Ribulose 1, 5-Bisphosphate Carboxylase/Oxygenase Activity and Photorespiration during the Ageing of Flag Leaves of Wheat

Photosynthesis, photorespiration, and ribulose bisphosphatecarboxylase/oxygenase activities were measured in intact flagleaves of wheat during ageing. Photorespiration declined verylittle as the leaves aged, and much less than photosynthesis.These changes could not be explained by changes in the ribulosebisphosphate carboxylase to oxygenase ratio of fraction 1 protein.As the leaves grew older the enzyme activities in extracts ofleaves declined in parallel so the ratio remained constant.     

Evidence for Effects on the in Vivo Activity of Ribulose-Bisphosphate Carboxylase/Oxygenase during Development of Mn Toxicity in Tobacco

The progressive decrease in net photosynthesis accompanying development of Mn toxicity in young leaves of burley tobacco (Nicotiana tabacum L. cv KY 14) is a result of effects on in vivo activity of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (rubisco, EC 4.1.1.39). This conclusion is supported by: (a) decrease in rates of CO₂ depletion during measurements of CO₂ compensation, (b) increase in leaf RuBP concentrations, (c) progressive decreases in rate-constants of RuBP loss (light to dark transition analyses) with progressive increases of leaf Mn concentrations, and (d) restoration of diminished rates of net photosynthesis to control rates by elevated CO₂ (5%). Moreover, elevated CO₂ (1100 microliters per liter) during culture of Mn-treated plants decreased elevated RuBP concentrations to control levels and alleviated foliar symptoms of Mn toxicity. These effects of Mn toxicity on in vivo activity of rubisco were not expressed by in vitro kinetic analyses of rubisco prepared under conditions to sequester Mn or to adsorb polyphenols or their oxidation products. Similarly, the in vitro activity of fructose bisphosphatase (EC 3.1.3.11) was unaffected by Mn toxicity.     

Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Content and Activity in Wheat, Rye and Triticale

Photosynthetic parameters were measured in triticale and its parents wheat and rye. Soluble protein content in leaves, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) content per fresh mass, total chlorophyll content, biomass yield, leaf area, leaf mass and specific leaf mass were higher but Rubisco content expressed as percentage of soluble protein, carboxylase activity, photosynthetic rate and stomatal conductance were significantly lower in rye than in wheat. Native-PAGE of Rubisco revealed that rye carboxylase was different from that of wheat. The difference was not related to either the small or large subunit of Rubisco but, may be, to the ionic and/or other properties of the Rubisco protein moiety. Triticale Rubisco was similar to wheat. For most of the studied physiological parameters, triticale showed much more similarity with wheat than with rye.     

Effect of Betaine on Enzyme Activity and Subunit Interaction of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase from Aphanothece halophytica

The presence of betaine, a quaternary ammonium compound, at a concentration (0.5 molar) reported to accumulate inside Aphanothece halophytica in response to increasing external salinity, slightly promoted ribulose-1,5-bisphosphate (RuBP) carboxylase activity. KCl at 0.25 molar inhibited RuBP carboxylase about 55%. Betaine relieved the inhibition by 0.25 m KCl and the original uninhibited activity was restored at 1 m betaine. Other osmoregulatory solutes such as sucrose and glycerol also reduced KCl inhibition, though to a lesser extent than betaine. Proline had no effect. The protective effect of betaine against KCl inhibition of RuBP carboxylase activity was also observed in other cyanobacteria, i.e. Synechococcus ACMM 323, Plectonema boryanum, and Anabaena variabilis, and in the photosynthetic bacterium Rhodospirillum rubrum but not in Chromatium vinosum. Apart from betaine, other quaternary ammonium compounds, i.e. sarcosine and trimethylamine-N-oxide (TMAO), but not glycine, also protected the enzyme against KCl inhibition and the effectiveness of such compounds appeared to correlate with the extent of N-methylation. Heat and cold inactivation of the enzyme could be protected by either betaine or KCl. However, best protection occurred when both betaine and KCl were present together. The K_m (CO₂) was not altered by either betaine or KCl, nor when they were present together. However, the K_m (RuBP) was increased about 5-fold by KCl, but was unaffected by betaine. The presence of betaine together with KCl lowered the KCl-raised K_m (RuBP) by about half. The extent of the dissociation of the enzyme molecule under the condition of low ionic strength was reduced by either betaine or KCl alone and more so when they were present together. Glycine, sarcosine, and TMAO were more effective than betaine or KCl in lowering the extent of the dissociation of the enzyme molecule.     

