Altered Growth of Transgenic Tobacco Lacking Leaf Cytosolic Pyruvate Kinase

Previously, we reported that transformation of tobacco (Nicotiana tabacum L.) with a vector containing a potato cytosolic pyruvate kinase (PK_c) cDNA generated two plant lines specifically lacking leaf PK_c (PK_c−) as a result of co-suppression. PK_c deficiency in these primary transformants did not appear to alter plant development, although root growth was not examined. Here we report a striking reduction in root growth of homozygous progeny of both PK_c− lines throughout development under moderate (600 μE m⁻² s⁻¹) or low (100 μE m⁻² s⁻¹) light intensities. When both PK_c− lines were cultivated under low light, shoot and flower development were also delayed and leaf indentations were apparent. Leaf PK activity in the transformants was significantly decreased at all time points examined, whereas root activities were unaffected. Polypeptides corresponding to PK_c were undetectable on immunoblots of PK_c− leaf extracts, except in 6-week-old low-light-grown PK_c− plants, in which leaf PK_c expression appeared to be greatly reduced. The metabolic implications of the kinetic characteristics of partially purified PK_c from wild-type tobacco leaves are discussed. Overall, the results suggest that leaf PK_c deficiency leads to a perturbation in source-sink relationships.     

Pyruvate-kinase isoenzymes from zygotic and microspore-derived embryos of Brassica napus

Polyclonal antibodies against castor-oil seed cytosolic and leucoplastic pyruvate kinases (PK_c and PK_p, respectively; EC 2.7.1.40) were utilized to examine the subunit compositions and developmental profiles of canola (Brassica napus L. cv. Topas) PK_c and PK_p over 6 d of seed germination and 35 d of culture of microspore-derived embryos. The PK_c from germinating seeds appears to be composed of a single type of 56-kDa subunit, whereas the enzyme from cultured embryos contains equal proportions of immunologically related 57- and 56-kDa subunits. The PK_p was immunologically undetectable in germinating seeds, while the enzyme from cultured embryos consisted of immunologically related 64- and 58-kDa subunits in a ratio of about 12, respectively. The large increase in PK activity that occurs between the second and fourth days of seed gemination is based upon de-novo synthesis of PK_c. Between 7 and 14 d of culture of microspore-derived embryos, the levels of PK_p and PK maximal activity increased approx. 3- and 2.5-fold, respectively. These increases were coincident with an approximately fourfold rise in the in-vivo pyruvate: phosphoenolpyruvate concentration ratio. Conversely, PK_c was not only far less abundant relative to PK_p, but its level remained constant over 35 d of microspore-embryo culture. Developing non-zygotic (microspore-derived) embryos strongly resembled ripening zygotic (seed) embryos in terms of PK specific activity as well as relative amounts and subunit compositions of PK_c and PK_p. The results indicate that the synthesis of PK isoenzymes in B. napus seeds is highly regulated and that this regulation follows a preset developmental program.Abbreviations IgG immunoglobulin G - IU international unit - PEP phosphoenolpyruvate - 3-PGA 3-phosphoglycerate - PK(s) pyruvate kinase(s) - PK_c cytosolic pyruvate kinase - PK_p plastidic pyruvate kinase - PYR pyruvate Plant Research Centre contribution No. 1374We wish to thank Ms. Kathryn Hovey and Ms. Suzanne Belliveau (Agriculture Canada) for their expert assistance in the culturing and harvesting of microspore-derived embryos of canola. This work was supported by a Strategic Grant from the Natural Sciences and Engineering Research Council of Canada.     

