Alterations in Photosystem II electron transport as revealed by thermoluminescence of Cu-poisoned chloroplasts

The Rubisco activase amino acid sequences of spinach and tobacco are 79% identical, yet the tobacco protein does not facilitate the activation of the uncarbamylated, ribulose bisphosphate bound form of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) and vice versa. In contrast, combinations of the spinach Rubisco activase with Rubisco from non-Solanaceae species and combinations of tobacco Rubisco activase with Rubisco from other Solanaceae species are almost as effective as the analogous combination. To examine the basis of the preference of an activase protein for either Solanaceae or non-Solanaceae Rubisco, several recombinant chimeric proteins were obtained by combining regions from the cDNAs of spinach and tobacco activase and expression in Escherichia coli. The chimeric proteins were analyzed for ATP hydrolysis and ability to activate spinach and tobacco Rubisco. Comparisons of Rubisco preference with composition of the various activase chimeras indicate that the major determinants of Rubisco preference seem to be localized in the carboxyl-terminal region.     

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.     

Activation of interspecies-hybrid Rubisco enzymes to assess different models for the Rubisco–Rubisco activase interaction

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is prone to inactivation from non-productive binding of sugar-phosphates. Reactivation of Rubisco requires conformational remodeling by a specific chaperone, Rubisco activase. Rubisco activase from tobacco and other plants in the family Solanaceae is an inefficient activator of Rubisco from non-Solanaceae plants and from the green alga Chlamydomonas reinhardtii. To determine if the Rubisco small subunit plays a role in the interaction with Rubisco activase, a hybrid Rubisco (SSNT) composed of tobacco small subunits and Chlamydomonas large subunits was constructed. The SSNT hybrid, like other hybrid Rubiscos containing plant small subunits, supported photoautotrophic growth in Chlamydomonas, but growth in air was much slower than for cells containing wild-type Rubisco. The kinetic properties of the SSNT hybrid Rubisco were similar to the wild-type enzyme, indicating that the poor growth in air was probably caused by disruption of pyrenoid formation and the consequent impairment of the CO₂concentrating mechanism. Recombinant Rubisco activase from Arabidopsis activated the SSNT hybrid Rubisco and hybrid Rubiscos containing spinach and Arabidopsis small subunits at rates similar to the rates with wild-type Rubisco. However, none of the hybrid Rubiscos was activated by tobacco Rubisco activase. That replacement of Chlamydomonas small subunits with plant small subunits does not affect the species-specific interaction between Rubisco and Rubisco activase suggests that the association is not dominated by the small subunits that surround the Rubisco central solvent channel. Therefore, the geometry of a side-on binding mode is more consistent with the data than a top-on or ring-stacking binding mode.     

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.     

Subunit interactions of Rubisco activase: polyethylene glycol promotes self-association, stimulates ATPase and activation activities, and enhances interactions with Rubisco.

The effect of polyethylene glycol (PEG) on the enzymatic and physical properties of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase was examined. In the presence of PEG, Rubisco activase exhibited higher ATPase and Rubisco activating activities, concomitant with increased apparent affinity for ATP and Rubisco. Specific ATPase activity, which was dependent on Rubisco activase concentration, was also higher in the presence of Ficoll, polyvinylpyrrolidone, and bovine serum albumin. The ability of Rubisco activase to facilitate dissociation of the tight-binding inhibitor 2-carboxyarabinitol 1-phosphate from carbamylated Rubisco was also enhanced in the presence of PEG. Mixing experiments with Rubisco activase from two different sources showed that tobacco Rubisco activase, which exhibited little activation of spinach Rubisco by itself, was inhibitory when included with spinach Rubisco activase. Polyethylene glycol improved the ability of tobacco and a mixture of tobacco plus spinach Rubisco activase to activate spinach Rubisco. Estimates based on rate zonal sedimentation and gel-filtration chromatography indicated that the apparent molecular mass of Rubisco activase was two- to fourfold higher in the presence of PEG. The increase in apparent molecular mass was consistent with the propensity of solvent-excluding reagents like PEG to promote self-association of proteins. Likewise, the change in enzymatic properties of Rubisco activase in the presence of PEG and the dependence of specific activity on protein concentration resembled changes that often accompany self-association. For Rubisco activase, high concentrations of protein in the chloroplast stroma would provide an environment conducive to self-association and cause expression of properties that would enhance its ability to function efficiently in vivo.     

The Effects of Chilling in the Light on Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activation in Tomato (Lycopersicon esculentum Mill.)