Fallover of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Activity : Decarbamylation of Catalytic Sites Depends on pH

Loss of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity during CO₂ fixation, called fallover, occurred with or without loss of activator CO₂ from catalytic sites depending on pH. At pH 7.5, but not at pH 8.5, the fraction of Rubisco sites that were carbamylated decreased during fallover. Inhibitors which formed during fallover were identified following NaBH₄ reduction and separation of the products by high performance anion-exchange chromatography and pulsed amperometric detection. They were xylulose 1,5-bisphosphate (XuBP) and 3-ketoarabinitol 1,5-bisphosphate. During fallover at pH 8.5, 3-ketoarabinitol-P₂ was the only inhibitor binding to Rubisco and this binding was at carbamylated sites, although both inhibitors were made. At pH 7.5, both inhibitors were bound to catalytic sites of Rubisco with XuBP bound tightly to decarbamylated sites, whereas 3-ketoarabinitol-P₂ bound to carbamylated sites. The pH during fallover also influenced the ratio of 3-ketoarabinitol-P₂ to XuBP formed. When fallover occurred at pH 7.5, both the formation of XuBP and its binding affinity to decarbamylated Rubisco sites were increased compared with those at pH 8.5. 3-Ketoribitol-P₂ was not found at either pH.     

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.     

Acclimation of Two Tomato Species to High Atmospheric CO(2): II. Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Phosphoenolpyruvate Carboxylase

Lycopersicon esculentum Mill. cv Vedettos and Lycopersicon chmielewskii Rick, LA 1028, were exposed to two CO₂ concentrations (330 or 900 microliters per liter) for 10 weeks. The elevated CO₂ concentrations increased the initial ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity of both species for the first 5 weeks of treatment but the difference did not persist during the last 5 weeks. The activity of Mg²⁺-CO₂-activated Rubisco was higher in 900 microliters per liter for the first 2 weeks but declined sharply thereafter. After 10 weeks, leaves grown at 330 microliters per liter CO₂ had about twice the Rubisco activity compared with those grown at 900 microliters per liter CO₂. The two species showed the same trend to Rubisco declines under high CO₂ concentrations. The percent activation of Rubisco was always higher under high CO₂. The phosphoenolpyruvate carboxylase (PEPCase) activity measured in tomato leaves averaged 7.9% of the total Rubisco. PEPCase showed a similar trend with time as the initial Rubisco but with no significant difference between nonenriched and CO₂-enriched plants. Long-term exposure of tomato plants to high CO₂ was previously shown to induce a decline of photosynthetic efficiency. Based on the current study and on previous results, we propose that the decline of activated Rubisco is the main cause of the acclimation of tomato plants to high CO₂ concentrations.     

Regulation of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase in vivo: Control by High CO2 Concentration

High CO₂ concentrations (HC) in air induce partial deactivation of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO, EC 4.1.1.39). Under saturating irradiance, increase in [CO₂] to 1 200 cm³ m^–3 reduces the concentration of operating carboxylation centres by 20–30 %. At a further increase in [CO₂], the activity remained on the same level. Under limiting irradiance, the lowest activity was reached at 600 cm³(CO₂) m^–3. The presence of oxygen diminished deactivation, but O₂ failed to stimulate reactivation under high CO₂. Conditions that favour oxygenation of ribulose-1,5-bisphosphate (RuBP) facilitated reactivation. Even HC did not act as an inhibitor. HC induces deactivation of RuBPCO by increasing the concentration of free reaction centres devoid of the substrate, which are more vulnerable to inhibition than the centres filled with substrates or products.     

