Targeting a nuclear anthranilate synthase alpha-subunit gene to the tobacco plastid genome results in enhanced tryptophan biosynthesis. Return of a gene to its pre-endosymbiotic origin.

Anthranilate synthase (AS), the control enzyme of the tryptophan (Trp) biosynthetic pathway, is encoded by nuclear genes, but is transported into the plastids. A tobacco (Nicotiana tabacum) cDNA (ASA2) encoding a feedback-insensitive tobacco AS alpha-subunit was transformed into two different sites of the tobacco plastid genome through site-specific insertion to obtain transplastomic plants with normal phenotype and fertility. A high and uniform level of ASA2 mRNA was observed in the transplastomic plants but not in the wild type. Although the plants with the transgene insertion at ndhF-trnL only expressed one size of the ASA2 mRNA, the plants with the transgene incorporated into the region between accD and open reading frame (ORF) 184 exhibited two species of mRNA, apparently due to readthrough. The transplastomic plants exhibited a higher level of AS alpha-subunit protein and AS enzyme activity that was less sensitive to Trp-feedback inhibition, leading to greatly increased free Trp levels in leaves and total Trp levels in seeds. Resistance to an AS inhibitor, 5-methyl-Trp, was found during seed germination and in suspension cultures of the transplastomic plants. The resistance to the selection agent spectinomycin and to 5-methyl-Trp was transmitted maternally. These results demonstrate the feasibility of modifying the biosynthetic pathways of important metabolites through transformation of the plastid genome by relocating a native gene from the nucleus to the plastid genome. Very high and uniform levels of gene expression can be observed in different lines, probably due to the identical insertion sites, in contrast to nuclear transformation where random insertions occur.

Characterization of the regulatory function of the 46-kDa isoform of Rubisco activase from Arabidopsis.

Arabidopsis Rubisco activase was recently shown to be regulated by redox changes in the larger (46-kDa) isoform specifically mediated by thioredoxin-f [Zhang and Portis (1999) Proc Natl Acad Sci USA 96: 9438-9443]. Reduction greatly increases the activity of the 46-kDa isoform and the native protein at physiological ATP/ADP ratios. In this study we conducted additional experiments to characterize the regulation of Rubisco activase by thioredoxin-f. The K(m) for both ATP hydrolysis and Rubisco activation by the 46-kDa isoform was lowered by 4 to 5-fold after reduction, but the maximum activity was increased by only 10%. Only 0.35 muM thioredoxin-f was required for a half-maximal activity change after a 10 min preincubation and activation with 1 muM was complete after 10 min. Equal amounts of 46-kDa and 43-kDa isoforms were required for a complete inhibition of the Rubisco activation activity after a reduction-oxidation cycle and assay at an ATP/ADP ratio of 3:1, whereas activity was only inhibited by 50% at a 2:1 ratio (43-/46-kDa) of the isoforms. This requirement is consistent with the fact that Arabidopsis normally contains about a 1:1 ratio of the two isoforms at both the mRNA and protein levels. Redox titrations indicated a midpoint potential of -344 mV for the 46-kDa isoform as compared to -342 mV for spinach fructose 1,6-bisphosphatase at pH 7.9, consistent with previous reports indicating that these proteins are co-regulated by light intensity in a similar manner.

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.

Activase region on chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase. Nonconservative substitution in the large subunit alters species specificity of protein interaction.

In the active form of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC ), a carbamate at lysine 201 binds Mg(2+), which then interacts with the carboxylation transition state. Rubisco activase facilitates this spontaneous carbamylation/metal-binding process by removing phosphorylated inhibitors from the Rubisco active site. Activase from Solanaceae plants (e.g. tobacco) fails to activate Rubisco from non-Solanaceae plants (e.g. spinach and Chlamydomonas reinhardtii), and non-Solanaceae activase fails to activate Solanaceae Rubisco. Directed mutagenesis and chloroplast transformation previously showed that a proline 89 to arginine substitution on the surface of the large subunit of Chlamydomonas Rubisco switched its specificity from non-Solanaceae to Solanaceae activase activation. To define the size and function of this putative activase binding region, substitutions were created at positions flanking residue 89. As in the past, these substitutions changed the identities of Chlamydomonas residues to those of tobacco. Whereas an aspartate 86 to arginine substitution had little effect, aspartate 94 to lysine Rubisco was only partially activated by spinach activase but now fully activated by tobacco activase. In an attempt to eliminate the activase/Rubisco interaction, proline 89 was changed to alanine, which is not present in either non-Solanaceae or Solanaceae Rubisco. This substitution also caused reversal of activase specificity, indicating that amino acid identity alone does not determine the specificity of the interaction.