Photosynthesis rate, ribulsoe-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation state, and ribulose bisphosphate concentration were reduced after exposing tomato (Lycopersicon esculentum Mill.) plants to light at 4[deg]C for 6 h. Analysis of lysed and reconsituted chloroplasts showed that activity of the thylakoid membrane was inhibited and that Rubisco, Rubisco activase, and other soluble factors were not affected. Leaf photosynthesis rates and the ability of chilled thylakoid membranes to promote Rubisco activation recovered after 24 h at 25[deg]C. Thylakoid membranes from control tomato plants were as effective as spinach thylakoids in activating spinach Rubisco in the presence of spinach Rubisco activase. This observation is in sharp contrast to the poor ability of spinach Rubisco activase to activate tomato Rubisco (Z.-Y. Wang, G.W. Snyder, B.D. Esau, A.R. Portis, and W.L. Ogren [1992] Plant Physiol 100: 1858-1862). The ability of thylakoids from chilled tomato plants to activate Rubisco in the assay system was greatly inhibited compared to control plants. These experiments indicate that chilling tomato plants at 4[deg]C interferes with photosynthetic carbon metabolism at two sites, thioredoxin/ferredoxin reduction (G.F. Sassenrath, D.R. Ort, and A.R. Portis, Jr. [1990] Arch Biochem Biophys 282: 302-308), which limits bisphosphatase activity, and Rubisco activase, which reduces Rubisco activation state.     

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.     

烟草Rubisco活化酶的纯化及其特性

利用35％饱和硫酸铵分部、DEAE－Sephacel和FPIC－MonoQ柱层析等步骤从烟草叶片中纯化了Rubisco活化酶,并制备了其专一性抗体。此法不仅快速,而且比活力高。以往认为菠菜和拟南芥Rubisco活化酶由两种亚基组成。通过快速制备的粗提液分析．发现烟草Rubisco活化酶由一种42kD的亚基组成。即使在有多种蛋白酶抑制剂存在的情况下,此亚基仍很易降解为39kD的亚基。ATP不仅对酶的活性所必需,而且也有利于维持酶的稳定性。该酶的热稳定性远比Rubisco差。     

Identification of critical arginine residues in the functioning of Rubisco activase

Rubisco activase is a member of the AAA(+) family in which arginines located in the Box VII and Sensor 2 domains are a recurrent feature and typically contribute to ATP-binding/hydrolysis or an inter-subunit interface. Replacement of R241 or R244 in Box VII or R294 or R296 in Sensor 2 with alanine in tobacco activase did not greatly alter the binding of ATP or ADP. However, ATP hydrolysis was minimal (R241A and R244A) or greatly diminished (R296A) and none of these mutants were able to activate Rubisco. R241, R244 and R296 were also required for nucleotide-dependent conformational changes detected by intrinsic fluorescence and limited proteolysis. ATP-induced oligomerization, monitored by gel filtration, was not observed with the wild type and mutant tobacco activases in contrast to spinach activase and a R239A mutant (corresponding to R244A in tobacco). Thus, there is not a strict correlation of oligomerization with ATP hydrolysis and intrinsic fluorescence.     

Reductions of Rubisco activase by antisense RNA in the C4 plant Flaveria bidentis reduces Rubisco carbamylation and leaf photosynthesis

To function, the catalytic sites of Rubisco (EC 4.1.1.39) need to be activated by the reversible carbamylation of a lysine residue within the sites followed by rapid binding of magnesium. The activation of Rubisco in vivo requires the presence of the regulatory protein Rubisco activase. This enzyme is thought to aid the release of sugar phosphate inhibitors from Rubisco's catalytic sites, thereby influencing carbamylation. In C3 species, Rubisco operates in a low CO2 environment, which is suboptimal for both catalysis and carbamylation. In C4 plants, Rubisco is located in the bundle sheath cells and operates in a high CO2 atmosphere close to saturation. To explore the role of Rubisco activase in C4 photosynthesis, activase levels were reduced in Flaveria bidentis, a C4 dicot, by transformation with an antisense gene directed against the mRNA for Rubisco activase. Four primary transformants with very low activase levels were recovered. These plants and several of their segregating T1 progeny required high CO2 (>1 kPa) for growth. They had very low CO2 assimilation rates at high light and ambient CO2, and only 10% to 15% of Rubisco sites were carbamylated at both ambient and very high CO2. The amount of Rubisco was similar to that of wild-type plants. Experiments with the T1 progeny of these four primary transformants showed that CO2 assimilation rate and Rubisco carbamylation were severely reduced in plants with less than 30% of wild-type levels of activase. We conclude that activase activity is essential for the operation of the C4 photosynthetic pathway.     

