Changes in Photosynthetic Capacity in Rice Leaves from Emergence through Senescence. Analysis from Ribulose-1,5-bisphosphate Carboxylase and Leaf Conductance

Changes in the rates of gas exchange and the amount of ribulose1,5-bisphosphate (RuBP) carboxylase protein were determinedin the 12th leaf blades of rice during the reproductive stages.RuBP carboxylase exhibited a large change similar to that inthe assimilation rate at 2% O₂ throughout the leaf's life, butits decrease during senescence was barely faster than the decreasein the assimilation rate. Consequently, the overall relationshipwas slightly curvilinear. By contrast, leaf conductance decreasedmore slowly than the assimilation rate which resulted in theintercellular CO₂ concentration increasing during senescence. In order to determine the maximum activity of RuBP carboxylaseat the intercellular CO₂ concentration, the kinetic parametersand their pH response were determined using purified, and completelyactivated, rice RuBP carboxylase. The maximum carboxylase activityat the intercellular CO₂ concentration was linearly correlatedwith the assimilation rate at 2% O₂ (r=0.989), and was veryclose to that needed to account for the assimilation rate. We conclude that changes in both the amount of RuBP carboxylaseprotein and leaf conductance reflect the change in photosynthesisduring the life span of the leaf. (Received November 26, 1983; Accepted February 20, 1984)     

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.

     

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.     

Interaction of Hydrogen Peroxide with Ribulose-1,5-bisphosphate Carboxylase/Oxygenase from Rice

The properties of rice-derived ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) in different concentrations of hydrogen peroxide (H2O2) solutions have been studied. The results indicate that at low H2O2 concentrations (0.2-10 mM), the properties of rubisco (e.g., carboxylase activities, structure, and susceptibility to heat denaturation) change slightly. However, at higher H2O2 concentrations (10-200 mM), rubisco undergoes an unfolding process, including the loss of secondary and tertiary structure, forming extended hydrophobic interface, and leading to cross-links between large subunits. High concentrations of H2O2 can also result in an increase in susceptibility of rubisco to heat denaturation. Further pre-treatments with or without reductive reagents to rubisco show that the disulfide bonds in rubisco help to protect the enzyme from damage by H2O2 as well as other reactive oxygen species.     

Purification and Storage of Ribulose 1,5-bisphosphate Carboxylase from Rice Leaves

Ribulose 1,5-bisphosphate (RuBP) carboxylase was purified fromrice leaves. By using a buffer containing 12.5% (v/v) glycerolthroughout purification, the enzyme was protected from coldlability and was obtained at a high yield (5.5 mg/g fresh wt).The purified enzyme exhibited different rates of CO₂/Mg²⁺-activationby temperature pretreatment/storage. The purified enzyme was stable for at least one year in phosphatebuffer containing 12.5% (v/v) glycerol at 4°C or 50% (v/v)glycerol at –20°C. (Received March 1, 1983; Accepted June 27, 1983)     

核酮糖-1,5-二磷酸羧化酶/加氧酶(Rubisco)

文章就核酮糖-1,5-二磷酸羧化酶/加氧酶(Rubisco)的分布、结构、性质、分类与功能的研究进展作了介绍。     

Ribulose-1,5-Diphosphate Carboxylase Protein during Flag Leaf Senescence

Ribulose-l,5-diphosphate (RuDP) carboxylase protein and activitywere determined in relation to net photosynthetic rate duringthe senescence of intact flag leaves of wheat on the plant.Initially the decrease in RuDP carboxylase activity was greaterthan the decline in net photosynthesis. The major decrease inRuDP carboxylase activity over this period resulted from a decreasein enzyme specific activity from 11 to 2 µmol CO₂ fixedh^–1 mg^–1 protein. Loss of RuDP carboxylase proteindid not occur until late in senescence by which time chlorophyllconcentration had decreased by more than 50%. Treatment of flagleaves at weekly intervals with either 1000 parts 10^–62-chloro-ethyltrimethylammonium chloride or 100 parts 10^–6gibberellic acid with 1 part 10^–6 kinetin did not significantlyaffect net photosynthetic rate, RuDP carboxylase protein oractivity during senescence.     

Vacuole Cysteine Proteases and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Degradation during Monocarpic Senescence in Cowpea Leaves

Characterisation of proteases degrading ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO, EC: 4.1.1.39) was studied in the cowpea leaf during monocarpic senescence 3 and 9 d after flowering (DAF), representing early and mid pod fill. The stage at 3 DAF coincided with decrease in the metabolic parameters characterising senescence, i.e., contents of total soluble proteins, RuBPCO, and leaf nitrogen. At 9 DAF, there was a decline in total soluble proteins and an appearance of a 48 kDa cysteine protease. Characterisation of the proteases was done using specific inhibitors. Subcellular localisation at 3 DAF was studied by following the degradation of RuBPCO large subunit (LSU) in the vacuole lysates using immunoblot analyses. Cysteine proteases played a predominant role in the degradation of RuBPCO LSU at the crude extract level. At 9 DAF, expression of cysteine protease isoforms was monitored using polyclonal antibodies against papain and two polypeptides of molecular masses 48 and 35 kDa were observed in the vacuole lysates. We confirmed thus the predominance of cysteine proteases in the vacuoles during different stages of pod development in cowpea leaf.     

Ozone-Induced Reduction in Quantity of Ribulose-1,5-bisphosphate Carboxylase in Alfalfa Foliage

The concentration of ribulose-1,5-bisphosphate carboxylase was measured in the foliage of `Moapa 69,' `Ladak,' and `Vernal' cultivars of alfalfa (Medicago sativa L.) after plants were exposed to 0.25 or 0.30 microliters per liter ozone for 2 hours. The quantity of ribulose-1,5-bisphosphate carboxylase decreased 80%, 68%, and 36% in leaves which did not exhibit ozone symptoms and to undetectable levels, 99% and 79% in symptomatic foliage for each cultivar, respectively.     

