Partial purification and characterization of a protein inhibitor of phosphoenolpyruvate carboxylase kinase.

The activity of phosphoenolpyruvate carboxylase (PEPCase) kinase in leaf extracts increased markedly on dilution. This was shown to be caused by the presence of a protein that inhibits the kinase. The inhibitor protein was separated from the kinase and purified partially. It inhibited the kinase reversibly, presumably by a direct interaction; it was neither a protease nor a protein phosphatase. The amounts of kinase and inhibitor in leaves were estimated following separation by hydrophobic chromatography. The amount of inhibitor in the crassulacean acid metabolism plant Kalanchoe fedtschenkoi Hamet et Perrier was sufficient to inhibit the basal level of kinase activity present during the light period and the early stages of the dark period. Similarly, the amount of inhibitor in the C4 plant Zea mays L. was sufficient to inhibit the low amount of kinase activity present in the dark and at moderate light intensity. Analogous to the role of the protein inhibitor of mammalian cyclic AMP-dependent protein kinase, the function of the PEPCase kinase inhibitor may be to inhibit the basal level of kinase present in conditions under which rapid flux through PEPCase is not required.

Phosphoenolpyruvate carboxylase kinase is a novel protein kinase regulated at the level of expression.

Phosphorylation of phosphoenolpyruvate carboxylase plays a key role in the control of plant metabolism. Phosphoenolpyruvate carboxylase kinase is a Ca2+-independent enzyme that is activated by a process involving protein synthesis in response to a range of signals in different plant tissues. The component whose synthesis is required for activation has not previously been identified, nor has the kinase been characterised at a molecular level. We report the cloning of phosphoenolpyruvate carboxylase kinase from the Crassulacean Acid Metabolism plant Kalanchoë fedtschenkoi and the C3 plant Arabidopsis thaliana. Surprisingly, phosphoenolpyruvate carboxylase kinase is a member of the Ca2+/calmodulin-regulated group of protein kinases. However, it lacks the auto-inhibitory region and EF hands of plant Ca2+-dependent protein kinases, explaining its Ca2+-independence. Its sequence is novel in that it comprises only a protein kinase catalytic domain with no regulatory regions; it appears to be the smallest known protein kinase. In K. fedtschenkoi, the abundance of phosphoenolpyruvate carboxylase kinase transcripts increases during leaf development. The transcript level in mature leaves is very low during the photoperiod, reaches a peak in the middle of the dark period and correlates with kinase activity. It exhibits a circadian oscillation in constant conditions. Protein kinases are typically regulated by second messengers, phosphorylation or protein/protein interactions. Phosphoenolpyruvate carboxylase kinase is an exception to this general rule, being controlled only at the level of expression. In K. fedtschenkoi, its expression is controlled both developmentally and by a circadian oscillator.

Decarboxylation of Malate in the Crassulacean Acid Metabolism Plant Bryophyllum (Kalanchoe) fedtschenkoi (Role of NAD-Malic Enzyme).

The role of NAD-malic enzyme (NAD-ME) in the Crassulacean acid metabolism plant Bryophyllum (Kalanchoe) fedtschenkoi was investigated using preparations of intact and solubilized mitochondria from fully expanded leaves. Intact, coupled mitochondria isolated during the day or night did not differ in their ability to take up [14C]malic acid from the surrounding medium or to respire using malate or succinate as substrate. However, intact mitochondria isolated from plants during the day decarboxylated added malate to pyruvate significantly faster than mitochondria isolated from plants at night. NAD-ME activity in solubilized mitochondrial extracts showed hysteretic kinetics and was stimulated by a number of activators, including acetyl-coenzyme A, fructose-1,6-bisphosphate, and sulfate ions. In the absence of these effectors, reaction progress curves were nonlinear, with a pronounced acceleration phase. The lag period before a steady-state rate was reached in assays of mitochondrial extracts decreased during the photoperiod and increased slowly during the period of darkness. However, these changes in the kinetic properties of the enzyme could not account for the changes in the rate of decarboxylation of malate by intact mitochondria. Gel-filtration experiments showed that mitochondrial extracts contained three forms of NAD-ME with different molecular weights. The relative proportions of the three forms varied somewhat throughout the light/dark cycle, but this did not account for the changes in the kinetics behavior of the enzyme during the diurnal cycle.

