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.Abbreviations CAM Crassulacean acid metabolism - FW fresh weight - PEPCase phosphoenolpyruvate carboxylase - RuBPCase ribulose-1,5-bisphosphate carboxylase To whom correspondence should be addressed.     

Mutual stimulation of temperature and light effects on C(4) phosphoenolpyruvate carboxylase in leaf discs and leaves of Amaranthus hypochondriacus

This article reports marked modulation of the activity and regulatory properties of phosphoenolpyruvate carboxylase (PEPC) by temperature and light in leaf discs as well as leaves of Amaranthus hypochondriacus. The activity of PEPC increased by 1.7-fold at 45 degrees C over 25 degrees C. Warm temperature also stimulated the photoactivation of PEPC. The activation by light of PEPC was 1.9-fold at 25 degrees C and increased to 2.2-fold at 45 degrees C. The sensitivity of PEPC to its inhibitor malate was less and the activation by glucose-6-phosphate (G-6-P) or inorganic phosphate (Pi) was more at 45 degrees C than that at 25 degrees C. These effects of temperature were quite pronounced in light. Similar responses were observed when detached leaves were exposed to varying ambient temperature (dry heat). The activity of PEPC increased by 1.6-fold at 45 degrees C over 25 degrees C in the dark. The activation of PEPC by light was 2.1-fold at 25 degrees C and increased to 2.6-fold at 45 degrees C. Inhibition by malate was less and activation by G-6-P or Pi was more at 45 degrees C than that at 25 degrees C. Thus, there was a marked modulation of not only the activity but also the regulatory properties of the enzyme by temperature and light, independently as well as cooperatively with each other. Further experiments suggested that PEPC was able to memorize to a significant extent the changes induced by warm temperature and that these changes were complemented by subsequent illumination. These effects were not due to changes in PEPC protein levels. We conclude that temperature and light can modulate PEPC activity and regulatory properties not only individually but also in a significantly cooperative manner with each other. As significant increases in temperature are common during daytime in tropical or subtropical conditions, we suggest that the synergistic effects of temperature and light are quite relevant in optimizing the activity of PEPC in leaves of C(4) plants.     

Rapid modulation of nitrate reductase in pea roots

The regulatory properties of nitrate reductase (NR; EC 1.6.6.1) in root extracts from hydroponically grown pea (Pisum sativum L. cv. Kleine Rheinländerin) plants were examined and compared with known properties of NR from spinach and pea leaves. Nitrate-reductase activity (NRA) extracted from pea roots decreased slowly when plants were kept in the dark, or when illuminated plants were detopped, with a half-time of about 4 h (= slow modulation in vivo). In contrast, the half-time for the dark-inactivation of NR from pea leaves was only 10 min. However, when root tip segments were transferred from aerobic to anaerobic conditions or vice versa, changes in NRA were as rapid as in leaves (= rapid modulation in vivo). Nitrate-reductase activity was low when extracted from roots kept in solutions flushed with air or pure oxygen, and high in nitrogen. Okadaic acid, a specific inhibitor of type-1 and type-2A protein phosphatases, totally prevented the in vivo activation by anaerobiosis of NR, indicating that rapid activation of root NR involved protein dephosphorylation. Under aerobic conditions, the low NRA in roots was also rapidly increased by incubating the roots with either uncouplers or mannose. Under these conditions, and also under anaerobiosis, ATP levels in roots were much lower than in aerated control roots. Thus, whenever ATP levels in roots were artificially decreased, NRA increased rapidly. The highly active NR extracted from anaerobic roots could be partially inactivated in vitro by preincubation of desalted root extracts with MgATP (2 mM), with a half-time of about 20 min. It was reactivated by subsequently incubating the extracts with excess AMP (2 mM). Thus, pea root NR shares many of the previously described properties of NR from spinach leaves, suggesting that the root enzyme, like the leaf enzyme, can be rapidly modulated, probably by reversible protein phosphorylation/ dephosphorylation.     

