Hydrolysis of Ribulose-1,5-bisphosphate Carboxylase by Endoproteinases from Senescing Barley Leaves

The hydrolysis of ¹⁴C-labeled ribulose-1,5-bisphosphate carboxylase (RuBPCase) by two partially purified endoproteinases from senescing barley (Hordeum vulgare v. Numar) leaves is described. The major thiol proteinase, EP₁, exhibits biphasic kinetics which appear to be caused by a region of the large subunit of RuBPCase that is highly sensitive to attack by EP₁. This proteinase further hydrolyzes both the large and small subunit to smaller peptides. A second proteinase, EP₂, appears to convert the small subunit of RuBPCase rapidly to a 13.7-kilodalton fragment during initial stages of hydrolysis and then to degrade both this fragment and the large subunit. The presence of a third endoproteinase, EP₃, was discovered when [¹⁴C]RuBPCase, which appeared to be homogeneous by sodium dodecyl sulfate polyacrylamide electrophoresis, seemed to undergo very low but significant rates of “autolysis.” The large molecular weight fragments produced by EP₃ were different from those of EP₁ and EP₂.     

Degradation of Ribulose-1, 5-Bisphosphate Carboxylase/Oxygenase in Wheat Leaves During Dark-induced Senescence

The degradation of Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) in wheat (Triticum aestivum L. cv. Yangmai 158) leaves during dark-induced senescence was studied. An in vivo degradation product of Rubisco large subunit (LSU) with molecular weight of 50 kD was detected by SDS-PAGE and immunoblotting with antibody against tobacco Rubisco. This fragment could also be detected in natural senescence. The result also suggested that the Rubisco holoenzyme had not dissociated when LSU hydrolyzed from 53 kD to 50 kD. And LSU could be fragmented to 50 kD at 30-35 ℃ and at pH 7.5 in crude enzyme extracts of wheat leaves dark-induced for 48 h, which suggested that maybe LSU was degraded to 50 kD by an unknown protease in chloroplast.     

Degradation of the Large Subunit of Ribulose-1, 5-Bisphosphate Carboxylase/Oxygenase in Wheat Leaves

The degradation of the large subunit (LSU) of ribulose- 1, 5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) in wheat (Triticum aestivum L. cv. Yangmai 158) leaves was investigated. A 50 kDa fragment, a portion of the LSU of Rubisco, was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with antibody against tobacco Rubisco in crude enzyme extract of young wheat leaves. The appearance of the 50 kDa fragment was most obvious at 30-35 ℃ and pH 5.5. The LSU and its 50 kDa fragment both existed when the crude enzyme extract was incubated for 60 min. The amount of LSU decreased with incubation time from 0 to 3 h in crude enzyme extract. However, the 50 kDa fragment could not be found any pH from 4.5 to 8.5 in chloroplast lysates of young wheat leaves. In addition,through treatment with various inhibitors, reactions were inhibited by cysteine proteinase inhibitor E-64 or leupeptin.     

Degradation of the Large Subunit of Ribulose-1, 5-Bisphosphate Carboxylase／Oxygenase in Wheat Leaves

The degradation of the large subunit (LSU) of ribulose- 1, 5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) in wheat (Triticum aestivum L. cv. Yangmai 158) leaves was investigated. A 50 kDa fragment, a portion of the LSU of Rubisco, was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with antibody against tobacco Rubisco in crude enzyme extract of young wheat leaves. The appearance of the 50 kDa fragment was most obvious at 30-35 ℃ and pH 5.5. The LSU and its 50 kDa fragment both existed when the crude enzyme extract was incubated for 60 min. The amount of LSU decreased with incubation time from 0 to 3 h in crude enzyme extract. However, the 50 kDa fragment could not be found any pH from 4.5 to 8.5 in chloroplast lysates of young wheat leaves. In addition,through treatment with various inhibitors, reactions were inhibited by cysteine proteinase inhibitor E-64 or leupeptin.     

Variability of Reaction Kinetics for Ribulose-1,5-bisphosphate Carboxylase in a Barley Population

The photosynthetic enzyme ribulose bisphosphate carboxylase-oxygenase [EC 4.1.1.39] (RuBPCase) plays a key role in the carbon reduction system of plants. In this study, we determined the kinetic variability of RuBPCase among 46 varieties of Hordeum vulgare L. at two ages. The V_max CO₂ and K_m CO₂ of RuBPCase was determined for each cultivar. Varietal differences were found in K_m CO₂ and V_max CO₂ for one and four genotypes, respectively. One variety exhibited atypical behavior in both K_m and V_max. A comparison of varieties and age showed a significant interaction between these factors for K_m but not for V_max. These data indicate the presence of kinetic variability in RuBPCase within the H. vulgare population and perhaps between plant ages.     