Circadian rhythms in the activity of a plant protein kinase.

Bryophyllum fedtschenkoi is a Crassulacean acid metabolism plant whose phosphoenolpyruvate carboxylase is regulated by reversible phosphorylation in response to a circadian rhythm. A partially purified protein kinase phosphorylated phosphoenolpyruvate carboxylase in vitro with a stoichiometry approaching one per subunit and caused a concomitant 5- to 10-fold decrease in the sensitivity of the carboxylase to inhibition by malate. The sites phosphorylated in vitro were identical to those phosphorylated in intact tissue. The activity of the protein kinase was controlled in a circadian fashion. During normal diurnal cycles, kinase activity appeared between 4 and 5 h after the onset of darkness and disappeared 2----3 h before the end of darkness. Kinase activity displayed circadian oscillations in constant environmental conditions. The activity of protein phosphatase 2A, which dephosphorylates phosphoenolpyruvate carboxylase, did not oscillate. Treatment of detached leaves with the protein synthesis inhibitors puromycin and cycloheximide blocked the nocturnal appearance of the protein kinase activity, maintained phosphoenolypyruvate carboxylase in the dephosphorylated state and blocked the circadian rhythms of CO2 output that is observed in constant darkness and CO2-free air. The simplest explanation of the data is that there is a circadian rhythm in the synthesis of phosphoenolpyruvate carboxylase kinase.

Illumination increases the phosphorylation state of maize leaf phosphoenolpyruvate carboxylase by causing an increase in the activity of a protein kinase.

Illumination of maize leaves increases the phosphorylation state of phosphoenolpyruvate carboxylase and reduces the sensitivity of the enzyme to feedback inhibition by malate. Red, white and blue light were each found to be equally potent, and the effect of light was blocked by 3(3,4-dichlorophenyl)-1,1-dimethylurea. A phosphoenolpyruvate carboxylase kinase was partially purified from illuminated maize leaves by a three-step procedure. Phosphorylation of phosphoenolpyruvate carboxylase by this protein kinase reached 0.7-0.8 molecules/subunit and correlated with a 3- to 4-fold increase in Ki for malate. The protein kinase was inhibited by L-malate, but was insensitive to a number of other potential regulators. Freshly prepared and desalted extracts of darkened maize leaves contained very little kinase activity, but the activity appeared when leaves were illuminated for 30-60 min before extraction. The catalytic subunit of protein phosphatase 2A from rabbit skeletal muscle, but not that of protein phosphatase 1, could dephosphorylate phosphoenolpyruvate carboxylase. The protein phosphatases 1 and 2A activities of maize leaves were not affected by illumination. It is suggested that the major means by which light stimulates the phosphorylation of phosphoenolpyruvate carboxylase is by an increase in the activity of the protein kinase.

The role of the epidermis in the generation of the circadian rhythm of carbon dioxide fixation in leaves of Bryophyllum fedtschenkoi.

The role of the epidermis in the generation of the endogenous circadian rhythm of CO2 exchange in leaves of Bryophyllum fedtschenkoi has been examined. At 25° C the rhythm of CO2 output exhibited by whole leaves kept in continuous darkness and an initially CO2-free air stream also occurs in isolated pieces of mesophyll. The sensitivity to light of the rhythms in whole leaves and in isolated mesophyll appears to be identical. At 15° C, however, no rhythm is observed in isolated mesophyll tissue, despite there being a conspicuous rhythm in intact leaves. The rhythm of net CO2 assimilation in whole leaves kept in continuous light and a stream of normal air at either 25° C or at 15° C is abolished by removal of the epidermis, although at 15° C and under the higher of the two light levels used, there is an indication that rhythmicity may begin to reappear after the third day of the experiment. Thus, only under certain environmental conditions is the rhythm of CO2 exchange in Bryophyllum leaves independent of the epidermis. The results indicate that the rhythm of carbon dioxide fixation in continuous darkness and CO2-free air is generated primarily in the mesophyll cells, whereas the rhythm in continuous light and normal air is generated in the stomatal guard cells or in an interaction of these cells with the mesophyll cells.

