Purification and species distribution of rubisco activase

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase, a soluble chloroplast protein which promotes light-dependent rubisco activation, was partially purified from spinach chloroplasts by ion-exchange and gel-filtration fast protein liquid chromatography. The protein could also be isolated using rate zonal centrifugation in sucrose gradients followed by conventional ion-exchange on DEAE-cellulose. The active enzyme was composed of 44 and 41 kilodalton subunits. Antibodies to the activase polypeptides were produced in tumor-induced mouse ascites fluid and used as probes for activase on immunoblots of soluble proteins from a number of species. One or both of the activase polypeptides were recognized in all higher plant species examined including Arabidopsis thaliana, soybean, kidney bean, pea, tobacco, maize, oat, barley, celery, tomato, pigweed, purslane, dandelion, sorghum, and crabgrass. The polypeptides were not present in a mutant of Arabidopsis which is incapable of activating rubisco in vivo. The activase polypeptides were also detected in cell extracts of the green alga Chlamydomonas reinhardii. Activase activity, which had been demonstrated previously in wild-type Arabidopsis and in spinach, was measured in protoplast extracts of Nicotiana rustica. The results suggest that control of rubisco by activase may be an ubiquitous form of regulation in eucaryotic photosynthetic organisms.     

Adenosine triphosphate hydrolysis by purified rubisco activase

Activation of ribulose bisphosphate carboxylase/oxygenase (rubisco) in vivo is mediated by a specific protein, rubisco activase. In vitro, activation of rubisco by rubisco activase is dependent on ATP and is inhibited by ADP. Purified rubisco activase hydrolyzed ATP with a specific activity of 1.5 mumol min-1 mg-1 protein, releasing approximately stoichiometric amounts of ADP and Pi. Hydrolysis was highly specific for ATP-Mg and had a broad pH optimum, with maximum activity at pH 8.0-8.5. ATPase activity was inhibited by ADP but not by molybdate, vanadate, azide, nitrate, or fluoride. Addition of rubisco in either the inactive or activated form had no significant effect on ATPase activity. Incubation of rubisco activase in the absence of ATP resulted in loss of both ATPase and rubisco activation activities. Both activities were also heat labile, with 50% loss in activity after 5 min at 38 degrees C and complete inhibition following treatment at 43 degrees C. Both activities showed a sigmoidal response to ATP concentration, with half-maximal activity at 0.053 mM ATP. Rubisco activation activity was dependent on the concentrations of both ATP and ADP. The results suggest that ATPase activity is an intrinsic property of rubisco activase.     

Influence of salicylic acid on rubisco and rubisco activase in tobacco plant grown under sodium chloride in vitro

The present study was designed to evaluate the influence of salicylic acid (SA) on the growth of salt stress (sodium chloride) induced in tobacco plants. In addition, quantification of rubisco and rubisco activase contents of the plants was also determined in treatments with the control, 10⁻⁴ mM SA, 50 mM NaCl, 100 mM NaCl, 150 mM NaCl, SA + 50 mM NaCl, SA + 100 mM NaCl and SA + 150 mM NaCl, respectively after in vitro culture for 5 weeks. The growth of the tobacco plant decreased in 50 mM and 100 mM NaCl when not treated with SA. However, the growth was accelerated by SA, and the growth retardation caused by NaCl was improved by SA. The content of rubisco was improved by SA only in plants treated with 50 mM NaCl, and the activity of rubisco was increased by SA resulting in the decreased effect of NaCl, but only in 50 mM NaCl treated plants. The content of rubisco activase decreased due to NaCl, and SA did not improve the effect caused by NaCl. The activity of rubisco activase was increased by SA resulting in decreased activity caused by NaCl, but increased effect by SA was not recovered to the level of NaCl untreated plants. The activity of rubisco and rubisco activase, which decreased due to denaturing agents, did not demonstrate significant improvement when compared to the control.     

