Changes in Levels of Phosphoenolpyruvate Carboxylase with Induction of Crassulacean Acid Metabolism in Mesembryanthemum crystallinum L.

Expanded leaves of Mesembryanthemum crystallinum L. performingC₃ photosynthesis were induced to perform pronounced Crassulaceanacid metabolism (CAM) by exposing the plant roots to higherNaCl concentration. Levels of phosphoenolpyruvate (PEP) carboxylaseactivity increased 10-fold during the 7-day induction period.Densitometric analysis of Coomassie-stained sodium dodecyl sulfate(SDS) polyacrylamide gradient slab gels of leaf extracts, preparedduring the course of CAM induction, revealed that at least fivebands of polypeptides increased in content (kilodalton valuesof 98, 91, 45, 41, 38). Higher levels of three additional polypeptides(kilodalton values of 102, 76, 33) became apparent after tissuehad been grown for 2 weeks at 400 mM NaCl. Of these polypeptides,that having a mass of 98 kilodaltons was identified as the subunitof PEP carboxylase by comparison with the corresponding bandfrom partially purified PEP carboxylase from the same tissue.Only a faint 98 kilodalton band was evident on SDS gels fortissue operating in the C₃ mode; staining intensity at thislocation increased with increasing NaCl-salinity in the rootingmedium until CAM was fully induced. These data provide evidencefor net synthesis of PEP carboxylase and several other proteinsduring the induction of CAM in M. crystallinum. ¹ Present address: USDA, P. O. Box 867 Airport Rd., Beckley,WV. 25801, U.S.A. ² Present address: Department of Botany, Washington State University,Pullman, Washington 99164, U.S.A. ³ Present address: Botanisches Institut der Universit?t, MittlererDallenbergweg 64, 8700 W?rzburg, W.-Germany. (Received October 27, 1981; Accepted March 15, 1982)     

Transport of Phosphoenolpyruvate by Chloroplasts from Mesembryanthemum crystallinum L. Exhibiting Crassulacean Acid Metabolism

Chloroplasts from CAM-Mesembryanthemum crystallinum can transport phosphoenolpyruvate (PEP) across the envelope. The initial velocities of PEP uptake in the dark at 4°C exhibited saturation kinetics with increasing external PEP concentration. PEP uptake had a V_max of 6.46 (±0.05) micromoles per milligram chlorophyll per hour and an apparent K_mpep of 0.148 (±0.004) millimolar. The uptake was competitively inhibited by Pi (apparent K_i = 0.19 millimolar), by glycerate 3-phosphate (apparent K_i = 0.13 millimolar), and by dihydroxyacetone phosphate, but malate and pyruvate were without effect. The chloroplasts were able to synthesize PEP when presented with pyruvate. PEP synthesis was light dependent. The prolonged synthesis and export of PEP from the chloroplasts required the presence of Pi or glycerate 3-phosphate in the external medium. It is suggested that the transport of pyruvate and PEP across the chloroplasts envelope is required during the gluconeogenic conversion of carbon from malate to storage carbohydrate in the light.     

Diurnal Ion Fluctuations in the Mesophyll Tissue of the Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum

Both laboratory- and field-grown Mesembryanthemum crystallinum plants exhibited large scale diurnal ion fluctuations. In mesophyll tissue, potassium and sodium levels varied in conjunction with acid levels while chloride levels varied in opposition. Thus, dark CO₂ fixation in this Crassulacean acid metabolism species seems analogous to the common plant process of malate synthesis to balance cation surplus. Sodium levels in the epidermis appeared to fluctuate in opposition to those in the mesophyll. It is proposed that inorganic cations cycle between mesophyll and epidermal tissue to balance malate accumulation and to produce stomatal opening in the dark.     