Suppressor mutations in the chloroplast-encoded large subunit improve the thermal stability of wild-type ribulose-1,5-bisphosphate carboxylase/oxygenase

A temperature-conditional, photosynthesis-deficient mutant of the green alga Chlamydomonas reinhardtii, previously recovered by genetic screening, results from a leucine 290 to phenylalanine (L290F) substitution in the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC ). Rubisco purified from mutant cells grown at 25 degrees C has a reduction in CO(2)/O(2) specificity and is inactivated at lower temperatures than those that inactivate the wild-type enzyme. Second-site alanine 222 to threonine (A222T) or valine 262 to leucine (V262L) substitutions were previously isolated via genetic selection for photosynthetic ability at the 35 degrees C restrictive temperature. These intragenic suppressors improve the CO(2)/O(2) specificity and thermal stability of L290F Rubisco in vivo and in vitro. In the present study, directed mutagenesis and chloroplast transformation were used to create the A222T and V262L substitutions in an otherwise wild-type enzyme. Although neither substitution improves the CO(2)/O(2) specificity above the wild-type value, both improve the thermal stability of wild-type Rubisco in vitro. Based on the x-ray crystal structure of spinach Rubisco, large subunit residues 222, 262, and 290 are far from the active site. They surround a loop of residues in the nuclear-encoded small subunit. Interactions at this subunit interface may substantially contribute to the thermal stability of the Rubisco holoenzyme.     

Effect of Nd3+ ion on carboxylation activity of ribulose-1,5-bisphosphate carboxylase/oxygenase of spinach

Neodymium (Nd), as a member of rare earth elements, proved to enhance the photosynthesis rate and organic substance accumulation of spinach through the increase in carboxylation activity of Rubisco. Although the oxygenase activity of spinach Rubisco was slightly changed with the Nd(3+) treatment, the specific factor of Rubisco was greatly increased. It was partially due to the promotion of Rubisco activase (R-A) activity but mainly to the formation of Rubisco-Rubisco activase super-complex, a heavier molecular mass protein (about 1200kD) comprising both Rubisco and Rubisco activase. This super-complex was found during the extraction procedure of Rubisco by the gel electrophoresis and Western-blot studies. The formation of Rubisco-R-A super-complex suggested that the secondary structure of the protein purified from the Nd(3+)-treated spinach was different from that of the control. Extended X-ray absorption fine structure study of the 'Rubisco' purified from the Nd(3+)-treated spinach revealed that Nd was bound with four oxygen atoms and two sulfur atoms of amino acid residues at the Nd-O and Nd-S bond lengths of 2.46 and 2.89A, respectively.     

The activity of Rubisco��s molecular chaperone, Rubisco activase, in leaf extracts

Rubisco frequently undergoes unproductive interactions with its sugar-phosphate substrate that stabilize active sites in an inactive conformation. Restoring catalytic competence to these sites requires the "molecular chiropractic" activity of Rubisco activase (activase). To make the study of activase more routine and physiologically relevant, an assay was devised for measuring activase activity in leaf extracts based on the ATP-dependent activation of inactive Rubisco. Control experiments with an Arabidopsis activase-deficient mutant confirmed that the rate of Rubisco activation was dependent on the concentration of activase in the extracts. Activase catalyzed Rubisco activation at rates equivalent to 9-14% catalytic sites per min in desalted extracts of Arabidopsis, camelina, tobacco, cotton, and wheat. Faster rates were observed in a transgenic line of Arabidopsis that expresses only the β-isoform of activase, whereas no activity was detected in a line that expresses only the α-isoform. Activase activity was also low or undetectable in rice, maize, and Chlamydomonas, revealing differences in the stability of the enzyme in different species. These differences are discussed in terms of the ability of activase subunits to remain associated or to reassociate into active oligomers when the stromal milieu is diluted by extraction. Finally, the temperature response of activase activity in leaf extracts differed for Arabidopsis, camelina, tobacco, and cotton, corresponding to the respective temperature responses of photosynthesis for each species. These results confirmed the exceptional thermal lability of activase at physiological ratios of activase to Rubisco.     

