Journal of Experimental Botany, Vol. 52, No. 356, pp. 565-576,
April 2001
© 2001 Oxford University Press
Using immunohistochemistry to study plant metabolism: the examples of its use in the localization of amino acids in plant tissues, and of phosphoenolpyruvate carboxykinase and its possible role in pH regulation
1 Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UK
2 Istituto di Coltivazioni Arboree, Universita degli Studi di Perugia, via BorgoXX Guigno, 74-06121 Perugia, Italy
Received 31 March 2000; Accepted 5 December 2000
 |
Abstract |
|---|
 Top Abstract IntroductionLocalizing amino acids in...How immunohistochemistry is...ConclusionsReferences |
|---|
To understand many aspects of the metabolism of complex plant structures such as leaves, fruit and roots it is important to understand how metabolic processes are comparmentalized between tissues. The aim of this article is to show how immunohistochemistry, in conjunction with biochemical and physiological studies, is useful in understanding both the function of an enzyme in a tissue and metabolic processes occurring in plant tissues. This is illustrated by two examples. Firstly, the use of immunohistochemisty in the localization of amino acids in plant tissues is described. Secondly, the use of immunohistochemistry in understanding the function of an enzyme in a tissue and the metabolic processes occurring within the tissue is described. To illustrate this the example of phosophoenolpyruvate carboxykinase (PEPCK), an enzyme which is present in many plant tissues in which its function is unknown, is used. Evidence is provided that PEPCK may play a role in pH regulation in tissues active in the metabolism of nitrogen.
Key words: Phosphoenolpyruvate carboxykinase, amino acid localization, immunohistochemistry, nitrogen metabolism, pH regulation.
|
Introduction |
|---|
|
TopAbstractIntroductionLocalizing amino acids in...How immunohistochemistry is...ConclusionsReferences |
|---|
Why is it important to understand how metabolism is comparmentalized between plant tissues?
The aim of this article is to show how immunohistochemistry, in conjunction with biochemical and physiological studies, is useful in understanding both the function of an enzyme in a tissue and metabolic processes occurring in plant tissues. Complex plant structures such as leaves, fruit and roots are composed of a number of different tissues which in turn contain several types of cell. The metabolic processes occurring within these different tissues and cells are likely to vary greatly and an understanding of this is important to both understanding the metabolism of the plant structure and the role of individual enzymes within it. A good example of this is the compartmentation of photosynthetic processes between the mesophyll and bundle sheath in leaves of C4 plants (Hatch et al., 1975
; Edwards et al., 2001). However, in many plant structures our understanding of how metabolic processes are comparmentalized between different tissues is poor. Perhaps a reflection of this is the fact that the function of many enzymes in plant tissues is poorly understood. For example, phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.49; Walker et al., 1999), invertase (EC 3.2.1.26; Kingston-Smith et al., 1999), isocitrate lyase (EC 4.1.3.1; Nieri et al., 1997), pyruvate, orthophosphate dikinase (EC 2.7.9.1; Hudspeth et al., 1986), and NADP-malic enzyme (EC 1.1.1.40; NADP-ME; Famiani et al., 2000) are present in many tissues in which their function is uncertain.
How do we study cell specific metabolism?
For abundant cell types, such as the photosynthetic cells of leaves, approaches that have used the entire leaf have been very successful. On the other hand it is much more difficult to study the metabolism of low abundance cell types because metabolic processes occurring within them are masked by those of more abundant cell types. Many approaches have been used to study cell specific metabolism. One approach has been to isolate specific cell types, the isolation of mesophyll and bundle sheath cells from leaves of C4 plants was valuable in studying the C4 cycle (Hatch et al., 1975) and the isolation of aleurone tissue from germinating cereal seeds has been important in studying the interactions between plant hormones and metabolism (Fincher, 1989). Unfortunately, it is not possible to isolate most cell types at high yield and purity from plant tissues. A way round this problem has been to sample the contents of individual cells using, for example, single cell sampling (Oulaw and Zhang, 2001) the aphid stylet technique (Winter et al., 1992), microelectrodes (Walker et al., 1998; Miller et al., 2001) or pressure probes (Tomos and Leigh, 1999; Tomos and Sharrock, 2001). A number of approaches that enable the constituents of different cells to be visualized under the microscope, such as the visualization of radiolabelled metabolites by microautoradiography (Fritz et al., 1983), the expression of reporter genes in transgenic plants (Feuillet et al., 1995), the visualization of mRNA by in situ hybridization (Marrison and Leech, 1994; Facchini and De Luca, 1995), and the visualization of antigens such as proteins using specific antibodies by immunohistochemistry (Famiani et al., 2000) or tissue printing (Kingston-Smith and Pollock, 1996) have been widely used to study cell specific metabolism.
Using immunohistochemistry to study compartmentation of metabolism between cells and tissues
Immunohistochemistry utilizes antibodies to visualize antigens in sections of tissue under the microscope. Although immunohistochemistry has been used since 1950 to localize antigens in animal tissues it was not until 1970 that this technique was first used in plants (Perrot-Rechenmann and Gadal, 1986). Immunohistochemistry may be used in conjunction with light or electron microscopy. Light microscopy often possesses sufficient resolution to determine the distribution of an antigen between tissues and cell types (Walker et al., 1999; Famiani et al., 2000). Electron microscopy offers higher resolution and is particularly useful in determining the distribution of an antigen within a cell (Voznesenskaya et al., 1999). To prepare tissue for immunohistochemistry it is fixed, embedded and then sectioned. Sections are then incubated with an antibody specific for the antigen (primary antibody) and antibody binding visualized (for a review, see Perrot-Rechenmann and Gadal, 1986). For light microscopy, several media such as paraffin (Ishiyama et al., 1998), polyethylene glycol (Marrison and Leech, 1992) and resin (Voznesenskaya et al., 1999), have been used to embed plant tissues. The ideal embedding medium should preserve both the structure of the tissue and antigenicity of the antigen whilst being easy to use. Under the light microcope binding of primary antibody may be visualized in a number of ways. The primary antibody itself may be prelabelled with, for example, a fluorochrome. More commonly, binding of the primary antibody is detected by incubation with either a protein A or a second antibody conjugate which bind to the primary antibody. This latter approach has the advantage that it introduces an amplification step and also avoids an initial conjugation of the fluorochrome to the primary antibody, which may lower its affinity (Perrot-Rechenmann and Gadal, 1986). Protein A or the second antibody may be conjugated to a fluorochrome (Marrison and Leech, 1992), gold particles (Voznesenskayaet al., 1999) or an enzyme such as phosphatase (Walker et al., 1999) which allow its subsequent visualization. Regardless of the procedure followed it is essential to ensure that signals observed are due to the presence of the antigen. The tissue itself may give rise to a signal by, for example, autofluorescence or the presence of endogenous peroxidase or phosphatase activity that have not been inactivated by the fixation and embedding procedures (Perrot-Rechenmann and Gadal, 1986). The specificity of the primary antiserum is crucial and it may require affinity purification (Fig. 1A). A useful control is to determine whether the pattern of labelling is similar with crude and affinity purified antiserum (Hayakawa et al., 1999). An indication of specificity of the antiserum may be obtained by performing an immunoblot of an SDS-PAGE gel using an extract of the tissue which is to be studied in immunohistochemistry (Voznesenskaya et al., 1999). The preimmune serum may give rise to a signal in immunohistochemistry which should be checked (Holthaus and Schmitz, 1991). The assessment of the specificity of signals seen in immunohistochemistry is discussed in more detail below.
