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     

Reaction of ribulosebisphosphate carboxylase from rhodospirlilum rubrum with the potential affinity label 3-bromo-1,4-dihydroxy-2-butanone 1,4-biphosphate.

Under mild conditions, 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate rapidly and irreversibly inactivates ribulosebisphosphate carboxylase from $\text{Rhodospirillum rubrum}$. The substrate ribulosebisphosphate protects the enzyme against inactivation. Incorporation of reagent has been quantitated by reduction of the modified carboxylase with [³H]NaBH₄. Based on the difference in the levels of incorporation found in the inactivated enzyme as compared with the protected enzyme, loss of enzymic activity results from the modification of about 0.4 residue per catalytic subunit. Analyses of hydrolysates demonstrate that both cysteinyl and lysyl derivatives are present in the inactivated carboxylase; the protected sample contains about the same amount of modified cysteine but little of the modified lysine. Thus, inactivation appears to correlate with derivatization of lysyl residues.     

Regulation of autotrophic and heterotrophic metabolism in Pseudomonas oxalaticus OX1: Growth on mixtures of oxalate and formate in continuous culture

Pseudomonas oxalaticus was grown in carbon- and energy-limited continuous cultures either with oxalte or formate or with mixtures of these substrates. During growth on the mixtures, simultaneous utilization of the two substrates occurred at all dilution rates tested. Under these conditions oxalate repressed the synthesis of ribulosebisphosphate carboxylase. The degree of this repression was dependent on the dilution rate and the ratio of oxalate and formate in the medium reservoir. At a fixed oxalate/formate ratio repression was greatest at intermediate dilution rates, whereas derepression occurred at both low and high dilution rates. Progressive depression of ribulosebisphosphate carboxylase synthesis and of autotrophic CO₂ fixation at low dilution rates was attributed to the decreasing concentration of intracellular repressor molecule(s), parallel to the decreasing concentration of the growth-limiting substrates in the culture. To account for the derepression at higher dilution rates, it is proposed that the rate of oxalyl-CoA production from oxalate limits the supply of metabolic intermediates and that additional energy and reducing power generated from formate drains the pools of metabolic intermediates sufficiently to lower the intracellular concentration of the repressor(s). During growth of Pseudomonas oxalaticus on the heterotrophic substrate oxalate alone, at dilution rates below 10% of the maximum specific growth rate, derepression of ribulosebisphosphate carboxylase synthesis and of autotrophic CO₂ fixation was observed to a level which was 50% of that observed during growth on formate alone at the same dilution rate. It is concluded that in Pseudomonas oxalaticus the synthesis of enzymes involved in autotrophic CO₂ fixation via the Calvin cycle is regulated by a repression/derepression mechanism and that the contribution of autotrophic CO₂ fixation to the biosynthesis of cell material in this organism is mainly controlled via the synthesis of these enzymes.Abbreviations RuBPCase ribulosebisphosphate carboxylase - PMS phenazine methosulphate - DCPIP 2,6-dichlorophenolindophenol - FDH formate dehydrogenase - S_R concentration of growth-limiting substrate in reservoir     

Light control of plastogenesis and ribulosebisphosphate carboxylase levels in mustard seedling cotyledons

We have studied the problem whether the phytochrome-mediated accumulation of ribulosebisphosphate carboxylase (carboxylase; EC 4.1.1.39) in the cotyledons of sinapis alba L. is related to size, ultrastructure, or organization of the plastid compartment. We have shown that under different light conditions (e.g. continuous far-red light, continuous white light) which lead to conspicuously different plastids the time course of the enzyme levels remains precisely the same. It is concluded that the onset and the rate of carboxylase accumulation is not related to the organizational state of the plastid compartment as discernible under the electron microscope.Abbreviations P _tr far-red absorbing form of the phytochrome system - carboxylase ribulosebisphosphate carboxylase (EC 4.1.1.39)     