Differential Modulation of Carboxylase and Oxygenase Activities of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Released from Freshly Ruptured Spinach Chloroplasts

The carboxylase activity of ribulose 1,5-bisphosphate (RuBP)carboxylase/oxygenase released from freshly ruptured spinachchloroplasts was stimulated preferentially by Mg²⁺ while oxygenaseactivity was higher with Mn²⁺. Only Mg²⁺ could reactivate eitheractivity of desalted enzyme. The results suggest that carboxylaseand oxygenase activities of RuBP craboxylase/oxygenase can bemodulated selectively by Mg²⁺ or Mn²⁺. ¹ Present address: Department of Botany, Sri Venkateswara University,Tirupati 517 502, India. (Received March 5, 1981; Accepted June 26, 1981)     

Measurement of the Concentration of the Active Centers of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase in vivo

Methods for in vivo measurement of the concentration of the reactive centers of ribulose-1,5,-bisphosphate carboxylase/oxygenase (Rubisco) are suggested that are based on saturation of the active centers with RuBP and determination of the concentration of the Rubisco–RuBP complex. The total concentration of potentially reactive centers is calculated from the dependence of the concentration of this complex on CO₂ concentration at a steady-state photosynthetic rate with further extrapolation of the carbon dioxide dependence curve to a zero CO₂ concentration. The concentration of centers that possessed a catalytic activity under given environmental conditions was measured after transferring leaves having a steady-state photosynthetic rate into a medium devoid of CO₂ and O₂. This procedure ensured the saturation of the carboxylation centers with RuBP. The carboxylation rates were measured during a short-term exposure to ¹⁴CO₂, and the concentration of the complex was calculated using the values of CO₂ concentration during the exposure time, as well as the carboxylation rate and constant. Rubisco activity was found to decrease at elevated CO₂ concentrations due to a lower concentration of catalytically active enzyme centers.     

Effect of Triacontanol on Chlamydomonas: I. Stimulation of Growth and Photosynthetic CO(2) Assimilation

Treatment of Chlamydomonas reinhardtii cells, cultured at 5% CO₂, with 1 to 1000 micrograms triacontanol (TRIA) per liter resulted in 21 to 35% increases in cell density, 7 to 31% increases in total chlorophyll, and 20 to 100% increases in photosynthetic CO₂ assimilation. The increase in CO₂ fixation with TRIA treatment occurred before, and was independent of, increases in total chlorophyll or cell number. Chlamydomonas cells responded to a broad range of TRIA concentrations that were at least one order of magnitude above the optimum concentration established for higher plants. The necessity for larger concentrations of TRIA may be due to destabilizing effects of Ca²⁺ and K⁺ present in the Chlamydomonas growth medium. These ions caused flocculation of the colloidally dispersed TRIA in apparent competition with binding of [¹⁴C]TRIA to Chlamydomonas cells. Octacosanol inhibited the effect of TRIA on photosynthetic CO₂ assimilation. TRIA treatment did not alter the distribution of ¹⁴C-label among photosynthetic products. The effect of TRIA on photosynthetic CO₂ assimilation increased with time after treatment up to 3 days. Chlamydomonas cells that had been grown at low-CO₂ (air) did not respond to TRIA, and transfer of high-CO₂ (5%) grown cells that had responded to TRIA to a low-CO₂ atmosphere resulted in a loss of the effect of TRIA. The effect of pH on photosynthetic CO₂ assimilation indicated that CO₂ is probably the species of inorganic carbon utilized by control and TRIA-treated Chlamydomonas cells.     