Involvement of thiol groups in the activity of phosphoenolpyruvate carboxylase from maize leaves

Purified maize leaf phosphoenolpyruvate carboxylase (EC 4.1.1.31) was completely inactivated by several thiol-modifying reagents, including, CuCl₂, CdCl₂ and N-ethylmaleimide. The inactivation by CuCl₂ could be reversed by dithiothreitol, suggesting the involvement of vicinal dithiols in the inactivation process.Complete inactivation of phosphoenolpyruvate carboxylase was correlated with the incorporation of two mol (³H)N-ethylmaleimide per 100-kilodalton subunit. The total protection of the enzyme against N-ethylmaleimide inactivation afforded by the substrate, phosphoenolpyruvate, was correlated with the protection of one mol (³H)N-ethylmaleimide reactive residue per mol subunit.The complete inactivation of phosphoenolpyruvate carboxylase by N-ethylmaleimide and the protection afforded by phosphoenolpyruvate against modification suggest the presence of an essential cysteine residue in the catalytic site of the C₄ leaf enzyme.Abbreviations PEP, phosphoenolpyruvate - Mops, 4-morpholinepropanesulphonic acid (Consejo Nacional de Investigaciones Científicas y Técnicas, Fundación M. Lillo y U.N. de Rosario).     

Effect of LiCl on phosphoenolpyruvate carboxylase kinase and the phosphorylation of phosphoenolpyruvate carboxylase in leaf disks and leaves of <Emphasis Type="Italic">Sorghum vulgare</Emphasis>

In the present work, the effect of LiCl on phosphoenolpyruvate carboxylase kinase (PEPCase-k), C₄ phosphoenolpyruvate carboxylase (PEPCase: EC 4.1.1.31) and its phosphorylation process has been investigated in illuminated leaf disks and leaves of the C₄ plant Sorghum vulgare. Although this salt induced severe damages to older leaves, it did not significantly alter the physiological parameters (photosynthesis, transpiration rate, intercellular CO₂ concentration) of young leaves. An immunological approach was used to demonstrate that the PEPCase-k protein accumulated rapidly in illuminated leaf tissues, consistent with the increase in its catalytic activity. In vivo, LiCl was shown to strongly enhance the light effect on PEPCase-k protein content, this process being dependent on protein synthesis. In marked contrast, the salt was found to inhibit the PEPCase-k activity in reconstituted assays and to decrease the C₄ PEPCase content and phosphorylation state in LiCl treated plants. Short-term (15 min) LiCl treatment increased IP₃ levels, PPCK gene expression, and PEPCase-k accumulation. Extending the treatment (1 h) markedly decreased IP₃ and PPCK gene expression, while PEPCase-k activity was kept high. The cytosolic protein synthesis inhibitor cycloheximide (CHX), which blocked the light-dependent up-regulation of the kinase in control plants, was found not to be active on this process in preilluminated, LiCl-treated leaves. This suggested that the salt causes the kinase turnover to be altered, presumably by decreasing degradation of the corresponding polypeptide. Taken together, these results establish PEPCase-k and PEPCase phosphorylation as lithium targets in higher plants and that this salt can provide a means to investigate further the organization and functioning of the cascade controlling the activity of both enzymes.     

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.Abbreviations B. Bryophyllum - PEPCase phosphoenolpyruvate carboxylase     

Control of the phosphorylation of phosphoenolpyruvate carboxylase in higher plants

Phosphoenolpyruvate (PEP) carboxylase is regulated by reversible phosphorylation in higher plants. Recently several genes encoding PEP carboxylase kinase have been cloned. The purpose of this article is to assess the contribution that information on the structure and expression of these genes is making to our understanding of the posttranslational control of PEP carboxylase activity.     

Intact mesophyll protoplasts from Zea mays as a source of phosphoenolpyruvate carboxylase unaffected by extraction: Advantages and limitations

Isolated intact mesophyll protoplasts from Zea mays L. were used as an enzyme source for studying properties of phosphoenolpyruvate (PEP) carboxylase (EC 4.1 1 31) just after release from cells into the reaction medium. After the injection of protoplasts into the assay mixture, an initial lag of activity was observed, mainly due to the time necessary for complete disruption of protoplasts by the osmotic shock. The final specific activity obtained was ca 18 μmol mg^-1 of liberated protein min^-1, a value comparable to that usually achieved after arduous purification. Under the assay conditions employed, the chloroplasts were not disrupted and the retention of their proteins, together with the use of purified mesophyll protoplasts, were obviously the reasons for the high specific activity obtained. The activity and properties of phosphoenolpyruvate carboxylase stored in isolated protoplasts were stable for at least 24 h at 5°C. The main difference between the protoplast-derived and the routinely extracted enzyme was the sensitivity to malate inhibition, which was partially lost in the extracted phosphoenolpyruvate carboxylase; no difference was found in the K_m(PEP). The stress imposed by the protoplast isolation procedure diminished the sensitivity of the enzyme to malate inhibition, so that it can be inferred that the real malate sensitivity of pbosphocnolpyruvale carboxylase is even greater and that it is grossly underestimated with routinely extracted enzyme.     

