Planta (2004) 219: 48–58 DOI 10.1007/s00425-004-1220-y
O R I GI N A L A R T IC L E
Zhi-Hui Chen Æ Robert P. Walker Æ La´szlo´ I. Te´csi Peter J. Lea Æ Richard C. Leegood
Phosphoenolpyruvate carboxykinase in cucumber plants is increased both by ammonium and by acidification, and is present in the phloem Received: 27 June 2003 / Accepted: 13 December 2003 / Published online: 26 February 2004 Ó Springer-Verlag 2004
Abstract In cucumber (Cucumis sativus L.), phosphoenolpyruvate carboxykinase (PEPCK) was shown by activity measurements and immunoblots to be present in leaves, stems, roots, flowers, fruit and seed. However, immunolocalisation showed that it was present only in certain cell types. PEPCK was present in the companion cells of the adaxial phloem of minor veins, the adaxial and abaxial phloem of larger veins, the internal and external phloem of vascular bundles in petioles and stems, the phloem in roots and the extra-fascicular phloem in leaves, cotyledons, petioles and stems. Immunohistochemical evidence suggests that both the extra-fascicular phloem and the adaxial phloem are involved in the transport of amino acids. In roots and stems, the abundance of PEPCK was greatly increased by watering plants with a solution of ammonium chloride at low, but not at high pH. PEPCK also increased in leaves, but not roots or stems, of seedlings grown in an atmosphere containing 5% CO2, and in roots and stems of seedlings watered with butyric acid. All these treatments are known to lower the pH of plant cells. Amino acid metabolism in the phloem may produce an excess of carbon skeletons, pH perturbations and an imbalance in the production/utilisation of NADH. This raises the possibility that PEPCK may function in the conversion of these carbon skeletons to PEP, which, depending on the energy requirements of the phloem, is subsequently utilised by either gluconeogenesis or the Krebs cycle, which both consume protons.
Z.-H. Chen Æ R. P. Walker Æ L. I. Te´csi Æ R. C. Leegood (&) Robert Hill Institute and Department of Animal and Plant Sciences, University of Sheffield, Sheffield, S10 2TN, UK E-mail:
[email protected] Tel.: +44-114-2220040 Fax: +44-114-2220050 P. J. Lea Department of Biological Sciences, Lancaster University, Lancaster, LA1 4YQ, UK
Keywords Cataplerosis Æ Cucumis Æ Nitrogen metabolism Æ Phosphoenolpyruvate carboxykinase Æ pH regulation Æ Redox (NAD/NADH) regulation Abbreviations Asp: Aspartate Æ Asn: Asparagine Æ Glu: Glutamate Æ Gln: Glutamine Æ NADP-ME: NADPmalic enzyme Æ OAA: Oxaloacetate Æ PEP: Phosphoenolpyruvate Æ PEPC: Phosphoenolpyruvate carboxylase Æ PEPCK: Phosphoenolpyruvate carboxykinase
Introduction Phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.49) in plants is a cytosolic enzyme that catalyses the reversible reaction: oxaloacetate + ATP $ PEP + ADP + CO2 This reaction lies at an important crossroads in metabolism. Metabolism of lipids, organic acids and amino acids can give rise to oxaloacetate, and phosphoenolpyruvate (PEP) can be utilised either in glyceroneogenesis, gluconeogenesis or in the synthesis of secondary metabolites or other organic and amino acids. In keeping with this pivotal position, PEPCK has recently been shown to be present in a wide range of plant tissues including developing seeds (Leegood and Walker 1999), trichomes (Leegood and Walker 1999) and roots (Kim and Smith 1994; Walker et al. 2001). Similarly, in recent years it has been shown that in vertebrates PEPCK is present in a wide range of tissues and that its metabolic role is more complex than originally thought (Croniger et al. 2002). In plants, evidence is accumulating that PEPCK is associated with tissues in which the metabolism of nitrogenous compounds is enhanced (Walker et al. 1999; Lea et al. 2001; Walker and Chen 2002). For example, in developing grape seeds, the maximum abundance of PEPCK coincides with the deposition of storage proteins, it is
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localised in tissues likely to be involved in the transfer of assimilates to the developing storage tissues, such as the chalaza and the seed coat, and its abundance is greatly affected by the form in which nitrogen is supplied to the seed. Thus it is strongly increased by asparagine (Walker et al. 1999). PEPCK may also be involved in the transport of nitrogenous assimilates out of senescing cucumber cotyledons (Chen et al. 2000). Similarly, in kinetoplastids (e.g. trypanosomes) and in vertebrates, PEPCK is known to play a role in the metabolism of nitrogenous compounds (Urbina et al. 1990; Owen et al. 2002) and, in vertebrates, such metabolism may be associated with pH regulation (Pogson et al. 1976; Hanson and Reshef 1997) and the regulation of energy metabolism (Jungas et al. 1992; She et al. 2000; Croniger et al. 2002). The aim of this paper was to determine the location of PEPCK in cucumber plants, and its occurrence in relation to their nitrogen supply and to factors that are likely to lead to a perturbation of intracellular pH.
