The Role of Ascorbate Free Radical as an Electron Acceptor to ...

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cytochrome b-mediated trans-plasma membrane electron trans- port is demonstrated. Addition of ascorbate free radical to ascor- bate-loaded plasma membrane ...

Plant Physiol. (1994) 104: 1455-1458

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The Role of Ascorbate Free Radical as an Electron Acceptor to Cytochrome 6-Mediated Trans-Plasma Membrane Electron Transport in Higher Plants' Nele Horemans, Han Asard*, and Roland J. Caubergs

Department of Biology, University of Antwerp (Universitair Centrum Antwerpen), Groenenborgerlaan 171, B-2020 Antwerp, Belgium ~~~




Ohashi, 1988; Legendre et al., 1993). On the other hand, ascorbic acid, ascorbate oxidase, and ascorbate peroxidase have been suggested to occur in the plant cell wall (Mertz, 1964; Chichiricco et al., 1989). These latter components are likely to play a role in cross-linking reactions of cell wall polymers and could also be involved in oxygen radical scavenging and host-parasite interactions (Pene1 and Castillo, 1991). Two successive one-electron oxidations of ascorbate yield ascorbate free radical and the highly unstable dehydroascorbate, respectively. Like molecular oxygen, ascorbate free radical has been considered a potential electron acceptor to NADH-mediated plasma membrane redox systems (Morri. et al., 1986; Luster and Buckhout, 1988). The free radical is also accepted as the physiological electron acceptor of Cyt b561 of chromaffin granule vesicles (Njus et al., 1987). In this paper the possible actions of ascorbate free radical and dehydroascorbate as electron acceptors to the plant plasma membrane b-type Cyt are investigated using ascorbate-loaded plasma membrane vesicles.

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 dehydroascorbatewas ineffective.

In highly purified plasma membrane fractions of at least six higher plant species, a specific high-potential b-type Cyt (redox potential at pH 7.0 between +120 and +160 mV, a band at 560-561 nm) has been detected (Asard et al., 1989; Askerlund et al., 1989). This component is reducible in vitro by sodium ascorbic acid and constitutes 60 to 80% of the total Cyt amount detectable in plant plasma membranes. Suggestions have been made conceming the possible role of this redox component in plasma membrane redox reactions (Buckhout and Luster, 1991; Crane et al., 1991); however, little experimental evidence is available so far to support these ideas. Recently we have used plant plasma membrane vesicles loaded with the electron donor sodium ascorbate to investigate the possible involvement of the Cyt in trans-membrane electron transport reactions (Asard et al., 1992). a-Band absorption measurements demonstrated that the Cyt was reduced by ascorbate trapped inside the vesicles and that it was likely to act as a carrier in electron transfer from intemal ascorbate to an artificial extemal electron acceptor such as ferricyanide (Asard et al., 1992).This transmembrane electron transport model gives rise to new ideas conceming the physiological role of the Cyt and, in particular, raises questions regarding the possible nature of the in vivo electron acceptor. Recent demonstrations of the production of active (reduced) oxygen molecules upon elicitation of plant cells point to a possible role of molecular oxygen as an electron acceptor during plasma membrane electron transport (Doke and


Hypocotyl hooks of 5-d-old etiolated bean (Phaseolus vul-

garis L. var Limburgse Vroege) were harvested and collected on ice. Generally 100 g of tissue were homogenized in 250 mL of cold Hepes-KOH buffer (330 m SUC,50 mM Hepes, 0.1% BSA at pH 7.5) supplemented with 1 mM DTT, 0.5 mM PMSF, and 0.36% insoluble PVP. Ascorbate-loaded plasma membrane vesicles were prepared as described earlier (Asard et al., 1989, 1992). Spectrophotometric determinations of the Cyt b were carried out on an Aminco DW2a dual-wavelength spectrophotometer. Cyt absorption spectra were scanned at 2 nm s-' at 4OC, relative to 570 nm (isosbestic point). Generation and disproportionation of ascorbate free radicals was monitored on an Aminco DW2000 at 360 nm (split beam). An extinction coefficient (360 nm) of 5000 M-' cm-' was used to calculate ascorbate free radical concentrations (Skotland and Ljones, 1980). Part of the measurements were carried out in a stirred cuvette to ensure that oxygen was not limited during enzyme reactions. All measurements were performed in 600-fiLsamd e s and in resumension buffer with additions as indicated