Nitrate reductase activity and nitrogen compounds in xylem exudate of Juglans nigra seedlings: relation to nitrogen source and supply

Nitrogen (N) limits plant productivity and its uptake and assimilation may be regulated by N source, N availability, and nitrate reductase activity (NRA). Knowledge of how these factors interact to affect N uptake and assimilation processes in woody angiosperms is limited. We fertilized 1-year-old, half-sib black walnut (Juglans nigra L.) seedlings with ammonium (NH₄ ⁺) [as (NH₄)₂SO₄], nitrate (NO₃ ⁻) (as NaNO₃), or a mixed N source (NH₄NO₃) at 0, 800, or 1,600 mg N plant⁻¹ season⁻¹. Two months following final fertilization, growth, in vivo NRA, plant N status, and xylem exudate N composition were assessed. Specific leaf NRA was higher in NO₃ ⁻-fed and NH₄NO₃-fed plants compared to observed responses in NH₄ ⁺-fed seedlings. Regardless of N source, N addition increased the proportion of amino acids (AA) in xylem exudate, inferring greater NRA in roots, which suggests higher energy cost to plants. Root total NRA was 37% higher in NO₃ ⁻-fed than in NH₄ ⁺-fed plants. Exogenous NO₃ ⁻ was assimilated in roots or stored, so no difference was observed in NO₃ ⁻ levels transported in xylem. Black walnut seedling growth and physiology were generally favored by the mixed N source over NO₃ ⁻ or NH₄ ⁺ alone, suggesting NH₄NO₃ is required to maximize productivity in black walnut. Our findings indicate that black walnut seedling responses to N source and level contrast markedly with results noted for woody gymnosperms or herbaceous angiosperms.     

A study of the in-vitro regulation of phosphoenolpyruvate carboxylase from the epidermis of Commelina communis by malate and glucose-6-phosphate

Phosphoenolpyruvate (PEP) carboxylase activity in epidermal extracts of Commelina communis has been compared in the presence of malate and glucose-6-phosphate. The activity of PEP carboxylase was inhibited by increasing malate concentrations at several substrate (PEP) concentrations and changes in both the apparent K _m (PEP) and V _max values in the presence of malate suggested the occurence of mixed-type inhibiton. In the presence of glucose-6-phosphate no increase in enzyme activity was observed, although there was a slight decrease in the K _m (PEP). However, glucose-6-phosphate appeared to alleviate the inhibition caused by malate. The possible implications of these properties in the control of malate production in guard cells is discussed.Abbreviations PEP phosphoenolpyruvate - Glc6P glucose-6-phosphate     

Effects of temperature on the activity of phosphoenolpyruvate carboxylase and on the control of CO2 fixation in Bryophyllum fedtschenkoi

The phosphorylation state and the malate sensitivity of phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) in Bryophyllum fedtschenkoi Hamet et Perrier are altered by changes in the ambient temperature. These effects, in turn alter the in-vivo activity of the enzyme. Low temperature (3 °C or less), stabilizes the phosphorylated form of the enzyme, while high temperature (30 °C) promotes its dephosphorylation. The catalytic activity of the phosphorylated and dephosphorylated forms of PEPCase increases with temperature, but the apparent K _i values for malate of both forms of the enzyme decrease. Results of experiments with detached leaves maintained in darkness in normal air indicate that the changes in malate sensitivity and phosphorylation state of PEPCase with temperature are of physiological significance. When the phosphorylated form of PEPCase is stabilized by reducing the temperature of leaves 9 h after transfer to constant darkness at 15 °C, a prolonged period of CO₂ fixation follows. When leaves are maintained in constant darkness at 15 °C until CO₂ output reaches a low steady-state level and the PEPCase is dephosphorylated, reducing the temperature to 3 °C results in a further period of CO₂ fixation even though the phosphorylation state of PEPCase does not change.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase We thank the Agricultural and Food Research Council for financial support for this work.     