|
Immunohistochemistry has been widely used to localize antigens in plant tissues. The localization of enzymes by immunohistochemistry has provided valuable information about how metabolism is comparmentalized between different tissues in many plant structures such as the vasculature (Holthaus and Schmitz, 1991
; Facchini and de Luca, 1995) developing seeds (Muhitch et al., 1995; Ruan et al., 1997), fruit (Famiani et al., 2000), leaves and stems (Voznesenskaya et al., 1999), and roots (Ishiyama et al., 1998). To illustrate how immunohistochemistry may be of use in studying plant metabolism two examples of recent work, its use in localizing amino acids in plant tissues and its use both in understanding the function of an enzyme in a tissue and the metabolic processes occurring within that tissue are described.
|
Localizing amino acids in plant tissues using immunohistochemistry |
|---|
|
TopAbstractIntroductionLocalizing amino acids in...How immunohistochemistry is...ConclusionsReferences |
|---|
Immunohistochemistry using antibodies specific for different amino acids has been widely used to localize amino acids in animal tissues (Storm-Mathison et al., 1983
; Aoki et al., 1988). This procedure has been valuable in studying the distribution of neurones that utilize different amino acids as neurotransmitters in the central nervous system (Conti and Minelli, 1994) and in studying the process of amino acid reabsorption in the kidney (Ma et al., 1994). In contrast, this technique has been little used in plants (Walker et al., 1999). To obtain antibodies that recognize specifically a given amino acid, the amino acid is conjugated to a carrier protein which is then used as an immunogen. The specificity of the resulting antiserum may be assessed by dot blotting on nitrocellulose membrane (Fig. 1A
). This procedure involves applying different amino acid protein conjugates to nitrocellulose membrane. The membrane is then probed with an antiserum raised against one of the amino acid protein conjugates and antibody binding visualized in the same way as immunoblots of SDS-PAGE gels (Walker and Leegood, 1996). Antisera obtained are generally not specific and need affinity purification (Aoki et al., 1988; Ma et al., 1994; Fig. 1A). This lack of specificity is probably because the number of epitopes possessed by an amino acid is small and different amino acids possess some common epitopes. An antiserum against glutamate was prepared using the method of Aoki et al. (Aoki et al., 1988) and its specificity assessed by dot blotting (Ma et al., 1994). The crude antiserum was not specific for glutamate (Fig. 1A; this lack of specificity was also shown by antisera raised against aspartate, glutamine and asparagine conjugates, data not shown). The antibody against glutamate was purified by incubating 5 µl of crude antiserum with 10 µl each of aspartate, glutamine and asparagine rabbit serum albumin conjugates (prepared as described by Aoki et al., 1988) in 300 µl of 100 mM potassium phosphate buffer pH 7.4 at 25 °C for 2 h. After affinity purification the antibody only recognized glutamate on a dot blot (Fig. 1A). To assess the specificity of the antibody in immunohistochemistry, cucumber leaves were fixed using 3% glutaraldehyde in the absence of any amino acid or in the presence of either 5 mM glutamate or 5 mM alanine. Tissue fixed in the presence of glutamate was much more heavily stained (Fig. 1B). This technique was used to localize glutamate in a range of tissues such as developing grape seeds (Walker et al., 1999). This technique is potentially useful in studying many metabolic processes involving amino acids in plants and a number of antisera to different amino acids are available from commercial sources (see for example, www.immunologics.com). It remains to be determined whether this technique possesses sufficient sensitivity and specificity to be used to localize amino acids which are at low abundance in plant tissues. |
How immunohistochemistry is useful in understanding the functioning of plant tissues and the function of enzymes within them: the example of PEPCK |
|---|
|
TopAbstractIntroductionLocalizing amino acids in...How immunohistochemistry is...ConclusionsReferences |
|---|
PEPCK is present in many plant tissues in which its function is uncertain
In the cytosol of plant cells PEPCK catalyses the ATP-dependent decarboxylation of oxaloacetate, a reaction that lies at an important interface between lipid, amino acid, organic acid, and sugar metabolism. Perhaps a reflection of the importance of this reaction in plant metabolism is the finding that the activity of PEPCK is regulated by an elegant mechanism that involves reversible protein phosphorylation (Walker and Leegood, 1995
; Walker and Leegood, 1996; RP Walker et al., unpublished data). For many years two clearly defined roles for PEPCK in plant metabolism have been known, as a decarboxylase both in the C4 cycle in one subgroup of C4 plants and in many CAM plants (Hatch et al., 1975; Dittrich et al., 1973) and in catalysing the gluconeogenic flux from lipids and protein in germinating seeds (Benedict and Beevers, 1961; Stewart and Beevers, 1967). Recently, PEPCK was found to be present in a large range of plant tissues such as leaves of a number of C4 grasses, including maize, belonging to the NADP-ME group of C4 plants (Walker et al., 1997), in developing seeds, in ripening fruit, in vascular tissues and in leaves of C3 plants (Leegood and Walker, 1999; Leegood et al., 1999; Walker et al., 1999). Similarly, in animals, PEPCK is present in many tissues in which its function is uncertain (Zimmer and Magnuson, 1990; Hanson and Reshef, 1997). It seemed likely that knowing the location of PEPCK in plant tissues would be of assistance in understanding its function.