Characterization and kinetics of isoenzymes of pyruvate kinase from developing castor bean endosperm

Isozymes of pyruvate kinase (PK) have been isolated from developing castor bean endosperm. One isozyme, PK_c, is localized in the cytosol, and the other, PK_p, is in the plastid. Both isozymes need monovalent and divalent cations for activity, requirements which can be filled by K⁺ and Mg²⁺. Both isozymes are inhibited by citrate, pyruvate, and ATP. PK_c has a much broader pH profile than PK_p and is also more stable. Both have the same K_m (0.05 millimolar) for PEP, but PK_p has a 10-fold higher K_m (0.3 millimolar) for ADP than PK_c (0.03 millimolar). PK_c also has a higher affinity for alternate nucleotide substrates than PK_p. The two isozymes have different kinetic mechanisms. Both have an ordered sequential mechanism and bind phosphoenolpyruvate before ADP. However, the plastid isozyme releases ATP first, whereas pyruvate is the first product released from the cytosolic enzyme. The properties of the two isozymes are similar to those of their counterparts in green tissue.     

Photosynthesis and Carbon Partitioning in Transgenic Tobacco Plants Deficient in Leaf Cytosolic Pyruvate Kinase

Whole-plant diurnal C exchange analysis provided a noninvasive estimation of daily net C gain in transgenic tobacco (Nicotiana tabacum L.) plants deficient in leaf cytosolic pyruvate kinase (PK_c−). PK_c− plants cultivated under a low light intensity (100 μmol m⁻² s⁻¹) were previously shown to exhibit markedly reduced root growth, as well as delayed shoot and flower development when compared with plants having wild-type levels of PK_c (PK_c+). PK_c− and PK_c+ source leaves showed a similar net C gain, photosynthesis over a range of light intensities, and a capacity to export newly fixed ¹⁴CO₂ during photosynthesis. However, during growth under low light the nighttime, export of previously fixed ¹⁴CO₂ by fully expanded PK_c− leaves was 40% lower, whereas concurrent respiratory ¹⁴CO₂ evolution was 40% higher than that of PK_c+ leaves. This provides a rationale for the reduced root growth of the PK_c− plants grown at low irradiance. Leaf photosynthetic and export characteristics in PK_c− and PK_c+ plants raised in a greenhouse during winter months resembled those of plants grown in chambers at low irradiance. The data suggest that PK_c in source leaves has a critical role in regulating nighttime respiration particularly when the available pool of photoassimilates for export and leaf respiratory processes are low.     

Cytosolic pyruvate kinase: subunit composition,activity, and amount in developing castor and soybean seeds,and biochemical characterization of the purified castor seed enzyme

Antibodies against Brassica napus cytosolic pyruvate kinase (PK_c) (EC 2.7.1.40) were employed to examine PK_c subunit composition and developmental profiles in castor and soybean seeds. A 56-kDa immunoreactive polypeptide was uniformly detected on immunoblots of clarified extracts from developing castor endosperm or soybean embryos. Maximal PK_c activities occurred early in castor oil seed (COS) and soybean development (7.1 and 5.5 (μmol of pyruvate produced/min) g⁻¹ FW, respectively) and were up to 25-fold greater than those of fully mature seeds. Time-course studies revealed a close correlation between extractable PK_c activity and the relative amount of the immunoreactive 56-kDa PK_c polypeptide. PK_c from developing COS was purified 1,874-fold to homogeneity and a final specific activity of 73.1 (μmol of pyruvate produced/min) mg⁻¹ protein. Gel filtration and SDS-PAGE indicated that this PK_c exists as a 230-kDa homotetramer composed of 56-kDa subunits. The mass fingerprint of tryptic peptides of the 56-kDa COS PK_c subunit best matched three putative PK_cs from Arabidopsis thaliana. The purified enzyme was relatively heat-stable and displayed a broad pH optimum of 6.4. However, more efficient substrate utilization (in terms of V _max /K _m for phosphoenolpyruvate or ADP) was observed at pH 7.4. Glutamate was the most effective inhibitor, whereas aspartate functioned as an activator by partially relieving glutamate inhibition. Together with our previous studies, the results: (1) allow a model to be formulated regarding the coordinate allosteric control of PKc and phosphoenolpyruvate carboxylase by aspartate and glutamate in developing COS, and (2) provide further biochemical evidence that castor plant PK_c exists as tissue-specific isozymes that exhibit substantial differences in their respective physical and regulatory properties.     

