbons), the cycle then turns releasing two molecules of CO2 and malate is reformed. To achieve complete oxidation, cataplerotic pathways such as the conversion of malate to acetyl CoA are required, and either phosphoenolpyruvate carboxykinase (PEPCK) or the malic enzymes can be used in this conversion (Leegood and Walker, 2003). Similarly, when malate/citrate are substrates, these enzymes are required for, or may function in, gluconeogenesis, ethanolic fermentation, anthocyanin synthesis, and amino acid metabolism.
In plant cells, PEPCK is only present in the cytosol, in which it catalyses the reaction
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). Although, the occurrence of PEPCK in most fruits is uncertain, studies have been done in grapes and tomatoes. In these PEPCK appears, or increases in abundance, in the flesh at the onset of ripening, and this led to the suggestion that PEPCK might function in the catabolism of malate and/or citrate (Ruffner et al., 1976; Bahrami et al., 2001). By contrast, there are a number of forms of malic enzyme in plants, there are cytosolic and plastidic forms of NADP-malic enzyme (NADP-ME) and a mitochondrial NAD-malic enzyme (Artus and Edwards, 1985; Drincovich et al., 2001). The malic enzymes have been studied in several fruits, and it has been proposed that NADP-ME may function in the catabolism of malic and citric acids in the flesh during ripening (Hulme and Rhodes, 1971; Ruffner, 1982; Drincovich et al., 2001). However, the abundance of each of these forms of malic enzyme has not been clearly established, and neither has the relative contribution of either PEPCK or the different forms of malic enzyme to the catabolism of malic and citric acid, in any fruit. The aims of this study were 2-fold. Firstly, to characterize the occurrence and function of PEPCK in fruit further. Secondly, to begin to gain an understanding of the underlying enzymatic machinery used in the catabolism of organic acids in the flesh of soft fruit.
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Materials and methods |
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Plant material
Strawberries (Fragaria vesca selection 99/122-11 Istituto di Frutticoltura di Forlì), raspberries (Rubus idaeus cv. Malling Merton), blueberries (Vaccinium corymbosum cv. Blueray), and red currants (Ribes rubrum cv. Junifer) were collected from plants growing in central Italy in 2003. Only healthy fruit were collected, and these were taken from several positions on the plant. The developmental stage of the fruit was based on their colour, and these were: 1, green; 2, onset of coloration; 3, half-maximum coloration; 4, maximum coloration. Barley (Hordeum vulgare cv. Maris Mink), broccoli (Brassica oleracea cv. Corvet), maize (Zea mays L. cv. Golden Giant), and turnip (Brassica rapa cv. Snowball) were grown in a field, and Panicum maximum, tomato (Lycopersicon esculentum Mill. cv. Moneymaker), and Hoya carnosa in a greenhouse, in Perugia, Italy. Cucumber (Cucumis sativus cv. Marketmore) seeds were germinated in the dark in perlite at 25 °C for 4 d. Mature barley leaves were detached and placed on moist filter paper in a Petri dish in the dark at 25 °C for 4 d.
Measurement of fresh and dry weights
For each species of fruit, and at each stage of development, the weights of 30 intact fruits were each measured separately. This was done for both freshly harvested fruit and for the same fruit after being dried to constant weight by incubation in a forced-air oven at 105 °C.
Measurement of solute content
Solute content of the flesh was measured using a refractometer. For each species of fruit, and at each stage of development, measurements were done on each of 30 fruits.
Measurement of titratable acidity
10 g of intact fruit was homogenized in a mortar containing 50 ml of distilled water, passed through Miracloth and the filtrate adjusted to neutrality, as measured by a pH meter, by adding 100 mM NaOH. For each species of fruit, and at each stage of development, this procedure was done three times. Results are expressed as a percentage of citric acid.
Measurement of anthocyanin content
10 g of intact fruit was homogenized in a mortar containing 40 ml of distilled water and then centrifuged at 10 000 g for 5 min. The supernatant was passed through Miracloth and 5 ml of the filtrate was then added to 10 ml of distilled water. The absorbance of this solution at 520 nm was determined using a spectrophotometer.
Preparation of a nitrogen powder
For electrophoresis, enzyme assays and metabolite measurements, a nitrogen powder was used. This powder was prepared by removing seeds, skin, and central vascular bundles from the flesh of 10 fruits that were frozen in liquid nitrogen. The flesh was then ground in a mortar containing liquid nitrogen, and the resulting powder used either immediately or after storage at –80 °C.