Materials and methods
Measurement of PEPCK activity PEPCK activity was measured in the carboxylation direction [in which oxaloacetate (OAA) formed is reduced to malate by NADH] at 25°C. The assay mixture contained 100 mM Hepes– KOH (pH 6.8), 100 mM KCl, 0.14 mM NADH, 25 mM DTT, 6 mM MnCl2, 6 mM PEP, 1 mM ADP, 90 mM KHCO3 and 6 U ml)1 malate dehydrogenase. To maintain the pH at 6.8, 5 ll of 50% HCl was added. One unit of PEPCK activity corresponds to the production of 1 lmol product per min at 25°C. To establish that the maximum activity of PEPCK was measured, recovery experiments were done in which tissues were co-extracted with a small amount of cucumber cotyledon from germinating seeds, a tissue for which we have carefully optimised the extraction and assay of PEPCK.
SDS–PAGE and immunoblotting Plant material was homogenised as described above. A 150-ll aliquot of the supernatant was added to 150 ll of 10% (w/v) trichloroacetic acid and centrifuged at 12,000 g for 5 min. The pellet was centrifuged at 12,000 g for 5 min and the pellet resuspended in 100 ll of 62.5 mM Tris–HCl (pH 6.8), 10% glycerol, 5% SDS, 5% 2-mercaptoethanol, 0.002% bromophenol blue. Samples were incubated at 100°C for 3 min and then stored until required. Before electrophoresis, insoluble material was removed by centrifugation at 12,000 g for 3 min. SDS–PAGE and immunoblotting were done as described by Walker and Leegood (1996). Protein was measured as described by Walker et al. (1995).
Plant material Cucumber (Cucumis sativus L. cv. Marketmore), turnip (Brassica rapa L. cv. Snowball) and tomato (Lycopersicon esculentum Mill. cv. Gold Star) were grown from seed in perlite (six seedlings per 10-cm pot) under a 12-h photoperiod and an illumination of 200 lmol quanta m)2 s)1 at 25°C. No nutrients were provided. For immunohistochemistry, 4-week old plants were used. For feeding with different nitrogen sources, 3-week old seedlings were watered with a solution of either 4 mM NH4Cl, 4 mM KNO3 or 5 mM Asp (adjusted to pH 5.0 with KOH) each day for 5 days. For treatment with butyric acid, 3-week old seedlings were watered with a solution of 1 mM butyric acid (pH 5.5) each day for 3 days. At each watering, plants were given enough liquid so that at least 50 ml of liquid flushed through the pot, which was then left to drain until the next watering. For treatment with CO2, 3week old seedlings were placed in an atmosphere containing either 0.03% (air) or 5% CO2 for 5 days under a 12-h photoperiod at an illumination of 200 lmol quanta m)2 s)1 at 25°C. Cucumber seedlings were also grown in aerated hydroponic culture in a greenhouse using 100% nitrate culture medium (4 mM KNO3 adjusted to pH 5.0 with KOH; Santamaria and Elia 1997). Three weeks after germination the solution was changed to 100% ammonium culture medium (4 mM NH4Cl adjusted to pH 5.0 with KOH; Santamaria and Elia 1997). This solution was changed each evening. For measurement of the amount of PEPCK present in different parts of cucumber, plants were grown in soil in a glasshouse during the summer in Bolton, UK. Plants were fed Growmore fertiliser (nitrate-based) once a week. Petal and carpel were sampled 2 days, and seed and fruit 28 days, after flower opening. Cotyledons were sampled 5 days and hypocotyl 7 days after imbibition of seed.
