Silicomolybdate and silicotungstate mediated dichlorophenyldimethylurea-insensitive photosystem II reaction: electron flow, chlorophyll a fluorescence and delayed light emission changes.

We have investigated the possible role of silicomolybdate and silicotungstate as acceptors of electrons in chloroplasts directly from Q, the primary electron acceptor of Photosystem II. Our data show: 1. Either of these compounds can accept electrons directly from Q in a 3-(3', 4'-dichlorophenyl)-1, 1-dimethylurea (DCMU)-insensitive electron transport; however, the DCMU insensitivity is only short-lived, so initial rates must be used exclusively. 2. High concentrations of these silico compounds act as direct chemical quenchers of chlorophyll a fluorescence, but lower concentrations which also mediate O2 evolution affect only the variable component of fluorescence in a manner suggestive of their electron-accepting capabilities. 3. Measurements of delayed light emission confirm the conclusions made from the fluorescence data. Also, they show the role of Q in delayed light emission as hydroxylamine data of other investigations have shown the role of Z, the electron donor of Photosystem II. 4. Silico compounds appear to be acting as electron acceptors and not as simple membrane modifiers allowing other acceptors to support a DCMU-insensitive electron transport.

Spectral analysis of allophycocyanin I, II, III and B from Nostoc sp. phycobilisomes.

Low temperature (-196C) and room temperature (25C) absorption spectra of a family of allophycocyanin spectral forms isolated from Nostoc sp. phycobilisomes as well as of the phycobilisomes themselves have been analyzed by Gaussian curve-fitting. Allophycocyanin I and B share long wavelength components at 668 and 679 nm, bands that are absent from allophycocyanin II and III. These long wavelength absorption components are apparently responsible for the 20 nm difference between the 680 nm fluorescence emission maximum of allophycocyanin I and B and the 660 nm maximum of II and III. This indicates that allophycocyanin I and B are the final acceptors of excitation energy in the phycobilisome and the excitation energy transfer bridge linking the phycobilisome with the chlorophyll-containing thylakoid membranes. These Gaussian components are also found in resolved spectra of phycobilisomes, are arguing against this family of allophycocyanin molecules being artifactual products of protein purification procedures.

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).

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.

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 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.

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.

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.

Isolation and Characterization of the Central Component of the Phycobilisome Core of Nostoc sp.

Freshly isolated allophycocyanin is recovered from linear sucrose gradients made in 0.75 molar potassium phosphate buffer (pH 7.0) in three sizes: 19s, 10.3s, and 5.5s. The largest aggregate is a complex of a 680 nm fluorescing allophycocyanin I in the form (alphabeta)(3)gamma, where gamma is the 95 kilodalton (kD) polypeptide, and two 660 nanometer fluorescing allophycocyanin II (alphabeta)(3) molecules; the complex, stabilized in high phosphate concentrations, fluoresces maximally at 675 nanometers. The 10.3s fraction is a hexamer of allophycocyanin of the 660 nanometer fluorescing type, perhaps attached through two polypeptides of 46 kD and 44 kD. The 5.5s component of the allophycocyanin pool is the usual trimeric form of allophycocyanin (alphabeta)(3). A similar 19s fraction is the major component of allophycocyanin I isolated under optimum conditions in the presence of the protease inhibitor, phenylmethylsulfonylfluoride. This 19s fraction is apparently a central component of the core of the phycobilisome with its 95 kD polypeptide the attachment point of the phycobilisome and membrane. The 95 kD polypeptide has both long wavelength absorption and fluorescence bands which seem to account for the long wavelength fluorescence properties of allophycocyanin I.

Cloning, nucleotide sequence and mutational analysis of the gene encoding the Photosystem II manganese-stabilizing polypeptide of Synechocystis 6803.

Affinity purified, polyclonal antibodies raised against the Photosystem II 33 kDa manganese-stabilizing polypeptide of the spinach oxygen-evolving complex were used to isolate the gene encoding the homologous protein from Synechocystis 6803. Comparison of the amino acid sequence deduced from the Synechocystis psb1 nucleotide sequence with recently published sequences of spinach and pea confirms the homology indicated by antigenic cross-reactivity and shows that the cyanobacterial and higher plant sequences are 43% identical and 63% conserved. Regions of identity, varying in length from 1 to 10 consecutive residues, are distributed throughout the protein. The 28 residues at the amino terminus of the psb1 gene product, characteristic of prokaryotic signal peptides, show homology with the carboxyl-terminal third of the transit sequences of pea and spinach and are most likely needed for the transport of the manganese-stabilizing protein across the thylakoid membrane to its destination of the lumen. Synechocystis mutants which contain a kanamycin resistance gene cassette inserted into the coding region for the 32 kDa polypeptide were constructed. These mutants contain no detectable 32 kDa polypeptide, do not evolve oxygen, and are incapable of photoautotrophic growth.

Immunological conservation of phycobilisome rod linker polypeptides.

Monospecific polyclonal antibodies raised against the phycobilisome rod linker polypeptides of Nostoc sp. were used to assess structural similarities among phycobilisome linker proteins from diverse, phycobilisome-containing organisms. While a remarkable conservation of antigenic determinants was demonstrated for two of these rod linker proteins, the third linker polypeptide appeared to be conserved only among closely related species.

Cloning and characterization of a cDNA encoding the chloroplastic copper/zinc-superoxide dismutase from pea.