Choice of localization technique
For the study of PEPCK function a technique was required that could be used with a wide range of species and tissues and would show the location and abundance of the protein. An advantage of immunohistochemistry is that it reveals the location and abundance of the protein whereas visualization of mRNA by in situ hybridization may not. This is because the abundance of a protein and the mRNA encoding it are not always correlated (Oliveira et al., 1997; Ruan et al., 1997) and mRNA may be synthesized in different cells from where the protein is present (Kühn et al., 1997). Immunohistochemistry was routinely used, however, in situ hybridization (Fig. 1C) was also used as its main purpose was to confirm any results obtained were not artefactual. For immunohistochemistry wax embedded sections fixed using 2% (v/v) formaldehyde were used, this procedure gave good preservation of antigenicity and possessed sufficient resolution for determining intracellular compartmentation (Walker et al., 1999). Antigen was visualized using a phosphatase-linked second antibody in conjunction with a coloured dye reaction which gave good resolution visible using a standard light microscope (Walker et al., 1999). Similar considerations apply to in situ hybridization.
Assessing the specificity of staining in immunohistochemistry
It is essential to evaluate whether signals observed in immunohistochemistry are due to the presence of the target antigen. Perhaps the most important factor in avoiding artefacts in immunohistochemistry is the specificity of the antiserum. On blots of SDS-PAGE gels the molecular weight of immunoreactive polypeptides can be compared with that of the target polypeptide. On the other hand, the molecular weight of antigens visualized in immunohistochemistry is unknown which makes it more difficult to assess specificity. One check is to determine specificity for the target protein on blots of SDS-PAGE gels loaded with an extract of the tissue used in immunohistochemistry (Chen et al., 2000). The antiserum used in this study was raised against the 62 kDa fragment of PEPCK purified from cotyledons of germinating cucumber which shows high sequence similarity to PEPCK from other angiosperms and yeast (Walker et al., 1995). When used at a dilution of 1:1000 this antiserum recognized PEPCK specifically in extracts of most plant tissues when its abundance was only 0.01% of total protein and also recognized the enzyme from every angiosperm and gymnosperm tested as well as PEPCK from yeast (data not shown). The dilution of the antiserum is often important in determining specificity on blots of SDS-PAGE gels, if it is too concentrated non-specific bands may appear. In immunohistochemistry a range of dilutions of the antiserum should be tested to determine whether a similar pattern of staining occurs. A further check of specificity is to confirm results obtained by immunohistochemisty using a different technique. One way is microdissection of the tissue, in which the antigen was localized using immunohistochemistry, and immunoblot analysis or activity measurements of extracts of the dissected tissue. This approach confirmed that PEPCK is present in trichomes in cucumber leaves (Chen et al., 2000). Another way is to show a similar distribution of mRNA by in situ hybridization and protein by immunohistochemistry as shown for PEPCK in maize leaves (Fig. 1C). A further check of specificity is to show that the antiserum labels specifically a tissue in immunohistochemistry in which the antigen is known to be abundant. For example, PEPCK is very abundant in the bundle sheath of leaves of some C4 grasses and an antiserum raised to PEPCK only labelled this tissue (Walker et al., 1997; Fig. 1C). On the other hand, an antiserum to phosphoenolpyruvate carboxylase (EC 4.1.1.31; PEPC; Famiani et al., 2000), an enzyme which is very abundant in the mesophyll of maize, only labelled this tissue (Fig. 1C). Similarly, in developing grape seeds the amount of staining in immunohistochemistry correlated with the abundance of PEPCK at different stages of development (Walker et al., 1999). It is also important to show that serum taken from the animal before immunization does not contain antibodies that give rise to staining on immunoblots of SDS-PAGE gels or in immunohistochemistry. To illustrate this point it was found that in stems and petioles of several species of the Umbelliferae, such as celery, the antiserum only labelled a layer of cells that line a system of ducts which ramify throughout the plant body (Esau, 1936). However, immunoblots of SDS-PAGE gels and measurements of PEPCK activity failed to detect PEPCK in these tissues. It was found that the preimmune serum labelled these cells showing that staining was not due to the presence of PEPCK (data not shown). Another cause of artefactual staining is the tissue section itself generating a signal. Peroxidase-linked second antibodies were found to be unsuitable because peroxidases present in plant tissues were not inactivated by the fixation and embedding procedures used (Walker et al., 1999). In our hands phosphatase-linked second antibodies did not present this problem, neither did they bind to sections not incubated with primary antiserum.
PEPCK is enriched in tissues active in the metabolism of nitrogen
Using immunohistochemistry it was found that, in many plants, PEPCK was present in tissues that are likely to be active in the metabolism of nitrogen, such as developing seeds and the vasculature, and in these it was particularly abundant in regions where metabolism of amino acids is likely to be enhanced (Walker et al., 1999). In developing grape seeds PEPCK was present within the vasculature of the seed, the chalaza which adjoins the developing storage tissues and in a layer of specialized cells, termed the palisade, which is thought to be involved in both metabolism and distribution of imported assimilates before uptake by the developing storage tissues (Walker et al., 1999). In the palisade it was most abundant when the rate of storage protein deposition was greatest and the abundance was greatly influenced by the form of nitrogen supplied to the seeds during in vitro culture (Walker et al., 1999). Similarly, in maize PEPCK was abundant in the pedicel (data not shown), a tissue where amino acids are metabolized before import into the kernel (Muhitch et al., 1995). In pea, amino acids undergo extensive metabolism within the pod before transport into the seed (Murray and Cordova-Edwards, 1984) and PEPCK was particularly abundant in the vasculature of the pod (data not shown). A similar situation is found in fruit such as peach (data not shown) and grape (Fig. 2) where PEPCK is present in the vasculature where it may be involved in transformations of amino acids before redistribution to the seed.
|
In leaves and stems of grape and cucumber PEPCK is associated with the phloem (Leegood and Walker, 1999
), however, in barley and tobacco, grown on nitrate it is not. A particularly striking example is provided by cucumber in which PEPCK is abundant in companion cells of the extrafascicular phloem (Chen et al., 2000). The vasculature of curcurbits is unusual because the extrafascicular phloem ramifies throughout the plant and is not contained within vascular bundles (Esau, 1965). The function of the extrafascicular phloem has remained uncertain, however, evidence has recently been provided that it is involved in the transport of amino acids (Leegood et al., 1999). In ammonium-fed roots of a range of plants PEPCK was associated with the vasculature (see below). In cucumber and tobacco PEPCK was present in trichomes (Leegood et al., 1999) and in Clusia aripoensis in cells lining the latex producing ducts (Borland et al., 1998). These tissues may be involved in the protection of the plant from herbivores and are often rich in secondary metabolites (Gershenzon et al., 1989). Reassimilation of ammonium released by the action of phenylalanine ammonium lyase, which is important in the synthesis of many secondary metabolites is likely to occur in these tissues.
