Robert P. Walker

Regulation and roles of phosphoenolpyruvate carboxykinase in plants.

Phosphoenolpyruvate carboxykinase (PCK) is probably ubiquitous in flowering plants, but is confined to certain cells or tissues. It is regulated by phosphorylation, which renders it less active by altering both its substrate affinities and its sensitivity to regulation by adenylates. In the leaves of some C4 plants, such as Panicum maximum, dephosphorylation increases its activity in the light. In other tissues such regulation probably avoids futile cycling between phosphoenolpyruvate and oxaloacetate. Although PCK generally acts as a decarboxylase in plants, its affinity for CO2 measured at physiological concentrations of metal ions is high and would allow it to be freely reversible in vivo. While its function in gluconeogenesis in seeds postgermination and in leaves of C4 and crassulacean acid metabolism plants is clearly established, the possible functions of PCK in other plant cells are discussed, drawing parallels with those in animals, including its integrated function in cataplerosis, nitrogen metabolism, pH regulation, and gluconeogenesis.

Inducibility of crassulacean acid metabolism (CAM) in Clusia species; physiological/biochemical characterisation and intercellular localization of carboxylation and decarboxylation processes in three species which exhibit different degrees of CAM

Abstract. The biochemical basis for photosynthetic plasticity in tropical trees of the genus Clusia was investigated in three species that were from contrasting habitats and showed marked differences in their capacity for crassulacean acid metabolism (CAM). Physiological, anatomical and biochemical measurements were used to relate changes in the activities/amounts of key enzymes of C3 and C4 carboxylation to physiological performance under severe drought stress. On the basis of gas-exchange measurements and day/night patterns of organic acid turnover, the species were categorised as weak CAM-inducible (C.aripoensis Britt.), C3-CAM intermediate (C. minor L.) and constitutive CAM (C.␣rosea Jacq. 9.). The categories reflect genotypic differences in physiological response to drought stress in terms of net carbon gain; in C. aripoensis net carbon gain was reduced by over 80% in drought-stressed plants whilst carbon gain was relatively unaffected after 10 d without water in C. rosea. In turn, genotypic differences in the capacity for CAM appeared to be directly related to the capacities/amounts of phosphoenolpyruvate carboxylase (PEPCase) and phosphoenolpyruvate carboxykinase (PEPCK) which increased in response to drought in both young and mature leaves. Whilst measured activities of PEPCase and PEPCK in well-watered plants of the C3-CAM intermediate C. minor were 5–10 times in excess of that required to support the magnitude of organic acid turnover induced by drought, close correlations were observed between malate accumulation/PEPCase capacity and citrate decarboxylation/PEPCK capacity in all the species. Drought stress did not affect the amount of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) protein in any of the species but Rubisco activity was reduced by 35% in the weak CAM-inducible C. aripoensis. Similar amounts of glycine decarboxylase (GDC) protein were present in all three species regardless of the magnitude of CAM expression. Thus, the constitutive CAM species C. rosea did not appear to show reduced activity of this key enzyme of the photorespiratory pathway, which, in turn, may be related to the low internal conductance to CO2 in this succulent species. Immuno-histochemical techniques showed that PEPCase, PEPCK and Rubisco were present in cells of the palisade and spongy parenchyma in leaves of species performing CAM. However, in leaves from well-watered plants of C. aripoensis which only performed C3 photosynthesis, PEPCK was localized around latex-producing ducts. Differences in leaf anatomy between the species suggest that the association between mesophyll succulence and the capacity for CAM in these hemi-epiphytic stranglers has been selected for in arid environments.

Phosphoenolpyruvate carboxykinase plays a role in interactions of carbon and nitrogen metabolism during grape seed development

Abstract. Phosphoenolpyruvate carboxykinase (PEPCK) was shown to be present in a range of developing seeds by measurement of its activity and by immunoblotting. Its function was investigated during grape (Vitis vinifera L.) seed development. The maximum abundance of PEPCK coincided with the deposition of storage reserves. At this stage of development, immunohistochemistry showed that PEPCK was very abundant in a layer of cells located at the boundary of developing storage tissues and in the chalaza (close to the termination of the vascular supply to the seed) and was also present in the palisade layer of the seed coat (the inner layer of the outer integument). Earlier in development PEPCK was also present in the developing palisade layer and in the inner region of the nucellus which surrounds the developing endosperm. At later stages of development, PEPCK was located in the outer region of the endosperm. However, PEPCK was present in the phloem of the seed at all stages of development. Feeding of asparagine to developing grape seeds led to a strong induction of PEPCK. We suggest that, in developing grape seeds, both the chalaza and palisade tissue may distribute imported assimilates from the vasculature to the developing storage tissues and that PEPCK may play a role in the metabolism of nitrogenous assimilates during their delivery from the vasculature to the storage tissues.

Phosphoenolpyruvate carboxykinase from higher plants: Purification from cucumber and evidence of rapid proteolytic cleavage in extracts from a range of plant tissues

Phosphoenolpyruvate carboxykinase (PEPCK) was purified 600-fold to homogeneity from the cotyledons of cucumber (Cucumis sativus L.) and a polyclonal antiserum raised. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the purified preparation contained a single polypeptide of 62 kDa, consistent with previous studies of this enzyme in C4 grasses. Immunoblots of crude extracts showed that a form of PEPCK of approximately this molecular mass predominated in cucumber cotyledons and in a range of plant tissues (cotyledons of fat-storing seedlings, leaves of C4 and Crassulacean acid metabolism plants). However, when these tissues were extracted in the presence of SDS and the extracts analysed by immunoblotting, a larger polypeptide of 68–77 kDa was detected. Thus the enzyme generally measured in crude extracts is a smaller form which arises by rapid proteolysis. This phenomenon means that the native form of PEPCK has never been purified from plants nor its properties determined.

