Regulation of the plant-type 5'-adenylyl sulfate reductase by oxidative stress.

5'-Adenylyl sulfate (APS) reductase (EC 1.8.4.9) catalyzes a key reaction in the plant sulfate assimilation pathway leading to the synthesis of cysteine and the antioxidant glutathione. In Arabidopsis thaliana APS reductase is encoded by a family of three genes. In vitro biochemical studies revealed that the enzyme product derived from one of them (APR1) is activated by oxidation, probably through the formation of a disulfide bond. The APR1 enzyme is 45-fold more active when expressed in a trxB strain of Escherichia coli than in a trxB(+) wild type. The enzyme is inactivated in vitro by treatment with disulfide reductants and is reactivated with thiol oxidants. Redox titrations show that the regulation site has a midpoint potential of -330 mV at pH 8.5 and involves a two-electron redox reaction. Exposure of a variety of plants to ozone induces a rapid increase in APS reductase activity that correlates with the oxidation of the glutathione pool and is followed by an increase in free cysteine and total glutathione. During the response to ozone, the level of immunodetectable APS reductase enzyme does not increase. Treatment of A. thaliana seedlings with oxidized glutathione or paraquat induces APS reductase activity even when transcription or translation is blocked with inhibitors. The results suggest that a posttranslational mechanism controls APS reductase. A model is proposed whereby redox regulation of APS reductase provides a rapidly responding, self-regulating mechanism to control the glutathione synthesis necessary to combat oxidative stress.

Overproduction of Ascorbate Peroxidase in the Tobacco Chloroplast Does Not Provide Protection against Ozone.

Transgenic tobacco (Nicotiana tabacum cv Bel W3) plants were used to test the hypothesis that protection from O3 injury could be conferred by overproduction of ascorbate peroxidase (APX) in the chloroplast. The 10-fold increase in soluble APX activity in the chloroplast was expected to alleviate an implied increase in oxidative potential and prevent damage caused by O3. Three different O3 exposure experiments (one acute and two chronic) with two replicates each were conducted. APX activity in nontransgenic plants increased in response to chronic O3 exposure. However, most responses to O3 were similar between transgenic and nontransgenic plants. These included reductions in net photosynthesis and stomatal conductance, increases in ethylene emission and visible injury, and a decline in the level of the small subunit of ribulose-1,5-biphosphate carboxylase/oxygenase mRNA transcripts observed in response to the air pollutant in the acute and/or chronic experiments. No O3-induced effect on ribulose-1,5-biphosphate carboxylase/oxygenase quantity was observed in the chronic experiments. O3 did not induce acceleration of senescence, as expected from studies with most other species; rather, the tobacco plants rapidly developed necrotic lesions. Thus, overproduction of APX in the chloroplast did not protect this cultivar of tobacco from O3.

Molecular cloning and characterization of the genes encoding the L1 and L2 components of hemolysin BL from Bacillus cereus.

Hemolysin BL, which is composed of a binding component, B, and two lytic components, L1 and L2, is the enterotoxin responsible for the diarrheal food poisoning syndrome caused by strains of Bacillus cereus. To further characterize the toxin, we sought to clone and sequence the genes encoding the L1 and L2 proteins. A genomic library was screened with polyclonal antibody to the L1 and L2 proteins to identify recombinant clones containing the genes. Five clones reacted with the antibody to L2, but none reacted with the antibody to L1. Southern hybridization analysis with oligonucleotide probes designed from the N-terminal amino acid sequences of the L1 and L2 proteins, in conjunction with immunoblot and nucleotide sequence analysis, revealed that the recombinant plasmid from one of the clones contained two genes, hblC and hblD, which encode L2 and L1, respectively. The two genes are arranged in tandem and are separated by only 37 bases. The gene which encodes the B component of hemolysin BL (hblA) is located immediately downstream from the gene encoding the L1 protein. Northern blot analysis of B. cereus RNA showed a 5.5-kb transcript which hybridized with DNA fragments internal to, or including a portion of, the coding sequences of the B, L1, and L2 genes, suggesting that the clustered genes which encode the components of hemolysin BL are cotranscribed and constitute an operon.

Salt and oxidative stress: similar and specific responses and their relation to salt tolerance in citrus.