Preparation of extracts for measurement of PEPCK activity 0.2 g of tissue was ground in a mortar containing 800 ll of ice-cold 200 mM Bicine–KOH (pH 9.0), 50 mM DTT. The homogenate was clarified by centrifugation at 12,000 g for 5 min and the supernatant stored at )80°C.
Immunohistochemical studies Sections were prepared according to Walker et al. (1997). For visualisation of structure, resin and rehydrated paraffin sections were either incubated in 2 g l)1 Toluidine Blue, then rinsed with water or sections were incubated in 20 g l)1 Safranin O (25°C, 3 min), rinsed in water and then incubated in 5 g l)1 Astra Blue in 20 g l)1 tartaric acid (25°C, 10 s). For immunolocalisation, PEPCK and amino acids were visualised either using Nitroblue Tetrazolium or Fast Red TR (Sigma; Walker et al. 1997). Controls run with nonimmunised rabbit serum remained unstained. For Fast Red staining, sections were incubated (37°C, 30 min) in blocking solution (20 g l)1 BSA in Tris-buffered saline (100 mM Tris, 150 mM NaCl, pH 7.5). The primary polyclonal antiserum to PEPCK was raised in rabbits and used at a dilution of 1:1,000 (37°C, 2 h). The primary polyclonal anti-rabbit aspartate (Asp) and anti-rabbit glutamate (Glu) antisera (Sigma) were used at a dilution of 1:10,000 (37°C, 2 h). Sections were then incubated with anti-rabbit goat antibody– alkaline phosphatase conjugate (1:1,000) in blocking solution (37°C, 1 h). Finally the red colour was developed at 25°C in a substrate solution of Fast Red (Sigma). The microscope slides were washed in Tris-buffered saline with 5 ml l)1 Tween 20 (37°C, 15 min) between each step. After washing in water, the sections were mounted in glycerol jelly (BDH, UK) or DePeX (Fluka, Switzerland).
Source of antibodies The antiserum specific for PEPCK was raised against the enzyme purified from cotyledons of germinating cucumber seedlings (Walker et al. 1995). The antiserum specific for phosphoenolpyruvate carboxylase (PEPC) was raised in a rabbit against the pure enzyme from Amaranthus edulis L. leaves. The antiserum specific for NADP-malic enzyme (NADP-ME) was raised against the purified enzyme from maize (Zea mays L.) leaves (Langdale et al. 1988). The specificity of the antiserum was tested by ensuring it only recognised a polypeptide of the same size as the
50 enzyme when crude extracts of the tissue used in immunohistochemistry were analysed by immunoblots of SDS–PAGE gels (Chen et al. 2000).
Results PEPCK is present in many parts of cucumber plants Measurement of PEPCK activity, together with immunoblots (data not shown), showed that although PEPCK was most abundant in cotyledons of germinating seeds, in which it catalyses a gluconeogenic flux from stored lipid and protein (Leegood and ap Rees 1978), it was also present in many other tissues (Table 1). Although its abundance in these other tissues was much less than in cotyledons of germinating seeds, PEPCK was restricted to certain cell types (see below), and in these the abundance was therefore high. Structure of cucumber vasculature The structure of veins in cucumber leaves and cotyledons is shown diagramatically in Fig. 1. In the minor veins (Figs. 1b, 3a), the abaxial phloem elements are in symplastic connection with the mesophyll while the adaxial phloem elements are not (Fig. 1b; Turgeon et al. 1975). The adaxial and abaxial phloem elements are also present in the larger veins and the vascular bundles of petioles and stems (Figs. 1a, 2a,b). A third type of phloem is a network of solitary extra-fascicular phloem elements (Fig. 1a; Esau 1965). This is present in the petioles (Fig. 2a,f), around the larger veins in the cotyledons (Fig. 2b), and in the stems. These elements lie outside the main vascular bundles (Fig. 2a,b) and are also interconnected to each other and to the fascicular phloem by commissural phloem elements (Figs. 1b, 2a; Esau 1965). In the roots there is a more typical arrangement of the phloem (Fig. 2h). The phloem companion cells stain bright red with Safranin O/Astra Blue (Fig. 2a,f) or green with Toluidine Blue (Fig. 2b).