Differences in the Activation State of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase in Barley,Pea, and Wheat at Two Altitudes

Activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) is an important parameter determining the rate of net photosynthesis (P _N) in situ for which no information is available with reference to altitude. We analyzed activation state along with P _N in three plant species and their cultivars grown at low (LA, 1 300 m) and high (HA, 4 200 m) altitudes. No significant change in P _N and the initial activity of RuBPCO was obtained with reference to altitude. However, activation state of RuBPCO was reduced significantly in the HA plants as compared to the LA ones. Hence low partial pressure of CO₂ prevailing at HA might be responsible for the lower activation state of RuBPCO.     

Photosynthesis, Ribulose-1,5-bisphosphate Carboxylase, Electron Transport, and Ribulose 1,5-Bisphosphate of Virescent and Normal Green Wheat Leaves

CO₂ gas exchange, ribulose-1,5-bisphosphate, and electron transport have been measured in leaves of a yellow-green mutant of wheat (Triticum durum var Cappelli) and its wild type strain grown in the field. All these parameters, expressed on leaf area basis, were similar in both genotypes except electron transport which was more than double in the wild type. These results, treated according to a recent photosynthesis model for C₃ plants, seem to indicate that the electron transport rate of mutant leaves is not sufficient to support the carboxylation derived through both the assimilation rate and the in vitro ribulose-1,5-bisphosphate carboxylase activity. It is suggested that under our experimental conditions photosynthetic electron transport is not the sole energy-dependent determinant of ribulose-1,5-bisphosphate regeneration in the mutant.     

Enzymic Properties of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Purified from Rice Leaves

The enzymic properties of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase purified from rice (Oryza sativa L.) leaves were studied. Rice RuBPcarboxylase, activated by preincubation with CO₂ and Mg²⁺ like other higher plant carboxylases, had an activation equilibrium constant (K_cK_Mg) of 1.90 × 10⁵ to 2.41 × 10⁵ micromolar² (pH 8.2 and 25°C). Kinetic parameters of carboxylation and oxygenation catalyzed by the completely activated enzyme were examined at 25°C and the respective optimal pHs. The K_m(CO₂), K_m(RuBP), and V_max values for carboxylation were 8 micromolar, 31 micromolar, and 1.79 units milligram⁻¹, respectively. The K_m(O₂), K_m(RuBP), and V_max values for oxygenation were 370 micromolar, 29 micromolar, and 0.60 units milligram⁻¹, respectively.

Comparison of rice leaf RuBP carboxylase with other C₃ plant carboxylases showed that it had a relatively high affinity for CO₂ but the lowest catalytic turnover number (V_max) among the species examined.

     

Carbon Dioxide Fixation and Ribulose-1, 5-bisphosphate Carboxylase Activity in Intact Leaves of Sugar Beet Plants Exposed to Salinity and Water Stress

The influence of salinity in the growing media on ribulose-1,5-bisphosphate (RuBP) carboxylase and on CO₂ fixation by intactsugar beet (Beta vulgaris) leaves was investigated. RuBP carboxylase activity was mostly stimulated in young leavesafter exposure of plants for 1 week to 180 mM NaCl in the nutrientsolution. This stimulation was more effective at the higherNaHCO₂ concentrations in the reaction medium. Salinity also enhanced CO₂ fixation in intact leaves mostlyat rate-limiting light intensities. A 60 per cent stimulationin CO₂ fixation rate was obtained by salinity under 450 µEm^–2 s^–1. At quantum flux densities of 150 µEm^–2 s^–1 (400–700 nm) this stimulation was280 per cent. Under high light intensities no stimulation bysalinity was found. In contrast, water stress achieved by directleaf desiccation or by polyethylene glycol inhibited enzymeactivity up to fourfold at –1.2 MPa. Beta vulgaris, sugar beet, ribulose-1, 5-bisphosphate carboxylase, salt stress, water stress, carbon dixoide fixation, salinity     

Relation between Nitrogen and Ribulose-1,5-bisphosphate Carboxylase in Rice Leaves from Emergence through Senescence

