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.     

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.     

Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Content, Assimilatory Charge, and Mesophyll Conductance in Leaves

The content of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (E_t; EC 4.1.1.39) measured in different-aged leaves of sunflower (Helianthus annuus) and other plants grown under different light intensities, varied from 2 to 75 μmol active sites m⁻². Mesophyll conductance (μ) was measured under 1.5% O₂, as well as postillumination CO₂ uptake (assimilatory charge, a gas-exchange measure of the ribulose-1,5-bisphosphate pool). The dependence of μ on E_t saturated at E_t = 30 μmol active sites m⁻² and μ = 11 mm s⁻¹ in high-light-grown leaves. In low-light-grown leaves the dependence tended toward saturation at similar E_t but reached a μ of only 6 to 8 mm s⁻¹. μ was proportional to the assimilatory charge, with the proportionality constant (specific carboxylation efficiency) between 0.04 and 0.075 μm⁻¹ s⁻¹. Our data show that the saturation of the relationship between E_t and μ is caused by three limiting components: (a) the physical diffusion resistance (a minor limitation), (b) less than full activation of Rubisco (related to Rubisco activase and the slower diffusibility of Rubisco at high protein concentrations in the stroma), and (c) chloroplast metabolites, especially 3-phosphoglyceric acid and free inorganic phosphate, which control the reaction kinetics of ribulose-1,5-bisphosphate carboxylation by competitive binding to active sites.Rubisco (EC 4.1.1.39) catalyzes the irreversible carboxylation of RuBP to form two PGA molecules (in this work the oxygenase reaction was not active since a low O₂ concentration was used). RuBP carboxylation is the major rate-determining reaction in photosynthetic CO₂ assimilation. All factors that influence the photosynthetic rate do so by influencing the activity of Rubisco and the concentration of its substrates, CO₂ and RuBP. E_t in leaves may be as high as 75 μmol m⁻², and for the extracted enzyme K_m(CO₂) = 9.4 μm (Makino et al., 1985a) and K_m(RuBP) = 30 to 40 μm (Yeoh et al., 1981). In leaves photosynthesizing under atmospheric conditions, the concentration of RuBP may increase to 10 to 15 mm (Badger et al., 1984; Sharkey et al., 1986), but the concentration of CO₂ is usually about 4 to 8 μm in leaf intercellular spaces, depending on stomatal conductance. This CO₂ concentration is well below the K_m(CO₂) of the enzyme, and it is the initial slope of the kinetic curve V_M/K_m(CO₂), termed carboxylation conductance, that becomes important.r_c limits the CO₂-fixation rate in series with the other resistances, r_g and r_md. The carboxylation rates are usually expressed in relation to C_i or C_w. C_c is usually about 20% to 30% lower than C_w because of concentration decrease generated by the carboxylation flux on r_md. Considering the above, the carboxylation conductance in intact leaves in vivo may be found as the initial slope of the A versus C_c graph at low C_c values. If C_c cannot be calculated because r_md is unknown, the closest approximation is a plot of A versus C_w or A versus C_i. The true parameters of the carboxylase can be found only from experiments carried out in nonphotorespiratory conditions (1%–2% O₂); otherwise the competing oxygenase reaction consumes a part of RuBP and partially inhibits carboxylase activity.Because of technical problems with the measurement of A versus C_w relationships, in many studies only the net photosynthetic rate under atmospheric conditions (21% O₂) was related to Rubisco activity or content. Nevertheless, good correlation has been found (Makino et al., 1983; Hudson et al., 1992; Jacob and Lawlor, 1992; Jiang and Rodermel, 1995; Nakano et al., 1997). These results indicated that the level of Rubisco protein could be a limiting factor in photosynthesis throughout the life span of the leaf under natural environmental conditions. On the other hand, when Rubisco levels in leaves exceeded 4 g m⁻² (60 μmol m⁻²), the in vivo Rubisco activity (measured as photosynthesis under pC_i = 20 to 30 Pa and 21% O₂) became curvilinearly correlated with E_t (Makino et al., 1994, 1997). When measurements were made over the whole life span of wheat leaves, the measured rates of photosynthesis were lower in young leaves, which had high protein content, than would have been expected from the amount and activity of Rubisco (Lawlor et al., 1989).During senescence the decrease in Rubisco activity was initially greater than the decrease in net photosynthesis (Hall et al., 1978). In a willow canopy, Rubisco-specific activity was higher when the apparent E_t (N content in leaves) was smaller (Vapaavuori and Vuorinen, 1989). A similar nonlinearity was found in our previous experiments (Eichelmann and Laisk, 1990), in which we obtained a saturating relationship when E_t exceeded 30 μmol m⁻². In the latter work the initial slope of the A versus C_w curves under nonphotorespiratory conditions (1.5% O₂) was assumed to represent the Rubisco activity in vivo and was compared with the E_t. We discovered that growth light had the strongest influence on the saturation of the relationship between μ and E_t. In the present work we present insight into this relationship, using not only plants grown under different light intensities but also leaves adapted to different light intensities.     