' This work was financially supported by the National Fund for ScientificResearch (H.A.) and by the Instituut voor Wetenschappelijk Onderzoek in Nijverheid en Landbouw (N.H.). The work described in this paper is also supported by the Institute for the Study of Biological Evolution. * Corresponding author; fax 32-3-218-04-17. 1455


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Plant Physiol. Vol. 104, 1994

fresh preparation to the ascorbate-loaded vesicles. Even at high concentrations (500 nmol) no measurable changes in the a-band absorption maximum of the Cyt could be observed. Dehydroascorbate was, therefore, excluded as a potential electron acceptor. Cyt Oxidation by Ascorbate Free Radical

i1 ro


1 min

Figure 1. Time course of a-band absorption changes induced by ascorbate oxidase (20 units) followed by addition of Na-ascorbate (Asc; 550 nmol) or ferricyanide (FeCN; 1 nmol) in t h e presence of ascorbate oxidase (AO).

in the figure legends. The protein concentration was about 0.5 mg in a11 experiments (Markwell et al., 1978). RESULTS ./

Effect of Dehydroascorbate on Cyt b Reduction in Ascorbate-Loaded Plasma Membrane Vesicles

Plasma membranes were purified by aqueous two-phase partitioning and consist mainly of "right side-out" membrane vesicles. In plasma membrane fractions prepared in the presente of ascorbate the high potential b-type Cyt is largely reduced (66% compared to the dithionite-reducible level) by ascorbate effectively trapped inside the vesicles (Asard et al., 1992). Addition of 550 nmol of Na-ascorbate to freshly prepared loaded plasma membranes results in a rapid further reduction of the Cyt of about 15% (70% maximal reduction relative to dithionite). If ascorbate oxidase (20 units) is added to the plasma membrane vesicles, the a-band .shows an irreversible 10% absorption decrease, indicating that a limited fraction of Cyt b is reduced by free ascorbate on the outside of the membrane vesicles. Nearly complete oxidation of the Cyt could be obtained by addition of high concentrations (1 pmol) of the membrane-impermeable electron acceptor ferricyanide (not shown). We were interested in testing the possible action of ascorbate free radical as an electron acceptor to the reduced Cyt. The radical is generated in vitro by mixing Na-ascorbate (550 nmol) with a high concentration of the enzyme ascorbate oxidase (20 units). However, the generated radicals readily disproportionate to ascorbate and the fully oxidized dehydroascorbate. The possible action of the latter molecule as an electron acceptor was examined by adding it directly from a

The effect of ascorbate free radical on the reduced Cyt b in ascorbate-loaded plasma membrane vesicles was examined by following the time course of changes in the a-band absorption maximum at 561 nm (relative to 570 nm). Radical generation was initiated by ascorbate oxidase (21) units) soon after the addition of ascorbate (550 nmol) to the vesicle suspension. An immediate decrease (within 10 s) of the aband absorption was observed, followed by a slower but total reversion within 1 min (Fig. 1). These absorption changes indicate a rapid oxidation of the Cyt and a subsequent rereduction. In contrast to observations from ascorbate-loaded chromaffin vesicle ghosts (Kelley and Njus, 1986), the addition of ascorbate oxidase alone did not result in a transient absorption decrease. Also lower concentrations of ascorbate and/or ascorbate oxidase were tested and were generally much less effective. Simultaneous addition of ascorbate and dehydroascorbate to generate ascorbate free radvcal (Gonzalez-Reyes et al., 1992) did not result in sufficiently high concentrations of the radical in our hands. In a control experiment Cyt b present in nonloaded plasma membrane vesicles was reduced by the addition of external ascorbate (550 nmol) (Fig. 2). In this fraction, addition of ascorbate oxidase (20 units) resulted in a rapid oxidation of the Cyt without a measurable re-reduction within 10 min. The amount of ascorbate oxidase used in these experiments is, therefore, sufficient to oxidize a11 external ascorbate, and