Immunohistochemistry proved to be useful in developing our understanding of the function of PEPCK in plants because it showed that PEPCK was present in many tissues likely to be active in the metabolism of nitrogen. However, to investigate what, if any, this function might be in nitrogen metabolism required immunohistochemistry to be used in combination with other approaches. Similarly, although the location of PEPCK has been determined by immunohistochemistry in many mammalian tissues (Zimmer and Magnuson, 1990), immunohistochemistry alone has not allowed its functions to be determined and the function of PEPCK in most mammalian tissues remains unknown (Hanson and Reshef, 1997). This is perhaps a reflection of the difficulty of elucidating enzyme function since PEPCK has been extensively studied in animals (Hanson and Reshef, 1997). This difficulty in understanding enzyme function may be a reflection of our lack of understanding of many metabolic processes.
|
Altering the nitrogen metabolism of roots changes the abundance of PEPCK
The association of PEPCK with many tissues active in the metabolism of nitrogen raised the possibility that PEPCK may have a role in nitrogen metabolism. If so, how should this role be determined? One approach is to determine whether the abundance of PEPCK is altered by changes in nitrogen metabolism and a good way to do this is to feed roots different forms of nitrogen such as ammonium or nitrate. Feeding NH4Cl as opposed to KNO3 led to a large increase in the abundance of PEPCK in the roots of several plants (Fig. 3A
) much less being induced in the shoot tissues (data not shown).
It is well established that presenting NH4+/NH3 to a wide range of organisms may result in changes in the internal pH of their tissues (Boyarsky et al., 1988; Pollock 1989; Gerendás and Ratcliffe, 2000). The effect of supplied NH4+/NH3 on intracellular pH is complex and dependent on a number of factors such as the pH of the external medium, which alters the ratio of NH4+:NH3, type of tissue and the duration of feeding. For example, in tissue culture experiments using mammalian cells NH4Cl is often used to induce intracellular acidosis (Boyarsky et al., 1988). On the other hand, presenting NH4+/NH3 at alkaline pH to maize root tips results in intracellular alkalinization because of a rapid influx of NH3 by free diffusion and its subsequent protonation (Gerendás and Ratcliffe, 2000). In contrast, presenting NH4+/NH3 at pH 5.0 resulted in no change in cytosolic pH in maize root hairs (Kosegarten et al., 1997). After absorption of NH4+/NH3, its subsequent metabolism is thought to release protons (Raven and Smith, 1976; Raven, 1988; Bungard et al., 1999) a process that has been proposed to lead to the lowering of cytoplasmic pH in maize root tips grown on acidic media (Gerendás et al., 1993). The pH of extracts of maize roots, shown in Fig. 3B fed 4 mM ammonium for 7 d as opposed to 4 mM nitrate, prepared by homogenizing 0.1 g of root in 1 ml of water were lower by about 0.5 pH units. This raised the possibility that the appearance of PEPCK in roots fed ammonium was related to a lowering of intracellular pH within the root.
Some support for this idea is provided by the observation that in many mammalian and bird species cytosolic PEPCK is induced in the kidney in response to acidosis resulting from either oral administration of NH4Cl, starvation or diabetes (Watford, 1989; Mapes and Watford, 1989; Pollock, 1989). The promoter of cytosolic PEPCK from mammals contains elements that increase its transcription in response to acidosis (Holcomb et al., 1996; Cassuto et al., 1997). In the proximal tubule cells of the kidney of acidotic mammals deamination of glutamine and glutamate releases NH4+, NH3 then diffuses into the tubule to titrate the acidity of the urine (Hanson and Patel, 1993). In the tubule cell the anionic products of ammoniagenesis are then converted to neutral compounds which removes protons from the organism (Silbernagl and Scheller, 1986). Co-ordinate with the induction of PEPCK in rat kidney, the malate pool declined, on the other hand in dog kidney, in which PEPCK is not induced by acidosis, malate content increased several fold, suggesting that PEPCK is involved in malate dissimilation (Vinay et al., 1980). The importance of PEPCK in the acidotic kidney is illustrated by the observation that feeding the inhibitor of PEPCK, 3-mercaptopicolinate, led to a virtual abolition of ammonia formation in the acidotic kidney of rat (Bennett and Alleyne, 1976).
Other treatments that cause acidification of plant tissues induce PEPCK
To investigate whether the presence of PEPCK was related to acidification, a means of acidifying plant tissues was needed that was less dependent on nitrogen metabolism. This was done in two ways, either by placing plants in an atmosphere containing 5% CO2 (although this will affect nitrogen metabolism associated with photorespiration) or by feeding roots 1 mM butyric acid. These treatments are known to cause acidification of both plant and animal tissues (for review see Kurkdjian and Guern, 1989). In a range of plant species 5% CO2 caused an induction of PEPCK in the leaves (Fig. 4A) and butyric acid caused an induction of PEPCK in the roots (Fig. 4B) and in neither was there an increase in the abundance of NADP-ME as detected by immunoblots of SDS-PAGE gels (data not shown) using an antibody specific for NADP-ME (Langdale et al., 1988). These results suggested that in these tissues PEPCK may appear in response to acidification.
|
Does PEPCK have a role in regulating intracellular pH?