Regulation of phosphoenolpyruvate carboxylase: an example of signal transduction via protein phosphorylation in higher plants.

There is now good evidence that the malate sensitivity of PEPc is regulated by phosphorylation/dephosphorylation in the leaf tissue of C4 and CAM plants. This statement is based on the assessment of the phosphorylation state of PEPc in [32P]-labeled intact tissue by immunoprecipitation and the correlation between phosphorylation state and malate sensitivity that has been observed during incubation of purified PEPc in vitro with protein kinases or protein phosphatases. The phosphorylation of PEPc in the CAM plant B. fedtschenkoi is controlled by an endogenous rhythm whereas that of PEPc in the C4 plant maize is triggered directly by light. In neither case has the mechanism of signal transduction been identified. It is hoped that further work on the protein kinases and protein phosphatases involved will reveal the nature of the signalling systems. Preliminary work suggests that plant protein phosphatases are very similar to their mammalian counterparts. It is also noteworthy that higher plant genes very similar to the genes encoding the cyclic nucleotide-dependent protein kinases and the protein kinase C family have recently been identified. It is interesting to speculate that the protein kinases and phosphatases involved in signal transduction systems in plants may prove to be closely related to well-studied mammalian enzymes.

Purification, oligomerization state and malate sensitivity of maize leaf phosphoenolpyruvate carboxylase.

A method was developed for the purification of phosphoenolpyruvate carboxylase from darkened maize leaves so that the enzyme retained its sensitivity to inhibition by malate. The procedure depended on the prevention of proteolysis by the inclusion of chymostatin in the buffers used during the purification. The purified enzyme was indistinguishable from that in crude extracts as judged by native polyacrylamide-gel electrophoresis. SDS/polyacrylamide-gel electrophoresis followed by immunoblotting, and Superose 6 gel filtration. Gel-filtration studies showed that the purified enzyme and the enzyme in extracts of darkened or illuminated leaves showed a concentration-dependent dissociation of tetrameric into dimeric forms. Purified phosphoenolpyruvate carboxylase and enzyme in crude extracts from darkened leaves were equally sensitive to inhibition by malate (Ki approx. 0.30 mM) under conditions where it existed in the tetrameric or dimeric forms, but the enzyme in crude extracts from illuminated leaves was less sensitive to malate inhibition (Ki approx. 0.95 mM) whether it was present as a tetramer or as a dimer. It is concluded that changes in the oligomerization state of phosphoenolpyruvate carboxylase are not directly involved in its regulation by light.

Phase resetting of the circadian rhythm of carbon dioxide assimilation inBryophyllum leaves in relation to their malate content following brief exposure to high and low temperatures, darkness and 5% carbon dioxide.

Leaves ofBryophyllum fedtschenkoi show a persistent circadian rhythm in CO2 assimilation when kept in continuous illumination and normal air at 15°C. The induction of phase shifts in this rhythm by exposing the leaves for four hours at different times in the circadian cycle to 40° C, 2° C, darkness and 5% CO2 have been investigated. Exposure to high temperature has no effect on the phase at the apex of the peak but is effective at all other times in the cycle, whereas exposure to low temperature, darkness or 5% CO2 is without effect between the peaks and induces a phase shift at all other times. The next peak of the rhythm occurs 17 h after a 40° C treatment and 7-10 h after a 2° C, dark or 5% CO2 treatment regardless of their position in the cycle. When these treatments are given at times in the cycle when they induce maximum phase shifts, they cause no change in the gross malate status of the leaf. The gross malate content of the leaf in continuous light and normal air at 15% shows a heavily damped circadian oscillation which virtually disappears by the time of the third cycle, but the CO2 assimilation rhythm persists for many days. The generation of the rhythm, and the control of its phase by environmental factors are discussed in terms of mechanisms that involve the synthesis and metabolism of malate in specific localised pools in the cytoplasm of the leaf cells.

Control of the circadian rhythm of carbon dioxide assimilation in Bryophyllum leaves by exposure to darkness and high carbon dioxide concentrations.

