Plant Physiol. (1994) 104: 1455-1458

Rapid Communicafion 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 , University of Antwerp (Universitair Centrum Antwerpen), Groenenborgerlaan 171, B-2020 Antwerp, Belgium

~~~ ~~ ~~~~ ~~~~~ Ohashi, 1988; Legendre et al., 1993). On the other hand, The action of ascorbate free radical as an electron acceptor to ascorbic acid, ascorbate oxidase, and ascorbate peroxidase cytochrome b-mediated trans-plasma membrane electron trans- have been suggested to occur in the plant cell wall (Mertz, port is demonstrated. Addition of ascorbate free radical to ascor- 1964; Chichiricco et al., 1989). These latter components are bate-loaded plasma membrane vesicles caused a rapid oxidation likely to play a role in cross-linking reactions of cell wall of the cytochrome, followed by a slower re-reduction. The fully polymers and could also be involved in radical scav- reduced dehydroascorbate was ineffective. enging and host-parasite interactions (Pene1 and Castillo, 1991). Two successive one-electron oxidations of ascorbate yield ascorbate free radical and the highly unstable dehy- In highly purified plasma membrane fractions of at least droascorbate, respectively. Like molecular oxygen, ascorbate six higher plant species, a specific high-potential b-type Cyt free radical has been considered a potential electron acceptor ( potential at pH 7.0 between +120 and +160 mV, a- to NADH-mediated plasma membrane redox systems (Morri. band at 560-561 nm) has been detected (Asard et al., 1989; et al., 1986; Luster and Buckhout, 1988). The free radical is Askerlund et al., 1989). This component is reducible in vitro also accepted as the physiological electron acceptor of Cyt by sodium ascorbic acid and constitutes 60 to 80% of the b561 of chromaffin granule vesicles (Njus et al., 1987). total Cyt amount detectable in plant plasma membranes. In this paper the possible actions of ascorbate free radical Suggestions have been made conceming the possible role of and dehydroascorbate as electron acceptors to the plant this redox component in plasma membrane redox reactions plasma membrane b-type Cyt are investigated using ascor- - (Buckhout and Luster, 1991; Crane et al., 1991); however, bate-loaded plasma membrane vesicles. little experimental evidence is available so far to support these ideas. Recently we have used plant plasma membrane vesicles MATERIALS AND METHODS loaded with the sodium ascorbate to investi- Hypocotyl hooks of 5-d-old etiolated bean (Phaseolus vul- gate the possible involvement of the Cyt in trans-membrane garis L. var Limburgse Vroege) were harvested and collected electron transport reactions (Asard et al., 1992). a-Band ab- on ice. Generally 100 g of tissue were homogenized in 250 sorption measurements demonstrated that the Cyt was re- mL of cold Hepes-KOH buffer (330 m SUC,50 mM Hepes, duced by ascorbate trapped inside the vesicles and that it 0.1% BSA at pH 7.5) supplemented with 1 mM DTT, 0.5 mM was likely to act as a carrier in electron transfer from intemal PMSF, and 0.36% insoluble PVP. Ascorbate-loaded plasma ascorbate to an artificial extemal electron acceptor such as membrane vesicles were prepared as described earlier (Asard ferricyanide (Asard et al., 1992). This transmembrane electron transport model gives rise to new ideas conceming the phys- et al., 1989, 1992). iological role of the Cyt and, in particular, raises questions Spectrophotometric determinations of the Cyt b were car- regarding the possible nature of the in vivo electron acceptor. ried out on an Aminco DW2a dual-wavelength spectropho- Recent demonstrations of the production of active (re- tometer. Cyt absorption spectra were scanned at 2 nm s-' at duced) oxygen molecules upon elicitation of plant cells point 4OC, relative to 570 nm (isosbestic point). Generation and to a possible role of molecular oxygen as an electron acceptor of ascorbate free radicals was monitored during plasma membrane electron transport (Doke and 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, ' This work was financially supported by the National Fund for Scientific Research (H.A.) and by the Instituut voor Wetenschappelijk 1980). Onderzoek in Nijverheid en Landbouw (N.H.). The work described Part of the measurements were carried out in a stirred in this paper is also supported by the Institute for the Study of cuvette to ensure that oxygen was not limited during enzyme Biological Evolution. reactions. All measurements were performed in 600-fiL sam- * Corresponding author; fax 32-3-218-04-17. des and in resumension buffer with additions as indicated 1455 1456 Horemans et al. 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 ob- served. Dehydroascorbate was, therefore, excluded as a po- tential electron acceptor.