Leucoplast Pyruvate Kinase from Developing Castor Oil Seeds : Characterization of the Enzyme's Degradation by a Cysteine Endopeptidase

Leucoplast pyruvate kinase (PK_p; EC 2.7.1.40) from endosperm of developing castor oil seeds (Ricinus communis L. cv Baker 296) appears to be highly susceptible to limited degradation by a cysteine endopeptidase during the purification of the enzyme or incubation of clarified homogenates at 4°C. Purified castor seed PK_p was previously reported to consist of immunologically related 57.5 and 44 kilodalton subunits (Plaxton WC, Dennis DT, Knowles VL [1990] Plant Physiol 94: 1528-1534). By contrast, immunoreactive polypeptides of about 63.5 and 54 kilodaltons were observed when a western blot of an extract prepared under denaturing conditions was probed with affinity purified rabbit anti-(castor seed PK_p) immunoglobulin G. Proteolytic activity against PK_p was estimated by the disappearance of the 63.5 and 54 kilodalton subunits and the concomitant appearance of lower molecular mass immunoreactive degradation products during the incubation of clarified homogenates at 4°C. The presence of 2 millimolar dithiothreitol accelerated the degradation of PK_p. The conservation of the 63.5 and 54 kilodalton subunits was observed after extraction of the enzyme in the presence of 1 millimolar p-hydroxymecuribenzoate, or 1 millimolar Nα-p-tosyl-l-lysine chloromethyl ketone, or 10 millimolar iodoacetate. These results reveal that a cysteine endopeptidase was responsible for the in vitro proteolysis of PK_p. This endopeptidase is present throughout all stages of endosperm development. Its PK_p-degrading activity, however, appears to be most pronounced in preparations from older endosperm. When lysates of purified leucoplasts were incubated at 4°C for up to 21 hours, no degradation of PK_p was observed; this indicated an extra-leucoplastic localization for the cysteine endopeptidase. Although the in vivo subunit structure of PK_p remains uniform throughout all stages of endosperm development, the large decrease in PK activity that accompanies castor seed maturation coincides with a marked reduction in the concentration of PK_p.     

Regulation of cytosolic carbon metabolism in germinating Ricinus communis cotyledons

The cytosolic pyruvate kinase (PK_C, EC 2.7.1.40) and phosphoenolpyruvate carboxylase (PEP-Case, EC 4.1.1.31) from cotyledons of 6-d-old castor seedlings (Ricinus communis L.) have been partially purified and characterized. PK_C was purified 370-fold to a specific activity of 20 mol · min ¹·(mg protein)^–1, and was shown to exist as a 237-kDa homotetramer. In addition, PK_C displayed hyperbolic substrate saturation kinetics and demonstrated pH-dependent modulation by several metabolite effectors including glutamine, glutamate, arginine, malate and 2-oxoglutarate. Most were inhibitors at pH 6.9, while activation by glutamine, asparagine and arginine and only weak inhibition for the rest were observed at pH 7.5. PEPCase was purified 33-fold to a final specific activity of 1 mol · min^–1 · (mg protein)^–1. The subunit and native M_r for the enzyme were shown to be 100 and 367 kDa, respectively, suggesting a homotetrameric native structure. PEPCase displayed a typical pH activity profile with an alkaline optimum and activity decreasing rapidly below pH 7.0. The enzyme was potently inhibited by malate, isocitrate, aspartate and glutamate at pH 7.0, whereas inhibition by these compounds was considerably diminished at pH 7.5. A model depicting the regulation of glycolytic carbon flow during amino-acid and sucrose import by castor cotyledons is proposed.Abbreviations IgG immunoglobulin G - I₅₀a inhibitor concentration producing 50 inhibition of enzyme activity - PK_C and PK_pa cytosolic and plastidic isoenzymes of pyruvate kinase, respectively - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase - 3-PGA 3-phosphoglycerate This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC).     