Salt damage to plants has been attributed to a combination of several factors including mainly osmotic stress and the accumulation of toxic ions. Recent findings in our laboratory showed that phospholipid hydroperoxide glutathione peroxidase (PHGPX), an enzyme active in the cellular antioxidant system, was induced by salt in citrus cells and mainly in roots of plants. Following this observation we studied the two most important enzymes active in elimination of reactive oxygen species, namely, superoxide dismutase (SOD) and ascorbate peroxidase (APX), to determine whether a general oxidative stress is induced by salt. While Cu/Zn-SOD activity and cytosolic APX protein level were similarly induced by salt and methyl viologen, the response of PHGPX and other APX isozymes was either specific to salt or methyl viologen, respectively. Unlike PHGPX, cytosolic APX and Cu/Zn-SOD were not induced by exogenously added abscisic acid. Salt induced a significant increase in SOD activity which was not matched by the subsequent enzyme APX. We suggest that the excess of H2O2 interacts with lipids to form hydroperoxides which in turn induce and are removed by PHGPX. Ascorbate peroxidase seems to be a key enzyme in determining salt tolerance in citrus as its constitutive activity in salt-sensitive callus is far below the activity observed in salt-tolerant callus, while the activities of other enzymes involved in the defence against oxidative stress, namely SOD, glutathione reductase and PHGPX, are essentially similar.

Kinetic and spectral properties of pea cytosolic ascorbate peroxidase.

Sufficient highly purified native pea cytosolic ascorbate peroxidase was obtained to characterize some of its kinetic and spectral properties. Its rate constant for compound I formation from reaction with H2O2 is 4.O x 10(7) M-1 s-1, somewhat faster than is typical for peroxidases. Compound I has the typical optical spectrum of an iron(IV)-porphyrin-pi-cation radical, despite considerable homology with yeast cytochrome c peroxidase. The rate constant for compound I reduction by ascorbate is extremely fast (8.0 x 10(7) M-1 S-1 at pH 7.8), again in marked contrast to the behavior of the yeast enzyme. The pH-rate profile for compound I formation indicates a pKa value of 5.0 for a group affecting the active site reaction.

Overexpression of Copper/Zinc Superoxide Dismutase in the Cytosol of Transgenic Tobacco Confers Partial Resistance to Ozone-Induced Foliar Necrosis.

Ozone damage to plants has been attributed to the action of oxygen free-radicals and other ozone degradation products against which cellular antioxidant systems have been considered to be a front-line defense. The activity of superoxide dismutase (SOD), one such antioxidant, has been shown to increase in ozonated plants. Past work with pea (Pisum sativum L.) in our laboratory showed that the cytosolic Cu/Zn-SOD isoform and its transcript were most responsive to ozone, compared to chloroplastic Cu/Zn-SOD. In the current work we tested the hypothesis that plants that constitutively overexpress cytosolic SOD are more tolerant of ozone. Pea cytosolic Cu/Zn-SOD was overproduced in the cytosol of two cultivars of transformed tobacco (Nicotiana tabacum), Bel W3 and Wisconsin 38. Young and recently expanded leaves of transgenic plants of both cultivars showed less foliar necrosis than nontransformed controls when exposed to acute doses of ozone. We suggest that this may demonstrate the importance of Cu/Zn-SOD in the cytosol as a protector of the integrity of the plasma membrane and possibly other cellular constituents.

Molecular cloning and characterization of a cDNA encoding pea monodehydroascorbate reductase.

Monodehydroascorbate radicals are generated in plant cells enzymatically by the hydrogen peroxide scavenging enzyme, ascorbate peroxidase, and nonenzymatically via the univalent oxidation of ascorbate by superoxide, hydroxyl, and various organic radicals. Regeneration of ascorbate is achieved by monodehydroascorbate reductase (EC 1.6.5.4) using NAD(P)H as an electron donor or, alternatively, by a set of two coupled reactions requiring dehydroascorbate reductase, glutathione reductase, glutathione, and NAD(P)H. As monodehydroascorbate reductase is a key enzyme in maintaining reduced pools of ascorbate, an important antioxidant, we undertook this study to learn more about its structure, function, and regulation. Herein we report the molecular cloning and characterization of a cDNA encoding monodehydroascorbate reductase of pea (Pisum sativum L.). The cDNA encodes a 433-amino acid polypeptide that shows, respectively, 73 and 87% identity with peptide fragments from soybean and cucumber monodehydroascorbate reductase. Monodehydroascorbate reductase contains the NAD(P)H and FAD binding domains of other flavin oxidoreductases. The cloned enzyme lacks a transit peptide, but the sequence of the carboxyl terminus is Ser-Lys-Ile, similar to the targeting motif found in peroxisomal proteins. When expressed in Escherichia coli fused to maltose-binding protein, monodehydroascorbate reductase has enzymatic properties comparable with purified soybean and cucumber monodehydroascorbate reductase. Northern blot analysis shows that the monodehydroascorbate reductase transcript is 1.6 kilobase in size and is expressed at relatively low levels in all plant tissues examined.

