The metabolism of organic acids by a marine pennate diatom

Cocconeis diminuta, a marine benthic diatom, metabolizes acetate and lactate-¹⁴C. In the light, the major product was lipid, whereas in the dark, CO₂ was the major product. Analysis of proteins synthesized in the presence of acetate or lactate showed that radioactivity was incorporated predominantly into the glutamate family of amino acids and those amino acids related directly to the substrate. Light and dark assimilation of substrate was inhibited slightly by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea and 2,4-dinitrophenol. 3-(3′,4′-Dichlorophenyl)-1,1-dimethylurea caused a pattern of metabolism of acetate in the light characteristic of that which occurs in the dark. Monofluoroacetic acid inhibited assimilation considerably in the dark, but less in the light. The level of enzymes of the tricarboxylic acid cycle and NADH-oxidase were found to be about the same as those in other autotrophs. The metabolism of acetate and lactate is discussed in relation to the autotrophic mode of nutrition of Cocconeis diminuta.     

Growth characteristics of the cyanobacterium Nostoc flagelliforme in photoautotrophic, mixotrophic and heterotrophic cultivation

Nostoc flagelliforme is a terrestrial cyanobacterium with high economic value. Dissociated cells separated from a natural colony of N. flagelliforme were cultivated for 7 days under either phototrophic, mixotrophic or heterotrophic culture conditions. The highest biomass, 1.67 g L⁻¹ cell concentration, was obtained under mixotrophic culture, representing 4.98 and 2.28 times the biomass obtained in phototrophic and heterotrophic cultures, respectively. The biomass in mixotrophic culture was not the sum as that in photoautotrophic and heterotrophic cultures. During the first 4 days of culture, the cell concentration in mixotrophic culture was lower than the sum of those in photoautotrophic and heterotrophic cultures. However, from the 5th day, the cell concentration in mixotrophic culture surpassed the sum of those obtained from the other two trophic modes. Although the inhibitor of photosynthetic electron transport DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] efficiently inhibited autotrophic growth of N. flagelliforme cells, under mixotrophic culture they could grow by using glucose. The addition of glucose changed the response of N.flagelliforme cells to light. The maximal photosynthetic rate, dark respiration rate and light compensation point in mixotrophic culture were higher than those in photoautotrophic cultures. These results suggest that photoautotrophic (photosynthesis) and heterotrophic (oxidative metabolism of glucose) growth interact in mixotrophic growth of N. flagelliforme cells.     

Initiation and characterization of a cotton (Gossypium hirsutum L.) photoautotrophic cell suspension culture

A heterotrophic cotton (Gossypium hirsutum L. cv. Stoneville 825) cell suspension culture was adapted to grow photoautotrophically. After two years in continuous photoautotrophic culture at 5% CO₂ (balance air), the maximum growth rate of the photoautotrophic cell line was a 400% fresh weight increase in eight days. The Chl concentration was approximately 500 g per g fresh weight.Elevated CO₂ (1%–5%) was required for culture growth, while the ambient air of the culture room (600 to 700 ul CO₂ 1^–1) or darkness were lethal. The cell line had no net photosynthesis at 350 ul 1^–1 CO₂, 2% O₂, and dark respiration ranged from 29 to 44 mol CO₂ mg^–1 Chl h^–1. Photosynthesis was inhibited by O₂. The approximate 1:1 ratio of ribulose 1,5-bisphosphate carboxylase (RuBPcase) to phosphoenolpyruvate carboxylase (PEPcase) (normally about 6:1 in mature leaves of C₃ plants) was due to low RuBPcase activity relative to that of C₃ leaves, not to high PEPcase activity. The PEPcase activity per unit Chl in the cell line was identical to that of spinach leaves, while the RuBPcase activity was only 15% of the spinach leaf RuBPcase activity. RuBPcase activity in the photoautotrophic cells was not limited by a lack of activation in vivo, since the enzyme in a rapidly prepared cell extract was 73% activated. No evidence of enzyme inactivation by secondary compounds in the cells was found as can be found with cotton leaves. Low RuBPcase activity and high respiration rates are most likely important factors in the low photosynthetic efficiency of the cells at ambient CO₂.Abbreviations Chl chlorophyll - COT heterotrophic cotton cell line - COT-P photoautotrophic cotton cell line - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - RuBPcase RuBP carboxylase - PEP phosphoenolpyruvate - PEPcase phosphoenolpyruvate carboxylase - MX Murashige and Skoog medium with 0.4 mg 1^–1 2,4-D - KT photomixotrophic medium with 1% sucrose - KT^o KT medium with no carbohydrate - KTP^o KT^o medium supplemented with 0.3 M Picloram - CER CO₂ exchange rate - PCER CO₂ exchange rate in the light     