1 min




Figure 2. Reduction of the b-type Cyt (a-band absorption)in nonloaded plasma membrane vesicles upon addition of 550 nmol of Na-ascorbate (Asc) and subsequent re-oxidation by ascorbate oxidase (AO; 20 units).

Cyt b-Mediated Ascorbate Free Radical Reduction the re-reduction observed with loaded plasma membrane vesicles occurs from ascorbate inside the vesicles. For comparison, Figure 1 also shows a-band absorption changes after addition of ferricyanide (1 nmol) to loaded vesicles. The oxidation and re-reduction kinetics are very similar to those obtained with ascorbate free radical. It is important to notice that the initial absorption decreases were similar when either ascorbate free radical or femcyanide was tested and are also similar to the levels obtained with an excess of ferricyanide (Asard et al., 1992). Ascorbate free radical is, therefore, capable of oxidizing the b-type Cyt to a similar extent as does ferricyanide. The time course of the Cyt oxidation and re-reduction by ascorbate free radical was not affected by addition of KCN (100 nmol) or salicylhydroxamic acid (100 nmol). Catalase (200 units) and superoxide dismutase (200 units) also had no effect (data not shown). Comparison of Electron Transport and Ascorbate Free Radical Disproportionation Kinetics

The generation and disproportionation of ascorbate free radicals can be monitored spectrophotometrically at 360 nm (Skotland and Ljones, 1980). Combined addition of 550 nmol of ascorbate and 20 units of ascorbate oxidase results in the very fast build up of about 3 nmol of ascorbate radical (Fig. 3, trace b). The kinetics of the Cyt b absorption changes upon ascorbate free radical addition to ascorbate-loaded plasma membrane fractions occur within a similar time course as the radical breakdown process (Fig. 3, trace a). Complete disappearance of ascorbate free radical and re-reduction of Cyt b both take about 1.2 min. The Cyt is, therefore, at least partially oxidized as long as the radical is present. With the kinetic resolution available on the current appa-

Figure 3. Comparison of the kinetics of Cyt b absorption changes (a) and generation of ascorbate free radical (b) upon addition (arrow) of ascorbate (550 nmol) and ascorbate oxidase (20 units).


ratus no significant differences could be found between the kinetics of ascorbate free radical breakdown in the absence of plasma membranes (disproportionation) or in the presence of either ascorbate-loaded or nonloaded membrane vesicles. Approximate half-life times between 4 and 10 s were found in a11 cases. DlSCUSSlON

