Partial purification and characterization of an ascorbate-reducible b-type cytochrome from the plasma membrane of Arabidopsis thaliana leaves.

The plant plasma membrane (PM) contains more than one b-type cytochrome. One of these proteins has a rather high redox potential (can be fully reduced by ascorbate) and is capable of transporting electrons through the PM. Four genes encoding proteins with considerable homology to the sequences of cytochrome b(561) proteins in the animal chromaffin granule membrane have recently been identified in the genome of Arabidopsis thaliana. In order to characterize the cytochrome b(561) located in the Arabidopsis PM, first PM vesicles were purified by aqueous polymer two-phase partitioning from the leaves of 9-week-old A. thaliana. PM proteins were solubilized by nonionic detergent, and the fully ascorbate-reducible b-type cytochrome was partially purified by anion-exchange chromatography steps. Potentiometric redox titration of the fraction, containing the fully ascorbate-reducible b-type cytochrome after the first anion-exchange chromatography step, revealed the presence of two hemes with redox potentials of 135 mV and 180 mV, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fractions containing the fully ascorbate-reducible b-type cytochrome after the second anion-exchange chromatography step revealed the presence of a single polypeptide band at about 120 kDa. However, heat treatment (15 min, 90 degrees C) before electrophoresis was able to split the 120 kDa band into two bands with molecular masses of about 23 and 28 kDa. These values are lower than the apparent molecular mass for the fully ascorbate-reducible b-type cytochrome purified from Phaseolus vulgarishypocotyls (about 52 kDa) but are in good agreement with those characteristic for the cytochrome b(561) proteins purified from chromaffin granule membranes (about 28 kDa) and the four polypeptides predicted from the Arabidopsis genome (24-31 kDa).

Copper-mediated oxidative burst in Nicotiana tabacum L. cv. Bright Yellow 2 cell suspension cultures.

In cell suspension cultures of Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) a rapid and concentration-dependent accumulation of H(2)O(2) is induced by excess concentrations of copper (up to 100 microM). This specific and early response towards copper stress was shown to be extracellular. Addition of 300 U of catalase per ml decreased the level of H(2)O(2). Superoxide dismutase (5 U/ml) induced an increase in H(2)O(2) production by 22.2%. This indicates that at least part of the H(2)O(2) is produced by dismutation of superoxide. Pretreatment of the cell cultures with the NAD(P)H oxidase inhibitors diphenylene iodonium (2 and 10 microM) and quinacrine (1 and 5 mM) prevented the generation of H(2)O(2) under copper stress for 90%. The influence of the pH on the H(2)O(2) production revealed the possible involvement of cell-wall-dependent peroxidases in the generation of reactive oxygen species after copper stress.

b-type cytochromes in plasma membranes of Phaseolus vulgaris hypocotyls, Arabidopsis thaliana leaves, and Zea mays roots.

The plasma membrane of higher plants contains more than one kind of b-type cytochromes. One of these has a high redox potential and can be fully reduced by ascorbate. This component, the cytochrome b561 (cyt b561), has its characteristic alpha-band absorbance close to 561 nm wavelength at room temperature. Cyt b561 was first isolated from etiolated bean hook plasma membranes by two consecutive anion exchange chromatography steps. During the first step performed at pH 8, cyt b561 did not bind to the anion exchange column, but other b-type cytochromes did. In the second step performed at pH 9.9, cyt b561 was bound to the column and was eluted from the column at an ionic strength of about 100 mM KCl. However, when the same protocol was applied to the solubilized plasma membrane proteins from Arabidopsis thaliana leaves and maize roots, the ascorbate-reducible cyt b561 bound already to the first anion exchange column at pH 8 and was eluted also at an ionic strength of about 100 mM KCl. Other b-type cytochromes than the ascorbate-reducible cyt b561 from the plasma membranes of Arabidopsis leaves and maize roots showed similar chromatographic characteristics to that of bean hypocotyls. These results demonstrate particular differences in the chromatographic behavior of cyt b561 from different sources.

Higher-plant plasma membrane cytochrome b561: a protein in search of a function.