Day/Night Changes in the Sensitivity of Phosphoenolpyruvate Carboxylase to Malate during Crassulacean Acid Metabolism

Phosphoenolpyruvate carboxylase (PEPC) was extracted from Mesembryanthemum crystallinum L. performing Crassulacean acid metabolism, at frequent intervals during a 12-hour light/12-hour dark cycle. Inhibition of PEPC by malate was followed at pH 8.0 and 7.5, 1 minute after homogenization of leaves. PEPC was more sensitive to malate during the light than during the dark periods and inhibition by malate was more pronounced at pH 7.5 than 8.0. For example, PEPC was not or only slightly inhibited by 0.5 millimolar malate during the dark period at both pH values and the rates per milligram chlorophyll were about the same. During the light period, 0.5 millimolar malate resulted in a 20 to 30% reduction of PEPC activity at pH 8.0 and a 80 to 90% reduction at pH 7.5. These and other experiments, in which plants were kept in prolonged dark periods, indicate that the increase in sensitivity of PEPC to malate is correlated with the change from acidification to deacidification in the tissue. These interactions account for apparent changes in pH response of PEPC in crude extracts assayed at different times of the day/night cycle.     

Metabolite Control Overrides Circadian Regulation of Phosphoenolpyruvate Carboxylase Kinase and CO(2) Fixation in Crassulacean Acid Metabolism

Phosphoenolpyruvate carboxylase (PEPc) catalyzes the primary fixation of CO₂ in Crassulacean acid metabolism plants. Flux through the enzyme is regulated by reversible phosphorylation. PEPc kinase is controlled by changes in the level of its translatable mRNA in response to a circadian rhythm. The physiological significance of changes in the levels of PEPc-kinase-translatable mRNA and the involvement of metabolites in control of the kinase was investigated by subjecting Kalanchoë daigremontiana leaves to anaerobic conditions at night to modulate the magnitude of malate accumulation, or to a rise in temperature at night to increase the efflux of malate from vacuole to cytosol. Changes in CO₂ fixation and PEPc kinase activity reflected those in kinase mRNA. The highest rates of CO₂ fixation and levels of kinase mRNA were observed in leaves subjected to anaerobic treatment for the first half of the night and then transferred to ambient air. In leaves subjected to anaerobic treatment overnight and transferred to ambient air at the start of the day, PEPc-kinase-translatable mRNA and activity, the phosphorylation state of PEPc, and fixation of atmospheric CO₂ were significantly higher than those for control leaves for the first 3 h of the light period. A nighttime temperature increase from 19°C to 27°C led to a rapid reduction in kinase mRNA and activity; however, this was not observed in leaves in which malate accumulation had been prevented by anaerobic treatment. These data are consistent with the hypothesis that a high concentration of malate reduces both kinase mRNA and the accumulation of the kinase itself.     

露花磷酸烯醇式丙酮酸羧化酶同功酶及其四级结构

PEPC的多态性在许多植物中都有报告。在CAM植物中,许多实验结果表明PEPC有两种分子量稍有不同的亚基（Hoffner等1989,Nimmo等1986,Muller和 Kludge1983,Muller等1982）。近年来,PEPC的多态性在基因水平也得到证实（Chollet等1996,Gehrig等1995）。冰叶日中花（Mesembtwcmptallinuzn）中,编码两个不同分子量PEPC多肽的基因已被克隆（Chollet等1996）。但这两个亚基究竟是相互结合而成异二聚体,还是以同聚方式各自缔合为两个同聚体酶目前尚有不同观点。有报告在有的CAM植物中,PEPC的聚合度有昼夜的变化,且这种变化引…     

Light Moderates the Induction of Phosphoenolpyruvate Carboxylase by NaCl and Abscisic Acid in Mesembryanthemum crystallinum

In Mesembryanthemum crystallinum, phosphoenolpyruvate carboxylase is synthesized de novo in response to osmotic stress, as part of the switch from C₃-photosynthesis to Crassulacean acid metabolism. To better understand the environmental signals involved in this pathway, we have investigated the effects of light on the induced expression of phosphoenolpyruvate carboxylase mRNA and protein in response to stress by 400 millimolar NaCl or 10 micromolar abscisic acid in hydroponically grown plants. When plants were grown in high-intensity fluorescent or incandescent light (850 microeinsteins per square meter per second), NaCl and abscisic acid induced approximately an eightfold accumulation of phosphoenolpyruvate carboxylase mRNA when compared to untreated controls. Levels of phosphoenolpyruvate carboxylase protein were high in these abscisic acid- and NaCl-treated plants, and detectable in the unstressed control. Growth in high-intensity incandescent (red) light resulted in approximately twofold higher levels of phosphoenolpyruvate carboxylase mRNA in the untreated plants when compared to control plants grown in high-intensity fluorescent light. In low light (300 microeinsteins per square meter per second fluorescent), only NaCl induced mRNA levels significantly above the untreated controls. Low light grown abscisic acid- and NaCl-treated plants contained a small amount of phosphoenolpyruvate carboxylase protein, whereas the (untreated) control plants did not contain detectable amounts of phosphoenolpyruvate carboxylase. Environmental stimuli, such as light and osmotic stress, exert a combined effect on gene expression in this facultative halophyte.     