Synthesis and assembly of ribulosebisphosphate carboxylase enzyme during greening of barley plants

The synthesis and activation of ribulosebisphosphate carboxylase was studied in etiolated barley leaves during increasing periods of light irradiation. Comparisons were made among enzymatic activity, ¹⁴C-amino acid incorporation into anti-ribulosebisphosphate carboxylase precipitable and 16S protein, and total mass of enzyme. A major portion of newly synthesized anti-ribulosebisphosphate carboxylase specific protein preceded light-induced increase in enzyme activity by a significant period of time. These findings are consistent with a model in which both subunits of ribulosebisphosphate carboxylase are synthesized in response to an early event in greening and subsequently become associated to active oligomeric carboxylase species.     

Enzymes of the autotrophic pathway in mating partners and transconjugants of Nocardia opaca 1 b and Rhodococcus erythropolis

Enzyme activities have been measured in the partners of a bacterial mating system consisting of the hydrogen autotroph Nocardia opaca (donor and Aut^- recipient), the heterotroph Rhodococcus erythropolis (recipient) and intra- and interspecies transconjugants after growth on fructose, pyruvate and under autotrophic conditions. Specific activities of each of the enzymes hydrogenase, phosphoribulokinase and ribulosebisphosphate carboxylase were high in autotrophically grown cells of the donor and the transconjugants: they amounted to only 10% after growth on pyruvate. The recipient cells did not grow autotrophically and the enzymes mentioned were not detectable even after growth on pyruvate. Other enzymes of the Calvin cycle were constitutively formed in all strains examined.The properties of hydrogenase (K _m for NAD, R_f in gel electrophoresis) and of ribulosebisphosphate carboxylase (K _m for RuBP and R_f) were the same in the donor and transconjugant cells. The properties of glucose-6-phosphate dehydrogenase (K _m for G-6-P and mode of inhibition by ATP and phosphoenolpyruvate) were the same in the recipient and the interspecies transconjugant cells and differed from those of the donor cells. The curves of growth under autotrophic conditions in batch culture of the donor and interspecies transconjugant were almost congruent. The specific activities of hydrogenase, phosphoribulokinase and ribulosebisphosphate carboxylase increased from 40% at the beginning to 100% at the end of the exponential growth phase; these enzymes were under coordinate control.The results are in accordance with genetic studies: the genetic information for autotrophic growth is localized on a so far unidentified genetic element and is transferred en bloc from N. opaca to Aut^- mutants of the same strain or to recipient bacteria such as R. erythropolis; expression in the wild type and transconjugant cells is the same.Abbreviations G-6-P glucose-6-phosphate - 6-PG 6-phosphogluconate - FBP fructose-1,6-bisphosphate - SBP sedoheptulose-1,7-bisphosphate - RuBP ribulose-1,5-bisphosphate     

Ultrastructural and biochemical development of the photosynthetic apparatus during callus induction in carrot root explants

During the first 8–12 days of cultivation in a nutrient solution containing IAA, inositol and kinetin freshly isolated carrot root explants develop into a chlorophyllous and photosynthetically active tissue culture. Electron microscopy, low temperature pigment absorption spectra and fluorescence induction profile recording as well as the determination of the activity of some enzymes (ribulosebisphosphate carboxylase, phosphoenolpyruvate carboxylase) and CO₂-fixation experiments were carried out. Based on the results, a sequence of developmental stages of the photosynthetic system will be proposed. During the autotropic period from about the 20th day of culture onward the light reaction system of these tissue cultures is quite comparable to that of carrot leaves; however, some differences in the CO₂-fixation mechanism were observed.     