Having shown that PEPCK is induced in several tissues that are likely to be acidotic the authors investigated whether PEPCK might have a function in acidotic tissues. This was done by using immunohistochemistry to localize PEPCK in maize root fed ammonium. It is thought that plant cells regulate their pH in two main ways, by proton coupled pumps at the plasma membrane and by a biochemical pH stat (Davies, 1986; Sakano, 1998). The biochemical pH stat involves the synthesis and often transport of malate between tissues followed by its decarboxylation. It is proposed that PEPC is responsible for the synthesis of malate and NAD(P)-malic enzyme for its decarboxylation (Davies, 1986). The decarboxylation of malate, subsequent respiratory oxidation of NADH (produced by the reaction of malic enzyme) and accompanying oxidative phosphorylation is a process which consumes protons (Sakano, 1998). As an example, leaves that are assimilating nitrate produce OH- as a consequence of the nitrate reductase reaction, malic acid is synthesized by PEPC in the leaf and malate is exported to the root as the neutral salt, this leaves protons behind in the leaf to neutralize alkalinity (Touraine et al., 1992). Similarly, acidification of plant cells often results in a rapid decrease in their malate content, and the consumption of protons associated with the decarboxylation of malate is proposed to counteract acidity (Mathieu et al., 1986; Sakano et al., 1998). The importance of the decarboxylation of organic acids in neutralizing acidity is also illustrated by the fact that the major source of alkali in the human diet are salts of organic acids contained in fruit whose decarboxylation produce alkalinity (Ganong, 1977).
A possibility is that in acidotic tissue PEPCK functions to decarboxylate oxaloacetate, which is derived from malate by the action of malate dehydrogenase (the conversion of malate to oxaloacetate produces NADH). This process would consume protons in a similar way to the malic enzyme reaction and PEPCK might therefore be functioning as a decarboxylase in the biochemical pH stat. This was investigated in more detail in maize. Supplying ammonium to maize roots led to a very large induction of PEPCK (Fig. 3B) and its specific activity (0.3 units activity mg-1 protein; 1 unit of activity is defined as 1 µmol of product produced min-1 at 25 °C) was similar to that in maize leaves in which it functions as a decarboxylase in the C4 cycle (Wingler et al., 1999). In the absence of ammonium PEPCK could not be detected (Fig. 3B). The abundance of PEPC also increased in these roots whereas the abundance of NADP-ME, which is proposed to act as the decarboxylase in the biochemical pH stat, declined (Fig. 3B). Immunohistochemistry showed PEPCK to be very abundant in the pericycle, a layer of cells enclosed by the endodermis (Fig. 2), in contrast, PEPC was more abundant in the cortex (Fig. 2; for structure see Esau, 1965). In roots not fed ammonium PEPCK was not detected in the pericycle in immunohistochemistry (data not shown). Preimmune sera for both antisera to PEPCK and PEPC did not give rise to a signal and in situ hybridization showed PEPCK mRNA to be localized in the pericycle (data not shown).
Feeding ammonium to rice roots results in a rapid induction of NADH-dependent glutamate synthase in the epidermis and exodermis, the two cell layers of the root surface. The presence of NADH-dependent glutamate synthase and glutamine synthase, together with the observation that rice root possesses a Casparian strip between the exodermis and cortex which may restrict apoplastic movement of water and solutes absorbed from the soil, suggests that a large proportion of ammonium is assimilated in these tissues (Ishiyama et al., 1998; Tobin and Yamaya, 2001). In maize roots grown in hydroponic culture, and therefore lacking an exodermis (Zimmermann et al., 2000), water and solutes may move through the apoplast in the outer cortical regions of the root before reaching the endodermis, which may limit water and solute flow to some extent. The pH of the apoplast of the cortex of maize roots is maintained at around pH 5 in the presence of either nitrate or ammonium and when the pH of the solution external to the roots is as high as 8.6 (Kosegarten et al., 1999). At pH 5.0 a large proportion of the ammonium within the apoplast of the root would be available as NH4+ and would not cause intracellular alkalinization (see above). It is possible that a proportion of ammonium fed to maize roots enters the symplast at the endodermis and is incorporated into amino acids within the pericycle which results in it becoming acidotic. Extrusion of protons from the pericycle may not be effective in their removal to the soil because of the potential barrier to apoplastic movement at the endodermis. The function of PEPCK in the pericycle of maize could be, in concert with malate dehydrogenase, to decarboxylate malate and hence neutralize acidity by consuming protons. The observation that PEPC is most abundant in the cortex raises the possibility that a proportion of malate is synthesized in the cortex by PEPC and then transported to the pericycle as the neutral salt which is then decarboxylated by PEPCK, a mechanism which is similar in some ways to the C4 photosynthetic cycle of maize leaves (Fig. 1C).
The question arises as to whether PEPCK might be involved in pH regulation in other tissues active in the metabolism of nitrogen such as developing seeds. PEPCK is abundant in many developing seeds such as grape in which its abundance correlates with both the rate of storage protein synthesis and form of amino acid supplied to the seed (Walker et al., 1999). In pea pods, imported amino acids are metabolized to different forms before export to the developing seed (Murray and Cordova-Edwards, 1984). In the vasculature of pea pod and other fruits such as peach and grape (Fig. 2) PEPCK is very abundant. A possibility is that in the vasculature of fruit tissues and the maternal tissues of some seeds such as grape (Walker et al., 1999) amino acids are converted to forms whose subsequent metabolism within the developing filial tissues of the seed does not lead to perturbations in pH. Similar to roots, a possibility is that within the maternal tissues of the developing seed and the vasculature of the fruit PEPCK might function as a decarboxylase in the biochemical pH stat.
|
Conclusions |
|---|
|
TopAbstractIntroductionLocalizing amino acids in...How immunohistochemistry is...ConclusionsReferences |
|---|
In this article it has been shown how immunohistochemistry is useful in determining the location of both amino acids and enzymes in plant tissues and how knowing the location of an enzyme helps us to understand both its function and the functioning of plant tissues. The example of PEPCK was used, an enzyme which has recently been shown to be present in many plant tissues in which its function is uncertain. Using immunohistochemistry in conjunction with biochemical approaches preliminary evidence was provided that PEPCK may be involved in the regulation of intracellular pH in tissues active in the metabolism of nitrogen. If PEPCK does act as a decarboxylase in the biochemical pH stat, then this raises the possibility that, as in C4 photosynthesis, either PEPCK, NAD-malic enzyme or NADP-malic enzyme may be used to decarboxylate organic acids, which one is used may depend on the tissue and the cause of acidosis.
|
Acknowledgments |
|---|
This research was supported by a BBSRC David Phillips Fellowship (RPW), a BBSRC Studentship to RPW (KEJ) and Research Grants CO5229 and RSP07804, and by the CNR (National Research Council of Italy) (FF).
|
Notes |
|---|
3 Joint first authors.
4 To whom correspondence should be addressed. Fax: +44 114 2220002. E-mail: rob.walker{at}sheffield.ac.uk
|
Abbreviations |
|---|
PEPC, phosphoenolpyruvate carboxylase; PEPCK, phosphoenolpyruvate carboxykinase; NADP-ME, NADP-malic enzyme.
|
References |
|---|
|
TopAbstractIntroductionLocalizing amino acids in...How immunohistochemistry is...ConclusionsReferences |
|---|
Aoki E, Semba R, Keino H, Kato K, Kashiwamata S. 1988. Glycine-like immunoreactivity in the rat auditory pathway. Brain Research 442, 63–71.[Web of Science][Medline]
Benedict CR, Beevers H. 1961. Formation of sucrose from malate in germinating castor beans. I. Conversion of malate to phosphoenolpyruvate. Plant Physiology 36, 540–544.