Table 1 Activity of PEPCK in different parts of cucumber (Cucumis sativus) plants. Measurements are means ± SE of three different extracts Plant part
Cotyledon following germination Mature leaf Root Petiole Hypocotyl Developing seed Petal Flesh of fruit Carpel
PEPCK activity U g)1 FW
U mg)1 protein
5.90±0.82
0.181±0.031
0.14±0.11 0.03±0.02 0.08±0.03 0.03±0.01 0.10±0.02 0.10±0.03 0.01±0.01 0.22±0.02
0.013±0.011 0.015±0.010 0.016±0.006 0.010±0.003 0.005±0.001 0.012±0.003 0.003±0.001 0.025±0.004
Fig. 1a, b Structure of a major vein (a) and a minor vein (b) of cucumber (Cucumis sativus)
PEPCK is present in cucumber phloem Immunolocalisation of PEPCK in cucumber leaves, photosynthetic cotyledons in which mobilisation of storage reserves was complete, and in petioles and stems, showed that it was prominent in the phloem. It was present throughout the extra-fascicular phloem elements of intermediate veins in cotyledons (Fig. 2d), in petioles (Fig. 2c,e) and stems (data not shown). In minor veins of both cotyledons (Fig. 3e,g) and of leaves (data not shown) it was present only in the adaxial, but not in the abaxial phloem. In larger veins of cotyledons (Fig. 2d) and leaves (data not shown), it was also present in the abaxial phloem. In petioles (Fig. 2c) and stems (data not shown) it was present in both internal and external fascicular phloem of vascular bundles. It c
Fig. 2a–h Structure of the vasculature and immunolocalisation of PEPCK in cucumber. The presence of PEPCK is indicated by blue (c–e, g) or red staining (h). a, c Vascular bundle in the petiole stained with Safranin and Fast Green (a) or for PEPCK protein (c). b, d Intermediate vein in a cotyledon stained with Toluidine Blue (b) or for PEPCK protein (d). e, f Longitudinal section of petiole showing a companion cell and sieve element in the extra-fascicular phloem stained for PEPCK protein (e) or with Safranin and Fast Green (f). All show the presence of PEPCK protein in the extrafascicular phloem. g, h Transverse section of a cucumber root, immunostained with Fast Red, showing the presence of PEPCK in the phloem companion cells (g) and a pre-immune control (h). ad Adaxial phloem of minor vein, ef extra-fascicular phloem, ce commissural element, c companion cell, se sieve element, x xylem, p fascicular phloem. Bars = 50 lm
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was also present in the phloem in the roots (Fig. 2h). PEPCK was particularly abundant in companion cells (Fig. 2e). PEPCK was also present in xylem parenchyma in intermediate veins of cotyledons (Fig. 2d) and vascular bundles of roots (Fig. 2h). At a dilution
of 1:1,000, the antibody for PEPCK was entirely specific for a polypeptide of the same molecular mass as PEPCK from cucumber cotyledons (Chen et al. 2000) and pre-immune rabbit serum gave no signal on immunoblots or sections (data not shown). PEPCK was
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Fig. 3a–h Structure of the vasculature and immunolocalisation of PEPCK, Asp and Glu, in cucumber. a Structure of a minor vein in the cotyledon, stained with Toluidine Blue. b Minor vein in a cotyledon immunostained for PEPCK protein, showing its presence in the adaxial phloem (also cut at a glancing angle on the right-hand side of the micrograph). c, d Immunolocalisation of Glu (c) and Asp (d) in the vascular bundle in the petiole. e–h Immunolocalisation of PEPCK (e, g), Glu (f) and Asp (h) in the minor vein in a cotyledon. ad Adaxial phloem of minor vein, ef extra-fascicular phloem, ic intermediary cell. Bars = 50 lm
not detected in any other cells in the roots, stems, cotyledon or leaves, except for the trichomes (Chen et al. 2000) and stomata. Amino acids are enriched in parts of the phloem Immunohistochemical methods have been widely used to visualise amino acids in vertebrates (Storm-Mathison
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Fig. 4 Increase of PEPCK in cucumber plants supplied with different nitrogen sources. Cucumber seedlings were grown in perlite for 5 days in the presence of either 4 mM NH4Cl, KNO3 or K-Asp. The abundance of PEPCK was assessed by both activity measurement and immunoblots of SDS–PAGE gels. The molecular mass of PEPCK was 74 kDa. Activity measurements (U g)1 fresh weight) are means of three separate extractions, for which standard errors were less than 10% of the mean. Loadings on gels correspond to 5 mg of tissue and antisera were used at a dilution of 1:1,000