Interactions between photoautotrophic and heterotrophic metabolism in photoheterotrophic cultures of Euglena gracilis

Interactions between photoautotrophic and heterotrophic metabolism in photoheterotrophic culture of Euglena gracilis were studied. Under a low light supply coefficient, these two metabolic activities seem to proceed independently. The cell growth rate in photoheterotrophic culture was about the sum of the growth rates in pure photoautotrophic and heterotrophic cultures. However under a high light supply coefficient, both photoautotrophic and heterotrophic (glucose assimilation) metabolic activities were inhibited, resulting in a low photoheterotrophic growth rate. The photoheterotrophic culture was more sensitive to photoinhibition compared to the pure photoautotrophic culture. Inhibition of glucose assimilation in the photoheterotrophic culture was due to both direct and indirect (through photosynthesis) effects of high light intensity. Cell growth, glucose assimilation and alpha-tocopherol content of the cells were higher when ambient air was used for aeration than when a mixture of carbon dioxide and air was used. Even when photosynthesis was inhibited by addition of 3-(3,4-dichlorophenyl)- 1,1-dimethylurea to photoheterotrophic culture, light stimulated alpha-tocopherol synthesis by E. gracilis.     

Hydrogen metabolism in the facultative anoxygenic cyanobacteria (blue-green algae) Oscillatoria limnetica and Aphanothece halophytica

Two facultative anoxygenic photoautotrophic cyanobacteria, Oscillatoria limnetica and Aphanothece halophytica were found capable of CO₂ photoassimilation using molecular hydrogen as electron donor in a photosystem I driven reaction. A. halophytica was also capable of evolving hydrogen from Na-dithionite reduced methylviologen in a light independent reaction.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DSPD Disallcylidenepropanediamine - FCCP Carbonylcyanide p-trifluoromethoxyphenyl hydrazone - Tricine N-tris(hydroxy methyl)-methylglycine     

Ribulose-1,5-bisphosphate carboxylase and phosphoenolpyruvate carboxylase activities of photoautotrophic callus of Platycerium coronarium (Koenig ex O.F. Muell.) Desv. under CO2 enrichment

The in vitro activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC) were measured in cell-free extracts of Platycerium coronarium callus cultured for up to 42 days under photoautotrophic conditions with CO₂ enrichment. With an increase in CO₂ in the culture environment to 10% (v/v) at low light, the apparent photoautotrophic fixation of CO₂ by Rubisco declined, whereas the non-photoautotrophic CO₂ fixation by PEPC activity was enhanced. Hence, photosynthesis appears to play a lesser role in providing carbon skeletons and energy with prolonged culture in a CO₂-enriched environment. Instead, the anaplerotic supply of C-skeletons by PEPC may be important under such a situation. Short-term H¹⁴CO₃-fixation experiments indicated that photoautotrophic callus cultured for 3 weeks with 10% CO₂ enrichment assimilated less ¹⁴CO₂ than the control (0.03% CO₂). Analyses of ¹⁴C-metabolites indicated that about 50% of the total soluble ¹⁴CO₂ fixed was in the organic acid fraction and 35% in the amino acid fraction. Despite the changes in the in vitro Rubisco/PEPC activity-ratio, no significant change in the ¹⁴C distribution pattern was apparent in response to increasing sucrose or CO₂ concentrations. The suppression of Rubisco activity and total chlorophyll content in high sucrose or elevated CO₂ concentrations suggests an inhibition of the capacity for photoautotrophic callus growth under these conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

H2-Photoproduction by green algae: The significance of anaerobic pre-incubation periods and of high light intensities for H2-photoproductivity ofChlorella fusca

