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W L Ogren

Publications and source records attributed to W L Ogren.

At least 37 records · Page 2Linked to original sources
PubMed1987

Photosynthetic characteristics of a photoautotrophic cell suspension culture of soybean.

A soybean suspension culture (SB-P) which can grow photoautotrophically in 5% CO(2) will not grow in ambient CO(2) levels. This elevated CO(2) requirement seems to be due to the additive effects of a number of factors. The in vivo activity of ribulose-1,5-bisphosphate carboxylase (RuBPcase) is much lower in the SB-P cells, compared to soybean plants. This may be due to the low light intensity used to culture the cells, which has been shown to decrease both the amount and activity in whole plants, resulting in a low rate of net photosynthesis. The RuBPcase activation level is also lowered in air CO(2) levels. The presence of the liquid medium raises the cells CO(2) compensation concentration (the CO(2) concentration reached when the rates of CO(2) fixed by photosynthesis and the CO(2) respired by the cells are equal). These factors, coupled with the high respiratory loss of CO(2) all contribute to reduced net photosynthesis in air, resulting in a photosynthetic capacity that is inadequate for cell survival. Active cell division, low photosynthetic capacity, elevated respiration, and a low ratio of RuBPcase(initial)/phosphoenolpyruvate carboxylase are traits that SB-P cells share with young leaf cells, indicating SB-P cell physiology may be comparable to that of young expanding leaves rather than to that of mature leaves.

Journal Article↗
PubMed1986

Purification of ribulose-1, 5-bisphosphate carboxylase/oxygenase with high specific activity by fast protein liquid chromatography.

A rapid procedure for the purification of ribulose-1, 5-bisphosphate carboxylase/oxygenase (rubisco) (EC 4.1.1.39) by fast protein liquid chromatography (FPLC) is described. Chloroplasts isolated mechanically from spinach leaves were used as the source of a stromal extract enriched in rubisco. By subsequent fractionation of this extract on ion-exchange FPLC, highly purified rubisco (sp act 2.10-2.76 mumol/mg protein X min) was obtained in less than 30 min. The high specific activity and excellent stability of the final preparation can be attributed to the use of chloroplasts as a starting material and the short time required for the chromatographic separation, both of which minimize proteolytic activity.

Chloroplasts↗
PubMed1986

Light and CO(2) Response of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activation in Arabidopsis Leaves.

The requirements for activation of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) were investigated in leaves of Arabidopsis wild-type and a mutant incapable of light activating rubisco in vivo. Upon illumination with saturating light intensities, the activation state of rubisco increased 2-fold in the wild-type and decreased in the mutant. Activation of fructose 1,6-bisphosphate phosphatase was unaffected by the mutation. Under low light, rubisco deactivated in both the wild-type and the mutant. Deactivation of rubisco in the mutant under high and low light led to the accumulation of high concentrations of ribulose 1,5-bisphosphate. Inhibiting photosynthesis with methyl viologen prevented ribulose 1,5-bisphosphate accumulation but was ineffective in restoring rubisco activation to the mutant. Net photosynthesis and the rubisco activation level were closely correlated and saturated at a lower light intensity in the mutant than in wild-type. At CO(2) concentrations between 100 and 2000 microliters per liter, the activation state was a function of the CO(2) concentration in the dark but was independent of CO(2) concentration in the light. High CO(2) concentration (1%) suppressed activation in the wild-type and deactivation in the mutant. These results support the concept that rubisco activation in vivo is not a spontaneous process but is catalyzed by a specific protein. The absence of this protein, rubisco activase, is responsible for the altered characteristics of rubisco activation in the mutant.

Journal Article↗
PubMed1986

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), MgCl(2), 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 Mg(2+) 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)(CO(2)) of rubisco activation in the reconstituted system was 4 micromolar CO(2), compared to a K(act)(CO(2)) of 25 to 30 micromolar CO(2) for the previously reported spontaneous CO(2)/Mg(2+) activation mechanism. The activation process characterized here explains the high degree of rubisco activation at the physiological concentrations of 10 micromolar CO(2) and 2 to 4 millimolar RuBP found in intact leaves, conditions which lead to almost complete deactivation of rubisco in vitro.

Journal Article↗
PubMed1985

Photorespiration-induced reduction of ribulose bisphosphate carboxylase activation level.