Role of signal peptides in targeting of proteins in cyanobacteria.

Proteins of cyanobacteria may be transported across one of two membrane systems: the typical eubacterial cell envelope (consisting of an inner membrane, periplasmic space, and an outer membrane) and the photosynthetic thylakoids. To investigate the role of signal peptides in targeting in cyanobacteria, Synechococcus sp. strain PCC 7942 was transformed with vectors carrying the chloramphenicol acetyltransferase reporter gene fused to coding sequences for one of four different signal peptides. These included signal peptides of two proteins of periplasmic space origin (one from Escherichia coli and the other from Synechococcus sp. strain PCC 7942) and two other signal peptides of proteins located in the thylakoid lumen (one from a cyanobacterium and the other from a higher plant). The location of the gene fusion products expressed in Synechococcus sp. strain PCC 7942 was determined by a chloramphenicol acetyltransferase enzyme-linked immunosorbent assay of subcellular fractions. The distribution pattern for gene fusions with periplasmic signal peptides was different from that of gene fusions with thylakoid lumen signal peptides. Primary sequence analysis revealed conserved features in the thylakoid lumen signal peptides that were absent from the periplasmic signal peptides. These results suggest the importance of the signal peptide in protein targeting in cyanobacteria and point to the presence of signal peptide features conserved between chloroplasts and cyanobacteria for targeting of proteins to the thylakoid lumen.

Regulation of pea cytosolic ascorbate peroxidase and other antioxidant enzymes during the progression of drought stress and following recovery from drought.

The molecular mechanism underlying the regulation of the expression of Apxl, the gene encoding cytosolic ascorbate peroxidase, as well as other antioxidant enzymes, was studied during the progression of drought stress and following recovery from drought. Increase in steady-state transcript levels of the cytosolic isozymes of ascorbate peroxidase and Cu/Zn-superoxide dismutase paralleled the increase in stomatal resistance during drought stress, but was even more dramatically enhanced following recovery from drought. Cytosolic Cu/Zn-superoxide dismutase and ascorbate peroxidase and chloroplastic Cu/Zn-superoxide dismutase protein and activity increased during drought stress and following recovery. In contrast, catalase activity increased during drought stress but returned to normal levels following recovery. During recovery from drought stress, cytosolic ascorbate peroxidase expression was regulated post-transcriptionally at the level of protein synthesis. The transcription rate of the Apxl gene, as determined by nuclear run-on assay, increased during drought stress and at 10 h following rewatering.

Molecular cloning and characterization of the hblA gene encoding the B component of hemolysin BL from Bacillus cereus.

Previous evidence suggests that hemolysin BL, which consists of a binding component, B, and two lytic components, L1 and L2, is the enterotoxin responsible for the diarrheal form of gastroenteritis caused by food-borne strains of Bacillus cereus. To prove that hemolysin BL and the enterotoxin are the same requires large amounts of these components free of other B. cereus proteins. For this purpose, we sought to clone the gene encoding the B component and to express it in Escherichia coli. A partial genomic library was constructed and a 29-base, 1,152-fold-degenerate oligonucleotide probe, designed from the N-terminal amino acid sequence of the B component, was used to identify recombinant clones containing the gene. Detection of gene products reactive with a monoclonal antibody specific for the B component and analysis of the nucleotide sequence confirmed that isolated clones, reactive with the oligonucleotide probe, did contain the gene encoding the B component. The protein, expressed in E. coli, apparently from the B. cereus promoter, produces a ring-shaped zone of hemolysis when combined with purified L components from B. cereus, a reaction typical of hemolysin BL. Northern (RNA) blot analysis of B. cereus RNA showed a large (5.1-kb) transcript which hybridized with a 500-bp probe internal to the B-component-coding sequence, suggesting that the hblA gene encoding the B component may be transcribed as part of a polycistronic message, possibly including the structural genes for the two lytic components. Higher levels of expression and disruption of the hblA gene are being pursued to resolve whether hemolysin BL is indeed the enterotoxin.

Detection of ascorbate peroxidase activity in native gels by inhibition of the ascorbate-dependent reduction of nitroblue tetrazolium.