During the past twenty years evidence has accumulated on the presence of a specific high-potential, ascorbate-reducible b-type cytochrome in the plasma membrane (PM) of higher plants. This cytochrome is named cytochrome b561 (cyt b561) according to the wavelength maximum of its alpha-band in the reduced form. More recent evidence suggests that this protein is homologous to a b-type cytochrome present in chromaffin granules of animal cells. The plant and animal cytochromes share a number of strikingly similar features, including the high redox potential, the ascorbate reducibility, and most importantly the capacity to transport electrons across the membrane they are located in. The PM cyt b561 is found in all plant species and in a variety of tissues tested so far. It thus appears to be a ubiquitous electron transport component of the PM. The cytochromes b561 probably constitute a novel class of transmembrane electron transport proteins present in a large variety of eukaryotic cells. Of particular interest is the recent discovery of a number of plant genes that show striking homologies to the genes coding for the mammalian cytochromes b561. A number of highly relevant structural features, including hydrophobic domains, heme ligation sites, and possible ascorbate and monodehydroascorbate binding sites are almost perfectly conserved in all these proteins. At the same time the plant gene products show interesting differences related to their specific location at the PM, such as potentially N-linked glycosylation sites. It is also clear that at least in several plants cyt b561 is represented by a multigene family. The current paper presents the first overview focusing exclusively on the plant PM cyt b561, compares it to the animal cyt b561, and discusses the possible physiological function of these proteins in plants.

Purification of cytochrome b-561 from bean hypocotyls plasma membrane. Evidence for the presence of two heme centers.

The high potential, ascorbate-reducible b-type cytochrome of plant plasma membranes, named cytochrome b-561, has been purified to homogeneity from etiolated bean hypocotyls. The pure protein migrated in denaturing electrophoresis as a broad band of approximately 55 kDa, and was found to be glycosylated. Optical redox titrations of partially purified cytochrome b-561 indicated that it contains two hemes with similar spectral features, but distinct midpoint redox potentials (E(m7)+135 mV and +206 mV, respectively). The presence of two heme centers in cytochrome b-561 is consistent with its role in electron transfer across plant plasma membranes.

Transport and action of ascorbate at the plant plasma membrane.

The plasmalemma is both a bridge and a barrier between the cytoplasm and the outside world. It is a dynamic interface that perceives and transmits information concerning changes in the environment to the nucleus to modify gene expression. In plants, ascorbate is an essential part of this dialogue. The concentration and ratio of reduced to oxidized ascorbate in the apoplast, for example, possibly modulates cell division and growth. The leaf apoplast contains millimolar amounts of ascorbate that protect the plasmalemma against oxidative damage. The apoplastic ascorbate-dehydroascorbate redox couple is linked to the cytoplasmic ascorbate-dehydroascorbate redox couple by specific transporters for either or both metabolites. Although evidence about the mechanisms driving ascorbate or dehydroascorbate transport remains inconclusive, these carrier proteins potentially regulate the level and redox status of ascorbate in the apoplast. The redox coupling between compartments facilitated by these transport systems allows coordinated control of key physiological responses to environmental cues.

Carrier mediated uptake of dehydroascorbate into higher plant plasma membrane vesicles shows trans-stimulation.

The activity of the ascorbate (Asc) carrier of purified Phaseolus plasma membranes is demonstrated to be highly stimulated when membrane vesicles are preloaded with Asc. Asc transport is inhibited by DTT but is not affected by glutathione or ferricyanide, indicating that dehydroascorbate (DHA) is the preferred species for uptake. Asc transport in the loaded vesicles showed saturable kinetics with an apparent affinity constant of 24 microM and maximal uptake rate of 94 pmol/mg/min. Addition of DHA stimulated the efflux of Asc molecules from the loaded vesicles. Together these results suggest the presence of an Asc/DHA exchange mechanism in higher plant plasma membranes.

The Ascorbate Carrier of Higher Plant Plasma Membranes Preferentially Translocates the Fully Oxidized (Dehydroascorbate) Molecule.

Recently, the uptake of 14C-labeled ascorbate (ASC) into highly purified bean (Phaseolus vulgaris L.) plasma membrane vesicles was demonstrated in our laboratory. However, the question of the redox status of the transported molecule (ASC or dehydroascorbate [DHA]) remained unanswered. In this paper we present evidence that DHA is transported through the plasma membrane. High-performance liquid chromatography analysis of the redox status of ASC demonstrated that freshly purified plasma membranes exhibit a high ASC oxidation activity. Although it is not yet clear whether this activity is enzymatic, it complicates the interpretation of ASC-transport experiments in vitro and in vivo. In an attempt to correlate the ASC redox status to transport of the molecule, the ability of different compounds to reduce DHA was analyzed and their effect on ASC-transport activity tested. Administering of various reductants resulted in different levels of inhibition of ASC uptake (dithiothreitol > dithioerythritol > [beta]-mercaptoethanol > [beta]-mercaptopropanol). Glutathione, cysteine, dithionite, and thiourea did not significantly affect ASC transport. Statistical analysis indicated a strong correlation of the Spearman rank correlation coefficient (Rs) of 0.919 (P = 0.0005, n = 9) between the level of ASC oxidation and the amount of transported molecules into the vesicles. The administering of ASC oxidants such as ferricyanide and ASC oxidase resulted in a stimulated ASC uptake into the plasma membrane vesicles. Together, our results demonstrate that a vitamin C carrier in purified bean plasma membranes translocates DHA from the apoplast to the cytosol.