Leaf photosynthesis and ribulose bisphosphate carboxylase activation level were inhibited in several mutants of the C(3) crucifer Arabidopsis thaliana which possess lesions in the photorespiratory pathway. This inhibition occurred when leaves were illuminated under a photorespiratory atmosphere (50% O(2), 350 microliters per liter CO(2), balance N(2)), but not in nonphotorespiratory conditions (2% O(2), 350 microliters per liter CO(2), balance N(2)). Inhibition of carboxylase activation level was observed in strains with deficient glycine decarboxylase, serine transhydroxymethylase, serine-glyoxylate aminotransferase, glutamate synthase, and chloroplast dicarboxylate transport activities, but inhibition did not occur in a glycolate-P phosphatase-deficient strain. Also, the photorespiration inhibitor aminoacetonitrile produced a decline in leaf and protoplast ribulose bisphosphate carboxylase activation level, but was without effect on intact chloroplasts. Fructose bisphosphatase, a light-activated enzyme which is strongly dependent on stromal pH and Mg(2+) for regulation, was unaffected by conditions which caused inhibition of ribulose bisphosphate carboxylase. Thus, the mechanism of inhibition does not appear to involve changes in stromal Mg(2+) and pH but rather is associated with metabolite flux through the photorespiratory pathway.

Journal Article↗
PubMed1983

Species variation in kinetic properties of ribulose 1,5-bisphosphate carboxylase/oxygenase.

Several kinetic parameters of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase from different species were measured and compared. The CO2/O2 specificity (VcKo/VoKc) was found to be about 80 in the enzymes from several C3 species and two C4 species. Specificity values of 58 and 70, respectively, were found in enzymes from the C4 plants Setaria italica and Sorghum bicolor. Two enzymes from cyanobacteria had values of about 50. Substitution of Mn2+ for Mg2+ reduced the CO2/O2 specificity by a factor of about 20 for all enzymes except that of Rhodospirillum rubrum, which was reduced by a factor of 10. Values for KMg2+(apparent) measured at 102 microM CO2 were found to vary by a factor of 8 between different RuBP carboxylase/oxygenase enzymes. Enzymes with high KMg2+(apparent) values generally had high Michaelis constants for CO2. The rate of CO2/Mg2+ activation was inhibited by RuBP in all enzymes, although the concentration of RuBP required to inhibit activation in the enzyme from the cyanobacterium Aphanizomenon flos-aquae was increased by an order of magnitude compared to other higher plant structural-type enzymes. The wide variation found in the kinetic properties of RuBP carboxylase/oxygenase isolated from diverse species appears to be determined in part by past evolutionary pressures and the present physicochemical environment in which the enzyme functions.

Enzyme Activation↗
PubMed1983

Rapid recovery of chloroplast mutations affecting ribulosebisphosphate carboxylase/oxygenase in Chlamydomonas reinhardtii.

Based on the unique ability of chloroplast genes to recombine in Chlamydomonas reinhardtii, a collection of acetate-requiring mutants was screened for recombination with a mutation affecting ribulose-1,5-bisphosphate carboxylase/oxygenase [Rbu-1,5-P(2) carboxylase/oxygenase; 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39]. This chloroplast mutation, rcl-u-1-10-6C, causes the absence of Rbu-1,5-P(2) carboxylase/oxygenase activities and alters the isoelectric point of the larger subunit. Several mutants that displayed little or no recombination with 10-6C were recovered, and two lacked carboxylase activity. These new chloroplast mutants lack both large and small Rbu-1,5-P(2) carboxylase/oxygenase subunits. The approach demonstrated here permits the routine recovery of chloroplast mutations affecting this enzyme. Multiple mutations in the Rbu-1,5-P(2) carboxylase/oxygenase large-subunit gene can be used to investigate the function and regulation of this enzyme and the regulation of chloroplast genes in general.

Journal Article↗
PubMed1983

An Arabidopsis thaliana mutant defective in chloroplast dicarboxylate transport.