A method for the detection of ascorbate peroxidase activity in native electrophoretic gels is described. The assay is based on the ability of ascorbate peroxidase to prevent the ascorbate-dependent reduction of nitroblue tetrazolium in the presence of H2O2. The method was found to be both sensitive (detection of less than 0.01 units of ascorbate peroxidase activity) and specific for ascorbate peroxidase activity. The application of the method for the detection of ascorbate peroxidase activity in protein extracts from several plant sources was investigated by comparing staining for activities of ascorbate peroxidase, horseradish peroxidase, and ascorbate oxidase and by immunodetection of ascorbate peroxidase in these extracts.

Molecular cloning and characterization of a gene encoding pea cytosolic ascorbate peroxidase.

A gene encoding cytosolic ascorbate peroxidase (ApxI) from pea (Pisum sativum L.) was isolated and its nucleotide sequence determined. By homologous alignment between the ApxI cDNA (Mittler, R., and Zilinskas, B. (1991) FEBS Lett. 289, 257-259) and the genomic clone, positions of introns and exons were determined. The isolated ApxI gene was found to contain 9 introns, the first of which was located within the 5'-untranslated region of the mRNA. Southern blot analysis of pea genomic DNA suggests that in pea cytosolic ascorbate peroxidase is encoded by a single copy gene. Steady state ApxI transcript levels were found to increase in response to several stresses imposed by drought, heat, and application of ethephon, abscisic acid, and the superoxide-generating agent paraquat. Increases in ascorbate peroxidase activity in response to stresses were less marked than changes observed in transcript levels; cytosolic ascorbate peroxidase protein levels measured by immunoblot analysis remained unchanged.

The Antiozonant Ethylenediurea Does Not Act via Superoxide Dismutase Induction in Bean.

It has been proposed that the mode of action of ethylenediurea, a very effective antiozonant, is via an increase in the antioxidant enzyme superoxide dismutase (EH Lee, JH Bennett [1982] Plant Physiol 69: 1444-1449). Data presented here refute that hypothesis. No ethylenediurea-associated increases in Cu/Zn-superoxide dismutase or Mn-superoxide dismutase activity, nor in steady-state Cu/Zn-superoxide dismutase protein levels, were found in soluble extracts of bean (Phaseolus vulgaris L. cv Bush Blue Lake 290) leaves. However, the cytosolic Cu/Zn-superoxide dismutase increased as a result of ozone fumigation and subsequent injury. Also noted was a developmentally related difference between chloroplastic and cytosolic Cu/Zn-superoxide dismutase, the latter declining during maturation of the leaf.

Purification and characterization of pea cytosolic ascorbate peroxidase.

The cytosolic isoform of ascorbate peroxidase was purified to homogeneity from 14-day-old pea (Pisum sativum L.) shoots. The enzyme is a homodimer with molecular weight of 57,500, composed of two subunits with molecular weight of 29,500. Spectral analysis and inhibitor studies were consistent with the presence of a heme moiety. When compared with ascorbate peroxidase activity derived from ruptured intact chloroplasts, the purified enzyme was found to have a higher stability, a broader pH optimum for activity, and the capacity to utilize alternate electron donors. Unlike classical plant peroxidases, the cytosolic ascorbate peroxidase had a very high preference for ascorbate as an electron donor and was specifically inhibited by p-chloromercurisulfonic acid and hydroxyurea. Antibodies raised against the cytosolic ascorbate peroxidase from pea did not cross-react with either protein extracts obtained from intact pea chloroplasts or horseradish peroxidase. The amino acid sequence of the N-terminal region of the purified enzyme was determined. Little homology was observed among pea cytosolic ascorbate peroxidase, the tea chloroplastic ascorbate peroxidase, and horseradish peroxidase; homology was, however, found with chloroplastic ascorbate peroxidase isolated from spinach leaves.

Molecular cloning and nucleotide sequence analysis of a cDNA encoding pea cytosolic ascorbate peroxidase.

A cDNA clone encoding the cytosolic ascorbate peroxidase of pea (Pisum sativum L.) was isolated and its nucleotide sequence determined. While ascorbate peroxidase shares limited overall homology with other peroxidases, significant homology with all known peroxidases was found in the vicinity of the putative active site.

Overproduction of petunia chloroplastic copper/zinc superoxide dismutase does not confer ozone tolerance in transgenic tobacco.

Transgenic tobacco (Nicotiana tabacum cultivar W38) plants that overproduce petunia chloroplastic Cu/Zn superoxide dismutase were exposed to ozone dosages that injure control tobacco plants. Based on foliar injury ratings, there was no consistent protection provided to the transgenic plants. These data indicate that an increase in the chloroplastic Cu/Zn superoxide dismutase alone is not sufficient to reduce ozone toxicity.

Construction and characterization of cyanobacterial mutants lacking the manganese-stabilizing polypeptide of photosystem II.

