Rubisco Activase Activity in Spinach Leaf Extracts

Rubisco activase is a chloroplast stromal protein that catalyzesthe activation of ribulose-1,5- bisphosphate carboxylase/oxygenase(rubisco) in vivo. Activation must occur before rubisco cancatalyze the photosynthetic assimilation of CO₂. In leaves,photosynthesis and rubisco activation increase with increasinglight intensity. Techniques are described that allow the activityof rubisco activase to be measured in extracts of spinach (Spinaceaoleracea L.) leaf tissue. In this context, rubisco activaseactivity is defined as the ability to promote activation ofthe inactive ribulose-1,5- bisphosphate-bound rubisco in anATP-dependent reaction. Determination of rubisco activase activityin extracts of dark and light treated leaf tissue revealed thatthe activation state of rubisco activase was independent oflight intensity. ¹Present address: Department of Biological Sciences, 213 Carson-TaylorHall, Louisiana Tech University, Ruston, Louisiana 71272, U.S.A.     

Multidisciplinary research in photosynthesis: A case history based on the green alga Chlamydomonas

This article examines the contribution of a unicellular green alga Chlamydomonas to progress in photosynthetic research. The objective is to focus on the aspects of Chlamydomonas that have provided an advantage over other photosynthetic organisms in investigating photosynthesis. To do this we discuss several examples that demonstrate the progress from a genetic study to a multidisciplinary approach that probes higher levels of complexity within the organism. These examples include the function and molecular regulation of electron transport components between photosystem II and photosystem I, the molecular genetics of the herbicide binding protein of photosystem II, and several different studies that have derived from a search for rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) mutants in Chlamydomonas, including chloroplast ribosome function, the regulation of the large subunit of rubisco, and the interaction between photosynthetic electron transport and carbon metabolism.     

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     

Quinoprotein D-glucose dehydrogenases inAcinetobacter calcoaceticus LMD 79. 41: Purification and characterization of the membrane-bound enzyme distinct from the soluble enzyme

Acinetobacter calcoaceticus is known to contain soluble and membrane-bound quinoprotein D-glucose dehydrogenases while other oxidative bacteria such asPseudomonas orGluconobacter contain only membrane-bound enzyme. The two different forms were believed to be the same enzyme or interconvertible. Present results show that the two different forms of glucose dehydrogenase are distinct from each other in their enzymatic and immunological properties as well as in their molecular size.The soluble and membrane-bound glucose dehydrogenases were separated after French press-disruption by repeated ultracentrifugation, and then purified to nearly homogeneous state. The soluble enzyme was a polypeptide of 55 Kdaltons, while the membrane-bound enzyme was a polypeptide of 83 Kdaltons which is mainly monomeric in detergent solution. Both enzymes showed different enzymatic properties including substrate specificity, optimum pH, kinetics for glucose, and reactivity for ubiquinone-homologues. Furthermore, the two enzymes could be distinguished immunochemically: the membrane-bound enzyme is cross-reactive with an antibody raised against membrane-bound enzyme purified fromPseudomonas but not with antibody elicited against the soluble enzyme, while the soluble enzyme is not cross-reactive with the antibody of membrane-bound enzyme.Data also suggest that the membrane-bound enzyme functions by linking to the respiratory chain via ubiquinone though the function of the soluble enzyme remains unclear.     

Catalysis of Ribulosebisphosphate Carboxylase/Oxygenase Activation by the Product of a Rubisco Activase cDNA Clone Expressed in Escherichia coli

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase activity was obtained from a partially purified extract of Escherichia coli transformed with a 1.6-kilobase spinach (Spinacia oleracea L.) cDNA clone. This activity was ATP-dependent. Catalysis of rubisco activation by spinach and cloned rubisco activase was accompanied by the same extent of carboxyarabinitol bisphosphate-trapped ¹⁴CO₂ as occurred in spontaneous activation, indicating that rubisco carbamylation is one facet of the rubisco activase reaction. The CO₂ concentration required for one-half maximal rubisco activase activity was about 8 micromolar CO₂. These observations are consistent with the postulated role of rubisco activase in regulating rubisco activity in vivo.     

