Phosphorylation of phosphoenolpyruvate carboxykinase in plants. Studies in plants with C4 photosynthesis and Crassulacean acid metabolism and in germinating seeds

We have previously shown that phosphoenolpyruvate carboxykinase (PEPCK) is phosphorylated in vivo in the cotyledons of darkened cucumber seedlings and that phosphorylation is reversed by light [Walker and Leegood (1995) FEBS Lett. 362, 70–74]. In this study the molecular mass of PEPCK was estimated in a range of gluconeogenic seedlings and in leaves of C4 plants and plants with Crassulacean acid metabolism (CAM). Phosphorylation of PEPCK was studied in these plants by feeding tissues with [32P]Pi and assessing phosphorylation by SDS/PAGE and autoradiography of either total proteins or of immunoprecipitated protein. In gluconeogenic seedlings and most CAM plants PEPCK had a molecular mass of 74 kDa, whereas in C4 grasses the molecular mass of PEPCK was always smaller and varied from 67–71 kDa. In all gluconeogenic seedlings and leaves of CAM plants PEPCK was phosphorylated, but it was not phosphorylated in all species of C4 grasses studied. In CAM plants, phosphorylation of PEPCK occurred at night and dephosphorylation occurred during the day. In C4 grasses phosphorylation occurred when leaves were darkened and the enzyme was dephosphorylated following illumination, but it was only phosphorylated in those plants with larger (71 kDa) molecular mass forms of PEPCK

Effects of Phosphorylation on Phosphoenolpyruvate Carboxykinase from the C4 Plant Guinea Grass

In the C4 plant Guinea grass (Panicum maximum), phosphoenolpyruvate carboxykinase (PEPCK) is phosphorylated in darkened leaves and dephosphorylated in illuminated leaves. To determine whether the properties of phosphorylated and non-phosphorylated PEPCK were different, PEPCK was purified to homogeneity from both illuminated and darkened leaves. The final step of the purification procedure, gel filtration chromatography, further separated phosphorylated and non-phosphorylated forms. In the presence of a high ratio of ATP to ADP, the non-phosphorylated enzyme had a higher affinity for its substrates, oxaloacetate and phosphoenolpyruvate. The activity of the non-phosphorylated form was up to 6-fold higher when measured at low substrate concentrations. Comparison of proteoloytically cleaved PEPCK from Guinea grass, which lacked its N-terminal extension, from yeast (Saccharomyces cerevisiae), which does not possess an N-terminal extension, and from the C4 plant Urochloa panicoides, which possesses an N-terminal extension but is not subject to phosphorylation, revealed similar properties to the non-phosphorylated full-length form from Guinea grass. Assay of PEPCK activity in crude extracts of Guinea grass leaves, showed a large difference between illuminated and darkened leaves when measured in a selective assay (a low concentration of phosphoenolpyruvate and a high ratio of ATP to ADP), but there was no difference under assay conditions used to estimate maximum activity. Immunoblots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showed no difference in the abundance of PEPCK protein in illuminated and darkened leaves. There were no light/dark differences in activity detected in maize (Zea mays) leaves, in which PEPCK is not subject to phosphorylation

Phosphoenolpyruvate Carboxykinase Assayed at Physiological Concentrations of Metal Ions Has a High Affinity for CO2

The effect of Mn2+/Mg2+ concentration on the activity of intact, homogeneous phosphoenolpyruvate carboxykinase (PEPCK) from leaves of the C4 grass, Guinea grass (Panicum maximum), have been investigated. Assay conditions were optimized so that PEPCK activity could be measured at concentrations of Mn2+/Mg2+ similar to those found in the cytosol (low micromolar Mn2+ and millimolar Mg2+). PEPCK activity was totally dependent on Mn2+ and was activated at low micromolar concentrations of Mn2+ by millimolar concentrations of Mg2+. Therefore, at physiological concentrations of Mn2+, PEPCK has a requirement for Mg2+. Assay at physiological concentrations of Mn2+/Mg2+ led to a marked decrease in its affinity for ATP and a 13-fold increase in its affinity for CO2. The Km (CO2) was further decreased by assay at physiological ATP to ADP ratios, reaching values as low as 20 μM CO2, comparable with the Km (CO2) of ribulose 1,5-bisphosphate carboxylase-oxygenase. This means that PEPCK will catalyze a reversible reaction and that it could operate as a carboxylase in vivo, a feature that could be particularly important in algal CO2-concentrating systems

Phosphoenolpyruvate Carboxykinase Is Involved in the Decarboxylation of Aspartate in the Bundle Sheath of Maize