Reduced Glutathione as an Effector of Phosphoenolpyruvate Carboxylase of the Crassulacean Acid Metabolism Plant Sedum praealtum D.C

Reduced glutathione, but not mercaptoethanol or dithiothreitol, inhibits phosphoenolpyruvate carboxylase (PEPC) in desalted leaf extracts from Sedum praealtum D.C. The inhibition is more evident at low pH values (< 7.2) and becomes increasingly smaller at higher pH. In the presence of the inhibitor, the hyperbolic rate curve of night PEPC is transformed to sigmoid and the S_0.5 is increased. When the enzyme is extracted during the day, the rate curve is sigmoid and it is not changed by the inhibitor, though the S_0.5 is further increased. Oxidized glutathione is completely inactive. Levels of reduced glutathione in leaf tissue are distinctly higher in the light. A role of photosynthetically reduced glutathione in the regulation of PEPC in Crassulacean acid metabolism species appears probable.     

Induction of Crassulacean Acid Metabolism in the Facultative Halophyte Mesembryanthemum crystallinum by Abscisic Acid

The facultative halophyte, Mesembryanthemum crystallinum, shifts its mode of carbon assimilation from the C₃ pathway to Crassulacean acid metabolism (CAM) in response to water stress. In this study, exogenously applied abscisic acid (ABA), at micromolar concentrations, could partially substitute for water stress in induction of CAM in this species. ABA at concentrations of 5 to 10 micromolar, when applied to leaves or to the roots in hydroponic culture or in soil, induced the expression of CAM within days (as indicated by the nocturnal accumulation of total titratable acidity and malate). After applying ABA there was also an increase in phosphoenolpyruvate carboxylase and NADP-malic enzyme activities. The degree and time course of induction by ABA were comparable to those induced by salt and water stress. Electrophoretic analyses of leaf soluble protein indicate that the increases in phosphoenolpyruvate carboxylase activity during the induction by ABA, salt, and water stress are due to an increase in the quantity of the enzyme protein. ABA may be a factor in the stress-induced expression of CAM in M. crystallinum, serving as a functional link between stress and biochemical adaptation.     

Changes in Properties of Phosphoenolpyruvate Carboxylase with Induction of Crassulacean Acid Metabolism (CAM) in the C4 Plant Portulaca Oleracea

Photosynthetica - Aiming at understanding the odd case of CAM expression by a C4 plant, some properties of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31, orthophosphate: oxaloacetate...     

Crassulacean Acid Metabolism and Crassulacean Acid Metabolism Modifications in Peperomia camptotricha

Peperomia camptotricha, a tropical epiphyte from Mexico, shows variable forms of Crassulacean acid metabolism (CAM). Young leaves exhibit CAM-cycling, while mature leaves show an intermediate type of metabolism, between CAM and CAM-cycling, having approximately the same amount of nighttime gas exchange as daytime. Metabolism of young leaves appears independent of daylength, but mature leaves have a tendency toward more CAM-like metabolism under short days (8 hours). Large differences in the physical appearance of plants were found between those grown under short daylengths and those grown under long daylengths (14 hours). Some anatomical differences were also detected in the leaves. Water stress caused a switch to CAM in young and mature leaves, and as water stress increased, they shifted to CAM-idling.     