Synthesis of the Small Subunit of Ribulose-1,5-bisphosphate Carboxylase by Soluble Fraction Polyribosomes of Pea Leaves

The products of amino acid incorporation by pea (Pisum sativum L.) leaf soluble fraction polyribosomes in the wheat germ system were examined by two-dimensional electrophoresis and fluorography.     

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.     

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)     

Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Content and Degradation in Diploid,Tetraploid, and Hexaploid Wheat Species during Monocarpic Senescence

Wheat provides a unique genetic system in which variable sink size is available across the ploidies. We characterized monocarpic senescence in diploid, tetraploid, and hexaploid wheat species in flag leaf from anthesis up to full grain maturity at regular intervals. Triticum tauschii Acc. cv. EC-331751 showed the fastest rate of senescence among the species studied and the rate of loss per day was highest in terms of photosynthesis rate, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) content, and flag leaf N content coupled with a higher rate of gain in grain N content. Cultivars Kundan and HD 4530 maintained high flag leaf N content throughout grain filling as compared to the diploids and showed a slower rate of senescence. RuBPCO content was higher in the diploids as compared to Kundan and HD 4530 at anthesis. However, the rate of decline in RuBPCO content per day was also higher in the diploids. This degradation in RuBPCO was mediated by high endoproteolytic activities in the diploids which in turn supported its higher rate of N mobilization as compared to the tetraploid and hexaploid wheat. Acidic endopeptidases were responsible for the mobilization of flag leaf nitrogen in wheat across ploidy levels (r=–0.582, p<0.01).     

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.     

烟草核酮糖-1,5-二磷酸羧化酶/加氧酶四级结构的研究(英文)

本文提出三种证据证明烟草核酮糖-1,5-二磷酸羧化酶/加氧酶(Rubisco)的大亚基伸展在小亚基的外面,小亚基排列在大亚基中间的概念。证据是:1.固定化胰蛋白酶在一定条件下可水解RubisCO的大亚基但不水解小亚基,而天然胰蛋白酶水解大亚基,也水解小亚基。2.固定化抗小亚基IgG-Sepharose可与游离的小亚基相结合,但不能与全酶结合。3.低浓度尿素处理可使固定化的RubisCO-Sepharose上的小亚基解离下来,而大亚基仍结合在载体上,这说明RubisCO是通过定位在分子表面上的大亚基的ε-氨基与Sepharose共价偶联的。当RubisCO中的小亚基全部被解离后,大亚基之间的结合进一步增强,这时解离大亚基所需的尿素浓度要比小亚基存在时高。任何RubisCO的四级结构模型都应将小亚基置于大亚基中间受保护的位置,一部份小亚基可暴露于全酶分子表面。     

Variations in the Specific Activity of Ribulose-1,5-bisphosphate Carboxylase between Species Utilizing Differing Photosynthetic Pathways

The in vitro specific activity of ribulose-1,5-bisphosphate carboxylase (RuBPCase) (micromoles CO₂ fixed per minute per milligram enzyme) from a number of C₃ and C₄ species and one green alga were measured. RuBPCases from species which utilize the C₄ pathway have a specific activity ~2-fold higher than those from C₃ species. RuBPCase from Chlamydomonas reinhardtii has a specific activity similar to the C₄ enzyme. Higher specific activity forms of RuBPCase are associated with a decreased enzyme affinity for CO₂ (increased K_m[CO₂]). A small but significant difference in the specific activity of RuBPCase from two C₄ decarboxylation types was also observed. The relationship between enzymic properties and the presence or absence of a CO₂ concentrating mechanism is discussed.     

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.

     

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。     

Photosynthesis and Ribulose 1,5-Bisphosphate Carboxylase in Rice Leaves: Changes in Photosynthesis and Enzymes Involved in Carbon Assimilation from Leaf Development through Senescence

Changes in photosynthesis and the ribulose 1,5-bisphosphate (RuBP) carboxylase level were examined in the 12th leaf blades of rice (Oryza sativa L.) grown under different N levels. Photosynthesis was determined using an open infrared gas analysis system. The level of RuBP carboxylase was measured by rocket immunoelectrophoresis. These changes were followed with respect to changes in the activities of RuBP carboxylase, ribulose 5-phosphate kinase, NADP-glyceraldehyde 3-phosphate dehydrogenase, and 3-phosphoglyceric acid kinase.

RuBP carboxylase activity was highly correlated with the net rate of photosynthesis (r = 0.968). Although high correlations between the activities of other enzymes and photosynthesis were also found, the activity per leaf of RuBP carboxylase was much lower than those of other enzymes throughout the leaf life. The specific activity of RuBP carboxylase on a milligram of the enzyme protein basis remained fairly constant (1.16 ± 0.07 micromoles of CO₂ per minute per milligram at 25°C) throughout the experimental period.

Kinetic parameters related to CO₂ fixation were examined using the purified carboxylase. The K_m(CO₂) and V_max values were 12 micromolar and 1.45 micromoles of CO₂ per minute per milligram, respectively (pH 8.2 and 25°C). The in vitro specific activity calculated at the atomospheric CO₂ level from the parameters was comparable to the in situ true photosynthetic rate per milligram of the carboxylase throughout the leaf life.

The results indicated that the level of RuBP carboxylase protein can be a limiting factor in photosynthesis throughout the life span of the leaf.