Fig. 6a–c Promotion of PEPCK in plant tissues by treatments that result in acidification of the cytosol. Seedlings were grown in perlite and no nutrients were provided unless stated. Leaves were placed in an atmosphere containing 5% CO2 for 5 days (a), and roots were fed either 4 mM NH4Cl for 5 days (b) or 1 mM butyric acid for 3 days (c). The abundance of PEPCK, PEPC and NADP-ME was assessed by immunoblots of SDS–PAGE gels. Activity measurements (U g)1 fresh weight) of PEPCK shown above the blots are the mean of three separate extractions, for which standard errors were less than 10% of the mean. Loadings on gels correspond to 5 mg of tissue and antisera were used at a dilution of 1:1,000
Fig. 5a, b Increase of PEPCK in cucumber roots supplied with ammonium. Cucumber seedlings were grown in hydroponic culture in an aerated nutrient solution with or without 4 mM ammonium. a Activity measurements are means ± SE of three separate extractions. b The abundance of PEPCK, PEPC and NADP-ME protein was assessed by immunoblots of SDS–PAGE gels. Loadings on gels correspond to 5 mg of tissue and antisera were used at a dilution of 1:1,000
et al. 1983; Aoki et al. 1988; Ma et al. 1994) and in a limited number of plant tissues (Walker et al. 2001). Antibodies are raised against amino acids that have been coupled to a carrier protein using glutaraldehyde. Tissue fixation also involves the use of glutaraldehyde or formaldehyde to cross-link endogenous amino acids to tissue proteins. Glu and Asp were immunolocalised using commercially available specific antibodies. Their
specificity was checked as previously described (Walker et al. 2001). This involved two processes: (i) Various amino acids were linked to BSA and dot-blotted. Crossreaction only occurred between an antibody and its corresponding amino acid antigen. (ii) Cucumber leaves were infiltrated with solutions of Asp and Glu, followed by fixation and the preparation of sections. Both Glu and Asp were particularly prominent in the extrafascicular phloem in petioles (Fig. 3c,d) and in the adaxial phloem of the minor veins (Fig. 3f,h), in which immunostaining coincided with that of PEPCK (Fig. 3e,g). PEPCK is increased in roots supplied with ammonium chloride at low pH Cucumber plants grown in perlite and not supplied with nutrients were fed three different forms of nitrogen
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(ammonium, nitrate or Asp). Ammonium fed at pH 5.0 led to an 8-fold increase in PEPCK activity in the roots and stems, but there was little change in the leaf (Fig. 4). There was some increase in leaves by nitrate and Asp. However, the amount of total protein in these leaves, unlike those fed ammonium, increased and the specific activity of PEPCK was therefore unchanged. In leaves a large proportion of PEPCK is present in the trichomes (Chen et al. 2000). A time-course for cucumber roots (Fig. 5) showed a maximum increase of PEPCK protein and activity by ammonium after 5 days. In contrast, the abundance of both NADP-ME and PEPC protein declined. A similar large increase of PEPCK by ammonium fed at low pH was also observed in roots of turnip and tomato (Fig. 6b), in which PEPCK protein and activity were undetectable in the absence of ammonium (Fig. 6c) or in the presence of nitrate (Fig. 6b). PEPCK is increased by other treatments that may lower cytosolic pH It is well established that presenting NH4+ to a wide range of organisms may result in a decrease in intracellular pH (for discussion, see Walker and Chen 2002). A means of acidifying plant tissues was therefore employed that was independent of nitrogen metabolism. This was done either by placing cucumber plants in an atmosphere containing 5% CO2 (Savchenko et al. 2000) (although this may influence the recycling of nitrogen associated with photorespiration), or by feeding roots 1 mM butyric acid (Kurkdjian and Guern 1989). In leaves of cucumber, in which PEPCK was already present in the phloem and trichomes (Fig. 6a; Chen et al. 2000), treatment with 5% CO2 caused a 50% increase of PEPCK, and in turnip and tomato leaves, in which PEPCK protein and activity was undetectable in control leaves, PEPCK was detected. In roots, butyric acid caused a large increase of both PEPCK protein and activity (Fig. 6c), similar to that brought about by ammonium (Fig. 6b). In contrast to PEPCK, there were no comparably large changes in the abundance of either PEPC or of NADP-ME protein.