Hydrogen-stimulated CO2 fixation and coordinate induction of hydrogenase and ribulosebiphosphate carboxylase in a H2-uptake positive strain of Rhizobium japonicum

H₂-uptake positive strains (122 DES and SR) and H₂-uptake negative strains SR2 and SR3 of Rhizobium japonicum were examined for ribulosebisphosphate (RuBP) carboxylase and H₂-uptake activities during growth conditions which induced formation of the hydrogenase system. The rate of ¹⁴CO₂ uptake by hydrogenase-derepressed cells was about 6-times greater in the presence than in the absence of H₂. RuBP carboxylase activity was observed in free-living R. japonicum strains 122 DES or SR only when the cells were derepressed for their hydrogenase system. Hydrogenase and RuBP carboxylase activities were coordinately induced by H₂ and both were repressed by added succinate. Hydrogenase-negative mutant strains SR2 and SR3 derived from R. japonicum SR showed no detecyable RuBP carboxylase activities under hydrogenase derepression conditions. No detectable RuBP carboxylase was observed in bacteroids formed by H₂-uptake positive strains R. japonicum 122 DES or SR. Propionyl CoA carboxylase activity was consistently observed in extracts of cells from free-living cultures of R. japonicum but activity was not appreciably influenced by the addition of H₂. Neither phosphoenolpyruvate carboxylase nor phosphoenolpyruvate carboxykinase activity was detected in extracts of R. japonicum.Abbreviations RuBP Ribulose 1,5-bisphosphate - (Na₂EDTA) (Ethylenedinitrilo)-tetraacetic acid, disodium salt - (propionyl CoA) Propionyl coenzyme A - (PEP) Phosphoenolpyruvate - (GSH) Reduced glutathione - (Tricine) N-tris(hydroxymethyl)-methylglycine     

Comparison of the Key Enzymes of Hyphomicrobium sp. 53-49 under Various Growth Conditions: Aerobic,Denitrifying and Autotrophic

For Hyphomicrobium 53-49 capable of growing under various conditions, aerobic methanol, anaerobic methanol (with denitrification), autotrophic (H₂-O₂-CO₂), aerobic ethanol and aerobic acetate, investigation and comparison of the specific activities of the following enzymes were performed: alcohol dehydrogenase (NAD-ethanol linked and NAD-methanol linked), primary alcohol dehydrogenase, formaldehyde dehydrogenase (NAD-GSH linked and DCPIP linked), formate dehydrogenase, serine hydroxymethyl transferase, hydroxypyruvate reductase, isocitrate lyase (icl), malate lyase, malate dehydrogenase, ribulosebisphosphate (RuBP) carboxylase, phos-phoenolpyruvate (PEP) carboxykinase (ADP linked), PEP carboxylase (phosphorylating), pyruvate carboxylase (NADH linked and NADPH linked) and α-ketoglutarate carboxylase (NADH linked and NADPH linked). On the basis of the data obtained, it was concluded that during growth on methanol, aerobically and anaerobically, the icl+ serine pathway operated, while during autotrophic growth on H₂-O₂-CO₂, CO₂ was incorporated through the RuBP pathway and others, and during growth on ethanol or acetate, neither the serine pathway nor the RuBP pathway operated. The organism changed its metabolism through the regulation of the metabolic enzymes according to the growth conditions.     

Blue light-induced synthesis of ribulosebisphosphate carboxylase in cultured plant cells

Upon illumination with blue light (350–550 nm) of suspension cultured cells (Nicotiana tabacum var. Samsun) the transition of leucoplasts to functional chloroplasts is induced. During the subsequent greening period chlorophylls as well as membrane and enzyme proteins are synthesized. Thus the amount of ribulosebisphosphate carboxylase (EC. 4.1.1.39) being small in leucoplasts increases dramatically due to de novo synthesis. This change is also reflected in the level of translatable messenger RNA specific for the small subunit of ribulosebisphosphate carboxylase which accumulates only in blue-irradiated cells; its in vitro translation product isolated by immunoprecipitation corresponds mainly to the precursor protein (Mr 20 000) of the small subunit. In contrast, red light (600–700 nm) does not induce synthesis of ribulosebisphosphate carboxylase. According to these findings it is proposed that blue light exerts its influence on ribulosebisphosphate carboxylase in cultured tobacco cells at a level below translation.     