Experiments using ascorbate-loaded plasma membrane vesicles have recently led to the hypothesis that a highpotential b-type Cyt could possibly mediate electron transfer from intravesicular ascorbate to an artificial externa1 electron acceptor such as ferricyanide (Asard et al., 1992). To point to the possible physiological function of this system it is necessary to identify the potential natural electron acceptor. The plasma membrane Cyt b is not autoxidizable, which seems to exclude molecular oxygen as a probable candidate. In addition to oxygen and iron chelates, ascorbate free radical has also been proposed as an in vivo oxidant to plasma membrane oxidoreductases (Morré et al., 1986; Luster and Buckhout, 1988). The radical might be generated in the cell wall matrix during cell growth processes and radical scavenging reactions. It was the aim of this work to test the idea that ascorbate free radical is an electron acceptor to Cyt b-mediated transmembrane electron transport using ascorbate as the electron donor. The data presented in ihis paper demonstrate that ascorbate free radical causes a rapid oxidation of the Cyt, followed by a slower re-reduction, in ascorbate-loaded plasma membrane vesicles. Like observations using femcyanide (Asard et al., 1992), the rapid oxidation of the Cyt b indicates an electron transfer on the extravesicular (cell wall) side of the membrane to nonpermeating ascorbate free radicals. Rereduction of the Cyt occurs only in ascorbate-loaded plasma membrane vesicles, indicating that intravesicular ascorbate can act as an electron donor. Dehydroascorbate, which is inevitably generated from disproportionation of the radicals, does not by itself seem to act as an electron acceptor in this system. Together these observations strongly suggest that ascorbate free radical can function as an electron acceptor in transmembrane electron transport involving a plant plasma membrane b-type Cyt. Ascorbic acid is a common constituent of plant cells, and it seems reasonable to accept this molecule as a natural cytoplasmic electron donor to the Cyt. Extracellular reduction of ascorbate free radical thus implies an "electron shuttle" function between the radical and cytoplasmic ascorbate. This model indicates a striking similarity to the function of adrenal granule Cyt b561, transporting electrons from cytoplasmic ascorbate to ascorbic acid free radical inside secretory vacuoles (Kelley and Njus, 1986; Wakefield et al., 1986). Neither ascorbate nor the free radical readily permeates a membrane, and reduction of ascorbate free radical effectively regenerates intravesicular ascorbate. By analogy to this system the plant plasma membrane Cyt b seems to serve to regenerate apoplastic ascorbate. Effective reduction in vivo of extracellular ascorbate radical at the expense of cytoplasmic ascorbate is favored by severa1 factors (Njus et al., 1987).The generally acidic cell wall matrix


Horemans e t al.


(pH about 5 compared to pH 7 in the cytoplasm) facilitates protonation of ascorbate radicals in the reduction to ascorbate. Also the low extracellular pH results in a more positive redox potential at pH 7.0 of the ascorbatelascorbate free radical redox couple (Iyanagi et al., 1985). The membrane potential of plant cells is generally about -60 to -120 mV (outside positive). This provides an additional driving force for electrons to the cell wall. Finally, oxidoreductases in the plant cell membrane may effectively maintain low intracellular ascorbate free radical concentrations. The latter argument is of particular interest because distinct NAD(P)H-dependent enzymes have been demonstrated in plant plasma membranes, including an NADH:ascorbate free radical reductase (Morré et al., 1986; Buckhout and Luster, 1991). It has also been suggested that the majority of these enzymes have both the electron donor and acceptor sites located on the cytoplasmic plasma membrane face (Askerlund et al., 1988). The plasma membrane NADH:ascorbate free radical reductase is, therefore, a potential candidate for involvement in intracellular radical scavenging and regeneration of cytosolic ascorbate at the plasma membrane. The high-potential Cyt b itself is only poorly reduced by NADH and should, therefore, be different from the NADH:ascorbate free radical reductase activity. This paper provides the first direct evidence for a transplasma membrane electron transport from ascorbate to ascorbate free radical. The high-potential plant plasma membrane Cyt b is suggested to mediate the electron transfer, providing an interesting further similarity to the animal chromaffin granule electron transport system. Received September 13, 1993; accepted January 4, 1994. Copyright Clearance Center: 0032-0889/94/104/1455/04. LITERATURE ClTED

Asard H, Horemans N, Caubergs RJ (1992) Transmembrane electron transport in ascorbate-loaded plasma membrane vesicles from higher plants involves a b-type cytochrome. FEBS Lett 306: 143-146 Asard H, Venken M, Caubergs RJ, Reijnders W, Oltmann FL, De Greef JA (1989) b-Type cytochromes in higher plant plasma membranes. Plant Physiol90: 1077-1083 Askerlund P, Larsson C, Widell S (1988) Localization of donor and acceptor sites of NADH dehydrogenase activities using inside-out and right-side-out plasma membrane vesicles from plants. FEBS Lett 239 23-28 Askerlund P, Larsson C, Widell S (1989) Cytochromes of plant