Mechanism of light regulation of Rubisco: a specific role for the larger Rubisco activase isoform involving reductive activation by thioredoxin-f.

Rubisco activase is a nuclear-encoded chloroplast protein that is required for the light activation of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) in vivo. In most plants examined to date, there are two isoforms of Rubisco activase arising from alternative splicing that differ only at the carboxyl terminus. Here we demonstrate with recombinant proteins that in Arabidopsis the larger isoform has a unique role in the regulation of Rubisco activity. At physiological ratios of ADP/ATP, the 46-kDa isoform has minimal ATP hydrolysis and Rubisco activation activity in comparison with the 43-kDa isoform. Analysis of a series of carboxyl-terminal deletion and Ala substitution mutants of the 46-kDa isoform revealed that the presence of Cys residues at positions 411 and 392 were essential to preserve a low ATP hydrolysis and Rubisco activation activity in the presence of ADP. Consequently, incubation of the 46-kDa isoform with DTT and thioredoxin-f increased both activities, whereas incubations with DTT alone or with thioredoxin-m were ineffective. Thioredoxin-f and DTT had no effect on the 43-kDa isoform. However, premixing both isoforms before conducting a reduction and oxidation cycle demonstrated that the activity of both isoforms could be regulated. Reduction and oxidation also modulated the activity of native activase proteins isolated from either Arabidopsis or spinach, but not tobacco, which only has the smaller isoform. These findings suggest that in plants containing both isoforms, Rubisco activase regulates the activity of Rubisco in response to light-induced changes in both the ADP/ATP ratio and the redox potential via thioredoxin-f.

Specificity for activase is changed by a Pro-89 to Arg substitution in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase.

Tobacco activase does not markedly facilitate the activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1. 39) from non-Solanaceae species, including the green alga Chlamydomonas reinhardtii. To examine the basis of this specificity, we focused on two exposed residues in the large subunit of Rubisco that are unique to the Solanaceae proteins. By employing in vitro mutagenesis and chloroplast transformation, P89R and K356Q substitutions were separately made in the Chlamydomonas enzyme to change these residues to those present in tobacco. Both mutants were indistinguishable from the wild type when grown with minimal medium in the light and contained wild-type levels of holoenzyme. Purified Rubisco was assessed for facilitated activation by spinach and tobacco activase. Both wild-type and K356Q Rubisco were similar in that spinach activase was much more effective than tobacco activase. In contrast, P89R Rubisco was not activated by spinach activase but was well activated by tobacco activase. Thus, the relative specificities of the spinach and tobacco activases for Chlamydomonas Rubisco were switched by changing a single residue at position 89. This result provides evidence for a site on the Rubisco holoenzyme that interacts directly with Rubisco activase.

ATP Hydrolysis Activity and Polymerization State of Ribulose-1,5-Bisphosphate Carboxylase Oxygenase Activase (Do the Effects of Mg2+, K+, and Activase Concentrations Indicate a Functional Similarity to Actin?).

The ATPase activity and fluoresence of ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) activase were determined over a range of MgCl2, KCl, and activase concentrations. Both salts promoted ADP release from ATP and intrinsic fluorescence enhancement by adenosine 5[prime]-[[gamma]-thio] triphosphate, but Mg2+ was about 10 times more effective than K+. ATPase and fluorescence enhancement both increased from zero to saturation within the same Mg2+ and K+ concentration ranges. At saturating concentrations (5 mM Mg2+ and 22 mM K+), the specific activity of ATPase (turnover time, about 1 s) and specific intrinsic fluorescence enhancement were maximal and unaffected by activase concentration above 1 [mu]M activase; below 1 [mu]M activase, both decreased sharply. These responses are remarkably similar to the behavior of actin. Intrinsic fluorescence enhancement of Rubisco activase reflects the extent of polymerization, showing that the smaller oligomer or monomer present in low-salt and activase concentrations is inactive in ATP hydrolysis. However, quenching of 1-anilinonapthaline-8-sulfonate fluorescence revealed that ADP and adenosine 5[prime]-[[gamma]-thio] triphosphate bind equally well to activase at low- and high-salt concentrations. This is consistent with an actin-like mechanism requiring a dynamic equilibrium between monomer and oligomers for ATP hydrolysis. The specific activation rate of substrate-bound decarbamylated Rubisco decreased at activase concentrations below 1 [mu]M. This suggests that a large oligomeric form of activase, rather than a monomer, interacts with Rubisco when performing the release of bound ribulose-1,5-bisphosphate from the inactive enzyme.