We recently showed that maize (Zea mays L.) leaves contain appreciable amounts of phosphoenolpyruvate carboxykinase (PEPCK; R.P. Walker, R.M. Acheson, L.I. Técsi, R.C. Leegood [1997] Aust J Plant Physiol 24: 459–468). In the present study, we investigated the role of PEPCK in C4 photosynthesis in maize. PEPCK activity and protein were enriched in extracts from bundle-sheath (BS) strands compared with whole-leaf extracts. Decarboxylation of [4-14C]aspartate (Asp) by BS strands was dependent on the presence of 2-oxoglutarate and Mn2+, was stimulated by ATP, was inhibited by the PEPCK-specific inhibitor 3-mercaptopicolinic acid, and was independent of illumination. The principal product of Asp metabolism was phosphoenolpyruvate, whereas pyruvate was a minor product. Decarboxylation of [4-14C]malate was stimulated severalfold by Asp and 3-phosphoglycerate, was only slightly reduced in the absence of Mn2+ or in the presence of 3-mercaptopicolinic acid, and was light dependent. Our data show that decarboxylation of Asp and malate in BS cells of maize occurs via two different pathways: Whereas malate is mainly decarboxylated by NADP-malic enzyme, decarboxylation of Asp is dependent on the activity of PEPCK

A spatial analysis of physiological changes associated with infection of cotyledons of marrow plants with cucumber mosaic virus

Changes in host primary metabolism associated with the compatible interaction between cucumber mosaic virus and cotyledons of the marrow plant (Cucurbita pepo L.) have been localized, first by measuring activities of key enzymes in infected and uninfected regions of the cotyledon, and second by histochemical techniques applied to tissue prints of the infected region. A series of progressive metabolic changes occurs within the expanding infected lesion. Virus replication and the synthesis of viral protein at the periphery creates a strong sink demand associated with increased activities of anaplerotic enzymes, increased photosynthesis, and starch accumulation. Inside the lesion, when the synthesis of virus has declined, photosynthesis is reduced, starch is mobilized, and the emphasis of metabolism is shifted toward glycolysis and mitochondrial respiration. These changes are associated spatially with the onset of chlorosis. A decrease in total protein synthesis in this inner zone could be instrumental in some or all of these changes, leading to symptoms of viral infection

Leaf-Atmosphere NH3 Exchange in Barley Mutants with Reduced Activities of Glutamine Synthetase

Mutants of barley (Hordeum vulgare L. cv Maris Mink) with 47 or 66% of the glutamine synthetase (GS) activity of the wild type were used for studies of NH3 exchange with the atmosphere. Under normal light and temperature conditions, tissue NH4+ concentrations were higher in the two mutants compared with wild-type plants, and this was accompanied by higher NH3 emission from the leaves. The emission of NH3 increased with increasing leaf temperatures in both wild-type and mutant plants, but the increase was much more pronounced in the mutants. Similar results were found when the light intensity (photosynthetic photon flux density) was increased. Compensation points for NH3 were estimated by exposing intact shoots to 10 nmol NH3 mol-1 air under conditions with increasing temperatures until the plants started to emit NH3. Referenced to 25[deg]C, the compensation points were 5.0 nmol mol-1 for wild-type plants, 8.3 nmol mol-1 for 47% GS mutants, and 11.8 nmol mol-1 for 66% GS mutants. Compensation points for NH3 in single, nonsenescent leaves were estimated on the basis of apoplastic pH and NH4+ concentrations. These values were 0.75, 3.46, and 7.72 nmol mol-1 for wild type, 47% GS mutants, and 66% GS mutants, respectively. The 66% GS mutant always showed higher tissue NH4+ concentrations, NH3 emission rates, and NH3 compensation points compared with the 47% GS mutant, indicating that NH4+ release was curtailed by some kind of compensatory mechanism in plants with only 47% GS activit

Regulation of Leaf Senescence by Cytokinin, Sugars, and Light. Effects on NADH-Dependent Hydroxypyruvate Reductase