The circadian rhythm of CO2 assimilation in detached leaves of Bryophyllum fedtschenkoi at 15° C in normal air and continuous illumination is inhibited both by exposure to darkness, and to an atmosphere enriched with 5% CO2. During such exposures substantial fixation of CO2 takes place, and the malate concentration in the cell sap increases from about 20 mM to a constant value of 40-50 mM after 16 h. On transferring the darkened leaves to light, and those exposed to 5% CO2 to normal air, a circadian rhythm of CO2 assimilation begins again. The phase of this rhythm is determined by the time the transfer is made since the first peak occurs about 24 h afterwards. This finding indicates that the circadian oscillator is driven to, and held at, an identical, fixed phase point in its cycle after 16 h exposure to darkness or to 5% CO2, and it is from this phase point that oscillation begins after the inhibiting condition is removed. This fixed phase point is characterised by the leaves having acquired a high malate content. The rhythm therefore begins with a period of malate decarboxylation which lasts for about 8 h, during which time the malate content of the leaf cells must be reduced to a value that allows phosphoenolpyruvate carboxylase to become active. Inhibition of the rhythm in darkness, and on exposure to 5% CO2 in continuous illumination, appears to be due to the presence of a high concentration of CO2 within the leaf inhibiting malic enzyme which leads to the accumulation of high concentrations of malate in the leaf cells. The malate then allosterically inhibits phosphoenolpyruvate carboxylase upon which the rhythm depends. The results give support to the view that malate synthesis and breakdown form an integral part of the circadian oscillator in this tissue.

Period and phase control by temperature in the circadian rhythm of carbon dioxide fixation in illuminated leaves of Bryophyllum fedtschenkoi.

The rhythm of CO2 assimilation exhibited by leaves of Bryophyllum fedtschenkoi maintained in light and normal air occurs only at constant ambient temperatures between 10°C and 30°C. Over this range the period increases linearly with increasing temperature from the extremely low value of 15.7 h to 23.3 h, but shows a considerable degree of temperature compensation. Outside the range 10°C-30°C the rhythm is inhibited but re-starts on changing the temperature to 15°C. Prolonged exposure of leaves to high (40°C) and low (2°C) temperature inhibits the rhythm by driving the basic oscillator to fixed phase points in the cycle which differ by 180°, and which have been characterised in terms of the malate status of the leaf cells. At both temperatures loss of the circadian rhythm of CO2 assimilation is due to the inhibition of phosphoenolpyruvate carboxylase (PEPCase) activity, but the inhibition is apparently achieved in different ways at 40°C and 2°C. High temperature appears to inhibit directly PEPCase activity, but not the activity of the enzymes responsible for the breakdown of malate, with the result that the leaf acquires a low malate status. In contrast, low temperature does not directly inhibit PEPCase activity, but does inhibit enzymes responsible for malate breakdown, so that the malate level in the leaf increases to a high value and PEPCase is eventually allosterically inhibited. The different malate status of leaves held at these two temperatures accounts for the phases of the rhythms being reversed on returning the leaves to 15°C. After exposure to high temperature, CO2 fixation by PEPCase activity can begin immediately, whereas after exposure to low temperature, the large amount of malate accumulated in the leaves has to be decarboxylated before CO2 fixation can begin.

Persistent circadian rhythms in the phosphorylation state of phosphoenolpyruvate carboxylase from Bryophyllum fedtschenkoi leaves and in its sensitivity to inhibition by malate.

Phosphoenolpyruvate carboxylase (EC 4.1.1.31; PEPCase) from Bryophyllum fedtschenkoi leaves has previously been shown to exist in two forms in vivo. During the night the enzyme is phosphorylated and relatively insensitive to feedback inhibition by malate whereas during the day the enzyme is dephosphorylated and more sensitive to inhibition by malate. These properties of PEPCase have now been investigated in leaves maintained under constant conditions of temperature and lighting. When leaves were maintained in continuous darkness and CO2-free air at 15°C, PEPCase exhibited a persistent circadian rhythm of interconversion between the two forms. There was a good correlation between periods during which the leaves were fixing respiratory CO2 and periods during which PEPCase was in the form normally observed at night. When leaves were maintained in continuous light and normal air at 15°C, starting at the end of a night or the end of a day, a circadian rhythm of net uptake of CO2 was observed. Only when these constant conditions were applied at the end of a day was a circadian rhythm of interconversions between the two forms of PEPCase observed and the rhythms of enzyme interconversion and CO2 uptake did not correlate in phase or period.