The relation between N content and ribulose-l,5-bisphosphate(RuBP) carboxylase protein was examined in the 12th leaf bladeof rice. Plants were grown under different amounts of N afterthe emergence of the 12th leaf blade. RuBP carboxylase proteinincreased with leaf N during leaf expansion. The synthesis ofRuBP carboxylase predominated during this period, and changesin the amounts of carboxylase synthesized until leaf death paralleledchanges in the N influx to the leaves. When the carboxylasereached its maximum content, the proportion of RuBP carboxylaseto leaf N was 27 to 28% irrespective of N treatment. As theleaf senesced, however, this proportion differed significantlywith the treatment. It was higher in the N-deficient leaf thanin the N-sufficient leaf. This was due to different patternsof RuBP carboxylase degradation for the treatments during senescence.RuBP carboxylase was degraded actively during the early stageof senescence in the N-sufficient leaf, whereas its degradationproceeded almost constantly in the N-deficient leaf during senescence. (Received October 17, 1983; Accepted January 27, 1984)     

Ribulose 1, 5-bisphosphate Carboxylase/ Oxygenase Activities in Leaves of Greenhouse Roses

An enzyme assay was developed to measure the initial and Mg²⁺–CO₂activated forms of Ribulose 1,5-bisphosphate Carboxylase/Oxygenase(Rubisco) in rose leaves. The assay was verified by co-extractionof the leaflets with partially purified spinach Rubisco andthrough correlation with net photosynthetic rates of individualleaflets (r²=0.7324). Changes in activities were measured asa function of depth of leaves in the canopy for two cultivarsof greenhouse hybrid tea roses. Initial Rubisco activity declinedwith increasing canopy depth for both cultivars. The activatedform of the enzyme, however, remained constant with canopy depthfor cv. Red Success; but increased with canopy depth, then declinedafter mid-canopy in the cv. Royalty. Rubisco activities werealso measured in the cv. Red Success grown in CO₂ enriched environments(100 mm³ dm^–3) at three humidity levels. The activitieswere not significantly affected by humidity treatment. However,there was a trend for plants grown at lower humidity to havehigher activated activities. Key words: Humidity, Rubisco, Rosa ? hybrida, Royalty, Red Success     

Relationship between the Kinetic Properties and the Small Subunit Composition of Nicotiana Ribulose-1, 5-bisphosphate Carboxylase

Genetic variability in the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase) in several Nicotiana species has been characterized by isoelectric focusing patterns. This heritable variation provides an opportunity to examine the functional role of each of these subunits. In this study, specifically designed RuBPCase enzymes composed of identical large subunits but different small subunits were constructed in vivo by interspecific hybridization between the species N. sylvestris, N. tabacum, N. glauca, N. glutinosa, N. plumbaginifolia, and N. tomentosiformis. Small subunit polypeptides were combined to form a sequence of one, two, three, and four polypeptides with the large subunit of N. sylvestris. Kinetic properties of these hybrid enzymes were compared. No differences in the specific activity of either carboxylation or oxygenation nor in K_m values for ribulose 1,5-bisphosphate, CO₂, or O₂ were detected among the RuBPCase enzymes from the various interspecific hybrids. Likewise, the ratio of carboxylation to oxygenation was constant.     

Differences between Wheat Genotypes in Specific Activity of Ribulose-1,5-bisphosphate Carboxylase and the Relationship to Photosynthesis

The in vitro ribulose-1,5-bisphosphate (RuBP) carboxylase activity per unit of leaf nitrogen was found to be 30% greater in Triticum aestivum than in T. monococcum. This was due to a higher specific activity of the enzyme from T. aestivum, as the amount of RuBP carboxylase protein per unit of total leaf nitrogen did not differ between the genotypes. The occurrence of higher specific activity of RuBP carboxylase is shown to correlate with possession of the large subunit derived from the B genome of wheat.

Despite the greater RuBP carboxylase activity per unit of leaf nitrogen in T. aestivum, the initial slopes of curves relating rate of CO₂ assimilation to intercellular p(CO₂) are similar in T. aestivum and T. monococcum for the same nitrogen content per unit leaf area. The similarity of the initial slopes is the result of a greater resistance to CO₂ transfer between the intercellular spaces and the site of carboxylation in T. aestivum than in T. monococcum.