Changes in Properties of Phosphoenolpyruvate Carboxylase with Induction of Crassulacean Acid Metabolism (CAM) in the C4 Plant Portulaca Oleracea

Aiming at understanding the odd case of CAM expression by a C₄ plant, some properties of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31, orthophosphate: oxaloacetate carboxylyase, phosphorylating) were comparatively studied in leaves of CAM-expressing and non-expressing Portulaca oleracea L. plants. CAM expression was induced by growing plants under an 8-h photoperiod and under water-stress. CAM induction in leaves of these plants (designated as CAM) is indicated by the nocturnal acidification and by the clear diurnal oscillation pattern and amplitude of acidity, malic acid, and PEPC activity characteristic of CAM plants. Treatment of the other plant group (designated as C₄) by growth under a 16-h photoperiod and well-watered conditions did not induce expression of the tested criteria of CAM in plants. In these C₄ plants, the mentioned CAM criteria were undetectable. PEPC from CAM and C₄ Portulaca responded differently to any of the studied assay conditions or effectors. For example, extent and timing of sensitivity of PEPC to pH change, inhibition by malate, activation by glucose-6-phosphate or inorganic phosphate, and the enzyme affinity to the substrate PEP were reversed with induction of CAM from the C₄-P. oleracea. These contrasting responses indicate distinct kinetic and regulatory properties of PEPC of the two modes. Thus by shifting to CAM in the C₄ Portulaca a new PEPC isoform may be synthesised to meet CAM requirements. Simultaneous occurrence of both C₄ and CAM is suggested in P. oleracea when challenged with growth under stress. This revised version was published online in August 2006 with corrections to the Cover Date.     

Salt Requirement for Crassulacean Acid Metabolism in the Annual Succulent, Mesembryanthemum crystallinum

In experiments with the facultative Crassulacean acid metabolism (CAM) species, Mesembryanthemum crystallinum, only plants which received high levels of inorganic salts fixed substantial amounts of CO₂ by the CAM pathway. Equivalent osmolarities of polyethylene glycol 6000 did not yield any CAM fixation. Plant water potential and turgor pressure had no detectable influence on the amount of CAM fixation. These observations rule out the possibility that the inorganic ions were acting as osmotic agents.     

Malate Decarboxylation by Mitochondria of the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum

Mitochondria isolated from leaves of Mesembryanthemum crystallinumoxidized malate by both NAD malic enzyme and NAD malate dehydrogenase.Rates of malate oxidation were higher in mitochondria from plantsgrown at 400 mil NaCl in the rooting medium and performing Crassulaceanacid metabolism (CAM) than in mitochondria from plants grownat 20 mM NaCl and exhibiting C₃-photosynthetic CO₂ fixation.The mitochondria isolated from plants both in the CAM and C₃modes were tightly coupled and gave high respiratory control.At optimum pH for malate oxidation (pH 7.0), pyruvate was themajor product in mitochondria from CAM-M. crystallinum, whereasmitochondria from C₃-M. crystallinum produced predominantlyoxaloacetate. Both the extracted NAD malic enzyme in the presenceof CoA and the oxidation of malate to pyruvate by the mitochondriafrom plants in the CAM mode had a pH optimum around 7.0 withactivity declining markedly above this pH. The activity of NAD-malicenzyme, expressed on a cytochrome c oxidase activity basis,was much higher in mitochondria from the CAM mode than the C₃mode. The results indicate that mitochondria of this speciesare adapted to decarboxylate malate at high rates during CAM. ¹Current address: Lehrstuhl für Botanik II, UniversitätWurzburg, Mittlerer Dallenbergweg 64, 8700 Würzburg, WestGermany. ²Current address: KD 120, Chemical Research Division, OntarioHydro, 800 Kipling Avenue, Toronto, Ontario M8Z5S4, Canada. ³Current address: Department of Botany, Washington State University,Pullman, Washington 99164-4230, U.S.A. (Received March 13, 1986; Accepted September 18, 1986)     

Phosphoenolpyruvate Carboxylase from Mesembryanthemum crystallinum: Its Isolation and Inactivation in vitro

Phosphoenolpyruvate carboxylase (PEPC) was isolated from leavesof Mesembryanthemum crystallinum, which performed Crassulaceanacid metabolism. PEPC was much more stable when extracted from expanded thanfrom expanding leaves. The inactivation of PEPC in desaltedextracts from expanding leaves was much faster at 25 than at0 °C, was stimulated by raising Mg²⁺ from 0.1 to 3.0 mM,and was reduced by bovine serum albumin. The same type of inactivationwas found after mixing extracts from the two types of leaves,and the decrease in PEPC activity then also included inactivationof the ’stable’ PEPC from the expanded leaves. Afterelution from DEAE-cellulose, PEPC from expanding leaves wasmuch more stable than in desalted, crude extracts. It is suggested that another enzyme is involved in this inactivationof PEPC, but this needs verification.     