Mutants of the cyanobacterium Synechocystis sp. Pasteur Culture Collection (PCC) 6803 that specifically lack the extrinsic 33-kDa manganese-stabilizing polypeptide of the photosystem II oxygen-evolving complex have been constructed by two independent methods. Cartridge mutagenesis was used to insertionally inactivate the psbO gene of one mutant and completely delete the psbO gene of the other mutant. These mutants have no detectable manganese-stabilizing polypeptide, but they do accumulate steady-state levels of the intrinsic photosystem II polypeptides D1, D2, and CP-43 that are comparable to wild-type, as determined by immunoblot analysis. Measurement of the evolution of the relative quantum yields of chlorophyll fluorescence following actinic flash excitation indicates that though the concentration of reaction centers in mutant cells is comparable to that of wild-type cells, approximately 40% of these centers harbor a fluorescence-quenching species other than P680+. The mutants are capable of photoautotrophic growth at a slower rate than that of wild-type. Under conditions of Ca2+ depletion where wild-type growth is unaffected, the mutants are unable to grow at all. The manganese-stabilizing protein, therefore, enhances the binding of Ca2+ or protects the reaction center at low Ca2+ concentrations. The mutant evolve oxygen at approximately 70% of the wild-type rate, but are completely photoinactivated by high light intensities. Our results indicate that the manganese-stabilizing polypeptide is not absolutely required for photosystem II assembly or function in cyanobacteria, but its absence does lead to an enhanced sensitivity to photoinhibition.

Orientation and linear dichroism of Mastigocladus laminosus phycocyanin trimer and Nostoc sp. phycocyanin dodecamer in stretched poly(vinyl alcohol) films.

The linear dichroism (LD) spectra of the C-phycocyanin (C-PC) trimer disks oriented in poly(vinyl alcohol) films (PVA) at room temperature and at 95 K were determined. Utilizing the known atomic coordinates of the chromophores (Schirmer, T., Bode, W. and Huber, R. (1987) J. Mol. Biol. 196, 677-695) and theoretical estimates of the orientations of the transition dipole moments relative to the molecular framework, the LD spectra were simulated using the pairwise exciton interaction model of Sauer and Scheer (Biochim. Biophys. Acta 936 (1988) 157-170); in this model, the alpha 84 and beta 84 transition moments are coupled by an exciton mechanism, while the beta 155 chromophore remains uncoupled. Linear dichroism spectra calculated using this exciton model, as well as an uncoupled chromophore (molecular) model, were compared with experimental LD spectra. Satisfactory qualitative agreement can be obtained in both the exciton and molecular models using somewhat different relative values of the theoretically estimated magnitudes of the beta 155 oscillator strength. Because the relative contributions of each of the chromophores (and thus exciton components) to the overall absorption of the C-PC trimer are not known exactly, it is difficult to differentiate successfully between the molecular and exciton models at this time. The linear dichroism spectra of PC dodecamers derived from phycobilisomes of Nostoc sp. oriented in stretched PVA films closely resemble those of the C-PC trimers from Mastigocladus laminosus, suggesting that the phycocyanin chromophores are oriented in a similar manner in both cases, and that neither linker polypeptides nor the state of aggregation have a significant influence on these orientations and linear dichroism spectra. The LD spectra of oriented phycocyanins in stretched PVA films at low temperatures (95 K) appear to be of similar quality and magnitude as the LD spectra of single C-PC crystals (Schirmer, T. and Vincent, M.G. (1987) Biochim. Biophys. Acta 893, 379-385).

The relationship between EDU pre-treatment and C2H4 evolution in ozonated pea plants.

A soil drench of [Formula: see text] (EDU) (150 ppm) applied to 'Progress No. 9' pea plants 24 h before an acute ozone exposure (0.25 ppm, 4 h) completely protected the foliage from visible symptoms normally induced by the pollutant. In the absence of ozone, EDU-treated plants were found to emit the same amount of C(2)H(4) as plants not treated with EDU. Based on this evidence, EDU-induced tolerance to ozone could not have been attributed to the prevention of an interaction between ethylene and ozone (sensu Mehlhorn and Wellburn). In the presence of ozone, EDU-treated plants did not emit the burst of C(2)H(4) that normally occurs (sensu Craker), extending the observation that EDU-treated plants do not exhibit the adverse physiological responses normally caused by ozone. The classic C(2)H(4) biosynthesis inhibitor aminoethoxyvinylglycine (AVG) did not prevent ozone phytotoxicity, although it significantly reduced ethylene emission from the ozonated tissue.