Using sodium-dithionite as an oxygen scavenger, the influences of different light intensities and periods of anaerobic pre-incubation in the dark on H₂-photoproductivity were studied with the green algaChlorella fusca. By measuring hydrogen production in the light using manometric and gas chromatographic methods the effectiveness of sodium dithionite in stabilizing photoproduction was established. For high rates of H₂-photoproduction high light intensities up to 30,000 lux (580 W m^-2) were necessary; these are comparable to those required for light saturation of oxygen photoproduction by this alga. AlthoughChlorella fusca produces H₂ immediately after transition to anaerobic conditions, the optimum rate of H₂ production was reached after a 5 h dark adaptation period only. The results obtained are discussed with respect to characteristics of H₂-photoproduction by green algae: the initial burst kinetics, the light saturation, and the obligate period of anaerobic adaptation. It is concluded that H₂-photoproduction byChlorella is an anaerobic photosynthetic process which occurs in the absence of CO₂ and can be experimentally stabilized by exogenous oxygen scavengers.Abbreviations DCMU (3-(3,4-Dichlorophenyl)-1,1-dimethylurea) - HEPES (2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid)     

Protein synthesis in Petunia hybrida chloroplasts isolated from leaves and cell cultures

Isolation and incubation conditions were established for Petunia hybrida chloroplasts capable of performing in vitro protein and RNA synthesis. Under these conditions, chloroplasts from leaves as well as from the non-photoautotrophic mutant green cell culture AK-2401 are able to incorporate labeled amino acids into polypeptides. Intact chloroplasts can use light as an energy source; photosynthetically-inactive chloroplasts require the addition for ATP for this protein synthesis. Sodium dodecylsulphate polyacrylamide slab gel electrophoresis shows that in isolated leaf chloroplasts at least twenty-five radioactive polypeptide species are synthesized. The three major products synthesized have molecular weights of 52,000, 32,000 and 17,000. Coomassie brilliant-bluestained polypeptide patterns from plastids isolated from the mutant green cell culture AK-2401 differ considerably from those obtained from leaf chloroplasts. The pattern of radioactive polypeptides synthesized in these isolated cell culture plastids also shows differences. These results indicate that the difference in developmental stage observed between plastids from the cell culture AK-2401 and leaves is reflected in an altered expression of the chloroplast DNA.Abbreviations CAP D-threo-chloramphenicol - 2,4-D 2,4-dichlorophenoxyacetic acid - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - RuBPCase ribulose-1,5-bisphosphate carboxylase - SDS sodium dodecylsulphate     

Photoautotrophic growth of soybean cells in suspension culture: I. Establishment of photoautotrophic cultures

Highly chlorophyllous photomixotrophic callus was visually selected from callus originating from soybean (Glycine max (L.) Merr. var. Corsoy) cotyledon. Suspension cultures initiated from this callus became photoautotrophic under continuous light with an atmosphere of 5% CO₂ (balance air). Dry weight increases of 1000 to 1400% in the 2-week subculture period have been observed. The cellular Chl content ranged from 4.4 to 5.9 micrograms per milligram dry weight which is about 75 to 90% of the Chl content in soybean leaves under equivalent illumination (300 micro-Einsteins per square meter per second).

No growth can be observed in the dark in sucrose-lacking medium or in the presence of 0.5 micromolar 3-(3,4-dichlorophenyl)-1,1-dimethylurea, a concentration which does not inhibit heterotrophic growth (on sucrose). Photoautotrophic growth has an absolute requirement for elevated CO₂ concentrations (>1%). During the 14-day subculture period, growth (fresh weight and dry weight) is logarithmic. Photosynthesis quickly increases after day 4, reaching a peak of 83 micromoles CO₂ incorporated per milligram Chl per hour while dark respiration decreases 90% from day 2 to day 6. The pH of the growth medium quickly drops from 7.0 to 4.5 before slowly increasing to 5.0 by day 14. At this pH range and light intensity (200-300 microEinsteins per square meter per second), no O₂ evolution could be detected although at high pH and light intensity O₂ evolution was recorded.