Molecular characterization of plastid pyruvate kinase from castor and tobacco

Clones encoding two different forms of plastid pyruvate kinase (PK_p; EC 2.7.1.40) have been isolated from both castor and tobacco seed cDNA libraries. One form, designated PK_pA, from castor was described in a previous report, and the tobacco homologue of PK_pA has now been isolated. In addition, a second cDNA, designated PK_pG, has been identified and sequenced in both species. Western blot analysis, using antibodies raised against protein overexpressed from these clones, indicates that they encode the two predominant polypeptides of plastid pyruvate kinase from developing castor endosperm. In castor, both PK_pA and PK_pG are encoded by single genes. In the allotetraploid Nicotiana tabacum, there are two copies of each, one derived from each of the progenitors of this species. The expression of the genes for PK_pA and PK_pG was examined in various tissues from both castor and tobacco. In castor, both forms are expressed in developing and germinating endosperm and in the root but neither is expressed in the leaf. In tobacco, both forms are expressed in developing seeds but in mature tissues, PK_pA is most abundant in roots and PK_pG in leaves.     

Metabolic and molecular responses in Nile tilapia,Oreochromis niloticus during short and prolonged hypoxia

The strictly aquatic breathing Nile tilapia, Oreochromis niloticus is an extremely hypoxia-tolerant fish. To augment our understanding of the effects of hypoxia on anaerobic glycolysis in the Nile tilapia, we studied the effect of short-term for 1 day (trial 1) and long-term for 30 days (trial 2) hypoxia on a selected glycolytic enzymes activity and mRNA expression in liver and white muscle. The hypoxic oxygen concentrations used in the two trials were 2, 1, and 0.5 mg O₂ L^?1 for comparison with a control normoxic group 8 mg O₂ L^?1. The activity of phosphofructokinase (PFK), pyruvate kinase (PK), and lactate dehydrogenase (LDH) in liver and white muscle except liver LDH decreased in trial 1 and increased in trial 2. Assessments of mRNA levels in trial 1 revealed that PFK was downregulated and LDH was upregulated in liver and white muscle, while PK fluctuated between upregulation in liver and downregulation in white muscle. Meanwhile, PK and LDH were upregulated while PFK was similar to control values in both tissues in trial 2. Comet assay results demonstrated an increase in DNA damage that was directly proportional to increasing hypoxic concentrations. This damage was more pronounced in trial 1. This suggests that the Nile tilapia cope better with long-term hypoxic conditions, possibly as an adaptive response.     

Relationship between the Subunits of Leucoplast Pyruvate Kinase from Ricinus communis and a Comparison with the Enzyme from Other Sources

Two cDNA clones, PK_pα and PK_pβ, for the leucoplast isozyme of pyruvate kinase have been isolated and characterized. A Southern blot of castor (Ricinus communis) DNA probed with PK_pα indicates the presence of a single gene for PK_p. Most (1610 base pairs) of the sequence of both cDNAs is identical. These 1610 base pairs begin with an ATG translation initiation codon, and have 248 base pairs of 3′-untranslated and 1362 base pairs of coding sequence. The sequences of the two clones 5′- to the identical regions are different but both encode peptides with a high percentage of hydrophobic amino acids. The derived sequence of PK_pα encodes eight amino acid residues which have been identified as the amino-terminus of one subunit of PK_p from castor seed leucoplasts when the enzyme is purified in the absence of cysteine endopeptidase inhibitors. The sequence upstream of these amino acids is possibly the transit peptide for this protein. When PK_p is extracted under conditions that eliminate its proteolytic degradation, its α-subunit has a relative molecular weight equal to the full-length coding sequence of PK_pα. The data indicate that the transit peptide for the subunit of leucoplast pyruvate kinase encoded by PK_pα is not cleaved until the protein is released from the plastid. The derived amino acid sequences of PK_pα and PK_pβ are most closely related to Escherichia coli pyruvate kinase. Although the residues involved in substrate binding are conserved in leucoplast pyruvate kinase, there is no phosphorylation site and only 5 of 15 amino acids in the E. coli fructose-1,6-bisphosphate binding site are conserved.     