Discussion We have shown that PEPCK is present in leaves, roots, stems, flowers, fruit and developing seed of cucumber. PEPCK was present in the companion cells of the phloem of leaves, stems and roots; however, it was not detectable in all types of phloem within these structures. PEPCK is also present in the phloem in grape (Walker et al. 1999), pea and Arabidopsis (unpublished data) and in the CAM plant, Clusia minor (Borland et al. 1998). The presence of PEPCK in phloem is dependent on the part of the plant and its developmental and physiological state, e.g it is present in pea under some circumstances, but not others (Leegood and Walker 1999, and
data not shown), as also shown for glutamine synthetase and glutamate synthase (Tobin and Yamaya 2001). The vascular anatomy of the curcurbits has long been studied (Fischer 1884; Crafts 1932; Whiting 1938; Turgeon et al. 1975). Vascular bundles of most curcurbits are bicollateral throughout the plant, even within minor veins (Esau 1969; Turgeon et al. 1975). In addition to this fascicular phloem, a unique feature of most curcurbits is the presence of an extra-fascicular phloem that is not contained in the vascular bundles (Fischer 1884; Esau 1969). The extra-fascicular phloem is interconnected by commissural elements that also connect with the fascicular phloem (Crafts 1932; Esau 1969). Minor veins consist of a highly ordered series of cells (Turgeon and Webb 1976). An abaxial phloem is present consisting of sieve tubes and modified companion cells, termed intermediary cells. In contrast to the abaxial phloem there are very few plasmodesmata connecting the adaxial phloem with the symplast of the mesophyll (Turgeon et al. 1975; Schmitz et al. 1987). Between the two types of phloem are a parenchyma cell and tracheids. The structure of the sieve elements and companion cells of the extra-fascicular phloem and fascicular phloem can be distinguished on the basis of their morphology (Whiting 1938; Turgeon et al. 1975) and distribution and content of P-proteins (Evert et al. 1973; Smith et al. 1987; Dannenhoffer et al. 1997). In these respects the adaxial phloem resembles the extra-fascicular phloem (Turgeon et al. 1975). The distribution of PEPCK within the phloem of cucumber further highlights these similarities. It is not known whether the sieve tubes of the adaxial phloem are connected to the extrafascicular phloem. Although it is possible that the extra-fascicular phloem is not involved in the long-distance transport of assimilates (Smith et al. 1987), it is labelled when leaves are exposed to 14CO2 (Webb and Gorham 1964; see also Evert et al. 1973). By exposing leaves to 14CO2, Webb and Gorham (1964) provided evidence that the extrafascicular phloem allows transport between vascular bundles, and that this enables any mature exporting leaf to supply any young importing leaf, which is not possible in many plants. The immunohistochemical data shown in Fig. 3 support the notion that the extra-fascicular phloem is involved in the transport of amino acids. The presence of PEPCK in the adaxial, but not the abaxial, phloem also raises the question of their functions. In minor veins of cucurbits it has been shown that the abaxial phloem transports sugars and is loaded symplastically from intermediary cells, in which raffinose-family oligosaccharides are synthesised (Turgeon and Hepler 1989; Haritatos et al. 1996; Schaffer et al. 1996; Turgeon 1996). In contrast, amino acids may be loaded into curcurbit minor veins by a process requiring membrane transport, because plasma membrane vesicles prepared from zucchini leaves are able to transport amino acids but not sugars (Hsiang and Bush 1992). Turgeon et al. (1975) suggested that the adaxial phloem
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was involved in loading assimilates from the apoplast and Turgeon and Webb (1976) suggested that it may play a role in the import of assimilates early in leaf development. In support of this, fluorescein dye travels towards the leaf in the adaxial phloem and away from the leaf in the abaxial phloem in a curcurbit (Peterson and Currier 1969). In squash, the abaxial phloem and adaxial phloem may transport assimilates in either direction depending on the developmental stage of the leaf (Webb and Gorham 1965). Pristupa (1983) used histochemistry to show that the adaxial phloem of pumpkin contains little sugar. Schmitz et al. (1987) exposed leaves to 14CO2 and determined in which cells radiolabelled assimilates were present using microautoradiography. The adaxial phloem was labelled much less than the abaxial phloem, and it was suggested that the adaxial phloem was not involved in sugar transport. However, this does not exclude the possibility that it is involved in either the loading or transport of amino acids, because these are