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 CO₂ concentrations between 100 and 2000 microliters per liter, the activation state was a function of the CO₂ concentration in the dark but was independent of CO₂ concentration in the light. High CO₂ 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.     

Electron Transport through photosystem I Stimulates Light Activation of Ribulose Bisphosphate Carboxylase/Oxygenase (Rubisco) by Rubisco Activase

The activation state of ribulose bisphosphate carboxylase/oxygenase (rubisco) in a lysed chloroplast system is increased by light in the presence of a saturating concentration of ATP and a physiological concentration of CO₂ (10 micromolar). Electron transport inhibitors and artificial electron donors and acceptors were used to determine in which region of the photosynthetic electron transport chain this light-dependent reaction occurred. In the presence of DCMU and methyl viologen, the artificial donors durohydroquinone and 2,6-dichlorophenolindophenol (DCPIP) plus ascorbate both supported light activation of rubisco at saturating ATP concentrations. No light activation occurred when DCPIP was used as an acceptor with water as electron donor in the presence of ATP and dibromothymoquinone, even though photosynthetic electron transport was observed. Nigericin completely inhibited the light-dependent activation of rubisco. Based on these results, we conclude that stimulation of light activation of rubisco by rubisco activase requires electron transport through PSI but not PSII, and that this light requirement is not to supply the ATP needed by the rubisco activase reaction. Furthermore, a pH gradient across the thylakoid membrane appears necessary for maximum light activation of rubisco even when ATP is provided exogenously.     

Transformation of eggplant (Solanum melongena L.) using a binary Agrobacterium tumefaciens vector

Summary Kanamycin resistant plants of Solarium melongena L. (eggplant) cv. Picentia were obtained following the cocultivation of leaf explants with Agrobacterium tumefaciens. A disarmed binary vector system containing the neomycin phosphotransferase (NPTII) gene as the selectable marker and chloramphenicol acetyltransferase (CAT) as a reporter gene was utilized. In vitro grown plants were used as sources of explants to produce transgenic plants on selective medium containing 100 mg/l kanamycin. The transformation and expression of the foreign genes was confirmed by DNA hybridizations, leaf disc assays, and by measuring NPTII and CAT enzyme activities. This technique is simple, rapid, efficient, and transgenic eggplants of this commercial cultivar have been transferred to soil where they have flowered and set seed.Abbreviations CAT chloramphenicol acetyltransferase - MS Murashige and Skoog - NPTII neomycin phosphotransferase - NOS nopaline synthase - ZEA zeatin     

Primary Structure of Chlamydomonas reinhardtii Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Activase and Evidence for a Single Polypeptide

Immunoblot analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) activase from the green alga Chlamydomonas reinhardtii indicated the presence of a single polypeptide. This observation contrasts with the Spinacea oleracea (spinach) and Arabidopsis thaliana proteins, in which two polypeptide species are generated by alternative pre-mRNA splicing. A Chlamydomonas rubisco activase cDNA clone containing the entire coding region was isolated and sequenced. The open reading frame encoded a 408 amino acid, 45 kilodalton polypeptide that included a chloroplast transit peptide. The presumptive mature polypeptide possessed 62% and 65% amino acid sequence identity, respectively, with the spinach and Arabidopsis mature polypeptides. The Chlamydomonas rubisco activase transit peptide possessed almost no amino acid sequence identity with the higher plant transit peptides. The nucleotide sequence of Chlamydomonas rubisco activase cDNA provided no evidence for alternative mRNA splicing, consistent with the immunoblot evidence for only one polypeptide. Genomic DNA blot analysis indicated the presence of a single Chlamydomonas rubisco activase gene. In the presence of spinach rubisco activase, a lower extent and rate of activation were obtained in vitro with Chlamydomonas rubisco than with spinach rubisco. We conclude Chlamydomonas rubisco activase comprises a single polypeptide which differs considerably from the higher plant polypeptides with respect to primary structure.     