Bennett FI, Alleyne GAO. 1976. Gluconeogenesis and ammoniagenesis in rat kidney: the effect of 3-mercaptopicolinic acid. FEBS Letters 65, 215–219.[Web of Science][Medline]
Borland A, Tecsi LI, Leegood RC, Walker RP. 1998. Inducibility of Crassulacean acid metabolism (CAM) in Clusia species; physiological/biochemical characterisation and intercellular localization of carboxylation processes in three species which exhibit different degrees of CAM. Planta 205, 342–351.
Boyarsky G, Ganz MB, Sterzel RB, Boron WF. 1988. pH regulation in single glomerular mesangial cells. I. Acid extrusion in abscence and presence of HCO3-. American Journal of Physiology 255, C844–C856.
Bungard RA, Wingler A, Morton JD, Andrews M, Press MC, Scholes JD. 1999. Ammonium can stimulate nitrate and nitrite reductase in the absence of nitrate in Clematis vitalba. Plant, Cell and Environment 22, 856–859.
Cassuto H, Olswang Y, Livoff AF, Nechushtan H, Hanson RW, Reshef L. 1997. Involvement of HNF-1 in the regulation of phosphoenolpyruvate carboxykinase gene expression in kidney. FEBS Letters 412, 597–602.[Web of Science][Medline]
Chen Z-H, Walker RP, Tecsi L, Wingler A, Leegood RC. 2000. Does phosphoenolpyruvate carboxykinase catalyse a gluconeogenic flux during the senescence of barley leaves or cucumber cotyledons? Plant Cell Physiology 41, 960–967.
Conti F, Minelli A. 1994. Glutamate immunoreactivity in rat cerebral cortex is reversibly abolished by 6-diazo-5-oxo-L-norleucine (DON), an inhibitor of phosphate-activated glutaminase. The Journal of Histochemistry and Cytochemistry 42, 717–726.
Davies DD. 1986. The fine control of cytosolic pH. Physiologia Plantarum 67, 702–706.
Dittrich P, Campbell WH, Black CC. 1973. Phosphoenolpyruvate carboxykinase in plants exibiting Crassulacean acid metabolism. Plant Physiology 52, 357–361.
Edwards GE, Franceschi VR, Ku MSB, Voznesenskaya EV, Pyankov VI, Andreo CS. 2001. Compartmentation of photosynthesis in cells and tissues of C4 plants. Journal of Experimental Botany 52, 577–590.
Esau K. 1936. Ontogeny and structure of collenchyma and of vascular tissues in celery petioles. Hilgardia 10, 431–476.
Esau K. 1965. Plant anatomy. New York: Wiley.
Facchini PJ, De Luca V. 1995. Phloem-specific expression of tyrosine/dopa decarboxylase genes and the biosynthesis of isoquinoline alkaloids in opium poppy. The Plant Cell 7, 1811–1821.[Abstract]
Famiani F, Walker RP, Tecsi L, Chen Z-H, Proietti P, Leegood RC. 2000. An immunohistochemical study of the compartmentation of metabolism during the development of grape (Vitis vinifera L.) berries. Journal of Experimental Botany 51, 675–683.
Feuillet C, Lauvergeat V, Deswarte C, Pilate G, Boudet A, Grima-Pettenati J. 1995. Tissue- and cell-specific expression of a cinnamyl alcohol dehydrogenase promoter in transgenic poplar plants. Plant Molecular Biology 27, 651–667.[Web of Science][Medline]
Fincher GB. 1989. Molecular and cellular biology associated with endosperm mobilization in germinating cereal grains. Annual Review of Plant Physiology and Molecular Biology 40, 305–346.[Web of Science]
Fritz E, Evert RF, Heyser W. 1983. Microautoradiographic studies of phloem loading and transport in the leaf of Zea Mays L. Planta 159, 193–206.
Ganong WF. 1977. Review of medical physiology, 8th edn. Los Altos: Lange Medical Publications.
Gerendás J, Ratcliffe RG. 2000. Intracellular pH regulation in maize root tips exposed to ammonium at high external pH. Journal of Experimental Botany 51, 207–219.
Gerendás J, Ratcliffe RG, Sattelmacher B. 1993. Relationship between intracellular pH and N metabolism in maize (Zea mays L.) roots. Plant and Soil 155/156, 167–170.
Gershenzon J, Maffei M. Croteau R. 1989. Biochemical and histochemical localization of monoterpene biosynthesis in the glandular trichomes of spearmint (Mentha spicata). Plant Physiology 89, 1351–1357.
Hanson RW, Patel YM. 1993. Phosphoenolpyruvate carboxykinase (GTP): the gene and the enzyme. Advances in Enzymology and Related Areas of Molecular Biology 69, 203–281.
Hanson RW, Reshef L. 1997. Regulation of phosphoenolpyruvate carboxykinase (GTP) gene expression. Annual Review of Biochemistry 66, 581–611.[Web of Science][Medline]
Hatch MD, Kagawa T, Craig S. 1975. Subdivision of the C4-pathway species based on differing C4 acid decarboxylating systems and ultrastructural features. Australian Journal of Plant Physiology 2, 111–128.
Hayakawa T, Hopkins L, Peat LJ, Yamaya T, Tobin AK. 1999. Quantitative intercellular localization of NADH-dependent glutamate synthase protein in different types of root cells in rice plants. Plant Physiology 119, 409–416.