盐胁迫对露花叶片的脱落酸含量及磷酸烯醇式丙酮酸羧化酶昼夜调节特性的影响

兼性CAM植物在转为CAM型后,CAM代谢的关键酶磷酸烯醇式丙酮酸（PEP）核化酶会出现昼夜调节特性的变化（Osmond1978）。关于PEP梭化酶昼夜调节特性的机理存在两种观点：1．PEPK化酶昼夜聚合度发生了变化,白天为二聚体PEPK化酶,对苹果酸抑制敏感;而夜间为四聚体,对苹果酸抑制不敏感（U和Wedding1985）。2·nsv＄化酶昼夜磷酸化状态发生变化,夜间PEPW化酶磷酸化,对苹果酸抑制不敏感;而白天PEP＄化酶脱磷酸化,对苹果酸抑制敏感（Nimmo等1986）。植物生长调节物质如ABA和细胞分裂素对兼性CAM植物PEP＆化酶的表达有诱…     

Properties of Leaf NAD-Malic Enzyme from the Inducible Crassulacean Acid Metabolism Species Mesembryanthemum crystallinum

NAD-malic enzyme (NAD-ME) functions to decarboxylate malatein the light in leaves of certain species displaying Crassulaceanacid metabolism (CAM). The properties of NAD-ME in desaltedextracts from the inducible CAM species, Mesembryanthemum crystallinumwere examined. The shapes of the malate saturation curve andthe activity versus pH curve at 10 mM malate were dependenton the presence of the activator CoA. The malate saturationcurve was sigmoidal in the absence of an activator and hyperbolicin the presence of CoA. The pH optimum with 10mM malate andMn²⁺ as cofactor was as low as 6.5 without an activator, andincreased to 7.2 in the presence of CoA. Fumarate activationwas synergistic with CoA above pH 7.2. The enzyme displayedhysteretic behavior under suboptimal assay conditions. Rapid extraction and desalting of the enzyme (<1.5 mim) followedimmediately by assay did not reveal any difference in the propertiesof the enzyme on a day/night basis. It is proposed that diurnalregulation of the enzyme in vivo is mediated by pH and malatelevel without a change in the oligomeric form of the enzyme.The molecular weight of the enzyme was approximately 350,000at pH 6.5 or 7.8. The enzyme obtained from M. crystallinum inthe C₃ mode was very similar to the CAM enzyme except that itdisplayed a lower V_max. ³ Current address: MSU-DOE Plant Research Lab, Michigan StateUniversity, E. Lansing, Michigan, U.S.A. 48824. (Received October 2, 1984; Accepted December 20, 1984)     

Salt Stress Increases the Level of Translatable mRNA for Phosphoenolpyruvate Carboxylase in Mesembryanthemum crystallinum

Mesembryanthemum crystallinum responds to salt stress by switching from C₃ photosynthesis to Crassulacean acid metabolism (CAM). During this transition the activity of phosphoenolpyruvate carboxylase (PEPCase) increases in soluble protein extracts from leaf tissue. We monitored CAM induction in plants irrigated with 0.5 molar NaCl for 5 days during the fourth, fifth, and sixth week after germination. Our results indicate that the age of the plant influenced the response to salt stress. There was no increase in PEPCase protein or PEPCase enzyme activity when plants were irrigated with 0.5 molar NaCl during the fourth and fifth week after germination. However, PEPCase activity increased within 2 to 3 days when plants were salt stressed during the sixth week after germination. Immunoblot analysis with anti-PEPCase antibodies showed that PEPCase synthesis was induced in both expanded leaves and in newly developing axillary shoot tissue. The increase in PEPCase protein was paralleled by an increase in PEPCase mRNA as assayed by immunoprecipitation of PEPCase from the in vitro translation products of RNA from salt-stressed plants. These results demonstrate that salinity increased the level of PEPCase in leaf and shoot tissue via a stress-induced increase in the steady-state level of translatable mRNA for this enzyme.