Measurement of soluble sugars
50 mg of frozen powder was added to an Eppendorf tube containing 1.5 ml of 20 mM HEPES-KOH (pH 7.1), 4 mM MgCl2, and 80% (v/v) ethanol, incubated at 80 °C for 1 h and then centrifuged at 12 000 g for 5 min. 150 µl of charcoal suspension (100 mg ml–l) was added to the supernatant, vortexed, and then centrifuged at 12 000 g for 5 min. The supernatant was stored at –20 °C until required. Glucose, fructose, and sucrose were measured in the supernatant using an enzyme-coupled spectrophotometric method (Jones et al., 1977; Antognozzi et al., 1996).
Measurement of malate and citrate
Malate and citrate were measured using enzyme-coupled spectrophotometric methods (Lowry and Passonneau, 1972). The following modifications were made to allow the use of a microplate reader. Each measurement used 50 mg of frozen powder. For malate, the assay mixture (250 µl) was 50 mM 2-amino-2-methylpropanol (pH 9.9), 40 mM glutamate, 1 mM NAD, 10 U aspartate aminotransferase, and 1 U of malate dehydrogenase. For citrate, the assay mixture (250 µl) was 540 mM glycyl glycine (pH 7.8), 6 mM NADH, 0.5 U of malate dehydrogenase, 0.25 U of lactate dehydrogenase, and 0.1 U of citrate lyase. All enzymes were from Sigma (Italy).
Enzyme assay
200 mg of frozen powder was added to 800 µl of 200 mM Bicine-KOH (pH 9.0) and 50 mM DTT in a mortar, ground using a pestle, and the homogenate clarified by centrifugation at 12 000 g for 5 min. Enzyme activity in the supernatants was measured immediately. The activity of PEPCK was measured in the carboxylation direction as described by Walker et al. (1999). PEPC activity was measured using an enzyme coupled assay as described by Ashton et al. (1990). One unit of enzyme activity is that which produces 1 µmol product min–1 at 25 °C.
SDS-PAGE and immunoblotting
For fruit 500 mg, and other tissues 60 mg, of frozen powder was added to 500 µl electrophoresis sample buffer (62 mM TRIS-HCl (pH 7.0), 5% 2-mercaptoethanol (v/v), 10% (w/v) SDS, 50 mM ascorbic acid, 0.002% (w/v) bromophenol blue, and 10% (w/v) glycerol) in a mortar and ground using a pestle. If the extract became yellow, several microlitres of 20% (w/v) NaOH were added until it just became blue. The extract was immediately transferred to an Eppendorf tube and placed in a boiling water-bath for 5 min. The sample was stored at room temperature, if it was to be run on a gel within an hour, or otherwise stored at –20 °C. The stability of the samples at –20 °C was dependent on the species and the stage of fruit development. In general, it was better to apply them to gels within two days. However, many samples were stable if stored for several weeks. Immediately before applying to SDS-PAGE gels, samples were centrifuged at 10 000 g for 5 min and 1–5 µl of supernatant was loaded on gels. SDS-PAGE and immunoblotting were done as described by Walker and Leegood (1996). Briefly SDS-PAGE was done using a Hoeffer mini-gel apparatus and western transfer using a Pharmacia Multiphor device in conjunction with Millipore Immobilon-P membrane. Immunoreactive polypeptides were detected using an Amersham enhanced chemiluminescence kit in conjunction with a peroxidase-conjugated second antibody. Protein was measured using a modified version of the Lowry method as described by Walker et al. (1995).
Source of antibodies
The PEPCK antisera were raised against the enzyme from either cotyledons of germinating cucumber (Walker et al., 1995) or from leaves of P. maximum (Walker et al., 2002). The phosphoenolpyruvate carboxylase (PEPC) was raised against the enzyme from leaves of P. maximum (RP Walker, unpublished work). The pyruvate, orthophosphate dikinase (PPDK) antiserum was raised to the enzyme from maize (Chastain et al., 2002) and had been affinity purified. The NADP-ME antiserum was raised against the enzyme from maize leaves and had been affinity purified (Maurino et al., 2001). The isocitrate lyase (ICL) antiserum was raised against the enzyme from castor bean endosperm (Maeshima et al., 1988).