Activation of ascorbate-glutathione cycle inArabidopsis leaves in response to aminotriazole

Aminotriazole(AT)-induced changes in growth, hydrogen peroxide content and activities of H₂O₂-scavenging antioxidant enzymes were investigated in the growing leaves ofArabidopsis plants (Arabidopsis thaliana cv Columbia). Catalase activity of rosette leaves was reduced by 65% with an application of 0.1 mM AT (a herbicide known as a catalase inhibitor), whereas the leaf growth and H₂O₂ content were almost unaffected. However, an approximate 1.6 to 2-fold increase in cytosolic ascorbate peroxidase (APX) activity concomitant with a substantial activation of glutathione reductase (GR) (approx. 22% increase) was observed during leaf growth in the presence of 0.1 mM AT. The activity of cytosolic APX in leaves was also increased by 1.8-fold with an application of exogenous 2 mM paraquat (an inducer of H₂O₂ production in plant cells) in the absence of AT. These results collectively suggest that (a) cytosolic APX and GR operate to activate an ascorbate-glutathione cycle for the removal of H₂O₂ under severe catalase deactivation, and (b) the expression of APX seems to be regulated by a change of the endogenous H₂O₂ level in leaf cells.     

Phosphoenolpyruvate metabolism in Teladorsagia circumcincta: A critical junction between aerobic and anaerobic metabolism

Nematodes which have adapted to an anaerobic lifestyle in their adult stages oxidise phosphoenolpyruvate (PEP) to oxaloacetate rather than pyruvate as the final product of glycolysis. This adaptation involves selective expression of the enzyme phosphoenolpyruvate carboxykinase (PEPCK), instead of pyruvate kinase (PK). However, such adaptation is not absolute in aerobic nematode species. We have examined the activity and kinetics of PEPCK and PK in larvae (L₃) and adults of Teladorsagia circumcincta, a parasite known to exhibit oxygen uptake. Results revealed that PK and PEPCK activity existed in both L₃s and adults. The enzymes had differing affinity for nucleotide diphosphates: while both can utilise GDP, only PK utilised ADP and only PEPCK utilised IDP. In both life cycle stages, enzymes showed similar affinity for PEP. PK activity was predominant in both stages, although activity of this enzyme was lower in adults. When combined, both the activity levels and the enzyme kinetics showed that pyruvate production is probably favoured in both L₃ and adult stages of T. circumcincta and suggest that metabolism of PEP to oxaloacetate is a minor metabolic pathway in this species.     

The response to oxidative stress induced by magnesium deficiency in kidney bean plants

To understand the plant response to oxidative stresses, we studied the influence of magnesium (Mg⁺⁺) deficiency on the formation of hydrogen peroxide (H₂O₂), malondialdehyde (MDA), and protease activity in kidney bean plants. The expression pattern of proteins under Mg⁺⁺ deficiency also was examined via two-dimensional electrophoresis. The formation of H₂O₂ and MDA increased in the primary leaves of plants grown in a nutrient solution deficient in Mg⁺⁺. Protease activity in Mg⁺⁺-deficient plants was also higher than in those grown with sufficient Mg⁺⁺. The expression pattern of the proteins showed that 25 new proteins were generated and 64 proteins disappeared under Mg⁺⁺-deficient conditions. Therefore, a deficiency in Mg⁺⁺ may cause oxidative stress and a change in protein expression. Some of these proteins may be related to the oxidative stress induced by Mg⁺⁺ deficiency.     