Species-dependent variation in the interaction of substrate-bound ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) and rubisco activase

Purified spinach (Spinacea oleracea L.) and barley (Hordeum vulgare L.) ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase supported 50 to 100% activation of substrate-bound Rubisco from spinach, barley, wheat (Triticum aestivum L.), soybean (Glycine max L.), pea (Pisum sativum L.), Arabidopsis thaliana, maize (Zea mays L.), and Chlamydomonas reinhardtii but supported only 10 to 35% activation of Rubisco from three Solanaceae species, tobacco (Nicotiana tabacum L.), petunia (Petunia hybrida L.), and tomato (Lycopersicon esculentum L.). Conversely, purified tobacco and petunia Rubisco activase catalyzed 75 to 100% activation of substrate-bound Rubisco from the three Solanacee species but only 10 to 25% activation of substrate-bound Rubisco from the other species. Thus, the interaction between substrate-bound Rubisco and Rubisco activase is species dependent. The species dependence observed is consistent with phylogenetic relationships previously derived from plant morphological characteristics and from nucleotide and amino acid sequence comparisons of the two Rubisco subunits. Species dependence in the Rubisco-Rubisco activase interaction and the absence of major anomalies in the deduced amino acid sequence of tobacco Rubisco activase compared to sequences in non-Solanaceae species suggest that Rubisco and Rubisco activase may have coevolved such that amino acid changes that have arisen by evolutionary divergence in one of these enzymes through spontaneous mutation or selection pressure have led to compensatory changes in the other enzyme.     

UV-B radiation affects chlorophyll and activation of rubisco by rubisco activase in<Emphasis Type="Italic">Canavalia ensiformis</Emphasis> L. leaves

We studied the influence of UV-B radiation on chlorophyll and rubisco activation by rubisco activase in the leaves of jackbean (Canavalia ensiformis). Chlorophyll content was decreased, indicating that the synthesis of those molecules may have been degraded or repressed after exposure. Rubisco content was significantly lower in radiated tissue compared with the untreated control; rubisco activity showed a similar pattern of change. Based on these data, we suggest that rubisco activity is associated with the level of rubisco protein, and that UV-B inhibits its activation and induction, as well as that of rubisco activase. Therefore, we propose that the inhibitory effect of rubisco by UV-B may be caused by rubisco activase.     

Influence of exogenous application of glutathione on rubisco and rubisco activase in heavy metal-stressed tobacco plant grown in vitro

The effect of glutathione on the influences of heavy metals affecting rubisco and rubisco activase was studied in tobacco plants grown in vitro where the shoot explants of the tobacco plant cultured on MS medium under aseptic conditions and two explants were placed in the control, 0.1 mM GSH, 1 mM GSH, 0.2 mM Cd, 0.2 mM Cu, 0.2 mM Zn, and a mixture of Cd and GSH, Cu and GSH, Zn and GSH, respectively. The effect of GSH on the growth of the tobacco plant was minimal, but the heavy metals clearly retarded its growth. GSH recovered the growth retarded by heavy metals, and the concentration of GSH required to recover the growth differed depending on the heavy metals. The content of chlorophyll in the plant increased through GSH and Zn, and decreased through Cd and Cu. The chlorophyll content which decreased due to Cd and Cu was recovered by GSH, and the content which increased due to Zn was decreased by 1 mM GSH. The content of rubisco decreased due to GSH and heavy metals, and the content which decreased due to heavy metals was recovered by GSH, and when GSH was treated with Zn, the increased rate was maximum compared to other heavy metals. The activity of rubisco was increased due to GSH and heavy metals, and the activity increased by Cd and Zn decreased through GSH. In the case of Cu, the activity of GSH increased even more. There was no effect of GSH on the influences of heavy metals on the content and activity of rubisco activase. The activity of rubisco decreased by thiourea among six denaturing agents, and increased by l-cysteine, and in most cases the activity level was recorded as high. The activity of rubisco activase all decreased as a result of six denaturing agents, and the effect caused by EDTA and guanidine-HCl was the greatest, while the effect caused by l-cysteine and urea was minimal.     