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Results |
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Changes in fresh and dry weights, soluble solids content, titratable acidity, and anthocyanin content during fruit development
For all fruits, both their fresh and dry weights and anthocyanin and soluble solids content increased during development (Fig. 1). Titratable acidity decreased the most in blueberries and raspberries, to a lesser extent in red currants, and changed little in strawberries (Fig. 1). Where comparable data exist, the results were similar to previous studies of black currants (Toldam-Andersen and Hansen, 1997
), blueberries (Kushman and Ballinger, 1967), raspberries (Perkins-Veazie and Nonnecke, 1992), and strawberries (Moing et al., 2001).
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Changes in the content of sugars and organic acids during fruit development
For measurement of both sugars and organic acids, recovery experiments were done in which a known amount of the metabolite was co-extracted with the flesh of each ripe soft fruit. For all fruits, recovery of metabolites was more than 90% (data not shown). During the development of the flesh of all fruits, there was a large increase in the abundance of soluble sugars both per fruit and g–1 FW (Fig. 2). Glucose and fructose were more abundant than sucrose in the flesh of blueberries, raspberries, and red currants, however, in strawberry flesh, a considerable amount of sucrose was also present (Fig. 2). These results are in agreement with previous studies (Green, 1971
; Darnell et al., 1994; Moing et al., 2001).
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In the fruits studied, citric, together with a smaller amount of malic acid, account for the bulk of their organic acid content (Whitting, 1958
; Green, 1971), therefore, only the anions of these were measured. The flesh of all fruits, at all stages of development, contained more citrate than malate (Fig. 2), however, more malate was present than previously found in blueberries (Markakis et al., 1963; Kushman and Ballinger, 1967). In the flesh of blueberries, raspberries, and red currants, the abundance of both malate and citrate g–1 FW was lower at the final stage of development than at the first stage, whereas, there was little change in that of strawberries (Fig. 2). When expressed on a per fruit basis, citrate content of the flesh increased throughout the development of strawberries and red currants, whereas, for blueberries and raspberries, it increased up to stage 3 and then decreased (Fig. 2). On a per fruit basis, malate increased in strawberry flesh and decreased in raspberry flesh throughout development, decreased in blueberry flesh between stages 2 and 4 and in red currant flesh it increased up to stage 3 and then decreased. These results are in agreement with previous studies of strawberries (Moing et al., 2001), and similar results to those for red currants were reported for black currants (Toldam-Andersen and Hansen, 1997).
When considering the decrease in malate and citrate content on a g–1 FW basis, it is important to allow for the decrease brought about by dilution arising from growth. In the fruit studied, most of the increase in volume during the period studied is a result of cell expansion (Coombe, 1976; Manning, 1993; Cano-Medrano and Darnell, 1997). To allow for this dilution, the results were adjusted using the equation: (amount g–1 FW at the present stage of development)–[(volume of whole fruit at previous stage of development/volume of whole fruit at present stage of development)xamount g–1 FW at the previous stage of development]. This difference gives the approximate amount of synthesis or dissimilation of malate or citrate, if the difference is positive it means that there has been synthesis and if it is negative there has been dissimilation. This assumes there is no import or export of these, and in the flesh of fruits in which it has been studied, such as grape, it is thought that the bulk of organic acids is synthesized within the fruit from imported sugars, and that these are not then exported (Ruffner, 1982). In the flesh of the soft fruits, only in blueberry and raspberry between stages 3 and 4 was there dissimilation of citrate, and there was dissimilation of malate between stages 2 and 4 in blueberry and raspberry and between stages 3 to 4 in red currant (Fig. 3).
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Enzyme abundance in the different fruits
For many fruits, it is difficult to prepare extracts that are suitable for SDS-PAGE (Famiani et al., 2000
), therefore, a number of extraction procedures were compared. Homogenization in a modified SDS-PAGE loading buffer was satisfactory for the fruits used in this study. The suitability of this method was shown by homogenizing maize leaves, whose polypeptide pattern after SDS-PAGE is known (Wingler et al., 1999), with the flesh of each fruit. Co-extraction did not lead to a loss of polypeptides present in maize leaves (data not shown). In addition, the flesh of soft fruits extracted alone produced a polypeptide pattern that was sharp and well separated (Fig. 6). The polypeptide composition of the flesh of the different fruits was not the same (Fig. 6).