Metabolic networks to generate pyruvate,PEP and ATP from glycerol in Pseudomonas fluorescens

Glycerol is a major by-product of the biodiesel industry. In this study we report on the metabolic networks involved in its transformation into pyruvate, phosphoenolpyruvate (PEP) and ATP. When the nutritionally-versatile Pseudomonas fluorescens was exposed to hydrogen peroxide (H₂O₂) in a mineral medium with glycerol as the sole carbon source, the microbe reconfigured its metabolism to generate adenosine triphosphate (ATP) primarily via substrate-level phosphorylation (SLP). This alternative ATP-producing stratagem resulted in the synthesis of copious amounts of PEP and pyruvate. The production of these metabolites was mediated via the enhanced activities of such enzymes as pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPC). The high energy PEP was subsequently converted into ATP with the aid of pyruvate phosphate dikinase (PPDK), phosphoenolpyruvate synthase (PEPS) and pyruvate kinase (PK) with the concomitant formation of pyruvate. The participation of the phospho-transfer enzymes like adenylate kinase (AK) and acetate kinase (ACK) ensured the efficiency of this O₂-independent energy-generating machinery. The increased activity of glycerol dehydrogenase (GDH) in the stressed bacteria provided the necessary precursors to fuel this process. This H₂O₂-induced anaerobic life-style fortuitously evokes metabolic networks to an effective pathway that can be harnessed into the synthesis of ATP, PEP and pyruvate. The bioconversion of glycerol to pyruvate will offer interesting economic benefit.     

Influence of Oxygen and Temperature on the Dark Inactivation of Pyruvate, Orthophosphate Dikinase and NADP-Malate Dehydrogenase in Maize

The influence of oxygen and temperature on the inactivation of pyruvate, Pi dikinase and NADP-malate dehydrogenase was studied in Zea mays. O₂ was required for inactivation of both pyruvate, Pi dikinase and NADP-malate dehydrogenase in the dark in vivo. The rate of inactivation under 2% O₂ was only slightly lower than that at 21% O₂. The in vitro inactivation of pyruvate, Pi dikinase, while dependent on adenine nucleotides (ADP + ATP), did not require O₂.

The postillumination inactivation of pyruvate, Pi dikinase in leaves was strongly dependent on temperature. As temperature was decreased in the dark, there was a lag period of increasing length (e.g. at 17°C there was a lag of about 25 minutes) before inactivation proceeded. Following the lag period, the rate of inactivation decreased with decreasing temperature. The half-time for dark inactivation was about 7 minutes at 32°C and 45 minutes at 17°C. The inactivation of pyruvate, Pi dikinase in vitro following extraction from illuminated leaves was also strongly dependent on temperature, but occurred without a lag period. In contrast, NADP-malate dehydrogenase was rapidly inactivated in leaves (half-time of approximately 3 minutes) during the postillumination period without a lag, and there was little effect of temperature between 10 and 32°C. The results are discussed in relation to known differences in the mechanism of activation/inactivation of the two enzymes.

     

Acetylcholinesterase molecular forms from rat and human erythrocyte membrane

Summary Inhibition of growth of PY815 mouse mastocytoma cells in vitro by N⁶, O²-dibutyryladenosine 3,5 cyclic monophosphate (DB cyclic AMP) was accompanied by increases in intracellular cyclic AMP and histamine and minor changes in cytosolic cyclic AMP-dependent histone kinase activity. However, DEAE-cellulose chromatography revealed substantial changes in the relative proportions of the principal cyclic AMP-dependent protein kinases and in free cyclic AMP-binding protein after DB cyclic AMP treatment. The activity of cytosolic cyclic AMP-dependent protein kinase type I (PK_I) decreased relative to cyclic AMP-dependent protein kinase type II (PK_II) and there was an increase in a cytosol cyclic AMP-binding protein with little associated protein kinase activity. The relative changes in activity of PK_I, PK_II and cyclic AMP binding protein after DB cyclic AMP treatment may reflect events important in the regulation of growth and differentiation of mast cells.Abbreviations DB cyclic AMP N⁶,O²-dibutyryladenosine-3, 5-cyclic monophosphate - cyclic AMP adenosine 3,5-cyclic monophosphate - PK_I type I cyclic AMP-dependent protein kinase - PK_II type II cyclic AMP-dependent protein kinase     

cGMP-stimulated protein kinase phosphorylates pyruvate kinase in an anoxia-tolerant marine mollusc

