RubisCO的研究进展

1,5-二磷酸核酮糖羧化酶/加氧酶(RubisCO)是调节光合和光呼吸,决定净光合作用的一个关键酶;也是植物可溶性蛋白质中含量最高的蛋白质.该酶广泛存在于植物及一些微生物体内.综述了近年来有关RubisCO的一些研究进展. 包括RubisCO的基本性质、结构与功能、酶基因工程、酶活性调节及其活化酶等.     

Photosynthetic Acclimation to Elevated CO2 Occurs in Transformed Tobacco with Decreased Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Content

Inhibition of net carbon assimilation rates during growth at elevated CO2 was studied in transgenic tobacco (Nicotiana tabacum L.) plants containing zero to two copies of antisense DNA sequences to the small subunit polypeptide (rbcS) gene of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). High- and low-Rubisco tobacco plants were obtained from the selfed progeny of the original line 3 transformant (S.R. Rodermel, M.S. Abbott, L. Bogorad [1988] Cell 55: 673-681). Assimilation rates of high- and low-Rubisco tobacco plants increased 22 and 71%, respectively, when transferred from 35- to 70-Pa CO2 chamber air at 900 [mu]mol m-2 s-1 photon flux density. However, CO2-dependent increases of net carbon assimilation rates of high- and low-Rubisco plants virtually disappeared after 9 d of growth in elevated CO2 chamber air. Total above-ground dry matter production of high- and low-Rubisco plants was 28 and 53% greater, respectively, after 9 d of growth at 70 Pa compared with 35 Pa CO2. Most of this dry weight gain was due to increased specific leaf weight. Rubisco activity, Rubisco protein, and total chlorophyll were lower in both high- and low-Rubisco plants grown in enriched compared with ambient CO2 chamber air. Soluble leaf protein also decreased in response to CO2 enrichment in high- but not in low-Rubisco tobacco plants. Decreased Rubisco activities in CO2-adapted high- and low-Rubisco plants were not attributable to changes in activation state of the enzyme. Carbonic anhydrase activities and subunit levels measured with specific antibodies were similar in high- and low-Rubisco tobacco plants and were unchanged by CO2 enrichment. Collectively, these findings suggested that photosynthetic acclimation to enriched CO2 occurred in tobacco plants either with or without transgenically decreased Rubisco levels and also indicated that the down-regulation of Rubisco in CO2-adapted tobacco plants was related to decreased specific activity of this enzyme.     

Exchange Properties of the Activator CO(2) of Spinach Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

The exchange properties of the activator CO₂ of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase were characterized both in vitro with the purified enzyme, and in situ within isolated chloroplasts. Carboxyarabinitol-1,5-bisphosphate, a proposed reaction intermediate analog for the carboxylase activity of the enzyme, was used to trap the activator CO₂ on the enzyme both in vitro and in situ. Modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in intact chloroplasts during a light/dark cycle was associated with a similar modulation in carboxyarabinitol-1,5-bisphosphate-trapped CO₂. The exchange kinetics of the activator CO₂ were monitored by activation of the enzyme to steady state in the presence of ¹²CO₂, followed by addition of ¹⁴CO₂ and determination of the amount of labeled CO₂ trapped on the enzyme by carboxyarabinitol-1,5-bisphosphate. Rate constants (K_obs) for exchange with both the purified enzyme (0.45 min⁻¹) and in illuminated chloroplasts (0.18 min⁻¹) were comparable to the observed rate constants for enzyme activation under the two conditions. A similar exchange of the activator CO₂ was not observed in chloroplasts in the dark. Kinetic analysis of the exchange properties of the purified enzyme were consistent with an equilibrium between active and inactive forms of the enzyme during steady state activation.     

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.     

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.     

Effects of O2 and CO2 on Nonsteady-State Photosynthesis (Further Evidence for Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Limitation)

The effects of CO2 and O2 on nonsteady-state photosynthesis following an increase in photosynthetic photon flux density (PPFD) were examined in Spinacia oleracea to investigate the hypotheses that (a) a slow exponential phase (the ribulose-1,5-bisphosphate carboxylase/oxygenase [Rubisco] phase) of nonsteady-state photosynthesis is primarily limited by Rubisco activity and (b) Rubisco activation involves two sequential, light-dependent processes as described in a previous study (I.E. Woodrow, K.A. Mott [1992] Plant Physiol 99: 298-303). Photosynthesis was found to be sensitive to O2 during the Rubisco phase in the approach of photosynthesis to steady state. Analyses of this sensitivity to O2 showed that the control coefficient for Rubisco was approximately equal to 1 during this phase, suggesting that Rubisco was the primary limitation to photosynthesis. O2 had almost no effect on the kinetics (described using a relaxation time, [tau] of the Rubisco phase for leaves starting in darkness or for leaves starting in low PPFD, but [tau] was substantially higher in the former case. CO2 was found to affect both the rate of photosynthesis and the magnitude of [tau] for the Rubisco phase. The [tau] value for the Rubisco phase was found to be negatively correlated with intercellular CO2 concentration (ci), and leaves starting in darkness had higher values of [tau] at any ci than leaves starting in low PPFD. The effects of CO2 and O2 on the Rubisco phase are consistent with the existence of two sequential, light-dependent processes in the activation of Rubisco if neither process is sensitive to O2 and only the second process is sensitive to CO2. The implications of the data for the mechanism of Rubisco activation and for the effects of stomatal conductance on nonsteady-state photosynthesis are discussed.     