The aim of this study was to investigate the interactions between cytokinin, sugar repression, and light in the senescence-related decline in photosynthetic enzymes of leaves. In transgenic tobacco (Nicotiana tabacum) plants that induce the production of cytokinin in senescing tissue, the age-dependent decline in NADH-dependent hydroxypyruvate reductase (HPR), ribulose-1,5-bisphosphate carboxylase/oxygenase, and other enzymes involved in photosynthetic metabolism was delayed but not prevented. Glucose (Glc) and fructose contents increased with leaf age in wild-type tobacco and, to a greater extent, in transgenic tobacco. To study whether sugar accumulation in senescing leaves can counteract the effect of cytokinin on senescence, discs of wild-type leaves were incubated with Glc and cytokinin solutions. The photorespiratory enzyme HPR declined rapidly in the presence of 20 mM Glc, especially at very low photon flux density. Although HPR protein was increased in the presence of cytokinin, cytokinin did not prevent the Glc-dependent decline. Illumination at moderate photon flux density resulted in the rapid synthesis of HPR and partially prevented the negative effect of Glc. Similar results were obtained for the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. It is concluded that sugars, cytokinin, and light interact during senescence by influencing the decline in proteins involved in photosynthetic metabolism

Oxygen Requirement and Inhibition of C4 Photosynthesis . An Analysis of C4 Plants Deficient in the C3 and C4 Cycles

The basis for O2 sensitivity of C4 photosynthesis was evaluated using a C4-cycle-limited mutant of Amaranthus edulis (a phosphoenolpyruvate carboxylase-deficient mutant), and a C3-cycle-limited transformant of Flaveria bidentis (an antisense ribulose-1,5-bisphosphate carboxylase/oxygenase [Rubisco] small subunit transformant). Data obtained with the C4-cycle-limited mutant showed that atmospheric levels of O2 (20 kPa) caused increased inhibition of photosynthesis as a result of higher levels of photorespiration. The optimal O2 partial pressure for photosynthesis was reduced from approximately 5 kPa O2 to 1 to 2 kPa O2, becoming similar to that of C3 plants. Therefore, the higher O2 requirement for optimal C4 photosynthesis is specifically associated with the C4 function. With the Rubisco-limited F. bidentis, there was less inhibition of photosynthesis by supraoptimal levels of O2 than in the wild type. When CO2 fixation by Rubisco is limited, an increase in the CO2 concentration in bundle-sheath cells via the C4 cycle may further reduce the oxygenase activity of Rubisco and decrease the inhibition of photosynthesis by high partial pressures of O2 while increasing CO2 leakage and overcycling of the C4 pathway. These results indicate that in C4 plants the investment in the C3 and C4 cycles must be balanced for maximum efficiency

Acclimation of photosynthesis to low temperature in Spinacia oleracea L. II. Effects of nitrogen supply

The photosynthetic capacity of leaves of N-sufficient plants of Spinacia oleracea L. increases following transfer a constant temperature of 10°C for 10 d compared to plants maintained at 25°C. The effects of nitrogen nutrition on this low temperature acclimation have been investigated in respect of CO2 assimilation, the activites and activation states of key enzymes and the partitioning of recently fixed carbon. N-deficiency greatly restricted acclimation of photosynthetic CO2 assimilation to low temperature at both ambient and at saturating CO2 concentrations, indicating a restriction on acclimatory changes in both ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) and the reactions of ribulose-1,5-bisphosphate regeneration. Nitrogen limitation led to an increase in the partitioning of recently-fixed carbon into starch. Total protein increased during acclimation in both N-sufficient and N-deficient leaves and was much less affected than were the activities of enzymes. Increases in the activation state of Rubisco and the stromal fructose-1,6-bisphosphatase occurred in response to low temperature, but increases in the activities of Rubisco, sucrose-phosphate synthase or the cytosolic fructose-1,6-bisphosphatase could not be sustained in N-deficient plants throughout the period of acclimation, although the activities of these enzymes declined less precipitately than in non-acclimated N-deficient plants. These data are all consistent with the view that increases in the activities of key enzymes of carbon assimilation are a pre-requisite for acclimation to low temperature and that these increases are restricted under N-limitation

Acclimation of photosynthesis to low temperature in Spinacia oleracea L. I. Effects of acclimation on CO2 assimilation and carbon partitioning

Acclimation of spinach plants grown at 25 °C to a temperature of 10°C for 10 d resulted in an increased capacity for leaf photosynthesis in saturating light and CO2, but not at ambient CO2 concentrations. Gas exchange and chlorophyll fluorescence measurements indicated that acclimation was accompanied by an increased capacity for the regeneration of ribulose-1,5-bisphosphate. Changes in starch, soluble carbohydrates and activities of sucrose-P synthase and ADP-glucose pyrophosphorylase were measured during the acclimation process. There was an initial increase in starch and sucrose during the first 2 d, but these then declined. There was an increase in the capacity for sucrose synthesis during low temperature acclimation, evidenced by an increase in the maximum activity of sucrose-P synthase activity and an increase in partitioning of 14CO2 into sucrose, but there was no increase in the activity of ADP-glucose pyrophosphorylase or carbon partitioning into starch