Summary A cystosolic protein kinase that phosphorylates pyruvate kinase (PK) in vitro has been identified in crude homogenates of heart, radular retractor, and foot muscle from the anoxia-tolerant marine whelk Busycon canaliculatum. Protein kinase action was measured by following changes in PK kinetic parameters: phosphorylated PK has a higher K _m value for phosphoenolpyruvate and a lower I₅₀ value for l-alanine. The crude protein kinase readily phosphorylated PK in a Mg²⁺-and ATP-dependent manner in the absence of any added effector. This activity was not affected by the addition of either cAMP (a stimulator of protein kinase A) or Ca²⁺ plus phorbol 12-myristate 13-acetate (stimulators of protein kinase C) to the incubation medium. Addition of cGMP to the homogenate, however, increased the rate of PK phosphorylation giving a 3–4-fold increase in the rate of change in PK kinetic parameters that was readily apparent after 5h. Complete time-courses of changes in PK kinetic parameters in the presence and absence of cGMP showed that cGMP increased the rate, but not the final extent, of PK phosphorylation. These results indicate that PK inactivation by enzyme phosphorylation in response to anoxia in whelk tissues may be mediated by a cyclic GMP stimulated protein kinase in response to changing levels of cGMP. This conclusion was further supported by data indicating that the total activity of protein kinase was the same in both anoxic and aerobic animals, and that the total PK phosphatase activity was also constant. Changes in PK phosphorylation during anoxia are not, therefore, the result of changes in the total amount of protein kinase or phosphatase.Abbreviations cAMP adenosine 3:5-monophosphate - cGMP guanosine 3:5-monophosphate - PK pyruvate kinase - PMA phorbol 12-myristate, 13-acetate - PEP phosphoenolpyruvate - K _m Michaelis constant - I ₅₀ inhibitor concentration that reduces enzyme activity by 50%     

Overexpression of Iron Superoxide Dismutase in Transformed Poplar Modifies the Regulation of Photosynthesis at Low CO2 Partial Pressures or Following Exposure to the Prooxidant Herbicide Methyl Viologen

Chloroplast-targeted overexpression of an Fe superoxide dismutase (SOD) from Arabidopsis thaliana resulted in substantially increased foliar SOD activities. Ascorbate peroxidase, glutathione reductase, and monodehydroascorbate reductase activities were similar in the leaves from all of the lines, but dehydroascorbate reductase activity was increased in the leaves of the FeSOD transformants relative to untransformed controls. Foliar H₂O₂, ascorbate, and glutathione contents were comparable in all lines of plants. Irradiance-dependent changes in net CO₂ assimilation and chlorophyll a fluorescence quenching parameters were similar in all lines both in air (21% O₂) and at low (1%) O₂. CO₂-response curves for photosynthesis showed similar net CO₂-exchange characteristics in all lines. In contrast, values of photochemical quenching declined in leaves from untransformed controls at intercellular CO₂ (Ci) values below 200 μL L⁻¹ but remained constant with decreasing Ci in leaves of FeSOD transformants. When the O₂ concentration was decreased from 21 to 1%, the effect of FeSOD overexpression on photochemical quenching at limiting Ci was abolished. At high light (1000 μmol m⁻² s⁻¹) a progressive decrease in the ratio of variable (F_v) to maximal (F_m) fluorescence was observed with decreasing temperature. At 6^oC the high-light-induced decrease in the F_v/F_m ratio was partially prevented by low O₂ but values were comparable in all lines. Methyl viologen caused decreased F_v/F_m ratios, but this was less marked in the FeSOD transformants than in the untransformed controls. These observations suggest that the rate of superoxide dismutation limits flux through the Mehler-peroxidase cycle in certain conditions.