Influence of Temperature on the Ratio of Ribulose Bisphosphate Carboxylase to Oxygenase Activities and on the Ratio of Photosynthesis to Photorespiration of Leaves

Lehnherr, B., Mächler, F. and Nösberger, J. 1985.Influence of temperature on the ratio of ribulose bisphosphatecarboxylase to oxygenase activities and on the ratio of photosynthesisto photorespiration of leaves.—J. exp. Bot. 36: 1117–1125. Rates of net and gross photosynthesis of intact white cloverleaves were measured by infrared gas analysis and by short termuptake of ¹⁴CO₂ respectively. Ribulose bisphosphate carboxylaseoxygenase (RuBPCO) was purified from young leaves and kineticproperties investigated in combined and separate assays. Theratio of carboxylase to oxygenase activities was compared withthe ratio of photosynthesis to photorespiration at various temperaturesand CO₂ concentrations. The ratio of photosynthesis to photorespiration at 30 Pa p(CO₂)was consistent with the ratio of carboxylase activity to oxygenaseactivity when each was measured above 20 °C. However, theratio of photosynthesis to photorespiration increased with decreasingtemperature, whereas the ratio of carboxylase to oxygenase activitywas independent of temperature. This resulted in a disagreementbetween the measurements on the purified enzyme and intact leafat low temperature. No disagreement between enzyme and leafat low temperature occurred, when the ratio of photosynthesisto photorespiration was determined at increased CO₂ concentrations. The results suggest an effect of low temperature and low CO₂concentration on the ratio of photosynthesis to photorespirationindependent of the enzyme. Key words: Ribulose bisphosphate carboxylase oxygenase, photorespiration, temperature     

Activities of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Phosphoenolpyruvate Carboxylase, and Oxygen Evolution in Transgenic Tobacco Plants

Three clones of tobacco transformed with the T-DNA of Agrobacterium rhizogenes Ri plasmid A4b cultivated in vitro on a solid agar medium neither showed pronounced morphological diversity nor significantly differed in chlorophyll (Chl) contents from control plants. However, the transformation caused a 27 to 83 % decay in leaf oxygen evolution and in both ribulose-1,5-bisphosphate carboxylase (RuBPC) and phosphoenolpyruvate carboxylase (PEPC) activities. Therefore, the transformation brought about a reduction of active PEPC as well as activable RuBPC amounts in plant tissues. Individual clones did not mutually differ. In tobacco transformed with A. rhizogenes strain TR101 and grown in soil only the mean leaf area tended to reduce. Chl contents, Chl a/b ratio, oxygen evolution, and activities of both RuBPC and PEPC were insignificantly affected by the transformation.     

Small Oligomers of Ribulose-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase Are Required for Biological Activity

Ribulose-bisphosphate carboxylase/oxygenase (Rubisco) activase uses the energy from ATP hydrolysis to remove tight binding inhibitors from Rubisco, thus playing a key role in regulating photosynthesis in plants. Although several structures have recently added much needed structural information for different Rubisco activase enzymes, the arrangement of these subunits in solution remains unclear. In this study, we use a variety of techniques to show that Rubisco activase forms a wide range of structures in solution, ranging from monomers to much higher order species, and that the distribution of these species is highly dependent on protein concentration. The data support a model in which Rubisco activase forms an open spiraling structure rather than a closed hexameric structure. At protein concentrations of 1 μm, corresponding to the maximal activity of the enzyme, Rubisco activase has an oligomeric state of 2–4 subunits. We propose a model in which Rubisco activase requires at least 1 neighboring subunit for hydrolysis of ATP.     