Activation of Ribulosebisphosphate Carboxylase/Oxygenase at Physiological CO(2) and Ribulosebisphosphate Concentrations by Rubisco Activase

The enzyme-catalyzed activation of ribulosebisphosphate carboxylase/oxygenase (rubisco) was investigated in an illuminated reconstituted system containing thylakoid membranes, rubisco, ribulosebisphosphate (RuBP), MgCl₂, carbonic anhydrase, catalase, the artificial electron acceptor pyocyanine, and partially purified rubisco activase. Optimal conditions for light-induced rubisco activation were found to include 100 micrograms per milliliter rubisco, 300 micrograms per milliliter rubisco activase, 3 millimolar RuBP, and 6 millimolar free Mg²⁺ at pH 8.2. The half-time for rubisco activation was 2 minutes, and was 4 minutes for rubisco deactivation. The rate of rubisco deactivation was identical in the presence and absence of activase. The K_act(CO₂) of rubisco activation in the reconstituted system was 4 micromolar CO₂, compared to a K_act(CO₂) of 25 to 30 micromolar CO₂ for the previously reported spontaneous CO₂/Mg²⁺ activation mechanism. The activation process characterized here explains the high degree of rubisco activation at the physiological concentrations of 10 micromolar CO₂ and 2 to 4 millimolar RuBP found in intact leaves, conditions which lead to almost complete deactivation of rubisco in vitro.     

CO2 donation by malate and aspartate reduces photorespiration in Panicum milioides,A C3C4 intermediate species

Oxygen inhibition of leaf slice photosynthesis in Panicum milioides increased from 20% to 30% at 21% O₂ in the presence of maleate, a phosphoenolpyruvate carboxylase inhibitor. The increased O₂ sensitivity was completely reversed by the addition of malate and aspartate, the stable products of the phosphoenolpyruvate carboxylase reaction. The C₄ acids, malate and aspartate, also reduced O₂ inhibition of photosynthesis by isolated bundle sheath strands, but not mesophyll protoplasts. Similarly, only bundle sheath strands exhibited an active C₄ acid-dependent O₂ evolution. Compartmentation of C₄ cycle enzymes, with pyruvate, Pi dikinase in the mesophyll and NAD-malic enzyme in the bundle sheath, was demonstrated. It is concluded that reduced photorespiration in P. milioides is due to a limited potential for C₄ photosynthesis permitting an increase in pCO₂ at the site of bundle sheath ribulosebisphosphate carboxylase.     

Glyoxylate Inhibition of Ribulosebisphosphate Carboxylase/Oxygenase Activation State in vivo

Photosynthetic CO₂ exchange in photorespiration mutants of Arabidopsisthaliana showed a time-dependent inhibition at 350 µl/literCO₂ in 50% O₂ but not in 2% O₂. In a glycolate-P phos-phatasedeficient mutant, inhibition of photosynthesis was due to adepletion of ribulosebisphosphate. In the remaining mutants,which have defects in photorespiratory enzymes which metabolizeamino acids, reduced photosynthesis was accompanied by a declinein the activation level of ribulosebisphosphate carboxylase/oxygenase(Chastain and Ogren 1985), a decline in ribulosebisphosphateconcentration, and an accumulation of glyoxylate. Addition ofglyoxylate at submillimolar concentrations to intact spinach(Spinacea oleracea L.) chloroplasts inhibited light activationof ribulosebisphosphate carboxylase/oxygenase (rubisco) andCO₂ fixation. Similar concentrations of glyoxylate had no effecton A. thaliana rubisco activity in vitro. These results suggestthat glyoxylate accumulation indirectly inhibited rubisco activationstate in vivo. The inhibition of photosynthesis in mutants whichaccumulate glyoxylate may be attributed to a decline in ribulosebisphosphateconcentration, a reduction in rubisco activation state, or acombination of both phenomena. ³Present address: CSIRO, Division of Plant Industry, GPO Box1600, Canberra, ACT 2601, Australia. (Received May 12, 1989; Accepted July 8, 1989)     

Dark fixation of CO2 during gluconeogenesis by the cotyledons of Cucurbita pepo L.