Two residues of rubisco activase involved in recognition of the Rubisco substrate

Rubisco activase is an AAA(+) protein, a superfamily with members that use a "Sensor 2" domain for substrate recognition. To determine whether the analogous domain of activase is involved in recognition of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39), two chimeric activases were constructed, interchanging a Sensor 2-containing region between activases from spinach and tobacco. Spinach chimeric activase was a poor activator of both spinach and tobacco Rubisco. In contrast, tobacco chimeric activase activated spinach Rubisco far better than tobacco Rubisco, similar to spinach activase. A point mutation, K311D, in the Sensor 2 domain of the tobacco chimeric activase abolished its ability to better activate spinach Rubisco. The opposite mutation, D311K, in wild type tobacco activase produced an enzyme that activated both spinach and tobacco Rubisco, whereas a second mutation, D311K/L314V, shifted the activation preference toward spinach Rubisco. The involvement of these two residues in substrate selectivity was confirmed by introducing the analogous single and double mutations in cotton activase. The ability of the two tobacco activase mutants to activate wild type and mutant Chlamydomonas Rubiscos was also examined. Tobacco D311K activase readily activated wild type and P89R but not D94K Rubisco, whereas the tobacco L314V activase only activated D94K Rubisco. The tobacco activase double mutant D311K/L314V activated wild type Chlamydomonas Rubisco better than either the P89R or D94K Rubisco mutants, mimicking activation by spinach activase. The results identified a substrate recognition region in activase in which two residues may directly interact with two residues in Rubisco.     

Decrease in carbamylation of rubisco by high CO2 concentration is due to decrease of rubisco activase in kidney bean

Decrease in rubisco activation at high CO₂ concentration was caused by decrease in carbamylation of rubisco (Rohet al., 1996). However, it is unclear whether decrease in carbamylation rate at high CO₂ concentration is due to decrease in activity itself or content of rubisco activase. To clarify this ambiguity, investigation was performed to determine effects of CO₂ concentration on rubisco activase with kidney bean (Phaseolus vulgaris L.) leaves grown at normal CO₂ (350 ppm) and high CO₂ (650 ppm) concentration. The analysis of Western blotting showed that the 50 and 14.5 kl) polypeptides were identified immunochemically as the large and small subunits of rubisco in the preparation, respectively. For the 14.5 kD small subunit, the degree of intensity at high CO₂ concentration was similar to that at normal CO₂ concentration. For the 50 kD large sububit, however, the intensity of a band at high CO, concentration was significantly higher than that at normal CO₂ concentration, indicating that only the large subunit is affected by high CO₂ concentration. The analysis of Western immunoblotting showed two major polypeptides at 46 and 42 kD which were identified as rubisco activase subunits. The intensities of two bands were shown to be higher at normal CO₂ than high CO₂ concentration. These data indicate that decrease of carbamylation resulting from increase of CO₂ concentration was caused by rubisco activase. Finally, by employing ATP hydrolysis assay and ELISA, we also observed a significant decrease in both activity and content of rubisco activase as CO₂ concentration was raised from normal to high CO₂ concentration. These results suggest that decrease in rubisco carbamylation at high CO₂ concentration is caused by activity itself and/or content of rubisco activase.     

Activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) by rubisco activase : effects of some sugar phosphates

The activation of purified ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) has been studied in the presence of sugar phosphates, and the effect of rubisco activase on this process determined. During an 11-minute time course at pH 7.7 and 11 micromolar CO₂, the activation of rubisco was strongly inhibited by ribulose-1,5-bisphosphate (4 millimolar), fructose-1,6-bisphosphate (1 millimolar) and ribose 5-phosphate (5 millimolar), but this inhibition was overcome by the addition of rubisco activase and activation then proceeded to a greater extent than spontaneous activation of rubisco. Glycerate 3-phosphate (20 millomolar) slowed the initial rate but not the extent of activation and rubisco activase had no effect on this. The activation of rubisco was shown to be affected by phosphoenolpyruvate (3 millimolar) but not by creatine phosphate (3 millimolar) or ATP (3 millimolar), and the creatine-phosphate/creatine phosphokinase system was used to generate the high ATP/ADP quotients required for rubisco activase to function. ATP was shown to be required for the rubisco activase-dependent rubisco activation in the presence of fructose-1,6-bisphosphate (1 millimolar). It is concluded that rubisco activase has a mixed specificity for some sugar phosphate-bound forms of rubisco, but has low or no activity with others. Some possible bases for these differences among sugar phosphates are discussed but remain to be established.     