Solubilization and Separation of a Plant Plasma Membrane NADPH-O2- Synthase from Other NAD(P)H Oxidoreductases.

Solubilization and ion-exchange chromatography of plasma membrane proteins obtained from bean (Phaseolus vulgaris L.) seedlings resulted in a single NAD(P)H-O2--synthase protein peak. This enzyme showed a high preference toward NADPH as a substrate (reaction rate, 27.4 nmol O2- produced min-1 mg-1 protein), whereas NADH reactions ranged from 0 to maximally 15% of the NADPH reactions. The protein functions as an oxidase and it was clearly resolved from NAD(P)H dehydrogenases identified with commonly used strong oxidants (ferricyanide, cytochrome c, DCIP, and oxaloacetate). The involvement of peroxidases in O2- production is excluded on the basis of potassium-cyanide insensitivity and NADPH specificity. The NADPH oxidase is only moderately stimulated by flavins (1.5-fold with 25 [mu]M flavine adenine dinucleotide and 2.5-fold with 25 [mu]M flavin mononucleotide) and inhibited by 100 [mu]M p-chloromercuribenzenesulfonic acid, 200 [mu]M diphenyleneiodonium, 10 mM quinacrine, 40 mM pyridine, and 20 mM imidazole. The presence of flavins was demonstrated in the O2-synthase fraction, but no b-type cytochromes were detected. The effect of these inhibitors and the detection of flavins and cytochromes in the plant O2- synthase make it possible to compare this enzyme with the NADPH O2- synthase of animal neutrophil cells.

The Role of Ascorbate Free Radical as an Electron Acceptor to Cytochrome b-Mediated Trans-Plasma Membrane Electron Transport in Higher Plants.

The action of ascorbate free radical as an electron acceptor to cytochrome b-mediated trans-plasma membrane electron transport is demonstrated. Addition of ascorbate free radical to ascorbate-loaded plasma membrane vesicles caused a rapid oxidation of the cytochrome, followed by a slower re-reduction. The fully reduced dehydroascorbate was ineffective.

Transmembrane electron transport in ascorbate-loaded plasma membrane vesicles from higher plants involves a b-type cytochrome.

The possible involvement of a high-potential b-type cytochrome in plasma membrane electron transport was tested using ascorbate-loaded membrane vesicles. Absorption spectra demonstrated that the cytochrome was about 89% reduced in these preparations. Use of ascorbate oxidase and washing of the vesicles further indicated that reduction was mediated by intra-vesicular ascorbate. Addition of low concentrations of ferricyanide caused a rapid cytochrome oxidation followed by a slower re-reduction. The kinetics of this response indicate that the electron acceptor was fully reduced before re-reduction of the cytochrome occurred. These observations suggest that the b-type cytochrome mediates transmembrane electron transfer.

b-Type Cytochromes in Higher Plant Plasma Membranes.

The composition and characteristics of b-type cytochromes from higher plant plasma membranes, purified using aqueous two-phase partitioning, were investigated. At least three different cytochromes were identified by their wavelength maxima and redox midpoint potentials (E(0)'). Cytochrome b-560.7 (E(0)' from + 110 to + 160 millivolts) was present in zucchini (Cucurbita pepo) hypocotyls and bean (Phaseolus vulgaris L.) hooks, although in different concentrations. The main component in cauliflower (Brassica oleracea L.) inflorescences (cytochrome b-558.8) is probably functionally similar to this cytochrome. The plasma membrane generally contains two to three cytochrome species. However, the occurrence and concentrations were species dependent. The high potential cytochrome can be reduced by ascorbate but not NADH, and may be involved in blue light perception.

Heterogeneity of auxin-accumulating membrane vesicles from Cucurbita and Zea: a possible reflection of cell polarity.

When microsomes from hypocotyls of Cucurbita pepo L. or coleoptiles of Zea mays L. were centrifuged on dextran-sucrose gradients a heterogeneity of auxin-accumulating vesicles was observed. Vesicles from the top part of the gradient showed saturable, specific accumulation of indole-3-acetic acid with only a small stimulation by phytotropins, and with very few binding sites for 1-N-naphthylphthalamic acid. In the vesicles from the lower part of the gradient, net accumulation of indole-3-acetic acid could be strongly increased by addition of phytotropins; binding of 1-N-naphthylphthalamic acid was high in this region. After two-phase partitioning, both kinds of vesicles were found in the upper-phase membrane fraction considered to be purified plasma membrane. The hypothesis is discussed that vesicles can be separated from the apical and basal parts of the cell's plasmalemma.