We did this work to discover the pathway of CO₂ fixation into sugars in the dark during gluconeogenesis by the cotyledons of 5-day-old seedlings of Cucurbita pepo L. We paid particular attention to the possibility of a contribution from ribulosebisphosphate carboxylase. The detailed distribution of ¹⁴C after exposure of excised cotyledons to ¹⁴CO₂ in the dark was determined in a series of pulse and chase experiments. After 4s in ¹⁴CO₂, 89% of the ¹⁴C fixed was in malate and aspartate. In longer exposures, and in chases in ¹²CO₂, label appeared in alanine, phosphoenolpyruvate, 3-phosphoglycerate and sugar phosphates, and accumulated in sugars. The transfer of label from C-4 acids to sugars was restricted by inhibition of phosphoenolpyruvate carboxykinase in vivo by 3-mercaptopicolinic acid. We conclude as follows. Initial fixation of CO₂ in the dark is almost entirely into phosphoenolpyruvate, probably via phosphoenolpyruvate carboxylase (EC 4.1.1.31) which we showed to be present in appreciable amounts. Incorporation into sugars occurs chiefly, if not completely, as a result of randomization of the carboxyl groups of the C-4 acids and subsequent conversion of the oxaloacetate to sugars via the accepted sequence for gluconeogenesis. Ribulosebisphosphate carboxylase appears to make very little contribution to sugar synthesis from fat.     

Glyoxylate inhibition of ribulosebisphosphate carboxylase/oxygenase activation in intact,lysed, and reconstituted chloroplasts

At bicarbonate concentrations equivalent to air levels of CO₂, activation of ribulosebisphosphate carboxylase/oxygenase (rubisco) was inhibited by micromolar concentrations of glyoxylate in intact, lysed, and reconstituted chloroplasts and in stromal extracts. The concentration of glyoxylate required for 50% inhibition of light activation in intact chloroplasts was estimated to be 35 micromolar. No direct inhibition by glyoxylate was observed with purified rubisco or rubisco activase at micromolar concentrations. Levels of ribulose 1,5-bisphosphate and ATP increased in intact chloroplasts following glyoxylate treatment. Results from experiments with well-buffered lysed and reconstituted chloroplast systems ruled out lowering of pH as the cause of inhibition. With intact chloroplasts, micromolar glyoxylate did not prevent activation of rubisco at high (10 mM) concentrations of bicarbonate, indicating that rubisco could be spontaneously activated in the presence of glyoxylate. These results suggest the existence of a component of the in vivo rubisco activation system that is not yet identified and which is inhibited by glyoxylate.Abbreviations PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - rubisco ribulosebisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate     

Formation of the small subunit in the absence of the large subunit of ribulose 1,5-bisphosphate carboxylase in 70 S ribosome-deficient rye leaves.

Immunological tests with monospecific antisera to ribulosebisphosphate carboxylase (EC 4.1.1.39) and to its large and small subunits indicated the presence of a protein with antigenic properties of the small subunit in the absence of the large subunit in the leaves of young rye plants (Secale cereale L.) with a high-temperature-induced (32 °C) deficiency of 70 S plastid ribosomes. The small subunit-like protein was isolated from crude extracts of plastid ribosome-deficient 32 °C-grown leaf tissue by the use of columns with immobilized antibody. The main polypeptide retained by the immobilized antibodies had the same mobility after electrophoresis on sodium dodecyl sulfate-polyacrylamide gels as the small subunit of ribulosebisphosphate carboxylase and was also immunologically identical to the small subunit. The small subunit-like protein was present in the supernatant as well as in the membrane fraction of isolated 70 S ribosome-deficient plastids. At very young stages of normal leaves grown at a permissive temperature (22 °C) an excess of small subunit was observed that was also not integrated into the complete ribulosebisphosphate carboxylase molecule. From the results, we conclude that the synthesis of the small subunit occurs on cytoplasmic ribosomes and is not strictly coordinated with the translation of the large subunit in the chloroplast. During early leaf development, the formation of the large subunit seems to be the ratelimiting step in the synthesis of ribulosebisphosphate carboxylase.     