Chloroplast protein synthesis elongation factor, EF-Tu, reduces thermal aggregation of rubisco activase

Chloroplast protein synthesis elongation factor, EF-Tu, has been implicated in heat tolerance in maize. The recombinant precursor of this protein, pre-EF-Tu, has been found to exhibit chaperone activity and protect heat-labile proteins, such as citrate synthase and malate dehydrogenase, from thermal aggregation. Chloroplast EF-Tu is highly conserved and it is possible that the chaperone activity of this protein is not species-specific. In this study, we investigated the effect of native wheat pre-EF-Tu on thermal aggregation of rubisco activase. Additionally, we investigated the effect of native and recombinant maize pre-EF-Tu on activase aggregation. Activase was chosen because it displays an exceptional sensitivity to thermal aggregation and constrains photosynthesis at high temperature. The native precursors of both wheat and maize EF-Tu displayed chaperone activity, as shown by the capacity of both proteins to reduce thermal aggregation of rubisco activase in vitro. Similarly, the recombinant maize pre-EF-Tu protected activase from thermal aggregation. This is the first report on chaperone activity of native pre-EF-Tu and the first evidence for thermal protection of a photosynthetic enzyme by this putative chaperone. The results are consistent with the hypothesis that chloroplast EF-Tu plays a functional role in heat tolerance by acting as a molecular chaperone.     

Immunoblot analysis of the expression of genes for barley rubisco activase inE. coli

Rubisco activity during photosynthesis is regulated by the rubisco activase, which facilitates the dissociation of RuBP and other inhibitory sugar phosphates from the active site of rubisco in an ATP-dependent reaction. In this paper, barleyRca genes (RcaA1,RcaA2 andRcaB) were expressed inE. coli and the activity of rubisco activase expressed was assayed biochemically by chromatography. Then the protein was identified electrophoretically by SDS-PAGE and detected immunologically by Western blot analysis using polyclonal antibodies raised against the kidney bean rubisco activase as probe. The band pattern of purified proteins on the polyacrylamide gel showed two polypeptides of 46 kD and 42 kD. Anti-rubisco activase antibodies reacted specifically with both polypeptides of 46 kD and 42 kD present in the crude extracts ofE. coli transformants. Therefore, it was found that the genes of barley rubisco activase was successfully expressed inE. coli as active forms of 46 kD and 42 kD.     

Relationship between the heat tolerance of photosynthesis and the thermal stability of rubisco activase in plants from contrasting thermal environments

Inhibition of net photosynthesis (Pn) by moderate heat stress has been attributed to an inability of Rubisco activase to maintain Rubisco in an active form. To examine this proposal, the temperature response of Pn, Rubisco activation, chlorophyll fluorescence, and the activities of Rubisco and Rubisco activase were examined in species from contrasting environments. The temperature optimum of Rubisco activation was 10 degrees C higher in the creosote bush (Larrea tridentata) compared with the Antarctic hairgrass (Deschampsia antarctica), resembling the temperature response of Pn. Pn increased markedly with increasing internal CO(2) concentration in Antarctic hairgrass and creosote bush plants subjected to moderate heat stress even under nonphotorespiratory conditions. Nonphotochemical quenching of chlorophyll fluorescence, the effective quantum yield of photochemical energy conversion (DeltaF/F(m)') and the maximum yield of PSII (F(v)/F(m)) were more sensitive to temperature in Antarctic hairgrass and two other species endemic to cold regions (i.e. Lysipomia pumila and spinach [Spinacea oleracea]) compared with creosote bush and three species (i.e. jojoba [Simmondsia chinensis], tobacco [Nicotiana tabacum], and cotton [Gossypium hirsutum]) from warm regions. The temperature response of activity and the rate of catalytic inactivation of Rubisco from creosote bush and Antarctic hairgrass were similar, whereas the optimum for ATP hydrolysis and Rubisco activation by recombinant creosote bush, cotton, and tobacco activase was 8 degrees C to 10 degrees C higher than for Antarctic hairgrass and spinach activase. These results support a role for activase in limiting photosynthesis at high temperature.     