Growth and photosynthesis under high and low irradiance of Arabidopsis thaliana antisense mutants with reduced ribulose-1,5-bisphosphate carboxylase/oxygenase activase content.

Photosynthesis and growth to maturity of antisense ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase Arabidopsis thaliana with reduced concentrations of activase relative to wild-type (Wt) plants were measured under low (200 mumol m-2 s-1) and high (600 mumol m-2 s-1) photosynthetic photon flux density growing conditions. Both growth and photosynthesis were significantly reduced in an Arabidopsis clone (R100) with 30 to 40% Wt activase, an effect that was more pronounced in high light. The aboveground biomass of the antisense clone R100 reached 80% of Wt under low light and 65% of Wt under high light. Decreased growth in the antisense plants was attributed to reduced relative rates of growth and leaf area expansion early in development; all plants attained similar values of relative rates of growth and leaf elongation by 21 d after planting. Reductions in photosynthesis were attributed to decreased Rubisco activation in the antisense plants. Rubisco constituted about 40% of total soluble protein in both Wt and clone R100 under both light regimes. Activase content was 5% and 1.4% of total soluble protein in Wt and clone R100, respectively, and also was unaffected by growth irradiance. The stoichiometry of Rubisco to activase was estimated at 20 Rubisco active sites per activase tetramer in Wt Arabidopsis and 60 to 80 in the transgenic clone R100. We conclude that Wt Arabidopsis does not contain Rubisco activase in great excess of the amount required for optimal growth.

Heat Denaturation Profiles of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) and Rubisco Activase and the Inability of Rubisco Activase to Restore Activity of Heat-Denatured Rubisco.

We compared the heat-denaturation profiles of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase and further examined the ability of Rubisco activase to restore the activity of heat-denatured Rubisco originally reported (E. Sanchez de Jimenez, L. Medrano, and E. Martinez-Barajas [1995] Biochemistry 34: 2826-2831). Rubisco was heat-treated in both the carbamylated and uncarbamylated forms and in the presence and absence of 10 mM dithiothreitol (DTT). Both forms were highly resistant to heat denaturation and further protection was gained in the presence of DTT. A 50% loss in total activity occurred after 1 h at 57.5 and 55.2[deg]C for uncarbamylated Rubisco and at 60.2 and 59.6[deg]C for carbamylated Rubisco, in each case with and without DTT, respectively. In contrast, Rubisco activase lost 50% activity after only 5 min at 33[deg]C and the loss in activity was not affected by the presence of Rubisco. When Rubisco, heat-denatured to various extents, was incubated at room temperature with Rubisco activase or bovine serum albumin as a control, Rubisco activase did not have a significant specific ability to restore Rubisco activity. We conclude that Rubisco activase alone does not have the ability to restore the activity of heat-denatured Rubisco and is unlikely to protect or restore Rubisco activity from heat denaturation in vivo because it is more heat-labile than Rubisco.

Differential effects of N- and C-terminal deletions on the two activities of rubisco activase.

Spinach (Spinacea oleracea) leaf ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase was subjected to limited proteolysis with trypsin and directed deletions were made by modifying the spinach rubisco activase cDNA and expressing the 41-kDa isoform in Escherichia coli. Protein exposed to trypsin displayed a more rapid loss of the ability to promote the activation of decarbamylated rubisco than ATP hydrolysis (e.g., 10 and 50% activity remaining, respectively, after 1 h). A series of N-terminal deletions exhibited near abolition of rubisco activation after the 12th residue, a conserved tryptophan, was deleted. Conversely, a deletion of 19 residues at the C-terminus increased rubisco activation with little effect on ATP hydrolysis, resulting in an increased efficiency of activation. The C-terminal deletion mutant was further modified by a site-directed mutation in the ATP binding region (Q109E) which was previously observed to increase the efficiency of activation (J. B. Shen and W. L. Ogren, 1991, Plant Physiol. 99, 1201-1207). The efficiency of activation with this double mutant was greater than that for either of the original mutants. The results indicate that a conserved tryptophan in the N-terminal portion of rubisco activase is critical for promotion of the activation of rubisco, consistent with a possible role in interaction with rubisco. The C-terminus appears to have a regulatory effect on both rubisco activation and ATP hydrolysis.

Subsaturating Ribulose-1,5-Bisphosphate Concentration Promotes Inactivation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) (Studies Using Continuous Substrate Addition in the Presence and Absence of Rubisco Activase).