Crown gall transformation of lentil (Lens culinaris Medik.) with virulent strains of Agrobacterium tumefaciens

Summary Four diverse strains of Agrobacterium tumefaciens (C58, Ach5, GV3111, and A281) were capable of inducing tumors at a high frequency on inoculated stems of lentil (Lens culinaris Medik. cultivar Laird) in vivo, and on excised shoot apices in vitro. GV3111 and Ach5 produced the largest and heaviest tumors in vivo, while A281 produced the heaviest tumors in vitro. Tumor formation and opine production are indicative of plant cell transformation and tumors produced appropriate opines: nopaline (C58), octopine (Ach5 and GV3111), and agropine and mannopine (A281). Southern analysis of DNA from a tumor line produced by strain C58 showed that a T-DNA fragment had been transferred into the lentil genome.     

Ribulose-1,5-bisphosphate carboxylase/oxygenase activase protein prevents the in vitro decline in activity of ribulose-1,5-bisphosphate carboxylase/oxygenase

The rate of CO₂ fixation by ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) following addition of ribulose 1,5-bisphosphate (RuBP) to fully activated enzyme, declined with first-order kinetics, resulting in 50% loss of rubisco activity after 10 to 12 minutes. This in vitro decline in rubisco activity, termed fall-over, was prevented if purified rubisco activase protein and ATP were added, allowing linear rates of CO₂ fixation for up to 20 minutes. Rubisco activase could also stimulate rubisco activity if added after fallover had occurred. Gel filtration of the RuBP-rubisco complex to remove unbound RuBP allowed full activation of the enzyme, but the inhibition of activated rubisco during fallover was only partially reversed by gel filtration. Addition of alkaline phosphatase completely restored rubisco activity following fallover. The results suggest that fallover is not caused by binding of RuBP to decarbamylated enzyme, but results from binding of a phosphorylated inhibitor to the active site of rubisco. The inhibitor may be a contaminant in preparations of RuBP or may be formed on the active site but is apparently removed from the enzyme in the presence of the rubisco activase protein.     

Purification and species distribution of rubisco activase

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase, a soluble chloroplast protein which promotes light-dependent rubisco activation, was partially purified from spinach chloroplasts by ion-exchange and gel-filtration fast protein liquid chromatography. The protein could also be isolated using rate zonal centrifugation in sucrose gradients followed by conventional ion-exchange on DEAE-cellulose. The active enzyme was composed of 44 and 41 kilodalton subunits. Antibodies to the activase polypeptides were produced in tumor-induced mouse ascites fluid and used as probes for activase on immunoblots of soluble proteins from a number of species. One or both of the activase polypeptides were recognized in all higher plant species examined including Arabidopsis thaliana, soybean, kidney bean, pea, tobacco, maize, oat, barley, celery, tomato, pigweed, purslane, dandelion, sorghum, and crabgrass. The polypeptides were not present in a mutant of Arabidopsis which is incapable of activating rubisco in vivo. The activase polypeptides were also detected in cell extracts of the green alga Chlamydomonas reinhardii. Activase activity, which had been demonstrated previously in wild-type Arabidopsis and in spinach, was measured in protoplast extracts of Nicotiana rustica. The results suggest that control of rubisco by activase may be an ubiquitous form of regulation in eucaryotic photosynthetic organisms.     

Effects of organism type and growth conditions on cell disruption by impingement

Data for disruption of C. utilis, S. cerevisiae and B. subtilis cells by impingement of a high velocity jet of suspended cells against a stationary surface are compared. Differences between organisms were observed, but there were no general differences found between yeast and bacteria. In addition, growth conditions were found to have an effect on disruption with cells grown at a high specific growth rate easier to disrupt than cells grown at a low rate.Nomenclature a exponent of pressure (dimensionless) - D dilution rate (h^\s-1) - K dimensional rate constant (Pa ^\s-) - N number of passes (dimensionless) - P operating pressure (Pa) - R fraction of cells disrupted (dimensionless) - u_m maximum specific growth rate (h^\s-1)     

Energy distribution in the photochemical apparatus of Porphyridium cruentum: Picosecond fluorescence spectroscopy of cells in state 1 and state 2 at 77 K