Reactions of the photorespiratory pathway of C(3) plants are found in three subcellular organelles. Transport processes are, therefore, particularly important for maintaining the uninterrupted flow of carbon through this pathway. We describe here the isolation and characterization of a photorespiratory mutant of Arabidopsis thaliana defective in chloroplast dicarboxylate transport. Genetic analysis indicates the defect is due to a simple, recessive, nuclear mutation. Glutamine and inorganic phosphate transport are unaffected by the mutation. Thus, in contrast to previous reports for pea and spinach, glutamine uptake by Arabidopsis chloroplasts is mediated by a transporter distinct from the dicarboxylate transporter. Both the inviability and the disruption of amino-group metabolism of the mutant under photorespiratory conditions suggest that the primary function of the dicarboxylate transporter in vivo is the transfer of 2-oxoglutarate and glutamate across the chloroplast envelope in conjunction with photorespiratory nitrogen metabolism. The role commonly ascribed to this transporter, conducting malate-aspartate exchanges for the indirect export of reducing equivalents from the chloroplast, appears to be a minor one.

Journal Article↗
PubMed1983

Nuclear Suppressors of the Photosensitivity Associated with Defective Photosynthesis in Chlamydomonas reinhardii.

Several nuclear mutations were recovered that suppress the photosensitivity associated with the Chamydomonas reinhardii chloroplast mutant rcl-u-1-10-6C, which is defective in ribulose-1,5-bisphosphate carboxylase/oxygenase. Two of the suppressor mutations affect other components of photosynthesis. These results show that suppressors of photosensitivity are sufficiently common to permit the recovery of photosensitive, photosynthesis-deficient mutants in bright light, and indicate that photosynthesis-deficient mutants selected and maintained in the light may accumulate suppressors which can confuse the biochemical analysis of lesions in photosynthesis. One of the suppressor mutations inhibits photosystem II activity, indicating that photosensitivity can be mediated by partial reactions of the photosynthetic electron transport chain.

Journal Article↗
PubMed1983

Ploidy Effects in Isogenic Populations of Alfalfa (Medicago sativa L.) : IV. Similarity in Physical and Kinetic Properties of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase) was isolated from isogenic diploid-tetraploid and tetraploid-octoploid sets of alfalfa (Medicago sativa L.) leaves. Molecular weights of RuBPCase subunits were similar across ploidy levels of both isogenic sets with subunits of 52,000 and 14,000. Apparent K(m)(CO(2)) values and substrate specificity factors (V(c)K(o)/V(o)K(c)) of RuBPCase were similar across ploidy levels of both isogenic sets. These results indicate that ploidy had no effect on the kinetic properties of RuBPCase in alfalfa.

Journal Article↗
PubMed1983

Carbonic Anhydrase-Deficient Mutant of Chlamydomonas reinhardii Requires Elevated Carbon Dioxide Concentration for Photoautotrophic Growth.

A mendelian mutant of the unicellular green alga Chlamydomonas reinhardii has been isolated which is deficient in carbonic anhydrase (EC 4.2.1.1) activity. This mutant strain, designated ca-1-12-1C (gene locus ca-1), was selected on the basis of a high CO(2) requirement for photoautotrophic growth. Photosynthesis by the mutant at atmospheric CO(2) concentration was very much reduced compared to wild type and, unlike wild type, was strongly inhibited by O(2). In contrast to a CO(2) compensation concentration of near zero in wild type at all O(2) concentrations examined, the mutant exhibited a high, O(2)-stimulated CO(2) compensation concentration. Evidence of photorespiratory activity in the mutant but not in wild type was obtained from the analysis of photosynthetic products in the presence of (14)CO(2). At air levels of CO(2) and O(2), the mutant synthesized large amounts of glycolate, while little glycolate was synthesized by wild type under identical conditions. Both mutant and wild type strains formed only small amounts of glycolate at saturating CO(2) concentration. At ambient CO(2), wild type accumulated inorganic carbon to a concentration several-fold higher than that in the suspension medium. The mutant cells accumulated inorganic carbon internally to a concentration 6-fold greater than found in wild type, yet photosynthesis was CO(2) limited. The mutant phenotype was mimicked by wild type cells treated with ethoxyzolamide, an inhibitor of carbonic anhydrase activity. These observations indicate a requirement for carbonic anhydrase-catalyzed dehydration of bicarbonate in maintaining high internal CO(2) concentrations and high photosynthesis rates. Thus, in wild type cells, carbonic anhydrase rapidly converts the bicarbonate taken up to CO(2), creating a high internal CO(2) concentration which stimulates photosynthesis and suppresses photorespiration. In mutant cells, bicarbonate is taken up rapidly but, because of a carbonic anhydrase deficiency, is not dehydrated at a rate sufficiently rapid to maintain a high internal CO(2) concentration.