Purification of the phosphorylated night form and dephosphorylated day form of phosphoenolpyruvate carboxylase from Bryophyllum fedtschenkoi.

Phosphoenolpyruvate carboxylase of Bryophyllum fedtschenkoi was shown to exist in two forms: a night form, which is phosphorylated and has low sensitivity to inhibition by malate, and a day form, which is dephosphorylated and 10 times more sensitive to malate. The day and night forms of the enzyme were purified retaining their distinct malate sensitivities and phosphorylation states. The purified enzymes contained a major protein (subunit Mr 112,000) and a minor protein (subunit Mr 123,000). The two polypeptides appeared to have closely related amino acid sequences and were present in a similar ratio in extracts that had been prepared rapidly. The phosphate present in the night form of the enzyme was covalently bound to serine. It was not a catalytic intermediate. Alkaline phosphatase removed the phosphate group in vitro and increased the malate sensitivity of the enzyme to that observed for the day form. Both the day and night forms of the enzyme were probably tetramers, and their apparent Mr was lowered by the presence of malate, but was unaffected by Mg2+ ions, EDTA, a rise in pH or a 10-fold change in enzyme concentration. The rapid loss of malate sensitivity, observed in extracts of leaves prepared during the day and at night, was shown to be due to proteolysis of the enzyme. It was slowed in the presence of malate and by phosphorylation of the enzyme.

Metabolism of [(14)C]indole-3-acetic acid by the cortical and stelar tissues of Zea mays L. roots.

Reverse-phase high-performance liquid chromatography was used to analyse (14)C-labelled metabolites of indole-3-acetic acid (IAA) formed in the cortical and stelar tissues of Zea mays roots. After a 2-h incubation in [(14)C]IAA, stelar segments had metabolised between 1-6% of the methanol-extractable radioactivity compared with 91-92% by the cortical segments. The pattern of metabolites produced by cortical segments was similar to that produced by intact segments bathed in aqueous solutions of [(14)C]IAA. In contrast, when IAA was supplied in agar blocks to stelar tissue protruding from the basal ends of segments, negligible metabolism was evident. On the basis of its retention characteristics both before and after methylation, the major metabolite of [(14)C]IAA in Zea mays root segments was tentatively identified by high-performance liquid chromatography as oxindole-3-acetic acid.

A rapid circadian rhythm of carbon-dioxide metabolism in Bryophyllum fedtschenkoi.

Leaves of Bryophyllum fedtschenkoi Hamet et Perrier maintained in a stream of normal air and at 15° C exhibit a circadian rhythm of CO2 uptake in continuous light but not in continuous darkness. The rhythm is unusual in that it persists for at least 10 d, and has a short period of approximately 18 h. The mechanism by which this rhythm is generated is discussed.

Gravity perception in decapped roots of Zea mays.

Time-lapse photography and light microscopy were used to determine whether or not sedimentation of the newly developed amyloplasts in the apex of Zea mays L. roots occurred at the time when geotropic responsiveness reappears following removal of the cap. All decapped roots exhibiting a geotropic response had some amyloplast sedimentation in the apical cortical cells. Exposing decapped roots to a centrifugal acceleration of 25 g for 4 h showed that amyloplasts of a similar size and development were not displaced within the cytoplasm when this treatment began 12 h after decapping, whereas displacement did occur when the treatment began 24 h after decapping. This finding indicates the occurrence of a change in the physical characteristics of the cytoplasm between 12 h and 24 h after removing of the cap, which allows amyloplast movement and thus restores gravity perception.

Kinetic analysis of effectors of phosphoenolpyruvate carboxylase from Bryophyllum fedtschenkoi.