Cyt Oxidation by Ascorbate Free Radical 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 a- band absorption was observed, followed by a slower but total reversion within 1 min (Fig. 1). These absorption changes i1 indicate a rapid oxidation of the Cyt and a subsequent re- rro reduction. In contrast to observations from ascorbate-loaded 1 min chromaffin vesicle ghosts (Kelley and Njus, 1986), the addi- tion of ascorbate oxidase alone did not result in a transient absorption decrease. Also lower concentrations of ascorbate Figure 1. Time course of a-band absorption changes induced by and/or ascorbate oxidase were tested and were generally ascorbate oxidase (20 units) followed by addition of Na-ascorbate (Asc; 550 nmol) or ferricyanide (FeCN; 1 nmol) in the presence of much less effective. Simultaneous addition of ascorbate and ascorbate oxidase (AO). dehydroascorbate to generate ascorbate free radvcal (Gonza- lez-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 in the figure legends. The protein concentration was about membrane vesicles was reduced by the addition of external 0.5 mg in a11 experiments (Markwell et al., 1978). ascorbate (550 nmol) (Fig. 2). In this fraction, addition of ascorbate oxidase (20 units) resulted in a rapid oxidation of RESULTS the Cyt without a measurable re-reduction within 10 min. The amount of ascorbate oxidase used in these experiments Effect of Dehydroascorbate on Cyt b Reduction in ./ is, therefore, sufficient to oxidize a11 external ascorbate, and Ascorbate-Loaded Plasma Membrane Vesicles Plasma membranes were purified by aqueous two-phase partitioning and consist mainly of "right side-out" membrane 1 min vesicles. In plasma membrane fractions prepared in the pres- - ente of ascorbate the high potential b-type Cyt is largely 18 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 fer- ricyanide (not shown). We were interested in testing the possible action of ascor- bate 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 Figure 2. Reduction of the b-type Cyt (a-band absorption) in non- disproportionate to ascorbate and the fully oxidized dehy- loaded plasma membrane vesicles upon addition of 550 nmol of droascorbate. The possible action of the latter molecule as an Na-ascorbate (Asc) and subsequent re-oxidation by ascorbate oxi- electron acceptor was examined by adding it directly from a dase (AO; 20 units). Cyt b-Mediated Ascorbate Free Radical Reduction 1457 the re-reduction observed with loaded plasma membrane ratus no significant differences could be found between the vesicles occurs from ascorbate inside the vesicles. kinetics of ascorbate free radical breakdown in the absence For comparison, Figure 1 also shows a-band absorption of plasma membranes (disproportionation) or in the presence changes after addition of ferricyanide (1 nmol) to loaded of either ascorbate-loaded or nonloaded membrane vesicles. vesicles. The oxidation and re-reduction kinetics are very Approximate half-life times between 4 and 10 s were found similar to those obtained with ascorbate free radical. It is in a11 cases. important to notice that the initial absorption decreases were similar when either ascorbate free radical or femcyanide was DlSCUSSlON tested and are also similar to the levels obtained with an excess of ferricyanide (Asard et al., 1992). Ascorbate free Experiments using ascorbate-loaded plasma membrane radical is, therefore, capable of oxidizing the b-type Cyt to a vesicles have recently led to the hypothesis that a high- similar extent as does ferricyanide. potential b-type Cyt could possibly mediate electron transfer The time course of the Cyt oxidation and re-reduction by from intravesicular ascorbate to an artificial externa1 electron ascorbate free radical was not affected by addition of KCN acceptor such as ferricyanide (Asard et al., 1992). To point to (100 nmol) or salicylhydroxamic acid (100 nmol). Catalase the possible physiological function of this system it is neces- (200 units) and superoxide dismutase (200 units) also had no sary to identify the potential natural electron acceptor. The effect (data not shown). plasma membrane Cyt b is not autoxidizable, which seems to exclude molecular oxygen as a probable candidate. In Comparison of Electron Transport and Ascorbate Free addition to oxygen and chelates, ascorbate free radical Radical Disproportionation Kinetics has also been proposed as an in vivo oxidant to plasma membrane (Morré et al., 1986; Luster and The generation and disproportionation of ascorbate free Buckhout, 1988). The radical might be generated in the cell radicals can be monitored spectrophotometrically at 360 nm wall matrix during cell growth processes and radical scaveng- (Skotland and Ljones, 1980). Combined addition of 550 nmol ing reactions. It was the aim of this work to test the idea that of ascorbate and 20 units of ascorbate oxidase results in the ascorbate free radical is an electron acceptor to Cyt b-me- very fast build up of about 3 nmol of ascorbate radical (Fig. diated transmembrane electron transport using ascorbate as 3, trace b). The kinetics of the Cyt b absorption changes upon the electron donor. ascorbate free radical addition to ascorbate-loaded plasma The data presented in ihis paper demonstrate that