Inhibition and Acclimation of Photosynthesis to Heat Stress Is Closely Correlated with Activation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

Increasing the leaf temperature of intact cotton (Gossypium hirsutum L.) and wheat (Triticum aestivum L.) plants caused a progressive decline in the light-saturated CO₂-exchange rate (CER). CER was more sensitive to increased leaf temperature in wheat than in cotton, and both species demonstrated photosynthetic acclimation when leaf temperature was increased gradually. Inhibition of CER was not a consequence of stomatal closure, as indicated by a positive relationship between leaf temperature and transpiration. The activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which is regulated by Rubisco activase, was closely correlated with temperature-induced changes in CER. Nonphotochemical chlorophyll fluorescence quenching increased with leaf temperature in a manner consistent with inhibited CER and Rubisco activation. Both nonphotochemical fluorescence quenching and Rubisco activation were more sensitive to heat stress than the maximum quantum yield of photochemistry of photosystem II. Heat stress led to decreased 3-phosphoglyceric acid content and increased ribulose-1,5-bisphosphate content, which is indicative of inhibited metabolite flow through Rubisco. We conclude that heat stress inhibited CER primarily by decreasing the activation state of Rubisco via inhibition of Rubisco activase. Although Rubisco activation was more closely correlated with CER than the maximum quantum yield of photochemistry of photosystem II, both processes could be acclimated to heat stress by gradually increasing the leaf temperature.     

The Two Forms of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activase Differ in Sensitivity to Elevated Temperature

Ribulose-1,5-bisphosphate carboxylase/oxygenase activase often consists of two polypeptides that arise from alternative splicing of pre-mRNA. In this study recombinant versions of the spinach (Spinacea oleracea L.) 45- and 41-kD forms of activase were analyzed for their response to temperature. The temperature optimum for ATP hydrolysis by the 45-kD form was 45[deg]C, approximately 13[deg]C higher than the 41-kD form. When the two forms were mixed, the temperature response of the hybrid enzyme was similar to the 45-kD form. In the absence of adenine nucleotide, preincubation of either activase form at temperatures above 25[deg}C inactivated ATPase activity. Adenosine 5[prime]-([gamma]-thio)triphosphate, but not ADP, significantly enhanced the thermostability of the 45-kD form but was much less effective for the 41-kD form. Intrinsic fluorescence showed that the adenosine 5[prime]-([gamma]-thio)triphosphate-induced subunit aggregation was lost at a much lower temperature for the 41-kD than for the 45-kD form. However, the two activase forms were equally susceptible to limited proteolysis after heat treatment. The results indicate that (a) the 45-kD form is more thermostable than, and confers increased thermal stability to, the 41-kD form, and (b) a loss of subunit interactions, rather than enzyme denaturation, appears to be the initial cause of temperature inactivation of activase.     

Activities of Phosphoenolpyruvate Carboxylase and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Leaves and Fruit Pericarp Tissue of Different Coffee (Coffea sp.) Genotypes

In order to study photosynthetic characteristics, phosphoenolpyruvate carboxylase (PEPC) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) activities as well as soluble protein and chlorophyll contents were determined in leaf and fruit pericarp samples from diverse coffee genotypes (Coffea arabica cv. Colombia, Caturra, Caturra Erecta, San Pacho, Tipica, C. stenophylla, C. eugenioides, C. congensis, C. canephora, C. canephora cv. Arabusta, C. arabica cv. Caturra×C. canephora and Hibrido de Timor. We found a slightly higher PEPC activity in fruit pericarp than in leaves, while RuBPCO activity was much lower in pericarp than leaf tissue. Partial purification of PEPC and RuBPCO was carried out from leaves of C. arabica cv. Caturra and Michaelis-Menten kinetics for RuBPCO (K_m CO₂ = 5.34 µM), (K_m RuBP = 9.09 µM) and PEPC (K_m PEP = 19.5 µM) were determined. Leaf tissues of Colombia, Hibrido de Timor, and Caturra consistently showed higher content of protein [55.4–64.4 g kg^–1 (f.m.)] than San Pacho, C. stenophylla, Tipica, Caturra Erecta, and Caturra×C. canephora [25.6–36.9 g kg^–1 (f.m.)] and C. canephora cv. Arabusta, Borbon, C. congensis, C. eugenioides, and C. canephora [16.1–21.1 g kg^–1 (f.m.)].     