Replacement of nitrate by ammonium as the nitrogen source increases the salt sensitivity of pea plants. I. Ion concentrations in roots and leaves

Pea seedlings (Pisum sativum L. cv ‘Kleine Rheinlän-derin’) were grown hydroponically in solutions containing either nitrate (3 or 14 mol m⁻³) or ammonium (3 mol m⁻³) as the nitrogen source. Ammonium nutrition as such had no negative effect on plant biomass production, but drastically increased the sensitivity to moderate salinity (50 mol m⁻³ NaCl). The reasons for this effect are investigated here and in a subsequent paper. The appearance of visible symptoms of salt damage (wilting of marginal leaf areas followed by progressive necrosis) was paralleled by the development of several characteristic modifications in the solute and metabolite contents. Major changes were: (i) high salt (NaCl) accumulation in leaves; (ii) accumulation of ammonium (up to 20 mol m⁻³) and amino acids (up to 110 mol m⁻³) in leaves, but at decreased ammonium uptake rates; and (iii) decreased protein content. In a comparison paper we report on the subcellular distribution of salts, ammonium and metabolites under the above conditions.     

Regulation of nitrate reductase and phosphoenolpyruvate carboxylase activities in barley leaf protoplasts

Barley leaf protoplasts were incubated in light or darkness in the presence of various inhibitors, metabolites or weak acids/bases. Nitrate reductase (NR) and phosphoenolpyruvate carboxylase (PEPCase) were rapidly extracted from the protoplasts and assayed under sub-optimal conditions, i.e. in the presence of Mg²⁺ and malate, respectively. Under these conditions changes in activities are thought to reflect changes in the phosphorylation states of the enzymes. The NR was activated by illumination to 90% of its maximal activity within 10 min. Photosynthetic electron transport appeared necessary for light activation of NR since activation was inhibited by the photosynthetic electron-transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and, additionally, an electron acceptor (HCO ₃ ^- ) was required. The PEPCase was also activated by light. However, this activation was not prevented by DCMU or lack of HCO ₃ ^- . Loading of protoplasts in the dark with a weak acid resulted in activation of both NR and PEPCase. For NR, full activation was completed within 5 min, whereas for PEPCase a slower, modest activation continued for at least 40 min. Incubation of protoplasts with a weak base also gave activation of PEPCase, but not of NR. On the contrary, base loading counteracted light activation of NR. Since several treatments tested resulted in the modulation of either NR or PEPCase activity, but not both, signal transduction cascades leading to changes in activities appear to be very different for the two enzymes.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron) - DMO 5,5-dimethyl-2,4 oxazolidinedione - NR nitrate reductase - PEPCase Phosphoenolpyruvate carboxylase This work was supported by the Norwegian Research Council by a Grant to C.L: L.H.S. was supported by the Biotechnology and Biological Sciences Research Council.     

Secretion of malate and citrate from roots is related to high Al-resistance in Lespedeza bicolor

Lespedeza bicolor (Lespedeza bicolor Turcz. cv. Jiangxi) is a leguminous shrub that is well adapted to acid infertile soils. However, the mechanisms of aluminum resistance in this species have not been established. This study aimed to assess the possible resistance mechanisms of this plant to Al. An Al-sensitive species of Lespedeza, sericea lespedeza [Lespedeza cuneata (Dum.-Cours.) G. Don cv. Zhejiang], was used as a reference. The roots of L. bicolor secreted both malate and citrate after exposure to Al, but roots of L. cuneata did not. The secretion of organic acids from L. bicolor was specific to Al; neither 15-day P starvation nor 50 μM lanthanum induced the secretion of these organic acid anions. Secretion of organic acid anions in L. bicolor was detected after 3–6 h exposure to Al, and the amount increased significantly after 6 h exposure, suggesting that this plant shows a pattern II-type organic acid secretion. This is supported by the finding that the secretion was significantly inhibited by a protein-synthesis inhibitor, cycloheximide. Two kinds of anion-channel inhibitors had different effects on Al-induced secretion of organic acids: 9-anthracene carboxylic acid completely inhibited secretion, phenylglyoxal had no effect. Root elongation in L. bicolor was more severely inhibited by Al in the presence of 9-anthracene carboxylic acid. All these results indicated that the secretion of malate and citrate is a specialized response to Al stress in L. bicolor roots, which might be one of the Al-resistance mechanisms in this species.     