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plasma membranes. Characterization by absorbance difference spectroscopy and redox titration. Physiol Plant 7 6 123-134 Buckhout TJ, Luster DG (1991) Pyridine nucleotide-dependent reductases of the plant plasma membrane. In FL Crane, DJ Morri., HE Low, eds, Oxidoreduction at the Plasma Membrane: Relation to Growth and Transport, Vol 2. CRC Press, Boca Raton, FL, pp 61-83 Chichiricco G, Ceru MP, DAlessandro A, Oratore A, Avigliano L (1989) Immunohistochemical localization of ascorbate oxidase in Cucurliita pepo medullosa. Plant Sci 6 4 61-66 Crane FL, Morré DJ, Low HE, Bottger M (1991) The oxidoreductase enzymes in plant plasma membranes. In FL Crane, DJ Morri., HE Low, eds, Oxidoreduction at the Plasma Membrane: Relation to Growth and Transport, Vol 2. CRC Press, Boca Raton, FL, pp 21-33 Doke N, Ohashi Y (1988) Involvement of an 02-generating system in the induction of necrotic lesions on tobacco leaves infected with tobacco mosaic virus. Physiol Mo1 Plant Pathol 32: 163-175 Gonzalez-Reyes JA, Doring O, Navas P, Obst G, Bottger M (1992) The effect of ascorbate free radical on the energy state of the plasma membrane of onion (Allium cepa L.) root cells: alteration of K+ efflux by ascorbate? Biochim Biophys Acta 1098: 177-183 Iyanagi T, Yamazaki I, Anan KF (1985) One-electron oxidationreduction properties of ascorbic acid. Biochim Biophys Acta 806 255-261 Kelley PlM, Njus D (1986) Cytochrome b,,, spectral changes associated with electron transfer in chromaffin-vesicle ghosts. J Biol Chem :!61: 6429-6432 Legendre L, Reuter S, Heinstein PF, PS Low (1993) Characterization of the oligogalacturonide-induced oxidative burst in culture soybean (Glycine max) cells. Plant PhysiollO2: 233-240 Luster DG, Buckhout TJ (1988) Characterization and partia1 purification of multiple electron transport activities in plasma membranes from maize (Zea mays L.) roots. Physiol Plant 173 339-347 Markwell. MAK, Haas SM, Bieber LL, Tolbert NE (1978) A modification of the Lowry procedure to simplify protein determinations in membrane and lipoprotein samples. Ana1 Biochem 87: 206-210 Mertz D (1964) Ascorbic acid oxidase in cell growth. Plant Physiol 3 9 398 Morré DJ, Navas P, Penel C, Castillo FJ (1986) Auxin-stimulated NADH oxidase (semidehydrosascorbate reductase) of soybean plasma membrane: role in acidification of cytoplasm? I’rotoplasma 133 196-197 Njus PM, Kelley PM, Harnadek GJ, Pacquing YV (1987) Mechanism of ascorbic acid regeneration by cytochrome b561. Ann NY Acad Sci 493: 108-119 Penel C, Castillo FJ (1991) Peroxidases of plant plasma membranes, apoplastic ascorbate, and relation of redox activities to plant pathology. I n FL Crane, DJ Morri., HE Low, eds, Oxidoreduction at the Plasma Membrane: Relation to Growth and Transport, Vol 2. CRC Press, Boca Raton, FL, pp 121-147 Skotland T, Ljones T (1980) Direct spectrophotometric detection of ascorbate free radical formed by dopamine P-monooxygenaseand ascorbate reductase. Biochim Biophys Acta 630 30-35 Wakefield LL, Cass AEG, Radda GK (1986) Electron transfer across the chrornaffin granule membrane. J Biol Chem 261: 9?46-9752

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