William L. Ogren

Phosphoglycolate production catalyzed by ribulose diphosphate carboxylase.

Abstract An oxygen-dependent production of phosphoglycolate is catalyzed by purified soybean ribulose diphosphate carboxylase and by crude extracts of soybean and corn leaves. It is suggested that the phosphoglycolate produced in this reaction is the source of glycolate metabolized in photorespiration.

Regulation of photorespiration in C3 and C4 species

SummaryPhotorespiration is a light-stimulated oxidation of photosynthesis intermediates to CO2. This process occurs primarily in higher plants which fix CO2 via the C3 pathway of photosynthesis (Calvin cycle). The details of the mechanism of photorespiration are controversial, but glycolic acid metabolism is thought to be involved. Photorespiration appears to be a wasteful process, and net photosynthesis at atmospheric CO2 concentration can be increased by approximately 50% by stopping photorespiration and an associated oxygen inhibition of photosynthesis. Since most crop species fix CO2 by the C3 pathway, control of photorespiration represents a major opportunity for increasing crop productivity. Photorespiration is not readily detected in a second group of higher plants, the C4 species. C4 plants have solved the problem of photorespiration through the evolution of a specialized leaf anatomy and compartmented enzyme complement, which act to reduce glycolic acid biosynthesis and to recapture photorespiratory CO2 before it can exit from the leaf. However, breeding experiments indicate that it will be difficult, if not impossible, to transform photorespiratory C3 species into non-photorespiratory C4 species. Control of photorespiration in C3 species will have to be achieved by isolating photorespiratory-deficient mutants of C3 plants or by identifying chemical compounds that specifically inhibit photorespiratory metabolism.ZusammenfassungLichtatmung ist eine durch Licht geförderte Oxydation von Zwischenprodukten der Photosynthese zu CO2. Dieser Prozess spielt sich hauptsächlich in höheren Pflanzen ab, die CO2 über den C3-Weg der Photosynthese fixieren (Calvin Cyclus). Der genaue Mechanismus der Lichtatmung ist umstritten, aber es wird angenommen, dass der Glykolsäure-Stoffwechsel dabei eine Rolle spielt. Die Lichtatmung ist ein unwirtschaftlicher Prozess, und die reine Photosynthese bei atmosphärischen CO2 Konzentrationen kann um etwa 50% erhöht werden, wenn die Lichtatmung und die damit verbundene Sauerstoffhemmung der Photosynthese unterbunden wird. Da die meisten Nutzpflanzen CO2 auf dem C3-Weg fixieren, stellt eine Einschränkung der Lichtatmung eine wichtige Möglichkeit der Erhöhung der Produktivität von Nutzpflanzen dar. Bei einer zweiten Gruppe von höheren Pflanzen, den C4-Arten, kann die Lichtatmung nicht so leicht nachgewiesen werden. C4-Pflanzen haben das Problem der Lichtatmung durch Ausbildung einer speziellen Blattanatomie und eines getrennten Enzym-Komplements gelöst, das die Glykolsäurebiosynthese verringert und das CO2 der Lichtatmung bindet, bevor dieses das Blatt verlässt. Zuchtexperimente haben allerdings gezeigt, dass es schwierig wenn nicht unmöglich sein wird, lichtatmende C3-Arten in nicht lichtatmende C4 -Arten umzuwandeln. Eine Einschränkung der Lichtatmung in C3 -Arten wird durch Isolierung von lichtatmungsarmen Mutanten von C3-Arten erfolgen müssen, oder durch die Identifizierung von chemischen Verbindungen, die den Lichtatmungsstoffwechsel spezifisch hemmen.

A soluble chloroplast protein catalyzes ribulosebisphosphate carboxylase/oxygenase activation in vivo.

Ribulosebisphosphate carboxylase/oxygenase (EC 4.1.1.39) (rubisco) must be fully activated in order to catalyze the maximum rates of photosynthesis observed in plants. Activation of the isolated enzyme occurs spontaneously, but conditions required to observe full activation are inconsistent with those known to occur in illuminated chloroplasts. Genetic studies with a nutant of Arabidopsisthaliana incapable of activating rubisco linked two chloroplast polypeptides to the activation process in vivo. Using a reconstituted light activation system, it was possible to demonstrate the participation of a chloroplast protein in rubisco activation. These results indicate that a specific chloroplast enzyme, rubisco activase, catalyzes the activation of rubisco in vivo.

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.

Correlations among leaf CO2-exchange rates, areas and enzyme activities among soybean cultivars

Soybean (Glycine max (L.) Merr.) genotypes varying in area per nodal unit (usually a trifoliolate) and maturity class were grown in plots at the University of Illinois experimental farm. Leaf CO2-exchange rates per unit area (CER) were measured under sunlight on intact plants. In addition to previously reported correlations with specific leaf weight and chlorophyll, CER was positively correlated with ribulose bisphosphate carboxylase (RuBPcase) activity, specific activity, and soluble protein, and was negatively correlated with area per leaf unit. The CER: chlorophyll correlation was destroyed by high CER values in 2 chlorophyll-deficient lines. CER values for 27 of the 35 lines tested fell within the range of those for isolines of cultivar Clark varying in leaf characteristics. The CER values were highest for fully expanded leaves during rapid pod fill. These results suggested that photoperiod (maturity) genes and genes for leaf area growth interact with genes controlling photosynthetic CO2-exchange to produce the major differences in CER values among soybean genotypes.