     

Nitrogen Fixation Dynamics of Two Diazotrophic Communities in Mono Lake, California

Two types of diazotrophic microbial communities were found in the littoral zone of alkaline hypersaline Mono Lake, California. One consisted of anaerobic bacteria inhabiting the flocculent surface layers of sediments. Nitrogen fixation (acetylene reduction) by flocculent surface layers occurred under anaerobic conditions, was not stimulated by light or by additions of organic substrates, and was inhibited by O₂, nitrate, and ammonia. The second community consisted of a ball-shaped association of a filamentous chlorophyte (Ctenocladus circinnatus) with diazotrophic, nonheterocystous cyanobacteria, as well as anaerobic bacteria (Ctenocladus balls). Nitrogen fixation by Ctenocladus balls was usually, but not always, stimulated by light. Rates of anaerobic dark fixation equaled those in the light under air. Fixation in the light was stimulated by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea and by propanil [N-(3,4-dichlorophenyl)propanamide]. 3-(3,4-Dichlorophenyl)-1,1-dimethyl urea-elicited nitrogenase activity was inhibited by ammonia (96%) and nitrate (65%). Fixation was greatest when Ctenocladus balls were incubated anaerobically in the light with sulfide. Dark anaerobic fixation was not stimulated by organic substrates in short-term (4-h) incubations, but was in long-term (67-h) ones. Areal estimates of benthic N₂ fixation were measured seasonally, using chambers. Highest rates (~29.3 μmol of C₂H₄ m⁻² h⁻¹) occurred under normal diel regimens of light and dark. These estimates indicate that benthic N₂ fixation has the potential to be a significant nitrogen source in Mono Lake.     

Light-Dependent Ammonium Ion Toxicity of Rice Leaves in Response to Phosphinothricin Treatment

Ammonium ion accumulation in detached rice leaves treated with phosphinothricin (PPT), an inhibitior of glutamine synthetase (GS), was investigated in the light and darkness. PPT treatment increased NH₄ ⁺ content and induced toxicity in rice leaves in the light but not in darkness, suggesting the importance of light in PPT-induced NH₄ ⁺ toxicity in detached rice leaves. PPT treatment in the light resulted in a decrease of activities of the cytosolic form of GS and the chloroplastic form of GS. The photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea reduced NH₄ ⁺ accumulation induced by PPT in the light. In darkness, PPT-induced NH₄ ⁺ accumulation and toxicity were observed in the presence of glucose or sucrose.     

The development of photophosphorylation and photosynthesis in greening bean leaves

Photophosphorylation and oxygen evolution were measured in 8-day-old dark-grown bean leaves (Phaseolus vulgaris) after various times of greening in far red light and in white light. The sequence of development was the same for both greening regimes, but the processes were much more rapid in white light. The capacity for photophosphorylation, as assayed by the firefly luciferase assay, appeared after 12 hours in far red light. At this stage and for times up to 24 hours, photophosphorylation was not inhibited by 10⁻⁵m 3-(3,4-dichlorophenyl)-1,1-dimethylurea. At 24 hours, the capacity for oxygen evolution appeared and photophosphorylation became partially inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea at concentrations which inhibited oxygen evolution. In white light photophosphorylation appeared after 15 minutes, and oxygen evolution at one hour. Photophosphorylation became partially sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea when oxygen evolution appeared. Carbonylcyanide m-chlorophenyl-hydrazone inhibited photophosphorylation and photosynthesis at low concentrations, 10⁻⁵m, with immature leaves, but the leaves developed resistance to carbonylcyanide m-chlorophenyl-hydrazone as they greened.     