We developed a continuous-addition method for maintaining subsaturating concentrations of ribulose-1,5-bisphosphate (RuBP) for several minutes, while simultaneously monitoring its consumption by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This method enabled us to observe the effects of subsaturating RuBP and CO2 concentrations on the activity of Rubisco during much longer periods than previously studied. At saturating CO2, the activity of the enzyme declined faster when RuBP was maintained at concentrations near its Km value than when RuBP was saturating. At saturating RuBP, activity declined faster at limiting than at saturating CO2, in accordance with previous observations. The most rapid decline in activity occurred when both CO2 and RuBP concentrations were subsaturating. The activity loss was accompanied by decarbamylation of the enzyme, even though the enzyme was maintained at the same CO2 concentration before and after exposure to RuBP. Rubisco activase ameliorated the decline in activity at subsaturating CO2 and RuBP concentrations. The results are consistent with a proposed mechanism for regulating the carbamylation of Rubisco, which postulates that Rubisco activase counteracts Rubisco's unfavorable carbamylation equilibrium in the presence of RuBP by accelerating, in an ATP-dependent manner, the release of RuBP from its complex with uncarbamylated sites.

Mg2+ and ATP or adenosine 5'-[gamma-thio]-triphosphate (ATP gamma S) enhances intrinsic fluorescence and induces aggregation which increases the activity of spinach Rubisco activase.

ATP and Mg2+ caused a transient increase in the intrisinc fluorescence of Rubisco activase which was inhibited by the presence of ADP. Only minor changes in fluorescence were observed with ATP or Mg2+ alone. The fluorescence increase was stabilized by addition of an ATP regenerating system or by substitution of ATP with a non-hydrolyzable analog, adenosine 5'-[gamma-thio]-triphosphate (ATP gamma S). The initial rate of increase in fluorescence also depended on the concentration of protein in a manner consistent with second-order kinetics. The concentration dependence for the effect of ATP gamma S was sigmoidal, although at pH 8 the half-saturation requirements for both ATP gamma S (12 microM) and Mg2+ (1.5 mM) were not too dissimilar to the binding affinities (6 microM and 2 mM, respectively) determined indirectly with the fluorescent probe, 1-anilinonapthalene-8-sulfonate. However, the concentration dependence of ATP was about 5-fold higher than its binding affinity, also sigmoidal and quite similar to the concentration responses of ATP hydrolysis and activation of Rubisco by the protein. These characteristics of the intrinsic fluorescence indicate that it monitors a conformational change in the protein occurring after binding of the nucleotide and associated with increased aggregation. Direct evidence of increased aggregation in the presence of Mg2+ and ATP or ATP gamma S was obtained by gel-filtration chromatography. However, the apparent molecular mass was heterogeneous and also varied with temperature. The increased aggregation of the protein resulted in altered kinetic properties. The ATP hydrolysis activity of the protein increased and the half-maximal ATP concentration decreased as the protein concentration was increased in the assay. Also, a brief pretreatment of the protein with ATP and Mg2+ to increase aggregation eliminated the otherwise observed time delays in the Rubisco activation and ATP hydrolysis kinetics.

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.

Dissociation of ribulose-1,5-bisphosphate bound to ribulose-1,5-bisphosphate carboxylase/oxygenase and its enhancement by ribulose-1,5-bisphosphate carboxylase/oxygenase activase-mediated hydrolysis of ATP.

Ribulose bisphosphate (RuBP), a substrate of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is an inhibitor of Rubisco activation by carbamylation if bound to the inactive, noncarbamylated form of the enzyme. The effect of Rubisco activase on the dissociation kinetics of RuBP bound to this form of the enzyme was examined and characterized with the use of (3)H-labeled RuBP and proteins purified from spinach (Spinacia oleracea L.) In the absence of Rubisco activase and in the presence of a large excess of unlabeled RuBP, the dissociation rate of bound [1-(3)H]RuBP was much faster after a short (30 second) incubation than after an extended incubation (1 hour). After 1 hour of incubation, the dissociation rate constant (K(off)) of the bound RuBP was 4.8 x 10(-4) per second, equal to a half-time of about 35 minutes, whereas the rate after only 30 seconds was too fast to be accurately measured. This time-dependent change in the dissociation rate was reflected in the subsequent activation kinetics of Rubisco in the presence of RuBP, CO(2), and Mg(2+), and in both the absence or presence of Rubisco activase. However, the activation of Rubisco also proceeded relatively rapidly without Rubisco activase if the RuBP level decreased below the estimated catalytic site concentration. High pH (pH 8.5) and the presence of Mg(2+) in the medium also enhanced the dissociation of the bound RuBP from Rubisco in the presence of RuBP. In the presence of Rubisco activase, Mg(2+), ATP (but not the nonhydrolyzable analog, adenosine-5'-O-[3-thiotriphosphate]), excess RuBP, and an ATP-regenerating system, the dissociation of [1-(3)H]RuBP from Rubisco was increased in proportion to the amount of Rubisco activase added. This result indicates that Rubisco activase-mediated hydrolysis of ATP is required for promotion of the enhanced dissociation of the bound RuBP from Rubisco. Furthermore, product analysis by ion-exchange chromatography demonstrated that the release of the bound RuBP, in an unchanged form, was considerably faster than the observed increase in Rubisco activity. Thus, RuBP dissociation was experimentally separated from activation and precedes the subsequent formation of active, carbamylated Rubisco during activation of Rubisco by Rubisco activase.