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In order both to evaluate antibody specificity and to compare enzyme abundance in the different ripe soft fruits, immunoblots were used. These used extracts of the flesh of ripe fruits and tissues in which the enzymes studied are known to be abundant. The antiserum raised to PEPCK, from either cucumber or P. maximum, recognized a 74 kDa polypeptide in extracts of blueberry, raspberry, and red currant flesh, turnip tubers, broccoli florets, and leaves of the PEPCK-type CAM plant H. carnosa, but not in extracts of strawberry flesh or broccoli leaves (Fig. 4). The mass and occurrence of this polypeptide in the non-fruit samples are consistent with previous studies (Walker and Chen, 2002
). The PEPC antiserum recognized a 110 kDa polypeptide in extracts of all tissues, however, it was most abundant in those of H. carnosa leaves (Fig. 4). These observations are consistent with previous studies of PEPC (McNaughton et al., 1989). The NADP-ME antiserum recognized a 72 kDa polypeptide in all tissues (Fig. 4). The mass and occurrence of this polypeptide was consistent with previous studies of C3 tissues (Maurino et al., 2001). In addition, the antiserum recognized predominantly a 62 kDa polypeptide in extracts of maize leaves (data not shown), and this is the subunit size of the enzyme in this tissue (Maurino et al., 2001). The PPDK antiserum recognized a 95 kDa polypeptide in extracts of leaves of maize, P. maximum, and tomato, however, no polypeptides were recognized in those of the flesh of ripe soft fruits (Fig. 5). The mass and distribution of this polypeptide in the non-fruit tissues are consistent with previous studies (Chen et al., 2000; Chastain et al., 2002). Co-extraction of the flesh of each fruit and either tomato or maize leaves did not lead to a loss of this polypeptide (data not shown). The ICL antiserum recognized a 65 kDa polypeptide in extracts of tomato leaves, senescing barley leaves, and cotyledons of germinating cucumber, however, no polypeptides were recognized in those of either leaves of maize or P. maximum or the flesh of ripe soft fruits (Fig. 5). The mass and distribution of this polypeptide in the non-fruit tissues are consistent with previous studies (Maeshima et al., 1988; Nieri et al., 1997; Chen et al., 2000). Co-extraction of the flesh of each fruit and senescing barley leaves did not lead to a loss of this polypeptide (data not shown).
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Changes in the abundance of PEPCK and other enzymes during fruit development
SDS-PAGE gels were loaded with extracts of the flesh of soft fruit from fruits at different stages of development, and after electrophoresis polypeptides were visualized using Coomassie Brilliant Blue dye. The intensity of staining of the tracks was similar (Fig. 6). This suggested that the protein content g–1 FW of the flesh of each fruit changed little during the stages of development investigated. In addition, it appeared that the flesh of these fruit species contained a similar amount of protein g–1 FW (Fig. 6). This was in agreement with the protein content of these tissues measured using the Lowry procedure, which showed that the protein content of the flesh was 1–2 mg g–1 FW (data not shown). A number of abundant polypeptides were present in the flesh, particularly in that of raspberries and ripe red currants. These polypeptides were not contaminating seed storage proteins because their masses were different (Figs 6, 7). During ripening, some changes in the polypeptide pattern of the flesh occurred, and this was most noticeable in strawberries and red currants (Fig. 6).
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Changes in enzyme abundance in the flesh of soft fruit during development was investigated by using immunoblots of SDS-PAGE gels. In addition, the activities of PEPCK and PEPC were measured. With the exception of strawberry flesh, in which PEPCK was not detected, there was an increase in the abundance of PEPCK during ripening (Figs 3, 6). In blueberry and red currant flesh, the dissimilation of malate occurred when PEPCK was most abundant (Figs 3, 6). In raspberry flesh, the dissimilation of both malate and citrate occurred when PEPCK was most abundant (Figs 3, 6). In the flesh of these fruits, the maximum activity of PEPCK was about 0.15 U g–1 FW (Fig. 3). The failure to detect PEPCK in strawberry flesh was not a result of loss of the enzyme after extraction, because co-extraction of strawberry and blueberry flesh did not lead to a loss of the enzyme (data not shown). PEPC was detected in the flesh of all fruits at all stages of development (Figs 3, 6). The abundance of PEPC increased slightly during the development of blueberries, however, in the other fruits its abundance changed little. The activity of PEPC was between 0.10 and 0.30 U g–1 FW (Fig. 3). In ripe blueberries, dissection showed that both PEPCK and PEPC were present in the skin, flesh, and central vascular bundles (Fig. 8). Neither PPDK or ICL was detected in the flesh of the soft fruits at any stage of development (Fig. 6). NADP-ME was detected in the flesh of all the soft fruits at all stages of development (Fig. 6). In strawberry and red currant flesh, its abundance decreased slightly during development, however, in the other fruit its abundance changed little (Fig. 6).