The activity of phosphoenolpyruvate carboxylase (orthophosphate:oxaloacetate carboxy-lyase (phosporylating) EC 4.1.1.31) purified from Bryophyllum fedtschenkoi has been measured in the presence of various concentrations of phosphoenolpyruvate, L-malate and glucose 6-phosphate. At high pH, the enzyme is competitively inhibited by L-malate and activated by glucose 6-phosphate. A reaction scheme describing the interaction of enzyme, substrate and effectors is proposed. Values for the appropriate equilibrium constants have been calculated for the enzyme acting at pH 7.8, which is one of its two pH optima. The kinetics are more complicated at low pH, partly because of non-linear reaction rates and partly because inhibition by L-malate is not competitive. Activation by glucose 6-phosphate is similar at high and low pH values. The behaviour of a wide range of other possible effectors is described briefly.

Inhibition of the circadian rhythm of CO2 metabolism in Bryophyllum leaves by cycloheximide and dinitrophenol.

The circadian rhythm of CO2 output in darkened leaves of Bryophyllum fedtschenkoi R. Hamet and Perrier can be inhibited by cycloheximide (≧10(-6) mol) and 2,4-dinitrophenol (≧10(-5) mol) applied via the transpiration stream. After having been suppressed by 10(-6) M cycloheximide, the rhythm can be reinitiated with a 12-h exposure to light. Experiments using (14)CO2 show that cycloheximide abolishes the rhythm by inhibiting the dark fixation of CO2. Cycloheximide inhibits malate accumulation and acidification of the leaves, but does not affect the amount of the CO2-fixing enzyme phosphoenol-pyruvate carboxylase (PEP-C, EC 4.1.1.31) which can be extracted from the leaves during the 45 h of the experiment. Cycloheximide has no direct effect on the activity of the enzyme as measured in the assay. PEP-C from desalted leaf extracts was inhibited by L-malate (Ki=0.4 mmol). The most likely explanation for the inhibitory effect of cycloheximide and dinitrophenol is that they cause changes in tonoplast properties which result in a redistribution of malate from the vacuole to the cytoplasm. An increase in malate concentration in the cytoplasm will lead to inhibition of PEP-carboxylase, and hence the suppression of the rhythm of CO2 output.

Phosphoenolpyruvate carboxylase from the crassulacean plant Bryophyllum fedtschenkoi Hamet et Perrier. Purification, molecular and kinetic properties.

Phosphoenolpyruvate carboxylase from the Crassulacean plant Bryophyllum fedtschenkoi has been purified to homogenetity by DEAE-cellulose treatment, (NH4)2SO4 fractionation,, and chromatography on DEAE-cellulose and hydroxyapatite. Poly(ethylene glycol) is required in the extraction medium to obtain maximum enzyme activity. The purified enzyme has a specific activity of about 26 units/mg of protein at 25 degrees C. It gives a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, corresponding to a mol.wt. of 105,000, and gives a single band on non-denaturing gel electrophoresis at pH8.4. Cross-linking studies at pH8.0 indicate that the subunit structure is tetrameric but that the dimer may also be an important unit of polymerization. Gel filtration results at pH6.7 confirm that the native enzyme is tetrameric with a concentration-dependent dissociation to a dimer. The kinetic behaviour is characterized by (i) relatively small variations in maximum velocity between pH5.5 and 9.0 with a double optimum, (ii) a reversible temperature-dependent inactivation between 30 and 45 degrees C, (iii) inhibition by malate, which is pH-sensitive, and (iv) almost Michaelis-Menten behaviour with phosphoenolpyruvate as the varied ligand but sigmoidal behaviour under suitable conditions with malate as the varied ligand. The findings are related to other studies to the possible role phosphoenolpyruvate carboxylase in controlling a circadian rhythm of CO2 fixation.

The circadian rhythm in Bryophyllum leaves: Phase control by radiant energy.

A 4-h exposure to white light from fluorescent lamps can shift the phase of the rhythm of CO2 output in leaves of Bryophyllum fedtschenkoi Hamet & Perr. otherwise kept in continuous darkness. The position in the cycle at which irradiation occurs determines the magnitude and direction of the phase shift. Red and white light induce similar advances or delays in the phase, but blue and far-red irradiation have no effect. Far-red irradiation given simultaneously with, or immediately after, exposure to red light, modifies the phase-shift induced by red light alone. Radiation in the red and far-red regions of the spectrum interacted in several experimental régimes, but complete red/far-red reversibility was not observed. The evidence suggests that phytochrome is the receptor molecule involved in the induction of phase-shifts by light.