Involvement of megaplasmids in heterotrophic derepression of the carbon-dioxide assimilating enzyme system in Alcaligenes spp.

The facultatively chemolithoautotrophic hydrogen-oxidizing bacteria Alcaligenes eutrophus and Alcaligenes hydrogenophilus partially derepressed the formation of phosphoribulokinase and ribulosebisphosphate carboxylase during heterotrophic growth on fructose or gluconate. We examined whether the indigenous magaplasmids in these bacteria that encode the ability to oxidize hydrogen affected this derepression. The results suggest an involvement of the plasmids in the derepression for the following reasons: (i) wild-type strains, except A. eutrophus TF93, exhibited the derepressible phenotype; (ii) plasmid-cured mutants formed the enzymes with formate as autotrophic growth substrate but did not derepress their formation during heterotrophic growth; (iii) the phenotype of the wild type was restored by transfer of the plasmids into plasmid-cured mutants. Plasmid pHG2 from strain TF93 differed from the other wild-type plasmids by conferring a non-derepressible phenotype onto the harboring strain. Mutants of A. eutrophus H16 carrying deletions in plasmid pHG1 showed a similar phenotype as that of the plasmid-cured mutants. We concluded that the plasmids from the various strains studied encode a regulatory ability to derepress phosphoribulokinase and ribulosebisphosphate carboxylase under heterotrophic growth conditions.Abbreviations PRK phosphoribulokinase - RuBPC ribulosebisphosphate carboxylase - Hox ability to oxidize hydrogen - Cfx ability to fix carbon dioxide autotrophically Dedicated to Prof. Dr. H. G. Schlegel on the occasion of his 60th birthday     

D-Ribulose-1,5-bisphosphate carboxylase/oxygenase from chemolithotrophically grown Rhizobium japonicum

Ribulose-1,5-bisphosphate carboxylase/oxygenase has been purified from chemolithotrophically grown Rhizobium japonicum SR and ribulose-5-phosphate kinase activity has also been detected in extracts of such cells. Electrophoretically homogeneous ribulosebisphosphate carboxylase/oxygenase purified in the presence of PMSF showed two types of large subunits of 55 000 and 53 000 daltons and small subunits of 14 200 daltons. The heterogeneity of large subunits was not observed when the enzyme was prepared in the presence of PMSF and DIFP. Ribulose-1,5-bisphosphate carboxylase from R. japonicum was inhibited by antibodies to this enzyme and a single precipitin band from the antibody-enzyme interaction was observed on double diffusion plates. Antibodies to R. japonicum enzyme did not cross-react on immunodiffusion plates with the ribulosebisphosphate carboxylase/oxygenases from wheat, spinach, soybean and tobacco.     

Autotrophic Growth of Gas Vacuolate Strains of Microcyclus aquaticus on Methanol and Hydrogen

Seven strains of Microcyclus aquaticus were found to be capable of growth on methanol and hydrogen as energy sources. Carbon was incorporated as CO₂ via the Calvin cycle, as shown by the presence of ribulosebisphosphate carboxylase activity in methanol-grown cells and by the absence of key enzymes of the ribulose monophosphate and serine pathways. In addition, incoporation of [¹⁴C]methanol into cells was diminished when cultures were incubated in gas atmospheres enriched with carbon dioxide.