小麦叶片暗诱导衰老期间1,5-二磷酸核酮糖羧化酶/加氧酶的降解

研究了小麦(Triticum aestivum L. cv.Yangmai 158)叶片暗诱导衰老过程中1,5-二磷酸核酮糖羧化酶/加氧酶(Rubisco EC 4.1.1.39)的降解.发现在此期间Rubisco大亚基(LSU)发生裂解,产生50 kD的降解条带,同时在自然衰老过程中也检测到这一产物.初步实验结果表明LSU发生这步裂解时Rubisco全酶没有解离.另外,在粗酶液中当温度在30～35℃,pH 7.5时,这一步裂解反应能有效进行.     

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.     

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⁺.     

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.     

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.     

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.     

The Activity and Content of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Wheat Plants as Affected by Water Stress and Kartolin-4

The carboxylating activity and content of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO, EC 4.1.1.39), and other soluble proteins in young seedlings and mature leaves of Lutescens-758, a drought-sensitive cultivar of soft spring wheat Triticum aestivum L., were studied under the conditions of drought and subsequent rehydration. Seedlings and mature plants preliminarily treated with the cytokinin-like compound kartolin-4 were compared to untreated plants. Drought-induced decrease in RuBPCO activity should be attributed not only to proteolytic decomposition of the enzyme protein itself but also to a partial inhibition of its catalytic activity. The decrease in RuBPCO activity was larger than that in RuBPCO content. Water stress induced a marked decrease in the soluble protein content. Kartolin-4 increased the resistance to drought.     

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.     

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.     

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。     

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.     

Diversity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Large-Subunit Genes from Groundwater and Aquifer Microorganisms

To test our hypothesis that microbial autotrophic CO2 fixation plays an important role in subsurface systems of two large groundwater remediation projects, several anaerobic/microaerobic aquifer and groundwater samples were taken and used to investigate the distribution and phylogenetic diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large-subunit genes. Two primer sets were designed for amplifying partial-subunit genes of RubisCO forms I and II from the DNA, directly extracted from the samples. PCR products were used to construct five clone libraries with putative RubisCO form I sequences, and two libraries of DNA amplified by form II primers. Selected clones were screened for variation by restriction fragment length polymorphism analysis, and a total of 28 clone inserts were sequenced and further analyzed. The phylogenies constructed from amino acid sequences derived from the partial RubisCO large-subunit sequences showed a distinct pattern. Diverse sequences affiliated to the cluster of green-like type IA RubisCO sequences were found, representing various obligate and facultative chemolithoautotrophic Proteobacteria, whereas type II RubisCO sequences detected were most closely related to those of thiobacilli species. An isolate obtained from aquifer enrichment culture, which has been provisionally named Halothiobacillus sp. RA13 on the basis of its 16S rDNA sequence, was found to contain both types of RubisCO genes, i.e., forms I and II. Physiological and ecological considerations are discussed in the context of additional microbial data and physicochemical properties.