Acclimation of Two Tomato Species to High Atmospheric CO(2): I. Sugar and Starch Concentrations

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. Tomato plants grown at 900 microliters per liter contained more starch and more sugars than the control. However, we found no significant accumulation of starch and sugars in the young leaves of L. esculentum exposed to high CO₂. Carbon exchange rates were significantly higher in CO₂-enriched plants for the first few weeks of treatment but thereafter decreased as tomato plants acclimated to high atmospheric CO₂. This indicates that the long-term decline of photosynthetic efficiency of leaf 5 cannot be attributed to an accumulation of sugar and/or starch. The average concentration of starch in leaves 5 and 9 was always higher in L. esculentum than in L. chmielewskii (151.7% higher). A higher proportion of photosynthates was directed into starch for L. esculentum than for L. chmielewskii. However, these characteristics did not improve the long-term photosynthetic efficiency of L. chmielewskii grown at high CO₂ when compared with L. esculentum. The chloroplasts of tomato plants exposed to the higher CO₂ concentration exhibited a marked accumulation of starch. The results reported here suggest that starch and/or sugar accumulation under high CO₂ cannot entirely explain the loss of photosynthetic efficiency of high CO₂-grown plants.     

草酸对氧化胁迫-水稻叶片中核酮糖-1，5-二磷酸羧化酶/加氧酶降解的保护作用

以杂交稻(汕优63)为试验材料,在木村B营养液中培养至三叶期,用草酸5mmol/L预处理水稻2d,再处以氧化胁迫(用0．1mmol／L浓度的活性氧诱发剂甲基紫精处理)。结果表明MV诱发的氧化胁迫下,Rubisco及其它可溶性蛋白快速降解。草酸预处理可明显缓解Rubisco及其它可溶性蛋白的降解,降解速率分别降低1／3和1／2左右。植株经草酸处理后其叶片中几种抗氧化酶如AsA-POD、SOD、CAT活性大大提高,这可能是草酸预处理可缓解氧化胁迫下Rubisco和其它可溶性蛋白降解的重要原因。既然草酸能有效地诱导植物的抗氧化防卫反应,它可能作为一种诱抗剂来提高植物的抗逆性。     

Phylogenetic Diversity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Large-Subunit Genes from Deep-Sea Microorganisms

The phylogenetic diversity of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO, E.C. 4.1.1.39) large-subunit genes of deep-sea microorganisms was analyzed. Bulk genomic DNA was isolated from seven samples, including samples from the Mid-Atlantic Ridge and various deep-sea habitats around Japan. The kinds of samples were hydrothermal vent water and chimney fragment; reducing sediments from a bathyal seep, a hadal seep, and a presumed seep; and symbiont-bearing tissues of the vent mussel, Bathymodiolus sp., and the seep vestimentiferan tubeworm, Lamellibrachia sp. The RuBisCO genes that encode both form I and form II large subunits (cbbL and cbbM) were amplified by PCR from the seven deep-sea sample DNA populations, cloned, and sequenced. From each sample, 50 cbbL clones and 50 cbbM clones, if amplified, were recovered and sequenced to group them into operational taxonomic units (OTUs). A total of 29 OTUs were recorded from the 300 total cbbL clones, and a total of 24 OTUs were recorded from the 250 total cbbM clones. All the current OTUs have the characteristic RuBisCO amino acid motif sequences that exist in other RuBisCOs. The recorded OTUs were related to different RuBisCO groups of proteobacteria, cyanobacteria, and eukarya. The diversity of the RuBisCO genes may be correlated with certain characteristics of the microbial habitats. The RuBisCO sequences from the symbiont-bearing tissues showed a phylogenetic relationship with those from the ambient bacteria. Also, the RuBisCO sequences of known species of thiobacilli and those from widely distributed marine habitats were closely related to each other. This suggests that the Thiobacillus-related RuBisCO may be distributed globally and contribute to the primary production in the deep sea.     

Low Activation State of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Carboxysome-Defective Synechococcus Mutants

The high-CO2-requiring mutant of Synechococcus sp. PCC 7942, EK6, was obtained after extension of the C terminus of the small subunit of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco). The carboxysomes in EK6 were much larger than in the wild type, but the cellular distribution of the large and small sub-units of Rubisco was not affected. The kinetic parameters of in vitro-activated Rubisco were similar in EK6 and in the wild type. On the other hand, Rubisco appeared to be in a low state of activation in situ in EK6 cells pretreated with an air level of CO2. This was deduced from the appearance of a lag phase when carboxylation was followed with time in cells permeabilized by detergent and subsequently supplied with saturating CO2 and RuBP. Pretreatment of the cells with high CO2 virtually abolished the lag. After low-CO2 treatment, the internal RuBP pool was much higher in mutant cells than in the wild-type cells; pretreatment with high CO2 reduced the pool in mutant cells. We suggest that the high-CO2-requiring phenotype in mutants that possess aberrant carboxysomes arises from the inactivated state of Rubisco when the cells are exposed to low CO2.     

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.     

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⁺.     

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.