Photosynthesis is required for induction of the CO2-concentrating system in Chlamydomonas reinhardii

Chlamydomonas reinhardii cells grown photoautotrophically at air levels of C@ (0.03-0.04%) exhibit a much higher affinity for inorganic carbon in photosynthesis than do cells grown at elevated (I-58) CO, concentrations [ 11. This is also true of other unicellular green algae [2,3]. The apparent Km (CO2) of air-adapted C. reinhardii is much lower than that of RuBP carboxylase from the same cells [ 11. Two explanations have been offered for this phenomenon, one being solely dependent on the activity of carbonic anhydrase [4,5] and the other based on a CO2 concentrating mechanism [6,7]. Although not well characterized, the proposed CO2 concentrating mechanism appears to involve active HCOytransport into the algal cells [6-81, raising the internal inorganic carbon concentration several-fold higher than that of the surrounding medium [6,8,9]. Carbonic anhydrase activity increases substantially in high COz-adapted algae upon transfer to air levels of CO2 [2,4,10], as does the apparent capacity for energy dependent HCOF transport [6]. This suggests that carbonic anhydrase may play a specific role in the CO2 concentrating mechanism of these algae. We have utilized photosynthesis-deficient mutants of Chlamydomonas reinhardii and manipulation of growth conditions in wild-type C. reinhardii to study regulation of carbonic anhydrase activity and the capacity for bicarbonate transport. We conclude that there is a coordinated regulation of carbonic anhydrase and bicarbonate transport in Chlamydomonas and that this regulation may be mediated by photosynthetic or photorespiratory metabolism.

Biochemical and genetic analysis of an RuBP carboxylase/oxygenase-deficient mutant and revertants of Chlamydomonas reinhardii

RuBP carboxylase/oxygenase (EC 4.1.1.39) is a chloroplast localized enzyme consisting of 2 nonidentical subunits, each present in 8 copies [ 11. The large subunit is coded by a chloroplast gene in Chlamydomonas reinhardii [2,3] and contains the active site of the enzyme [4]. The function of the nuclear-encoded [5] small subunit remains unknown. The gaseous substrates of the enzyme, CO2 and 02, compete at the same active site [6]. Carboxylation of RuBP initiates C3 photosynthesis. Oxygenation of RuBP yields phosphoglycolate, which leads to the release of CO2 via photorespiration. Enhanced rates of CO2 fixation may be achieved if oxygenase activity could be reduced or eliminated [7]. The function of photorespiration is unknown. However, mutants of Arabidopsis that lack photorespiration have been recovered as COz-requiring strains [8]. These mutants demonstrate that photorespiration is not essential, and indicate that attempts to abolish photorespiration at ambient levels of CO2 must focus on reducing the oxygenase activity of RuBP carboxylase/oxygenase. The ratio of carboxylase to oxygenase activity varies in phylogenetically diverse species, apparently increasing during evolution 191. This observation suggests that the ratio of the two activities may be amenable to genetic alteration within a species. In [lo], a light-sensitive, acetate-requiring chloroplast mutant of Chlamydomonas reinhardii was described that lacks RuBP carboxylase activity and produces an enzyme with an altered large subunit isoelectric point. Here, we describe the isolation of photosynthetically competent revertants of this mutant in studies designed to explore the possibility of recovering enzymes with altered ratios of carboxylase/oxygenase activity. All revertants possess an enzyme restored to wild-type function. These results show that the defective RuBP carboxylase/oxygenase protein can be changed back to its original state, indicating that genetic approaches to modifying the enzyme are feasible.

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.

ACTION SPECTRA FOR ACCUMULATION OF INORGANIC CARBON IN THE CYANOBACTERIUM, Anabaena variabilis

Abstract— Action spectra for accumulation of inorganic carbon were obtained for Anabaena variabilis, strainM–2, in the presence and absence of photosynthetic CO2 fixation. The action spectrum for inorganic carbon accumulation in the presence of CO2 fixation showed a peak around 684 nm, corresponding to chlorophyll a absorption in PS 1, while that for CO2 fixation showed a peak around 630 nm, corresponding to phycocyanin absorption in PS 2. The action spectra obtained in the presence of iodoacetamide or diuron, which inhibit CO2 fixation, showed two peaks, one at about 684 nm and the other at 630 nm, with the 630 nm peak height 80 to 90% of the 684 nm peak. These results indicate that inorganic carbon transport in A. variabilis can be driven with near equal efficiency by energy derived from absorption in photosystem 1 alone and with energy transferred to PS 1 after absorption by PS 2.

Evidence for a saturable transport component in the inorganic carbon uptake of Chlamydomonas reinhardii

not received bicarbonate transport Chlamydomonas A/gal transport