Holcomb T, Liu WL, Snyder R, Shapiro R, Curthoys NP. 1996. Promoter elements that mediate the pH response of PCK mRNA in LLC-PK1-F+ cells. American Journal of Physiology—Renal Fluid and Electrolyte Physiology 271, F340–F346.
Holthaus U, Schmitz K. 1991. Distribution and immunolocalization of stachyose synthase in Cucumis melo L. Planta 185, 479–486.
Hudspeth RL, Glackin CA, Bonner J, Grula JW. 1986. Genomic and cDNA clones for maize phosphoenolpyruvate carboxylase and pyruvate, orthophosphate dikinase: Expression of different gene-family members in leaves and roots. Proceedings of the National Academy of Sciences, USA 83, 2884–2888.
Ishiyama K, Hayakawa T, Yamaya T. 1998. Expression of NADH-dependent glutamate synthase protein in the epidermis and exodermis of rice roots in response to the supply of ammonium ions. Planta 204, 288–294.[Web of Science][Medline]
Kingston-Smith AH, Pollock CJ. 1996. Tissue-level localization of acid invertase in leaves and its potential for the regulation of carbon export. New Phytologist 134, 423–432.
Kingston-Smith AH, Walker RP, Pollock CJ. 1999. Invertase in leaves: conundrum or control point? Journal of Experimental Botany 50, 735–743.
Kosegarten H, Grolig F, Wieneke J, Wilson G, Hoffmann B. 1997. Differential ammonia-elicited changes of cytosolic pH in root hair cells of rice and maize as monitored by 2',7'-bis-(2-carboxyethyl)-5 (and-6)-carboxyfluorescein- fluorescence ratio. Plant Physiology 113, 451–461.[Abstract]
Kosegarten H, Grolig F, Esch A, Glüsenkamp K-H, Mengel K. 1999. Effects of NH4+, NO3- and HCO3- on apoplast pH in the outer cortex of root zones of maize, as measured by the fluorescence ratio of fluorescein boronic acid. Planta 209, 444–452.[Web of Science][Medline]
Kühn C, Franceschi VR, Schulz A, Lemoine R, Frommer WB. 1997. Macromolecular traffiking indicated by localization and turnover of sucrose transporters in enucleate sieve elements. Science 275, 1298–1300.
Kurkdjian A, Guern J. 1989. Intracellular pH: measurement and importance in cell activity. Annual Review of Plant Physiology and Molecular Biology 40, 271–303.[Web of Science]
Langdale JA, Rothermel BA, Nelson T. 1988. Cellular patterns of photosynthetic gene expression in developing maize leaves. Genes and Development 2, 105–115.
Leegood RC, Walker RP. 1999. Phosphoenolpyruvate carboxykinase in plants: its role and regulation. In: Bryant JA, Burrell MM, Kruger NJ, eds. Plant carbohydrate biochemistry. Oxford: Bios Scientific Publishers, 201–213.
Leegood RC, Acheson RM, Técsi LI, Walker RP. 1999. The many-faceted function of phosphoenolpyruvate carboxykinase in plants. In: Kruger NJ, Hill SA, Ratcliffe RG, eds. Regulation of primary metabolic pathways in plants. Dordrecht: Kluwer Academic Publishers, 37–51.
Ma N, Aoki E, Semba R. 1994. An immunohistochemical study of aspartate, glutamate and taurine in rat kidney. The Journal of Histochemistry and Cytochemistry 42, 621–626.
Mapes RE, Watford M. 1989. Effects of metabolic acidosis and diabetes on the abundance of specific renal mRNAs. International Journal of Biochemistry 21, 297–305.
Marrison JL, Leech RM. 1992. Co-immunolocalization of topoisomerase II and chloroplast DNA in developing, dividing and mature wheat chloroplasts. The Plant Journal 2, 783–790.
Marrison JL, Leech RM. 1994. The subcellular and intra-organelle recognition of nuclear and chloroplast transcripts in developing leaf cells. The Plant Journal 6, 605–614.
Mathieu Y, Guern J, Pean M, Pasquier C, Beloeil J-C, Lallemand J-Y. 1986. Cytoplasmic pH regulation in Acer pseudoplatanus cells. II. Possible mechanisms involved in pH regulation during acid-load. Plant Physiology 82, 846–852.
Miller AJ, Cookson SJ, Smith SJ, Wells DM. 2001. The use of microelectrodes to investigate compartmentation and the transport of metabolized inorganic ions in plants. Journal of Experimental Botany 52, 541–549.
Muhitch MJ, Felker FC, Taliercio EW, Chourey PS. 1995. Immunolocalization of a unique form of maize kernel glutamine synthetase using a monoclonal antibody. Plant Physiology 107, 757–763.[Abstract]
Murray DR, Cordova-Edwards M. 1984. Amino acid and amide metabolism in the hulls and seeds of developing fruits of garden pea, Pisum sativum L. II. Asparagine. New Phytologist 97, 253–260.
Nieri B, Ciurli A, Pistelli L, Smith SM, Alpi A, De Bellis L. 1997. Glyoxylate cycle enzymes in seedlings and in mature plants of tomato (Lycopersicon esculentum Mill). Plant Science 129, 39–47.
Oliveira DO, Nacimento JR, Cordenunsi BR, Lajolo FM, Alcocer MJC. 1997. Banana sucrose-phosphate synthase gene expression during fruit ripening. Planta 203, 283–288.[Web of Science][Medline]
Outlaw Jr WH, Zhang S. 2001. Single-cell dissection and microdroplet chemistry. Journal of Experimental Botany 52, 605–614.
Perrot-Rechenmann C, Gadal P. 1986. Enzyme immunocytochemistry. In: Wang TL, ed. Immunology in plant science. Cambridge: Cambridge University Press, 59–88.
Pollock AS. 1989. Induction of renal phosphoenolpyruvate carboxykinase mRNA: suppressive effect of glucose. American Journal of Physiology 257, F145–F151.
Raven JA. 1988. Acquisition of nitrogen by the shoots of land plants: its occurrence and implications for acid-base regulation. New Phytologist 109, 1–20.
Raven JA, Smith FA. 1976. Nitrogen assimilation and transport in vascular land plants in relation to intracellular pH regulation. New Phytologist 76, 415–431.