A cDNA encoding the chloroplastic copper/zinc-superoxide dismutase of pea (Pisum sativum L.) was isolated from a cDNA library constructed in lambda gt11 from leaf mRNA. Nucleotide sequence analysis of the 875-base-pair clone revealed that it contained the complete coding sequence of the mature superoxide dismutase isozyme subunit, along with sequence information for a 48-amino acid N-terminal transit peptide. The deduced amino acid sequence of the mature subunit proved to be 64-87% homologous with amino acid sequences of copper/zinc-superoxide dismutases from other plant species. In vitro transcription, followed by cell-free translation, of the cDNA resulted in the formation of a 23.5-kDa precursor polypeptide, which, upon incubation with isolated pea chloroplasts, was imported and processed to its mature subunit molecular mass of 17.4 kDa.

Reversal of 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition of carbon dioxide fixation in spinach chloroplasts and protoplasts by dicarboxylic acids.

3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) inhibition of (14)CO(2) fixation in isolated intact spinach (Spinacia oleracea L.) chloroplasts was reversed (by about 34%) by l-malate but not by oxaloacetate (OAA). However, OAA reversed the DCMU inhibition in spinach protoplasts indicating an extrachloroplastic enzyme requirement. Extrachloroplastic OAA reduction was coupled with external dihydroxyacetone phosphate (DHAP) oxidation, and the malate formed from such coupling might then enter the chloroplasts. Evidence was presented using ruptured protoplasts that the export of recently formed 3-phosphoglyceric acid (PGA) out of chloroplasts in exchange for external DHAP was reversed by excess OAA. The PGA/DHAP shuttle across the chloroplast envelope was found to be regulated by the external concentrations of DHAP and OAA.

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.

Noncovalent Intermolecular Forces in Phycobilisomes of Porphyridium cruentum.

Using sensitized fluorescence as a measure of intactness of phycobilisomes isolated from Porphyridium cruentum, the effects of various environmental perturbations on phycobilisome integrity were investigated. The rate of phycobilisome dissociation in 0.75 ionic strength sodium salts proceeds in the order: SCN(-) > NO(3) (-) > Cl(-) > C(6)H(5)O(7) (3-) > SO(4) (2-) > PO(4) (3-), as predicted from the lyotropic series of anions and their effects on hydrophobic interactions in proteins. Similarly, increasing temperature (to 30 C) and pH values approaching the isoelectric points of the biliproteins stabilize phycobilisomes. Deuterium substitution at exchangeable sites on the phycobiliproteins decreases the rate of phycobilisome dissociation, while substitution at nonexchangeable sites increases rates of dissociation. It is concluded that hydrophobic intermolecular interactions are the most important forces in maintaining the phycobilisome structure. Dispersion forces also seem to contribute to phycobilisome stabilization. The adverse effects of electrostatic repulsion must not be ignored; however, it seems that the requirement of phycobilisomes of high salt concentrations is not simply countershielding of charges on the proteins.

Chlorophyll-Protein Complexes of the Cyanophyte, Nostoc sp.

Four chlorophyll-protein complexes have been resolved from the cyanophyte, Nostoc sp., by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis at 4 C. Complexes solubilized by SDS from Spinacia oleracea were run for comparison. As has been well documented, the P700-chlorophyll a-protein complex from the higher plant and blue-green algal samples are similar, and the light-harvesting pigment protein complex is present only in the former. Most noteworthy are two closely migrating chlorophyll proteins in Nostoc sp. which have approximately the same mobility as a single chlorophyll-protein band resolvable from spinach. The absorption maximum of the complex from spinach is at 667 nanometers, and those of the two complexes from Nostoc sp. are at 667 and 669 nanometers; the fluorescence emission maximum at -196 C is at 685 nanometers, and the 735 nanometer fluorescence peak, characteristic of the P700-chlorophyll a-protein complex, is absent. The apoproteins of these new complexes from Nostoc sp. and spinach are in the kilodalton range. It appears that at least one of these two chlorophyll-protein complexes from Nostoc sp. compares with those recently described by others from higher plants and green algae as likely photosystem II complexes, perhaps containing P680, although no photochemical data are yet available.

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.

Role of the Colorless Polypeptides in Phycobilisome Assembly in Nostoc sp.

We have identified the function of the ;extra' polypeptides involved in phycobilisome assembly in Nostoc sp. These phycobilisomes, as those of other cyanobacteria, are composed of an allophycocyanin core, phycoerythrin- and phycocyanin-containing rods, and five additional polypeptides of 95, 34.5, 34, 32, and 29 kilodaltons. The 95 kilodalton polypeptide anchors the phycobilisome to the thylakoid membrane (Rusckowski, Zilinskas 1982 Plant Physiol 70: 1055-1059); the 29 kilodalton polypeptide attaches the phycoerythrin- and phycocyanin-containing rods to the allophycocyanin core (Glick, Zilinskas 1982 Plant Physiol 69: 991-997). Two populations of rods can exist simultaneously or separately in phycobilisomes, depending upon illumination conditions. In white light, only one type of rod with phycoerythrin and phycocyanin in a 2:1 molar ratio is synthesized. Associated with this rod are the 29, 32, and 34 kilodalton colorless polypeptides; the 32 kilodalton polypeptide links the two phycoerythrin hexamers, and the 34 kilodalton polypeptide attaches a phycoerythrin hexamer to a phycocyanin hexamer. The second rod, containing predominantly phycocyanin, and the 34.5 and 29 kilodalton polypeptides, is synthesized by redlight-adapted cells; the 34.5 kilodalton polypeptide links two phycocyanin hexamers. These assignments are based on isolation of rods, dissociation of these rods into their component biliproteins, and analysis of colorless polypeptide composition, followed by investigation of complexes formed or not formed upon their recombination.

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.