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Discussion |
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In this study, the possibility that PEPCK might function in the dissimilation of malate/citrate in the flesh of soft fruit during ripening was investigated. This was done by determining the abundance of PEPCK and the amount of dissimilation of malate/citrate in the flesh of several soft fruit at different stages of development. In addition, to understand the metabolism of the flesh of these fruits more clearly, the abundance of a number of other enzymes was investigated.
PEPC was detected at all stages of development in the flesh of all the soft fruits (Figs 3, 6), and this is consistent with the view that it functions both in the synthesis of stored malate and citrate and in the anaplerotic replenishment of the Krebs cycle in fruits (Law and Plaxton, 1995; Guillet et al., 2002). PPDK was not present in the flesh of any of the soft fruits (Figs 5, 6) precluding gluconeogenesis from pyruvate. The possibility has been raised that ICL may function in the gluconeogenic conversion of ethanol to sugars (Tadege et al., 1999). However, although ethanol is synthesized and re-metabolized in some fruits such as grape (Terrier and Romieu, 2001), the absence of ICL in the flesh of the soft fruits appears to preclude the possibility of gluconeogenesis from any ethanol present (Figs 5, 6). The NADP-ME antiserum recognized a 72 kDa polypeptide in the flesh of all fruit (Figs 4, 6). Previous studies have shown that the subunit size of plastidic NADP-ME in C3 tissues is usually 72 kDa, and that this form of NADP-ME is widely distributed in plants (El-Shora and ap Rees, 1991; Maurino et al., 2001). The subunit size of the cytosolic form of NADP-ME is usually smaller, and this form of NADP-ME is of a more limited occurrence in C3 plants (El-Shora and ap Rees, 1991; Maurino et al., 2001). This suggests that the 72 kDa polypeptide is plastidic NADP-ME. Plastidic NADP-ME is thought to function in biosynthetic processes associated with growth (El-Shora and ap Rees, 1991), and the observation that the 72 kDa polypeptide was present throughout the growth of the fruits is also consistent with it being a plastidic form of NADP-ME (Fig. 6). In grape berries, a cytosolic NADP-ME of subunit size 63 kDa is present, and it has been proposed that this enzyme functions in the dissimilation of malate during ripening (Ruffner, 1982; Franke and Adams, 1992, 1995). The specific activity (enzyme activity/enzyme protein) of C3 plastidic NADP-ME is less than that of cytosolic NADP-ME (Drincovich et al., 2001; Franke and Adams, 1992). This means for a given amount of activity less cytosolic NADP-ME protein would be present, and this raises the possibility that, in the present study, the antibody used was too weak to detect the cytosolic enzyme. Visualization of total polypeptides showed that there were a number of abundant polypeptides in the flesh of all the fruits (Fig. 6). Although the identities of these are unknown they may be vegetative storage proteins because these are abundant in the flesh of a number of fruits. These have been implicated in both the prevention of fungal and insect attack and in the storage of nitrogen (Tattersall et al., 1997; Peumans et al., 2002).