Effects of sulfite on phosphoenolpyruvate carboxylase and nicotinamide adenine dinucleotide phosphate-dependent malate dehydrogenase in epidermal peels in two cultivars of pea

We have identified a differential response of stomatal conductance to sulfur dioxide in two cultivars of pea ( Pisum sativum L. cvs P715 and Nugget). The response to sulfite exposure of PEPC activities present in epidermal peels obtained from the two cultivars was qualitatively in agreement with the results obtained for stomatal conductance. With epidermal tissue isolated from the more sensitive cultivar, we have investigated the effect of light and sulfite on guard cell phosphoenolpyruvate carboxylase (E.E. 4.1.1.31.) and NADP-dependent malate dehydrogenase (E.C. 1.1.1.82), two enzymes of the malate biosynthetic pathway. No difference was found between the substrate-saturated activity of phosphoenolpyruvate carboxylase in epidermal tissue incubated in the light or in the dark under the same conditions. Substratesaturated NADP-dependent malate dehydrogenase activity increased nearly 3-fold during a 60 min incubation in the light. Incubations of epidermal tissue in the light in the presence of sulfite resulted in a decrease in the activity of both enzymes. Our results suggest that the inhibition of these two enzymes of the malate biosynthetic pathway may be one cause of sulfur dioxide-mediated stomatal closure.     

Recombinant thermoactive phosphoenolpyruvate carboxylase (PEPC) from Thermosynechococcus elongatus and its coupling with mesophilic/thermophilic bacterial carbonic anhydrases (CAs) for the conversion of CO2 to oxaloacetate

With the continuous increase of atmospheric CO₂ in the last decades, efficient methods for carbon capture, sequestration, and utilization are urgently required. The possibility of converting CO₂ into useful chemicals could be a good strategy to both decreasing the CO₂ concentration and for achieving an efficient exploitation of this cheap carbon source. Recently, several single- and multi-enzyme systems for the catalytic conversion of CO₂ mainly to bicarbonate have been implemented. In order to design and construct a catalytic system for the conversion of CO₂ to organic molecules, we implemented an in vitro multienzyme system using mesophilic and thermophilic enzymes. The system, in fact, was constituted by a recombinant phosphoenolpyruvate carboxylase (PEPC) from the thermophilic cyanobacterium Thermosynechococcus elongatus, in combination with mesophilic/thermophilic bacterial carbonic anhydrases (CAs), for converting CO₂ into oxaloacetate, a compound of potential utility in industrial processes. The catalytic procedure is in two steps: the conversion of CO₂ into bicarbonate by CA, followed by the carboxylation of phosphoenolpyruvate with bicarbonate, catalyzed by PEPC, with formation of oxaloacetate (OAA). All tested CAs, belonging to α-, β-, and γ-CA classes, were able to increase OAA production compared to procedures when only PEPC was used. Interestingly, the efficiency of the CAs tested in OAA production was in good agreement with the kinetic parameters for the CO₂ hydration reaction of these enzymes. This PEPC also revealed to be thermoactive and thermostable, and when coupled with the extremely thermostable CA from Sulphurhydrogenibium azorense (SazCA) the production of OAA was achieved even if the two enzymes were exposed to temperatures up to 60 °C, suggesting a possible role of the two coupled enzymes in biotechnological processes.     