Genetic engineering of the biosynthesis of glycinebetaine enhances photosynthesis against high temperature stress in transgenic tobacco plants

Genetically engineered tobacco (Nicotiana tabacum) with the ability to synthesis glycinebetaine was established by introducing the BADH gene for betaine aldehyde dehydrogenase from spinach (Spinacia oleracea). The genetic engineering enabled the plants to accumulate glycinebetaine mainly in chloroplasts and resulted in enhanced tolerance to high temperature stress during growth of young seedlings. Moreover, CO2 assimilation of transgenic plants was significantly more tolerant to high temperatures than that of wild-type plants. The analyses of chlorophyll fluorescence and the activation of Rubisco indicated that the enhancement of photosynthesis to high temperatures was not related to the function of photosystem II but to the Rubisco activase-mediated activation of Rubisco. Western-blotting analyses showed that high temperature stress led to the association of Rubisco activase with the thylakoid membranes from the stroma fractions. However, such an association was much more pronounced in wild-type plants than in transgenic plants. The results in this study suggest that under high temperature stress, glycinebetaine maintains the activation of Rubisco by preventing the sequestration of Rubisco activase to the thylakoid membranes from the soluble stroma fractions and thus enhances the tolerance of CO2 assimilation to high temperature stress. The results seem to suggest that engineering of the biosynthesis of glycinebetaine by transformation with the BADH gene might be an effective method for enhancing high temperature tolerance of plants.     

Rubisco, Rubisco activase, and global climate change

Global warming and the rise in atmospheric CO(2) will increase the operating temperature of leaves in coming decades, often well above the thermal optimum for photosynthesis. Presently, there is controversy over the limiting processes controlling photosynthesis at elevated temperature. Leading models propose that the reduction in photosynthesis at elevated temperature is a function of either declining capacity of electron transport to regenerate RuBP, or reductions in the capacity of Rubisco activase to maintain Rubisco in an active configuration. Identifying which of these processes is the principal limitation at elevated temperature is complicated because each may be regulated in response to a limitation in the other. Biochemical and gas exchange assessments can disentangle these photosynthetic limitations; however, comprehensive assessments are often difficult and, for many species, virtually impossible. It is proposed that measurement of the initial slope of the CO(2) response of photosynthesis (the A/C(i) response) can be a useful means to screen for Rubisco activase limitations. This is because a reduction in the Rubisco activation state should be most apparent at low CO(2) when Rubisco capacity is generally limiting. In sweet potato, spinach, and tobacco, the initial slope of the A/C(i) response shows no evidence of activase limitations at high temperature, as the slope can be accurately modelled using the kinetic parameters of fully activated Rubisco. In black spruce (Picea mariana), a reduction in the initial slope above 30 degrees C cannot be explained by the known kinetics of fully activated Rubisco, indicating that activase may be limiting at high temperatures. Because black spruce is the dominant species in the boreal forest of North America, Rubisco activase may be an unusually important factor determining the response of the boreal biome to climate change.     

Antisense RNA inhibition of Rubisco activase expression

Ribulose bisphosphate carboxylase (Rubisco) activase catalyzes the activation of Rubisco in vivo. Activase antisense DNA mutants of tobacco have been generated to explore the control that activase exerts on the photosynthetic process. These mutants have up to 90% reductions in activase protein levels as a consequence of an inhibition of activase mRNA accumulation. It is shown that photosynthesis, measured as the rate of CO₂ exchange (CER), is modestly decreased in plants exposed to high irradiances. The decreases in CER in the transgenic plants are accompanied by corresponding decreases in Rubisco activation, indicating that activase has a direct effect on photosynthetic rates in the antisense plants by influencing the activation state of Rubisco. It is concluded that in high light conditions, control of photosynthesis is largely shared between Rubisco and activase. Plant growth is also impaired in mutant plants that have severe reductions in activase. The inhibition of activase in the antisense plants does not have an impact on the accumulation of Rubisco large subunit or small subunit mRNAs or proteins. This indicates that the concerted expression of the genes for activase (Rca) and Rubisco (rbcL and rbcS) in response to light, developmental factors and circadian controls is not due to feedback regulation of rbcL or rbcS by the amount of activase protein.     

Light-dependent changes in ribulose bisphosphate carboxylase activase activity in leaves

An assay for the activity of ribulose bisphosphate carboxylase (Rubisco) activase in crude leaf extracts was developed. The assay is based on a spectrophotometric assay of Rubisco, and activase activity (in nanomoles activated Rubisco per minute per milligram chlorophyll) was calculated from the rate of increase in Rubisco activity over time. Activase activity measurements were made using samples from spinach (Spinacia oleracea) leaves undergoing (a) steady-state photosynthesis at various photon flux density (PFD) values and (b) nonsteady-state photosynthesis following an increase from darkness to a high PFD. Analysis of these samples showed that steady-state Rubisco activase activity was relatively low in darkness, increased with PFD, and saturated below 300 micromoles per square meter per second. Rubisco activity (measured spectrophotometrically) was also found to be low in darkness and to increase with PFD, but it saturated at much higher PFD values (approximately 1000 micromoles per square meter per second) along with the rate of photosynthesis. Following an increase in PFD from darkness to 650 micromoles per square meter per second, activase activity increased more or less linearly over a period of 5 to 6 minutes, after which it was constant. Rubisco activity, however, increased more slowly. The light-dependence of Rubisco activase is consistent with previous gas-exchange data showing two interdependent processes in the activation of Rubisco following an increase in PFD.     