usually far less abundant in phloem than sugars (Winter et al. 1992; Lohaus et al. 1994). The immunohistochemical data shown in Fig. 3 offer support for the view that the adaxial and extrafascicular phloem are involved in the loading/transport of amino acids, either synthesised in the leaf or delivered to the leaf in the xylem (Fritz et al. 1983). To investigate the function of PEPCK in cucumber vasculature, experiments were done to determine whether its abundance was altered in response to changes in growth conditions. The abundance of PEPCK was greatly increased in roots and stems of seedlings fed NH4Cl below pH 5.0. In contrast, if plants were fed NH4Cl at above pH 6.0 there was no increase of PEPCK. The cytosolic pH of maize roots fed ammonium at pH 4.0 falls whereas, when fed at higher pH, cytosolic pH increases (Gerenda´s et al. 1990; Gerenda´s and Ratcliffe 2000). When ammonium is fed to roots in a solution at high pH a greater proportion exists as NH3; this diffuses into the cell and equilibrates with NH4+ by consumption of a proton. However, at low pH, uptake of NH4+ predominates. Assimilation of NH4+ into amino acids generates protons, which can lead to a lowering of cytosolic pH (for discussion see Gerenda´s and Ratcliffe 2000; Walker and Chen 2002). In stems and roots of cucumber fed NH4Cl at pH 5.0 the intensity of staining within the vasculature increased (data not shown). In addition, in roots there was an increase of PEPCK within the vascular bundle similar to that observed in the pericycle in maize and in these it may serve a similar function as that proposed for maize (Walker et al. 2001; Walker and Chen 2002). PEPCK was also increased in roots and stems fed butyric acid, and in leaves of plants grown in an atmosphere containing 5% CO2. These treatments are known to lower cytosolic pH of plant cells (Kurkdjian and Guern 1989). These acidifying treatments led to no increase of PEPC or of NADP-ME, which are other enzymes proposed to be involved in pH regulation in plants (Davies 1986); indeed, the amount of NADP-ME declined in roots
treated with ammonium. In a diverse range of organisms, PEPCK functions as a carboxylase in fermentation pathways under anaerobic conditions (see Walker and Chen 2002). However, anaerobiosis, which also leads to acidification, did not lead to an increase of PEPCK in roots of cucumber or maize (data not shown), consistent with the observation that malate and its derivatives are not a significant product of anaerobic fermentation in roots (Smith and ap Rees 1979). These and previous results (Walker et al. 1999, 2001; Walker and Chen 2002) indicate an association between the presence of PEPCK, the metabolism of nitrogenous assimilates and pH regulation. This raises the question as to the function of PEPCK in these processes. In both plants and animals the metabolism of nitrogenous compounds has a major effect on acid–base balance and production and consumption of NADH (Raven and Smith 1976; Atkinson and Bourke 1984; Jungas et al. 1992). In many vertebrates, acidosis leads to an induction of PEPCK in the proximal tubule cells of the kidney (Hanson and Reshef 1997). In these glutamine (Gln) is deaminated to ammonium and 2-oxoglutarate, ammonia then diffuses into the urine and combines with a proton to form ammonium. Protons remaining in the cell are consumed by the dissimilation of 2-oxoglutarate. This is achieved by its conversion to OAA, which is decarboxylated to PEP by PEPCK. This process (cataplerosis) involves the removal of carbon from the Krebs cycle, and is required when 4-, 5-, or 6-carbon intermediates enter the Krebs cycle, during the catabolism of amino and organic acids. If they were not removed, the concentration of cycle intermediates would increase, because each turn of the cycle only liberates two molecules of CO2 (Leegood and Walker 1999, 2003; Owen et al. 2002). PEP is then utilised by either gluconeogenesis or the Krebs cycle and both processes consume protons. In vertebrates, catabolism of amino acids is compartmentalized between organs. One reason for this is that it enables the amount of NADH produced by this process to be coordinated with an organ’s requirement (Jungas et al. 1992; Brosnan 2000). This involves the interconversion of amino acids in one organ and their transport to another, and PEPCK plays a role in a number of these interconversions. Lipogenesis in animals is similarly limited by the tissue’s ability to utilise the NADH generated (Flatt 1970). In sinks in plants, the metabolism of amino acids is quantitatively often a major process (Murray 1987). As in animals, it is important that the spectrum of nitrogenous compounds delivered to a tissue is such that their subsequent metabolism does not produce large pH perturbations and that the tissue is able to deal with the required production or consumption of NADH. The import of different amino acids and their conversion to those required for protein synthesis place different demands on NADH production/consumption. For example, although the import of either asparagine (Asn) or Gln into a tissue and their conversion to the amino acids present in proteins are equivalent in terms of