Demonstration of Both a Photosynthetic and a Nonphotosynthetic CO(2) Requirement for NH(4) Assimilation in the Green Alga Selenastrum minutum

Nitrogen-limited and nitrogen-sufficient cell cultures of Selenastrum minutum (Naeg.) Collins (Chlorophyta) were used to investigate the dependence of NH₄⁺ assimilation on exogenous CO₂. N-sufficient cells were only able to assimilate NH₄⁺ maximally in the presence of CO₂ and light. Inhibition of photosynthesis with 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron also inhibited NH₄⁺ assimilation. These results indicate that NH₄⁺ assimilation by N-sufficient cells exhibited a strict requirement for photosynthetic CO₂ fixation. N-limited cells assimilated NH₄⁺ both in the dark and in the light in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron, indicating that photosynthetic CO₂ fixation was not required for NH₄⁺ assimilation. Using CO₂ removal techniques reported previously in the literature, we were unable to demonstrate CO₂-dependent NH₄⁺ assimilation in N-limited cells. However, employing more stringent CO₂ removal techniques we were able to show a CO₂ dependence of NH₄⁺ assimilation in both the light and dark, which was independent of photosynthesis. The results indicate two independent CO₂ requirements for NH₄⁺ assimilation. The first is as a substrate for photosynthetic CO₂ fixation, whereas the second is a nonphoto-synthetic requirement, presumably as a substrate for the anaplerotic reaction catalyzed by phosphoenolpyruvate carboxylase.     

Establishment and characterization of photoautotrophic protoplast-derived cultures ofNicotiana plumbaginifolia

A procedure is described for the rapid establishment of photoautotrophic protoplast-derived cultures ofNicotiana plumbaginifolia. Photoautotrophic growth was induced by lowering the glucose concentration to 2.5 g.l^–1 in the protoplast culture medium and by omitting glucose from the subsequent dilution medium. Four week-old highly viable suspensions were plated on an agar-medium without glucose in unsealed Petri dishes and kept in illuminated chambers flushed with 0.05 % or 2 % CO₂. Air-grown calli had net photosynthesis rates of 1.8 and 17 moles CO₂.g^–1 fresh wt.h^–1 in air at 0.034 % CO₂ and in air enriched with 1 % CO₂, respectively. Calli grown in 2 % CO₂ exhibited lower rates of net photosynthesis at the two CO₂ concentrations tested (0 and 7.5 moles CO₂.g^–1 fresh wt.h^–1, respectively). The contribution of photosynthesis to growth was estimated to be 80 % in air-grown calli and more than 90 % in calli grown in 2 % CO₂. The suitability of this photoautotrophic culture procedure is discussed with regard to the screening of photosynthetic mutants or transformants from protoplasts.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - IAA indoleacetic acid - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase     

Evidence for two different herbicide-binding proteins at the reducing side of Photosystem II

The binding behaviour at the thylakoid membrane of the radioactively labelled phenolic inhibitors 2-iodo-4-nitro-6-[2′,3′-³H]isobutylphenol and 3,5-diiodo-4-hydroxy[U-¹⁴C]benzonitrile (ioxynil) has been studied. As judged from displacement experiments with other herbicides, phenolic herbicides and herbicides as represented best by 3-(3,4-dichlorophenyl)-1,1-dimethylurea have different binding sites at the reducing side of Photosystem II. The binding parameters of phenolic herbicides are not, or only slightly, affected by trypsin treatment of chloroplasts. In chloroplasts, besides free pigments, lipids, and the light-harvesting chlorophyll $\text{a}\text{b}$ protein complex, a protein of molecular weight 41 000 is radioactively labelled by the photoaffinity label 4-nitro-2-azido-6-[2′,3′-³H]isobutylphenol. The amount of radioactivity bound to the 41 kDa protein is diminished if chloroplasts are incubated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea prior to addition of the photoaffinity label, but not if the 2,4-dinitrophenyl ether of 2-iodo-4-nitrothymol is used instead. These two compounds are characteristic representatives of inhibitiors acting at the reducing or the oxidizing site of plastoquinone, respectively. Based on these results, a model for two different herbicide-binding proteins at the reducing side of Photosystem II is presented.     