A fluorometric study with 1-anilinonaphthalene-8-sulfonic acid (ANS) of the interactions of ATP and ADP with rubisco activase.

The interactions of ATP and ADP with rubisco activase purified from spinach were investigated by measuring enhanced fluorescence due to ANS-binding to the protein. Evidence of conformational changes was observed from the differences in the interaction of ANS with rubisco activase in the presence of excess ATP and ADP. Fluorescent changes associated with the titration of a rubisco activase-ANS mixture with ATP and ADP indicated that the binding of ADP to rubisco activase was much tighter than that of ATP. The concentration of Mg2+ and pH had significant effects on the affinities of rubisco activase for ATP and ADP, with higher pH and Mg2+ concentration facilitating the binding of ATP to rubisco activase in the presence of ADP. The physiological implications of the binding characteristics of ATP and ADP with rubisco activase on the light-dark regulation of rubisco are discussed.

Partial reduction in ribulose 1,5-bisphosphate carboxylase/oxygenase activity by carboxypeptidase A.

Treatment with carboxypeptidase A of ribulose bisphosphate carboxylase/oxygenase (rubisco) from spinach and Chlamydomonas, but not tobacco, reduced activity by 60-70%. Further studies with the spinach enzyme indicated that only one amino acid from each of the large (valine) and small (tyrosine) subunits was removed and the loss of activity was correlated with modification of the large subunit. The modified enzyme also had a two-fold greater Km for RuBP but CO2/O2 specificity was only 5% lower and may not be significantly different. The relative rates of release of valine and tyrosine also depended on the presence or absence of RuBP or CO2 plus Mg during treatment. The results indicate that the C-terminal amino acid in the large subunit of spinach, which is not located near the active site region, plays a previously unrecognized role in determining the catalytic activity of the enzyme.

Impaired reductive activation of stromal bisphosphatases in tomato leaves following low-temperature exposure at high light.

Photosynthesis in domestic tomato (Lycopersicon esculentum L.) is highly sensitive to low temperature, particularly when accompanied by high light. Since previous studies have established that the inhibited plants retain photosynthetic electron transfer and ATP formation competence, we sought to identify specific steps in the photosynthetic carbon reduction pathway that could account for the lost photosynthetic capacity. Measurements of steady-state photosynthetic metabolite pool sizes showed an accumulation of fructose 1,6-bisphosphate and sedohepulose 1,7-bisphosphate following chilling in the light. Measurements of in vivo turnover rates of the metabolite pools accompanied by direct determinations of enzymatic activity showed that the capacity of the stromal bisphosphatases was substantially reduced following chilling in the light and was the cause of the bisphosphate accumulation. The time course of the loss of phosphatase activity closely mimicked that of the inhibition of net photosynthesis, further indicating that impaired phosphatase function is the underlying cause of the sensitivity of photosynthesis in tomato to light and chilling. Fructose 1,6-bisphosphatase extracted from inhibited tomato plants could be fully activated in the presence of dithiothreitol, indicating that chilling in the light disrupts the normal, thioredoxin-dependent, activation pathway of the stromal bisphosphatases. This disruption could involve a change in the redox potential of the functional disulfide on the phosphatases.

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 CO(2), 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.

Activity of ribulose 1,5-bisphosphate carboxylase oxygenase as a function of storage conditions.

Optimal storage conditions to retain ribulose 1, 5-bisphosphate carboxylase/oxygenase (Rubisco) activity were investigated. The soluble spinach (Spinacia oleracea) enzyme was pretreated with its activators, Mg(2+) and HCO(3) (-), and then stored for up to 30 days at 4 or -18 degrees C or in liquid N(2). Cold inactivation and conformational changes were suggested to be involved during Rubisco storage in the cold, leading to its inactivation. Pretreatment of the enzyme with Mg(2+) and CO(2) and subsequent storage at either 4 degrees C or in liquid N(2) or flushing the samples with N(2) and rapid freezing and storage in liquid N(2) are recommended as storage procedures. These storage treatments will prevent inactivation, so that full original specific activity will be preserved.