     

Evidence for in vivo Light-induced Synthesis of Ribulose-1,5-diP Carboxylase and Phosphoribulokinase in Greening Barley Leaves

When actinomycin D, puromycin, streptomycin, chloramphenicol, and cycloheximide, known inhibitors of protein synthesis, were applied to leaves of intact seedlings or detached leaves of barley prior to their greening, the same general response resulted: the light-induced increase in activity of ribulose 1,5-diphosphate carboxylase was prevented while that of phosphoribulokinase was only partially suppressed; synthesis of chlorophyll was arrested. This is taken as preliminary evidence that de novo synthesis of protein may be responsible for the observed increase in ribulose-1,5-diphosphate carboxylase activity during greening. However, other factors may be involved with the light-induced stimulation of phosphoribulokinase.

Carbohydrate metabolites and substrates of the enzymes failed to induce the formation of ribulose-1,5-diphosphate carboxylase and phosphoribulokinase in the dark. No evidence was found for the presence of inhibitors in etiolated seedlings or activators in illuminated leaves of barley. Carboxylase activity almost equal to that of the illuminated water control was stimulated by MgCl₂ in the dark; MgCl₂ had no effect on the activity of the kinase.

     

变色酸显色法同时测定核酮糖-1,5-二磷酸羧化酶/加氧酶活性

提出一个用变色酸-硫酸显色浊同时测定核酮糖-1,5-二磷酸(RuBP)羧化酶/加氧酶活性的方法:RuBP羧化酶/加氧酶与底物作用后,用碱性磷酸酯酶将其产物水解生成乙醇酸和甘油酸,然后与变色酸试剂在1:5的体积比下,沸水浴中显色反应90min,乙醇酸与变色酸反应生成红紫色化合物,甘油酸生成淡棕色化合物,分别在573nm,745nm各有一特征吸收峰。根据A_(573),A_(745)与乙醇酸和甘油酸浓度间的函数关系式,求出RuBP羧化酶/加氧酶活性。     

Induction of the Inactivation and Degradation of Phosphoenolpyruvate Carboxylase and Ribulose-1,5-bisphosphate Carboxylase/Oxygenase in Maize Leaves by Freezing and Thawing

When frozen leaves of 24-day-old maize (Zea mays L.) plant werethawed on moist filter paper at 26°C (freeze-thaw treatment)several enzymes, including phosphoenolpyruvate carboxylase (PEPC)and ribulose-1,5-bisphosphate carboxylase (RuBPC), were rapidlyinactivated and degraded. The kinetics of the inactivation anddegradation were pseudo first-order, and the halftimes for inactivationof PEPC and RuBPC were 3.2 and 2.4 min, respectively. The effectof the freeze-thaw treatment on the inactivation and degradationdiffered among various enzymes: the residual activities of RuBPC,PEPC, hydroxypyruvate reductase, Cyt c oxidase, NADP-malic enzymeand a-mannosidase 10 min after the start of the thawing treatmentwere 7, 16, 54, 64, 97 and 98% of the initial respective levels.Thirty min after the starting of thawing treatment, the amountsof total soluble protein, the large subunit of RuBPC, the smallsubunit of RuBPC, the PEPC subunit and the NADP-malic enzymesubunit had fallen to 61, 2, 16, 8, and 66% of the initial respectiveamounts. The effect of freeze-thaw treatment on PEPC was greater in oldleaves than in young leaves. There was a steady increase ofthe rate of degradation of PEPC by freeze-thaw treatment asplants aged from 6 to 24 days. These results are discussed inthe context of protein degradation in plant cells. (Received August 9, 1993; Accepted January 10, 1994)     

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.     

Light Dependency of Carboxylation Efficiency and Ribulose-1, 5-Bisphosphate Carboxylase Activation in Trifolium subterraneum L. Leaves

The effect of photosynthetic photon flux density (PPFD) on carboxylationefficiency, estimated as the initial slope (IS) of net CO₂ assimilationrate versus intercellular CO₂ partial pressure response curve,as well as on ribulose-1, 5-bisphosphate carboxylase (Rubisco)activation was measured in Trifolium subterraneum L. leavesunder field conditions. The relationship between IS and PPFDfits a logarithmic curve. Rubisco activation accounts for theIS increase only up to a PPFD of 550 µmol photons m^-2s^-1. Further IS increase, between 550 and 1000 µmol photonsm^-2 s^-1, could be related to a higher ribulose fcwphosphate(RuBP) availability. The slow, but sustained IS increase above1000 µmol photons m^-2 s^-1 could be explained by the mesophyllCO₂ diffusion barriers associated with the high chlorophylland protein content in field developed leaves. Key words: Photosynthesis, initial slope, ribulose-1, 5-bissphosphate carboxylase activation, light response, Trifolium subterraneum L     

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.