Excitation energy distribution in Porphyridium cruentum in state 1 and state 2 was investigated by time resolved 77 K fluorescence emission spectroscopy. The fluorescence rise times of phycoerythrin, phycocyanin and allophycocyanin (in cells in state 1 and state 2) were very similar in contrast to the emission from chlorophyll a (Chl a) associated with the two photosystems. In state 2 photosystem II (PSII) Chl a fluorescence emission rose faster than the PSI Chl a emission and decayed more rapidly, and the converse was observed in state 1. These kinetic data support the concept of increased energy transfer from PSII Chl a to PSI Chl a in state 2 in P. cruentum.Abbreviations APC allophycocyanin - Chl a chlorophyll a - PSII photosystem II - PC phycocyanin - PE phycoerythrin     

Protein-bound ribulose bisphosphate correlates with deactivation of ribulose bisphosphate carboxylase in leaves

Previous reports indicate that ribulose 1,5-bisphosphate (RuBP) binds very tightly to inactive ribulose bisphosphate carboxylase (rubisco) in vitro. Therefore, we decided to investigate whether there was evidence for tight binding of RuBP associated with deactivation of rubisco in vivo. We modified a technique for rapidly separating `free' metabolites from those bound to high molecular compounds. Arabidopsis thaliana plants were illuminated at various irradiances before freezing the leaves in liquid N₂ and assaying rubisco activity and RuBP. The percentage activation of rubisco varied from 37% at low irradiance (45 micromoles quanta per square meter per second) to 100% at high irradiance (800 micromoles quanta per square meter per second). The total amount of RuBP did not vary much with irradiance, but bound RuBP changed from 36% of the total at low irradiance to none at high irradiance. Bound RuBP was significantly correlated with the estimated number of inactive rubisco sites, with a ratio of about 1:1. After a step increase in irradiance, rubisco activation increased and total RuBP increased transiently, but steady levels of both occurred by 10 minutes. The amount of bound RuBP decreased with a similar time course to the estimated decrease in inactive rubisco sites. After a step decrease in irradiance, rubisco deactivated slowly for at least 25 minutes. Bound RuBP increased gradually but did so more slowly than the estimated increase in inactive rubisco sites.     

Fertile somatic hybrid between sexually incompatible Hyoscyamus muticus and Hyoscyamus albus

Summary Somatic hybrid plants have been regenerated from fused protoplasts of a chlorophyll deficient mutant of H. muticus (2n=28) with wild type protoplasts of H. albus (2n=68). The inability of protoplasts of H. albus to regenerate was utilized in complementation with achlorophyllous, but regenerating, protoplasts of H. muticus for the selection of green somatic hybrid colonies and plants. The somatic hybrid plants showed intermediate morphological characters, and possessed 82–120 chromosomes, with a modal number of 96 which is also the amphidiploid complement of the two species. The isozyme patterns indicated the presence and expression of genes from both parents. The hybrid plants produced 33–78% viable pollen and set viable seeds upon selfing and backcrossing in a directional manner.     

Somatic cell genetic studies inBrassica species

In vitro culture ofBrassica alba anthers on a growth medium containing inorganics of KB5 and organics, iron, sucrose and hormones of B5 resulted in a very high response of anthers (93.75%) towards callus induction. All the calli transferred to regeneration media responded favourably even after six months of callus induction. Numerous torpedo-shaped embryoids developed in clusters at many sites from each callus mass. Secondary embryogenesis and multiple shoot formation was also observed in many cases. The number of embryoids and plantlets produced by one embryogenic anther were as high as 169.8 and 17 respectively. 87% of the regenerated plants were haploids.     

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.     

Identification of a penicillin V acylase processing fungus

In our studies with the penicillin V acylase of Bovista plumbea strains NRRL 3501 and NRRL 3824, we wanted to receive spores of these fungi. Surprisingly the fruiting bodies obtained in our work were not identical with those characteristic for Bovista plumbea. We identified them as Pleurotus ostreatus. For this reason we have to correct the name of the fungi known as Bovista plumbea NRRL 3501 and NRRL 3824.