Phosphoenolpyruvate carboxykinase in cucumber plants is increased both by ammonium and by acidification, and is present in the phloem.

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.

Does phosphoenolpyruvate carboxykinase have a role in both amino acid and carbohydrate metabolism

Summary. Phosphoenolpyruvate carboxykinase (PEPCK) catalyses the reversible decarboxylation of oxaloacetate to yield phosphoenolpyruvate and CO2. The role of the enzyme in gluconeogenesis and anaplerotic reactions in a range of organisms is discussed, along with the important function in C4 and CAM photosynthesis in higher plants. In addition, new data are presented indicating that PEPCK may play a key role in amino acid metabolism. It is proposed that PEPCK is involved in the conversion of the carbon skeleton of asparagine/aspartate (oxaloacetate) to that of glutamate/glutamine (2-oxoglutarate). This metabolism is particularly important in the transport system, seeds and fruits of higher plants.

Purification, and phosphorylation in vivo and in vitro, of phosphoenolpyruvate carboxykinase from cucumber cotyledons

Phosphoenolpyruvate carboxykinase (PEPCK) with a subunit molecular mass of 74 kDa has been purified 450‐fold to homogeneity from the cotyledons of cucumber (Cucumis sativus L.). This is the first purification of the native form of the enzyme from any plant tissue. Incubation of the purified enzyme with [γ‐32P]ATP and either phosphoenolpyruvate‐carboxylase kinase or mammalian cAMP‐dependent protein kinase led to labelling of the enzyme in a part of the molecule separate from the active site. This was reversed by incubation with protein phosphatase 2A. Cotyledons of cucumber seedlings were also supplied with 32Pi. Homogenates of such cotyledons contained a heavily labelled polypeptide which was confirmed as PEPCK by immunoprecipitation. Labelling of PEPCK by 32Pi in darkened cotyledons was reversed by illumination.

Phosphoenolpyruvate carboxykinase: Structure, function and regulation

Abstract The aim of this article is to outline our understanding of the enzyme phosphoenolpyruvate carboxykinase (PEPCK). Although emphasis is placed on the enzyme derived from flowering plants, other organisms are also considered, because comparative studies provide invaluable information. The following points are considered in detail. Firstly, the possibility that PEPCK in all organisms arose from a common ancestor, and that the extension of about 12 kDa at the N-terminus of PEPCK-ATP from flowering plants, which is not possessed by PEPCK-ATP from other organisms, is homologous to the N-terminal region of PEPCK-GTP. Secondly, the regulation of PEPCK activity in flowering plants by reversible protein phosphorylation is described. Phosphorylation of the N-terminal extension possessed by PEPCK from flowering plants reduces its catalytic velocity several-fold at physiological concentrations of oxaloacetate. How this is likely to contribute to regulation of PEPCK in vivo is described. Thirdly, it is proposed that in flowering plants PEPCK plays a widespread role as a component of a mechanism that counteracts intracellular acidification. The proposed role of PEPCK in this mechanism is similar to that in the kidney during acidosis.

Coordinate Regulation of Phosphoenolpyruvate Carboxylase and Phosphoenolpyruvate Carboxykinase by Light and CO2 during C4 Photosynthesis

The aim of this study was to investigate the relationship between the phosphorylation and activation states of phosphoenolpyruvate carboxykinase (PEPCK) and to investigate how the phosphorylation states of PEPCK and phosphoenolpyruvate carboxylase (PEPC) are coordinated in response to light intensity and CO2 concentration during photosynthesis in leaves of the C4 plant Guinea grass (Panicum maximum). There was a linear, reciprocal relationship between the phosphorylation state of PEPCK and its activation state, determined in a selective assay that distinguishes phosphorylated from nonphosphorylated forms of the enzyme. At high photon flux density and high CO2 (750 μL L−1), PEPC was maximally phosphorylated and PEPCK maximally dephosphorylated within 1 h of illumination. The phosphorylation state of both enzymes did not saturate until high light intensities (about 1,400 μmol quanta m−2 s−1) were reached. After illumination at lower light intensities and CO2 concentrations, the overall change in phosphorylation state was smaller and it took longer for the change in phosphorylation state to occur. Phosphorylation states of PEPC and PEPCK showed a strikingly similar, but inverse, pattern in relation to changes in light and CO2. The protein phosphatase inhibitor, okadaic acid, promoted the phosphorylation of both enzymes. The protein synthesis inhibitor, cycloheximide, blocked dark phosphorylation of PEPCK. The data show that PEPC and PEPCK phosphorylation states are closely coordinated in vivo, despite being located in the mesophyll and bundle sheath cells, respectively.

The many-faceted function of phosphoenolpyruvate carboxykinase in plants

Phosphoenolpyruvate carboxykinase (PEPCK) undergoes light- or diurnally-regulated changes in phosphorylation in C3 and CAM plants but only in some C4 plants. The characteristics of this phosphorylation of PEPCK and its possible regulatory significance are discussed. The molecular mass of PEPCK from C4 plants is often smaller, and more variable, than the molecular mass of the enzyme in C3 and CAM plants. These differences probably reflect differences in the size of the N-terminal extension found in the enzyme from higher plants. This N-terminal extension contains the phosphorylation site and it is readily cleaved following extraction. PEPCK has four well-defined roles in plants: in photosynthesis in C4 and CAM plants, in the CO2-concentrating mechanism of certain algae and in gluconeogenesis following germination of fat-storing seedlings, but it has now also been located in the trichomes and in the phloem of some plants, such as cucurbits, suggesting a larger number of roles in plant metabolism than has hitherto been recognised. These possible roles are discussed, and in particular we discuss the general role of the phloem elements in cucurbits.