Reduction of ribulose biphosphate carboxylase activase levels in tobacco (Nicotiana tabacum) by antisense RNA reduces ribulose biphosphate carboxylase carbamylation and impairs photosynthesis.

The in vivo activity of ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) is modulated in response to light intensity by carbamylation of the active site and by the binding of sugar phosphate inhibitors such as 2'-carboxyarabinitol-1-phosphate (CA 1P). These changes are influenced by the regulatory protein Rubisco activase, which facilitates the release of sugar phosphates from Rubisco's catalytic site. Activase levels in Nicotiana tabacum were reduced by transformation with an antisense gene directed against the mRNA for Rubisco activase. Activase-deficient plants were photosynthetically impaired, and their Rubisco carbamylation levels declined upon illumination. Such plants needed high CO2 concentrations to sustain reasonable growth rates, but the level of carbamylation was not increased by high CO2. The antisense plants had, on average, approximately twice as much Rubisco as the control plants. The maximum catalytic turnover rate (k cat) of Rubisco decreases in darkened tobacco leaves because of the binding of CA 1P. The dark-to-light increase in k cat that accompanies CA 1P release occurred to similar extents in antisense and control plants, indicating that normal levels of activase were not essential for CA 1P release from Rubisco in the antisense plants. However, CA 1P was released in the antisense plants at less than one-quarter of the rate that it was released in the control plants, indicating a role for activase in accelerating the release of CA 1P.     

Alteration of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase activase activities by site-directed mutagenesis

Site-directed mutagenesis was performed on the 1.6 and 1.9 kilobase spinach (Spinacea oleracea) ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase cDNAs, encoding the 41 and 45 kilodalton (kD) isoforms of the enzyme, to create single amino acid changes in the putative ATP-binding site of Rubisco activase (Lys-107, Gln-109, and Ser-112) and in an unrelated cysteine residue (Cys-256). Replacement of Lys-107 with Met produced soluble protein with reduced Rubisco activase and ATPase activities in both isoforms. Substituting Ala or Arg for Lys-107 produced insoluble proteins. Rubisco activase activity increased in the 41-kD isoform when Gln-109 was changed to Glu, but activity in the 45-kD isoform was similar to the wild-type enzyme. ATPase activity in the Glu-109 mutations did not parallel the changes in Rubisco activase activity. Rather, a higher ratio of Rubisco activase to ATPase activity occurred in both isoforms. The mutation of Gln-109 to Lys inactivated Rubisco activase activity. Replacement of Ser-112 with Pro created an inactive protein, whereas attempts to replace Ser-112 with Thr were not successful. The mutation of Cys-256 to Ser in the 45-kD isoform reduced both Rubisco activase and ATPase activities. The results indicate that the two activities of Rubisco activase are not tightly coupled and that variations in photosynthetic efficiency may occur in vivo by replacing the wild-type enzyme with mutant enzymes.     

Rubisco Activase mRNA Expression in Spinach: Modulation by Nanoanatase Treatment

Characterized by a photocatalysis property, nanoanatase is closely related to the photosynthesis of spinach. It could not only improve light absorbance, transformation from light energy to electron energy, and active chemical energy, but also promote carbon dioxide (CO₂) assimilation of spinach. However, the molecular mechanism of carbon reaction promoted by nanoanatase remains largely unclear. In this study, we report that the amounts of Rubisco activase (rca) mRNA in the nanoanatase-treated spinach were increased by about 51%, whereas bulk-TiO₂ treatment produced an increase of only 5%. Accordingly, the protein level of Rubisco activase from the nanoanatase-treated spinach was increased by 42% compared with the control; however, bulk-TiO₂ treatment resulted in a 5% improvement. Further analysis indicated that the activity of Rubisco activase in the nanoanatase-treated spinach was significantly higher than the control by up to 2.75 times, and bulk-TiO₂ treatment had no such significant effects. Together, one of the molecular mechanisms of carbon reaction promoted by nanoanatase is that the nanoanatase treatment results in the enhancement of rca mRNA expressions, protein levels, and activities of Rubisco activase, thereby leading to the improvement of Rubisco carboxylation and the high rate of photosynthetic carbon reaction.