Evolution of enzymes involved in carbon metabolism (phosphoenolpyruvate and ribulose-bisphosphate carboxylases,phosphoenolpyruvate carboxykinase) during the light-induced greening of Euglena gracilis strains Z and ZR

Phosphoenolpyruvate carboxykinase activity decreases when Euglena gracilis Z and ZR undergo light-induced chloroplast development in batch resting medium lacking utilizable organic carbon and CO₂. This enzyme is present in heterotrophically grown cells (Briand et al. 1981) and assures gluconeogenesis. It was consistently more active in strain ZR. Decreased carboxykinase activities were accompanied by parallel increases in the activities of ribulose bisphosphate carboxylase and phosphoenolpyruvate carboxylase. The rates of O₂ evolution in light were much lower than those of CO₂ fixed simultaneously. The incorporation of ¹⁴CO₂ into early C-4 dicarboxylic acids was higher in green cells than in etiolated cells, and it was even higher in green cells assayed in light in the presence of (DCMU). A hypothesis has been proposed, according to which there is a possible cooperation of phosphoenolpyruvate carboxylase in photosynthetic CO₂ fixation, especially under conditions of limiting CO₂.High temperatures (34° C) depress carboxylation enzyme activities to a greater extent than that of the carboxykinase without a great effect on cellular chlorophyll content. In the presence of 25 m DCMU, however, chlorophyll accumulation is reduced without any detectable changes in enzyme activities in the Z strain. The ZR strain displayed its characteristic resistance to DCMU.Abbreviations PEP phosphoenolpyruvate - RuBP ribulose bisphosphate - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea To whom all correspondence and reprint request should be addressed     

Optical characteristics of the harmful dinoflagellate Alexandrium tamarense in response to different nitrogen sources

The chlorophyll a specific absorption coefficient a_ph^* and absorption ratio (a_ph ratio) of Alexandrium tamarense at five concentrations of nitrate, ammonium and urea over a range from 6 to 100 μM were examined. The experimental results were compared to two coastal diatom species and a prymnesiophyte to identify differences in absorption ratios. Cells exposed to increasing nitrate concentrations were characterized by an increase in a_ph^* at 443, 490, 510, 555 and 675 nm. However, ammonium and urea induced low a_ph^* values at their lowest and highest concentrations. The a_ph relative to 510 or 555 nm was constant regardless of the concentration of the N source, but dependent on the N source. Oxidized N induced a lower a_ph ratio than the reduced form. Comparisons of the a_ph ratio among taxonomic groups revealed significant differences. The a_ph ratio of A. tamarense was 20–30 and >50% lower than those of two diatoms and a prymnesiophyte, respectively. The a_ph ratio of the present study could assist in increasing the capability for detecting harmful species such as A. tamarense.     

Short-term modulation of nitrate reductase activity by exogenous nitrate in Nicotiana plumbaginifolia and Zea mays leaves

Maize (Zea mays L.) grown on low (0.8 mM) NO ₃ ^- , as well as untransformed and transformed Nicotiana plumbaginifolia constitutively expressing nitrate reductase (NR), was used to study the effects of NO ₃ ^- on the NR activation state. The NR activation state was determined from the relationship of total activity extracted in the presence of ethylenediaminetetracetic acid to that extracted in the presence of Mg²⁺. Light activation was observed in both maize and tobacco leaves. In the tobacco lines, NO ₃ ^- did not influence the NR activation state. In excised maize leaves, no correlation was found between the foliar NO ₃ ^- content and the NR activation state. Similarly, the NR activation state did not respond to NO ₃ ^- . Since the NR activation state determined from the degree of Mg²⁺-induced inhibition of NR activity is considered to reflect the phosphorylation state of the NR protein, the protein phosphatase inhibitor microcystin LR was used to test the importance of protein phosphorylation in the NO ₃ ^- -induced changes in NR activity. In-vivo inhibition of endogenous protein phosphatase activity by microcystin-LR decreased the level of NR activation in the light. This occurred to the same extent in the presence or absence of exogenous NO ₃ ^- . We conclude that NO ₃ ^- does not effect the NR activation state, as modulated by protein phosphorylation in either tobacco (a C₃ species) or maize (a C₄ species). The short-term regulation of NR therefore differs from the NO ₃ ^- -mediated responses observed for phosphoenolpyruvate carboxylase and sucrose phosphate synthase.Abbreviations Chl chlorophyll - MC microcystin-LR - PEP-Case phosphoenolpyruvate carboxylase - SPS sucrose-phosphate synthase We are indebted to Madeleine Provot and Nathalie Hayes for excellent technical assistance. This work was funded by EEC Biotechnology Contract No. BI02 CT93 0400, project of technical priority, Network D — Nitrogen Utilisation and Efficiency.