Journal Article↗
PubMed1983

Reduced Inorganic Carbon Transport in a CO(2)-Requiring Mutant of Chlamydomonas reinhardii.

A mendelian mutant of the unicellular green alga Chlamydomonas reinhardii has been isolated that is deficient in inorganic carbon transport. This mutant strain, designated pmp-1-16-5K (gene locus pmp-1), was selected on the basis of a requirement of elevated CO(2) concentration for photoautrophic growth. Inorganic carbon accumulation in the mutant was considerably reduced in comparison to wild type, and the CO(2) response of photosynthesis indicated a reduced affinity for CO(2) in the mutant. At air levels of CO(2) (0.03-0.04%), O(2) inhibited photosynthesis and stimulated the synthesis of photorespiratory intermediates in the mutant but not in wild type. Neither strain was significantly affected by O(2) at saturating CO(2) concentration. Thus, the primary consequence of inorganic carbon transport deficiency in the mutant was a much lower internal CO(2) concentration compared to wild type. From these observations, we conclude that enzyme-mediated transport of inorganic carbon is an essential component of the CO(2) concentrating system in C. reinhardii photosynthesis.

Journal Article↗
PubMed1982

Mutants of the cruciferous plant Arabidopsis thaliana lacking glycine decarboxylase activity.

A mutant of Arabidopsis thaliana (L.) Heyn. (a small plant in the crucifer family) that lacks glycine decarboxylase activity owing to a recessive nuclear mutation has been isolated on the basis of a growth requirement for high concentrations of atmospheric CO2. Mitochondria isolated from leaves of the mutant did not exhibit glycine-dependent O2 consumption, did not release 14CO2 from [14C]glycine, and did not catalyse the glycine-bicarbonate exchange reaction that is considered to be the first partial reaction associated with glycine cleavage. Photosynthesis in the mutant was decreased after illumination under atmospheric conditions that promote partitioning of carbon into intermediates of the photorespiratory pathway, but was not impaired under non-photorespiratory conditions. Thus glycine decarboxylase activity is not required for any essential function unrelated to photorespiration. The photosynthetic response of the mutant in photorespiratory conditions is probably caused by an increased rate of glyoxylate oxidation, which results from the sequestering of all readily transferable amino groups in a metabolically inactive glycine pool, and by a depletion of intermediates from the photosynthesis cycle. The rate of release of 14CO2 from exogenously applied [14C]glycollate was 14-fold lower in the mutant than in the wild type, suggesting that glycine decarboxylation is the only significant source of photorespiratory CO2.

Amino Acid Oxidoreductases↗
PubMed1982

A Mutant of Arabidopsis thaliana Which Lacks Activation of RuBP Carboxylase In Vivo.

A mutant of Arabidopsis thaliana has been isolated in which ribulose-1,5-bisphosphate carboxylase is present in a nonactivatable form in vivo. The mutation appears to affect carboxylase activation specifically, and not any other enzyme of the photosynthesis or photorespiratory cycles. The effect of the mutation on carboxylase activation is indirect, inasmuch as the properties of ribulose-1,5-bisphosphate carboxylase purified from the mutant are not distinguishable from those of the wild type enzyme. The mutant requires high levels of atmospheric CO(2) for growth because photosynthesis is severely impaired in atmospheres containing normal levels of CO(2), irrespective of the atmospheric O(2) concentration. In this respect, the mutant is distinguished from previously described high-CO(2) requiring mutants of Arabidopsis which have defects in photorespiratory carbon or nitrogen metabolism.

Journal Article↗
PubMed1981

A Sensitive Assay Procedure for Simultaneous Determination of Ribulose-1,5-bisphosphate Carboxylase and Oxygenase Activities.