Ruan Y-L, Chourey PS, Delmer DP, Perez-Grau L. 1997. The differential expression of sucrose synthase in relation to diverse patterns of carbon partitioning in developing cotton seed. Plant Physiology 115, 375–385.[Abstract]
Sakano K. 1998. Revision of biochemical pH-stat: involvement of alternative pathway metabolisms. Plant Cell Physiology 39, 467–473.
Sakano K, Kiyota S, Yazaki Y. 1998. Degradation of endogenous organic acids induced by Pi uptake in Catharanthus roseus cells: involvement of the biochemical pH-stat. Plant Cell Physiology 39, 615–619.
Santamaria P, Elia A. 1997. Producing nitrate-free endive heads: effect of nitrogen form on growth, yield and ion composition of endive. Journal of the American Society of Horticultural Science 122, 140–145.
Silbernagl S, Scheller D. 1986. Formation and excretion of NH3- NH4+. New aspects of an old problem. Klinische Wochenschrift 64, 862–870.[Web of Science][Medline]
Stewart CR, Beevers H. 1967. Gluconeogenesis from amino acids in germinating castor bean endosperm and its role in transport to the embryo. Plant Physiology 42, 1587–1595.
Storm-Mathison J, Leknes AK, Bore AT, Vaaland JL, Edminson P, Haug F-MS, Ottersen OP. 1983. First visualization of glutamate and GABA in neurones by immunocytochemistry. Nature 301, 517–520.[Medline]
Tobin AK, Yamaya T. 2001. Cellular compartmentation of ammonium. Journal of Experimental Botany 52, 591–604.
Tomos AD, Leigh RA. 1999. The pressure probe: a versatile tool in plant cell physiology. Annual Review of Plant Physiology and Plant Molecular Biology 50, 447–472.[Web of Science]
Tomos AD, Sharrock RA. 2001. Cell sampling and analysis (SiCSA): metabolites measured at single cell resolution. Journal of Experimental Botany 52, 623–630.
Touraine B, Muller B, Grignon C. 1992. Effect of phloem-translocated malate on NO-3 uptake by roots of intact soybean plants. Plant Physiology 99, 1118–1123.
Vinay P, Allignet E, Pichette C, Watford M, Lemieux G, Gougoux A. 1980. Changes in renal metabolite profile and ammoniagenesis during acute and chronic metabolic acidosis in dog and rat. Kidney International 17, 312–325.[Web of Science][Medline]
Voznesenskaya EV, Franceschi VR, Pyankov VI, Edwards GE. 1999. Anatomy, chloroplast structure and compartmentation of enzymes relative to photosynthetic mechanisms in leaves and cotyledons of species in the tribe Salsoleae (Chenopodiaceae). Journal of Experimental Botany 50, 1779–1795.
Walker RP, Acheson RM, Técsi LI, Leegood RC. 1997. Phosphoenolpyruvate carboxykinase in C4 plants: its role and regulation. Australian Journal of Plant Physiology 24, 459–468.
Walker DJ, Black CR, Miller AJ. 1998. The role of cytosolic potassium and pH in the growth of barley roots. Plant Physiology 118, 957–964.
Walker RP, Chen Z-H, Técsi LI, Famiani F, Lea PJ, Leegood RC. 1999. Phosphoenolpyruvate carboxykinase plays a role in interactions of carbon and nitrogen metabolism during grape seed development. Planta 210, 9–18.[Web of Science][Medline]
Walker RP, Leegood RC. 1995. Purification and phosphorylation in vivo and in vitro, of phosphoenolpyruvate carboxykinase from cucumber cotyledons. FEBS Letters 362, 70–74.[Web of Science][Medline]
Walker RP, Leegood RC. 1996. Phosphorylation of phosphoenolpyruvate carboxykinase in plants: studies in plants with C4 photosynthesis and Crassulacean acid metabolism and in germinating seeds. Biochemical Journal 317, 653–658.
Walker RP, Trevanion SJ, Leegood RC. 1995. Phosphoenolpyruvate carboxykinase from higher plants: purification from cucumber and evidence of rapid proteolytic cleavage in extracts from a range of plant tissues. Planta 196, 58–63.
Watford M. 1989. Hormonal and nutritional regulation of phosphoenolpyruvate carboxykinase mRNA levels in chicken kidney. Journal of Nutrition 119, 319–322.
Wingler A, Walker RP, Chen Z-H, Leegood RC. 1999. Phosphoenolpyruvate carboxykinase is involved in the decarboxylation of aspartate in the bundle sheath of maize. Plant Physiology 120, 539–545.
Winter H, Lohaus G, Heldt HW. 1992. Phloem transport of amino acids in relation to their cytosolic levels in barley leaves. Plant Physiology 99, 996–1004.
Zimmer DB, Magnuson MA. 1990. Immunochemical localization of P-enolpyruvate carboxykinase in adult and developing mouse tissues. Journal of Histochemistry and Cytochemistry 38, 171–178.[Abstract]
Zimmermann HM, Hartmann K, Schreiber L, Steudle E. 2000. Chemical composition of apoplastic transport barriers in relation to radial hydraulic conductivity of corn roots (Zea mays L.). Planta 210, 302–311.[Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Malone, Z.-H. Chen, A. R. Bahrami, R. P. Walker, J. E. Gray, and R. C. Leegood Phosphoenolpyruvate Carboxykinase in Arabidopsis: Changes in Gene Expression, Protein and Activity during Vegetative and Reproductive Development Plant Cell Physiol., March 1, 2007; 48(3): 441 - 450. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. E. Lunn Compartmentation in plant metabolism J. Exp. Bot., January 1, 2007; 58(1): 35 - 47. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. L. Rylott, A. D. Gilday, and I. A. Graham The Gluconeogenic Enzyme Phosphoenolpyruvate Carboxykinase in Arabidopsis Is Essential for Seedling Establishment Plant Physiology, April 1, 2003; 131(4): 1834 - 1842. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z.-H. Chen, R. P. Walker, R. M. Acheson, and R. C. Leegood Phosphoenolpyruvate Carboxykinase Assayed at Physiological Concentrations of Metal Ions Has a High Affinity for CO2 Plant Physiology, January 1, 2002; 128(1): 160 - 164. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. P. Walker, Z.-H. Chen, R. M. Acheson, and R. C. Leegood Effects of Phosphorylation on Phosphoenolpyruvate Carboxykinase from the C4 Plant Guinea Grass Plant Physiology, January 1, 2002; 128(1): 165 - 172. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||