Effects of Light and Inhibitors of Photosynthesis and Respiration on the Multiplication of Tobacco Mosaic Virus in Tobacco Protoplasts

The multiplication rate of tobacco mosaic virus (TMV) in tobaccoprotoplasts in light was several times than in the dark. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea(DCMU) at 10^–5M completely antagonized this illuminationeffect. KCN at 10^–4 M and antimycin A at 10^–5 M,which prevented the protoplasts from surviving in the dark,did not block TMV multiplication in light. Inhibitor experimentsshowed that photosynthesis and respiration were indirectly associatedwith the TMV multiplication. Either of them was found to benecessary for TMV multiplication but neither was indispensable.They play complementary roles in the supply of energy and materialsrequired for virus production. (Received August 2, 1984; Accepted July 9, 1985)     

Light-dependent hydrogen evolution by Scenedesmus

Summary The effect of glucose and the uncoupler Cl-CCP upon hydrogen production was studied in adapted cells of Scenedesmus obliquus D₃. Cl-CCP at 10^-5M concentration completely inhibited the evolution of H₂ in the dark and increased the apparent rate of H₂ evolution in the light. At 10^-5M Cl-CCP, photosynthesis and photoreduction by anaerobically adapted algae were only temporarily inhibited; O₂ evolution reappeared after approximately 1 hr of illumination if CO₂ was present. Increasing the Cl-CCP concentration to 5 x 10^-5M led to a maximum rate of photohydrogen production and fully inhibited H₂ evolution, photoreduction and dark H₂ evolution. H₂ evolution was accompanied by a release of varying amounts of CO₂ in the light, as well as in the dark. Dark CO₂ production was stimulated by Cl-CCP. H₂ evolution in the light was stimulated by adding glucose to autotrophically grown cells or by growing the cells heterotrophically with glucose; starvation had an opposite effect. Adapted cells released ¹⁴CO₂ from the 3 and/or 4 position of specifically labeled glucose, indicating that degradation occurred via the Embden-Meyerhof pathway. The amount of H₂ released by autotrophically grown cells was the same either with continuous illumination or with short periods of light, followed by darkness. Scenedesmus mutant No. 11, which is unable to evolve O₂ was not inhibited in its capacity to evolve H₂ in the light. These data indicate that the evolution of H₂ in the light by adapted Scenedesmus depends upon the degradation of organic material and does not require the production of free O₂ by photosystem II.The following abbreviations are used: Cl-CCP = carbonyl cyanide m-chlorophenylhydrazone; DCMU = 3-(3,4-dichlorophenyl)-1,1-dimethylurea, DNP = 2,4-dinitrophenol.This work was supported by contract AT-(40-1)-2687 from the U.S. Atomic Energy Commission.     

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.     

Growth characteristics of hormone and vitamin independent photoautotrophic cell suspension cultures fromChenopodium rubrum

Summary This communication reports the photoautotrophic growth of hormone and vitamin independent cell suspension cultures ofChenopodium rubrum. The transfer of cells from stationary growth into fresh culture medium results in a high protein formation, followed by an exponential phase of cell division, whereas the onset of rapid chlorophyll formation is delayed for 4 days. At the stage of most rapid cell division there is no net synthesis of starch and sugar. When the cells enter stationary growth, there is a progressive accumulation of chlorophyll, sugar, and starch.Photoautotrophic cell cultures assimilate about 80–90 mol CO₂/mg chlorophyll X hour. Dark CO₂ fixation is about 3.7% to 2.2% of the light values during exponential and stationary growth, respectively. As shown by short-term¹⁴CO₂ fixation, CO₂ is predominantly assimilated through ribulosebisphosphate carboxylase via the Calvin pathway. There is a significant increase in the¹⁴C label of C₄ carboxylic acids in exponentially dividing cells as compared to cells from stationary growth. Thein vitro activity of phosphoenolpyruvate carboxylase and ribulosebisphosphate carboxylase is almost equal during exponential cell division. A decrease in cell division activity is accompanied by a significant change in the specific activities of both carboxylation enzymes. In non dividing cells from stationary growth the activity of ribulosebisphosphate carboxylase is greately enhanced and that of phosphoenolpyruvate carboxylase is reduced, documenting the development of carboxylation capacities typical for C₃-plants.The experimental results provide evidence that phosphoenolpyruvate carboxylase activity might be regulated by ammonia and could be involved in anaplerotic CO₂ fixation which supplies carbon skeletons of the citric acid cycle.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - EDTA ethylene-diamine-tetraacetic acid - FDP fructose bisphosphate - F-6-P fructose-6-phosphate - G-6-P glucose-6-phosphate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PGA 3-phosphoglyceric acid - PEP phosphoenolpyruvate - RuDP ribulosebisphosphate