A sensitive assay procedure is described for the simultaneous determination of ribulose-1,5-bisphosphate (RuBP) carboxylase and oxygenase activities. In this assay, [1-(3)H]RuBP is incubated with (14)CO(2) and O(2). Carboxylation rate is determined from (14)CO(2) incorporation and oxygenation rate is determined from [2-(3)H]glycolate-phosphate production. The assay was found to be suitable at all CO(2) and O(2) concentrations examined, which ranged from 0 to 300 micromolar CO(2) (20 millimolar NaHCO(3)) and 0 to 1.15 millimolar (100%) O(2). In combination with a polarographic assay, the stoichiometry of the RuBP oxygenase reaction was found to be RuBP-O(2)-glycolate phosphate-glycerate phosphate (1:1:1:1).The ratio of soybean (Merr.) RuBP carboxylase and oxygenase activities was measured at various CO(2) and O(2) concentrations and in the presence of several compounds which have been reported to alter differentially the rate of the two reactions. The ratio of the two activities was a linear function of the ratio of the CO(2) and O(2) concentrations. The substrate specificity factor V(c)K(o)/V(o)K(c), which determines relative carboxylase and oxygenase reaction rates as a function of the CO(2) and O(2) concentrations, was found to be 73 in the presence of Mg(2+). Of the several compounds which have been reported to alter differentially the two reactions, we found that only Mn(2+) substitution for Mg(2+) was effective. Compared to Mg(2+), Mn(2+) reduced the K(m)(O(2)) from 690 to 36 micromolar O(2) and reduced the specificity factor to about 4. The K(m)(CO(2)) was about 20 micromolar CO(2) in the presence of both Mg(2+) and Mn(2+). Comparison of reaction rates in the presence of activated and inactivated enzyme allowed a direct determination that both carboxylase and oxygenase activities are similarly activated by CO(2), with an activation equilibrium constant of about 1.3 millimolar NaHCO(3) (27 micromolar CO(2)) at pH 7.85 and 10 millimolar Mg(2+).

Journal Article↗
PubMed1981

Photorespiration-deficient Mutants of Arabidopsis thaliana Lacking Mitochondrial Serine Transhydroxymethylase Activity.

Three allelic mutants of Arabidopsis thaliana which lack mitochondrial serine transhydroxymethylase activity due to a recessive nuclear mutation have been characterized. The mutants were shown to be deficient both in glycine decarboxylation and in the conversion of glycine to serine. Glycine accumulated as an end product of photosynthesis in the mutants, largely at the expense of serine, starch, and sucrose formation. The mutants photorespired CO(2) at low rates in the light, but this evolution of photorespiratory CO(2) was abolished by provision of exogenous NH(3). Exogenous NH(3) was required by the mutants for continued synthesis of glycine under photorespiratory conditions. These and related results with wild-type Arabidopsis suggested that glycine decarboxylation is the sole site of photorespiratory CO(2) release in wild-type plants but that depletion of the amino donors required for glyoxylate amination may lead to CO(2) release from direct decarboxylation of glyoxylate. Photosynthetic CO(2) fixation was inhibited in the mutants under atmospheric conditions which promote photorespiration but could be partially restored by exogenous NH(3). The magnitude of the NH(3) stimulation of photosynthesis indicated that the increase was due to the suppression of glyoxylate decarboxylation. The normal growth of the mutants under nonphotorespiratory atmospheric conditions indicates that mitochondrial serine transhydroxymethylase is not required in C(3) plants for any function unrelated to photorespiration.

Journal Article↗
PubMed1980

Photorespiration mutants of Arabidopsis thaliana deficient in serine-glyoxylate aminotransferase activity.

Three mutants of the crucifer Arabidopsis thaliana (Linnaeus) Heynhold were isolated that are completely lacking in activity catalyzed by serine-glyoxylate aminotransferase (EC 2.6.1.45), a peroxisomal enzyme involved in photorespiratory carbon metabolism. These mutants were viable and exhibited normal photosynthesis under conditions that suppressed photorespiration, but they were inviable and photosynthesized at greatly reduced rates under conditions that promoted photorespiration. Serine and glycine accumulated as end products of photosynthesis in the mutants, mostly at the expense of starch and sucrose. The mutants are allelic, and the segregation patterns of plant viability, photosynthetic activity, and enzyme activity in the F(1) and F(2) generations indicated that all the observed effects were caused by a single recessive nuclear mutation. This conclusion was confirmed by the isolation of seven revertants in which viability, photosynthetic capacity, and enzyme activity were simultaneously restored. Mutants of the type described here, in which photorespiration is changed from a merely wasteful process into one that is lethal, may permit the direct selection of secondary mutations that reduce photorespiration.

Journal Article↗