Effect of Mg2+ on the Structure and Function of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

Mg²⁺ in various concentrations was added to purified Rubisco in vitro to gain insight into the mechanism of molecular interactions between Mg²⁺ and Rubisco. The enzyme activity assays showed that the reaction between Rubisco and Mg²⁺ was two order, which means that the enhancement of Rubisco activity was accelerated by low concentration of Mg²⁺ and slowed by high concentration of Mg²⁺. The kinetics constant (K _m) and V _max was 1.91 μM and 1.13 μmol CO₂ mg⁻¹ protein∙min⁻¹, respectively, at a low concentration of Mg²⁺, and 3.45 μM and 0.32 μmol CO₂∙mg⁻¹ protein∙min⁻¹, respectively, at a high concentration of Mg²⁺. By UV absorption and fluorescence spectroscopy assays, the Mg²⁺ was determined to be directly bound to Rubisco; the binding site of Mg²⁺ to Rubisco was 0.275, the binding constants (K _A) of the binding site were 6.33 × 10⁴ and 5.5 × 10⁴ l·mol⁻¹. Based on the analysis of the circular dichroism (CD) spectra, it was concluded that the binding of Mg²⁺ did not alter the secondary structure of Rubisco, suggesting that the observed enhancement of Rubisco carboxylase activity was caused by a subtle structural change in the active site through the formation of the complex with Mg²⁺.     

Linearity and Functioning Forms in the Carboxylase Reaction of Spinach Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase

The reaction of spinach RuBisCO activated with CO₂ and Mg²⁺proceeded in two phases, an initial burst for a few minutesand the subsequent linear phase, in the presence of saturatingconcentrations of CO₂, ribulose 1,5-bisphosphate (RuBP), andMg²⁺. The percentage of the activity in the linear phase tothat in the initial burst was 55% with RuBisCO prepared withpolyethylene glycol, and very close to the value with the enzymereleased immediately from isolated chloro-plasts. RuBisCO preparedwith ammonium sulfate had a much larger decrease of the activityin the linear phase. The Euglena enzyme had a linear courseof reaction with time for up to 20 minutes. The K_m for CO₂ of spinach RuBisCO activated beforehand was 20µM in the initial burst, and 28 µM in the linearphase. In the carboxylase reaction initiated with inactive enzyme,the activity was initially negligible, but in 5 minutes increasedto the level observed in the linear phase of the activated enzyme.The K_m for CO₂ in the linear phase of the pre-inactivated enzymewas 70 µM. The concentration of RuBP was the immediate cause of the two-phasiccourse of the carboxylase reaction of spinach RuBisCO. The curvatureof the time course was not observed below 35 µM RuBP.The enzyme required over 88 µM RuBP for the conventionaltwo-phasic course. Further increase of the concentration ofRuBP increased the extent of the curvature, but did not startthe curvature sooner after the start of the reaction. Even ifspinach RuBisCO was in the linear phase, dilution of RuBP orits consumption by the enzymatic reaction to less than 30 µMcaused the enzyme to show the resumed biphasic reaction courseafter addition of a high concentration of RuBP. ¹This paper is the twenty-fourth in a series on PhotosyntheticCarbon Metabolism in Euglena gracilis. (Received September 19, 1988; Accepted November 25, 1988)     

蔗糖对水稻幼苗叶片谷氨酰胺合成酶和1,5-二磷酸核酮糖羧化酶/加氧酶的影响

报道了在光照和暗处培养下,不同的浓度的蔗水稻幼苗叶片GS及其同工酶、1,5－二磷酸核酮糖羧化酶／加氧酶（Rubisco）的影响。无论是在光照或在暗处,蔗糖对GS活性均有抑制作用,尤其是在较高蔗糖下作用更为明显;虽然Rubisco及可溶性蛋白的水平在光照和暗处有显著的差别,但蔗糖对其未见明显影响。NativePAGE与活性染色表明,在光照下或在暗处,蔗糖对GS2的抑制蔗糖浓度升同而加强,但对GS1未有明显影响。这些结果提示,在水稻幼苗生长中,蔗糖不能象不光一样诱导叶水GS活性及其同工酶表达。