Phosphorylation pattern of Rubisco activase in Arabidopsis leaves

Rubisco activase (RCA) is an ancillary photosynthetic protein essential for Rubisco activity. Some data suggest that post‐translational modifications (such as reduction of disulphide bridges) are involved in the regulation of RCA activity. However, despite the key role of protein phosphorylation in general metabolic regulation, RCA phosphorylation has not been well characterised. We took advantage of phosphoproteomics and gas exchange analyses with instant sampling adapted to Arabidopsis rosettes to examine the occurrence and variations of phosphopeptides associated with RCA in different photosynthetic contexts (CO₂ mole fraction, light and dark). We detected two phosphopeptides from RCA corresponding to residues Thr 78 and Ser 172, and show that the former is considerably more phosphorylated in the dark than in the light, while the latter show no light/dark pattern. The CO₂ mole fraction did not influence phosphorylation of either residue. Phosphorylation thus appears to be a potential mechanism associated with RCA dark inactivation, when Rubisco‐catalysed carboxylation is arrested. Since Thr 78 and Ser 172 are located in the N and Walker domains of the protein, respectively, the involvement of phosphorylation in protein–protein interaction and catalysis is likely.     

Overexpression of the rubisco activase gene improves growth and low temperature and weak light tolerance in Cucumis sativus

Rubisco activase (RCA) is an important enzyme that can catalyze the carboxylation and oxygenation activities of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco), which is involved in the photosynthetic carbon reduction cycle. Here, we studied the effects of changes in RCA activity on photosynthesis, growth and development, as well as the low temperature and weak light tolerance of RCA overexpressing transgenic cucumber (Cucumis sativus) plants. CsRCA overexpression increased the plant height, leaf area and dry matter, and decreased the root/top ratio in transgenic cucumber plants compared with the wild‐type (WT) plants. Low temperature and low light stress led to decreases in the CsRCA expression and protein levels, the photosynthetic rate (Pn) and the stomatal conductance (Gs), but an increase in the intercellular CO₂ (Ci) concentration in cucumber leaves. The actual photochemical efficiency and maximal photochemical efficiency of photosystem II in cucumber seedlings also declined, but the initial fluorescence increased during low temperature and weak light stress. Transgenic plants showed a lower decrease in the CsRCA expression level and actual and maximal photochemical efficiencies, as well as increases in the Ci and initial fluorescence relative to the WT plants. Low temperature and low light stress resulted in a significant increase in the malondialdehyde (MDA) content; however, this increase was reduced in transgenic plants compared with that in WT plants. Thus, the overexpression of CsRCA may promote the growth and low temperature and low light tolerance of cucumber plants in solar greenhouses.     

Alteration of the adenine nucleotide response and increased Rubisco activation activity of Arabidopsis rubisco activase by site-directed mutagenesis

Arabidopsis Rubisco was activated in vitro at rates 2- to 3-fold greater by recombinant Arabidopsis 43-kD Rubisco activase with the amino acid replacements Q111E and Q111D in a phosphate-binding loop, G-G-K-G-Q-G-K-S. However, these two mutant enzymes had only slightly greater rates of ATP hydrolysis. Activities of the Q111D enzyme were much less sensitive and those of Q111E were somewhat less sensitive to inhibition by ADP. Both mutant enzymes exhibited higher Rubisco activation activities over the physiological range of ADP to ATP ratios. Enzymes with non-polar, polar, and basic residues substituted at position Gln-111 exhibited rates of Rubisco activation less than the wild-type enzyme. Estimates of the relative affinity of the wild type and the Q111D, Q111E, and Q111S enzymes for adenosine nucleotides by a variety of methods revealed that the nucleotide affinities were the most diminished in the Q111D enzyme. The temperature stability of the Q111D and Q111E enzymes did not differ markedly from that of the 43-kD recombinant wild-type enzyme, which is somewhat thermolabile. The Q111D and Q111E enzymes, expressed in planta, may provide a means to better define the role of the ADP to ATP ratio in the regulation of Rubisco activation and photosynthesis rate.