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acid–base balance, different rearrangements of carbon skeletons are necessary to support the synthesis of Asp and Glu which are precursors for the biosynthesis of nucleotides and many amino acids. More NADH is generated by the conversion of Asn, rather than Gln, to Asp and Glu. Cucumber plants grown on nitrate contained much less PEPCK in roots and stems than those grown on ammonium supplied at low pH. In stems, petioles and roots of cucumber, the xylem and phloem, in addition to functioning in the transport of assimilates through them, will provide assimilates for their own maintenance and for the growth of surrounding tissues. The composition of nitrogenous compounds in the phloem or xylem is dependent on the form in which nitrogen is supplied to the root (Pate 1980). If cucumber roots are fed ammonium, the form of nitrogen available from both xylem and phloem will be largely Gln, Asn and citrulline (Lindt and Feller 1987; Mitchell et al. 1992; Cramer et al. 1993). There is evidence that amino acids are catabolised within the vasculature. In rice, the ratio of Gln to Glu and of Asn to Asp is much higher in phloem close to the developing seeds than in the phloem of the leaf (Hayashi and Chino 1990). Lohaus et al. (1994) showed conversion of Glu to Gln and a net consumption of Glu and Gln in the phloem. Some of the ammonia released would likely diffuse out of these cells and be assimilated in nearby sinks. It is important to note that the synthesis of most amino acids from ammonia and glucose is proton neutral, whereas their synthesis from ammonium would produce a proton (Walker and Chen 2002). The protons arising in the phloem from loss of ammonia could then be consumed by metabolism of the carbon skeletons by either gluconeogenesis or the Krebs cycle. In contrast, if cucumber plants are fed nitrate this will be present in the xylem and may be utilised by stems and petioles for growth (Pate 1980; Lindt and Feller 1987). The assimilation of nitrate into proteins, unlike ammonium, produces hydroxyl ions and nitrate reduction requires NADH. In many plants, these hydroxyl ions are consumed by the synthesis of malic acid in the tissue assimilating nitrate, and malate is exported to the root. In the root, metabolism of malate produces hydroxyl ions, which are excreted to the soil (Raven and Smith 1976). In this situation there would be a lower requirement for PEPCK in stems or petioles because the metabolism of malate within them would generate alkalinity. Thus a number of factors, such as the potential for pH perturbations and for the over-production or underutilisation of NADH, mean that the form in which nitrogenous compounds are exported from a source may not be suitable for a sink. This means that extensive transformations may be required en route. It is likely that PEPCK functions in a number of transformations; in which it has three potential roles. The first, cataplerosis, is required, for example, when Asn or Gln are metabolized by the Krebs cycle. The second role is in redox balance, e.g. increases in NADH will result when
carbon skeletons from Asn are partially oxidized by the Krebs cycle rather than being utilized in gluconeogenesis. Evidence for these first two functions comes from developing pea seeds, in which extensive conversion of Asn to Gln, alanine and ammonium occurs during the transport of Asn to tissues in which proteins are synthesized, but a significant proportion of the carbon skeletons are converted to sugars by gluconeogenesis (Murray 1987). Similarly, in young maize plants, metabolism of Asn translocated to the shoots leads to the accumulation of neutral sugars (Cramer et al. 1993) The third role is in pH regulation, since gluconeogenesis and metabolism by the Krebs cycle are processes that consume protons and can therefore counteract acidity deriving from the metabolism of ammonium or amino acids (Walker and Chen 2002). PEPCK can potentially perform all these roles in concert, and we suggest that the use of amino acids as either respiratory or gluconeogenic substrates is far more widespread in plants than generally thought (Leegood and Walker 2003). Acknowledgments This research was supported by the Biotechnology and Biological Sciences Research Council, UK (Research Grant RSP07804, and by a David Phillips Fellowship to R.P.W.). Z.-H. Chen and R.P. Walker are joint first authors.
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