PEPCK was present in the flesh of blueberries, raspberries, and red currants but not strawberries. The maximum amount of PEPCK in these was about 0.15 U g–1 FW. Expressed on this basis, this is considerably less than in the bundle sheath of leaves of the C4 plant P. maximum which contains about 8 U g–1 FW. In these, PEPCK acts as a decarboxylase in the photosynthetic C4 cycle and flux through PEPCK is up to 5 µmol min–1 g–1 FW (Walker and Chen, 2002). However, this comparison does not take into account either the differences in the structure of the cells in which PEPCK is present, or the rate of decarboxylation of oxaloacetate by PEPCK in these tissues. In many fruits, the parenchyma cells of the flesh contain larger vacuoles than bundle sheath cells, and these vacuoles, which contain little protein and no PEPCK, account for most of the tissues fresh weight. For this reason, it is perhaps more useful to compare the amount of enzyme per unit total protein, in this case the fruit contain 0.1–0.15 U mg–1 total protein, whereas P. maximum leaves contain 0.3–0.7 U mg–1 total protein (Walker et al., 1999, 2002). On this basis, these fruits can be considered to contain a considerable amount of PEPCK. In grapes and tomatoes, the rate of dissimilation of citrate and/or malate is much lower than the amount of PEPCK activity present. For example, in grapes the rate of dissimilation of malate is 0.005 µmol min–1 g–1 FW and the activity of PEPCK is 0.15 U g–1 FW (Ruffner, 1982; Walker and Chen, 2002). In the flesh of blueberries, raspberries, and red currants, the rate of dissimilation of malate and the amount of PEPCK present were similar to grape (Fig. 3). One reason for this excess of PEPCK activity could be that, as in grapes, there is a continuous synthesis and dissimilation of citrate and/or malate (Diakou et al., 2000; Terrier and Romieu, 2001). In addition, it is possible that in non-C4 plant tissues the concentration of oxaloacetate is such that PEPCK would work well below its maximum catalytic rate (Walker and Chen, 2002; Walker et al., 2002). In the flesh of blueberries, raspberries, and red currants the abundance of PEPCK increased during ripening, a period when there was dissimilation of malate, whereas in strawberry flesh there was no dissimilation and no PEPCK was detected (Figs 3, 6). In raspberry flesh, PEPCK was most abundant when citrate was dissimilated (Figs 3, 6). These observations are consistent with PEPCK having a function in the catabolism of citrate and/or malate as has been proposed in grapes (Ruffner and Kliewer, 1975) and tomatoes (Bahrami et al., 2001). However, smaller amounts of PEPCK were also present in the flesh of blueberries, raspberries, and red currants when there was no dissimilation of malate and this raised the possibility that PEPCK might have additional functions. For example, in young grapes it has been suggested that PEPCK might function both as a carboxylase (Ruffner and Kliewer, 1975) and in the metabolism of nitrogenous compounds in the vasculature (Walker et al., 1999).
In blueberries, raspberries, and red currants both PEPC and PEPCK were present in the flesh during ripening and in blueberry and red currants, as in grape (Ruffner et al., 1976), similar amounts were present (Fig. 3). These enzymes are only present in the cytosol, in which PEPC catalyses the carboxylation of phosphoenolpyruvate to oxaloacetate and PEPCK catalyses the reverse reaction (Walker and Chen, 2002). To investigate whether the enzymes were present in different cells, ripe blueberries were dissected to give skin, which contains parenchyma cells but no vasculature, flesh that contains both parenchyma cells and vascular bundles (Cano-Medrano and Darnell, 1997), and central vascular bundles from which flesh was carefully removed by washing. PEPCK and PEPC were present in all tissues (Fig. 8), and this suggests that, as in leaves of many CAM plants, both enzymes are present in the same cells. In this situation, control of flux through PEPCK and PEPC might be achieved by regulatory mechanisms, which includes reversible phosphorylation of both enzymes, that are thought to be important in leaves of CAM plants (Walker and Chen, 2002). This raises the question as to why both PEPCK and PEPC are probably present in the cytosol of the same cells. In ripening grapes, there are periods when either malate or sugar is the prefered substrate for the Krebs cycle (Ruffner, 1982; Terrier and Romieu, 2001). The presence of both PEPCK and PEPC allows flexibility with respect to which substrate is used by the Krebs cycle. The presence of PEPC allows the anaplerotic replenishment of the cycle, necessary for biosynthesis, when sugars are being used as the substrate, without having to utilize stored malate. The presence of PEPCK allows malate to be used in the synthesis of Krebs cycle intermediates for biosynthesis, without having to utilize sugars. In addition, the complete oxidation of malate/citrate by the Krebs cycle requires a cataplerotic reaction such as that catalysed by PEPCK (see Introduction). In addition, evidence has been provided that gluconeogenesis from malate occurs in the flesh, during the ripening of grapes and tomatoes (Ruffner, 1982; Halinska and Frenkel, 1991). Gluconeogenesis from malate/citrate requires the presence of either PEPCK or PPDK, and the latter is not present in the flesh of ripening blueberries, raspberries, or red currants (Fig. 6). Therefore, a further possibility is that PEPCK might function in gluconeogenesis in some soft fruits.
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Footnotes |
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Abbreviations: ICL, isocitrate lyase; NADP-ME, NADP-malic enzyme; PEPCK, phosphoenolpyruvate carboxykinase; PEPC, phosphoenolpyruvate carboxylase; PPDK, pyruvate, orthophosphate dikinase.
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References |
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