ascor- membrane fractions occur within a similar time course as the bate free radical causes a rapid oxidation of the Cyt, followed radical breakdown process (Fig. 3, trace a). Complete disap- by a slower re-reduction, in ascorbate-loaded plasma mem- pearance of ascorbate free radical and re-reduction of Cyt b brane vesicles. Like observations using femcyanide (Asard et both take about 1.2 min. The Cyt is, therefore, at least al., 1992), the rapid oxidation of the Cyt b indicates an partially oxidized as long as the radical is present. electron transfer on the extravesicular (cell wall) side of the With the kinetic resolution available on the current appa- membrane to nonpermeating ascorbate free radicals. Re- .. reduction 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 vacu- oles (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 apo- plastic ascorbate. Figure 3. Comparison of the kinetics of Cyt b absorption changes Effective reduction in vivo of extracellular ascorbate radical (a) and generation of ascorbate free radical (b) upon addition (arrow) at the expense of cytoplasmic ascorbate is favored by severa1 of ascorbate (550 nmol) and ascorbate oxidase (20 units). factors (Njus et al., 1987). The generally acidic cell wall matrix 1458 Horemans et al. Plant Physiol. Vol. 104, 1994

(pH about 5 compared to pH 7 in the cytoplasm) facilitates plasma membranes. Characterization by absorbance difference protonation of ascorbate radicals in the reduction to ascor- spectroscopy and redox titration. Physiol Plant 76 123-134 Buckhout TJ, Luster DG (1991) Pyridine nucleotide-dependent re- bate. Also the low extracellular pH results in a more positive ductases of the plant plasma membrane. In FL Crane, DJ Morri., redox potential at pH 7.0 of the ascorbatelascorbate free HE Low, eds, Oxidoreduction at the Plasma Membrane: Relation radical redox couple (Iyanagi et al., 1985). The membrane to Growth and Transport, Vol 2. CRC Press, Boca Raton, FL, pp potential of plant cells is generally about -60 to -120 mV 61-83 Chichiricco G, Ceru MP, DAlessandro A, Oratore A, Avigliano L (outside positive). This provides an additional driving force (1989) Immunohistochemical localization of ascorbate oxidase in for electrons to the cell wall. Finally, oxidoreductases in the Cucurliita pepo medullosa. Plant Sci 6461-66 plant cell membrane may effectively maintain low intracel- Crane FL, Morré DJ, Low HE, Bottger M (1991) The lular ascorbate free radical concentrations. enzymes in plant plasma membranes. In FL Crane, DJ Morri., HE The latter argument is of particular interest because distinct Low, eds, Oxidoreduction at the Plasma Membrane: Relation to Growth and Transport, Vol 2. CRC Press, Boca Raton, FL, pp NAD(P)H-dependent enzymes have been demonstrated in 21-33 plant plasma membranes, including an NADH:ascorbate free Doke N, Ohashi Y (1988) Involvement of an 02-generating system radical reductase (Morré et al., 1986; Buckhout and Luster, in the induction of necrotic lesions on tobacco leaves infected with 1991). It has also been suggested that the majority of these tobacco mosaic virus. Physiol Mo1 Plant Pathol 32: 163-175 Gonzalez-Reyes JA, Doring O, Navas P, Obst G, Bottger M (1992) enzymes have both the electron donor and acceptor sites The effect of ascorbate free radical on the state of the located on the cytoplasmic plasma membrane face (Asker- plasma membrane of onion (Allium cepa L.) root cells: alteration lund et al., 1988). The plasma membrane NADH:ascorbate of K+ efflux by ascorbate? Biochim Biophys Acta 1098: 177-183 free radical reductase is, therefore, a potential candidate for Iyanagi T, Yamazaki I, Anan KF (1985) One-electron oxidation- involvement in intracellular radical scavenging and regener- reduction properties of ascorbic acid. Biochim Biophys Acta 806 255-261 ation of cytosolic ascorbate at the plasma membrane. The Kelley PlM, Njus D (1986) Cytochrome b,,, spectral changes asso- high-potential Cyt b itself is only poorly reduced by NADH ciated with electron transfer in chromaffin-vesicle ghosts. J Biol and should, therefore, be different from the NADH:ascorbate Chem :!61: 6429-6432 free radical reductase activity. Legendre L, Reuter S, Heinstein PF, PS Low (1993) Characterization of the oligogalacturonide-induced oxidative burst in culture soy- This paper provides the first direct evidence for a trans- bean (Glycine max) cells. Plant PhysiollO2: 233-240 plasma membrane electron transport from ascorbate to as- Luster DG, Buckhout TJ (1988) Characterization and partia1 purifi- corbate free radical. The high-potential plant plasma mem- cation of multiple electron transport activities in plasma mem- brane Cyt b is suggested to mediate the electron transfer, branes from maize (Zea mays L.) roots. Physiol Plant 173 339-347 Markwell. MAK, Haas SM, Bieber LL, Tolbert NE (1978) A modi- providing an interesting further similarity to the animal chro- fication of the Lowry procedure to simplify protein determinations maffin granule electron transport system. in membrane and lipoprotein samples. Ana1 Biochem 87: 206-210 Mertz D (1964) Ascorbic acid oxidase in cell growth. Plant Physiol Received September 13, 1993; accepted January 4, 1994. 39 398 Morré DJ, Navas P, Penel C, Castillo FJ (1986) Auxin-stimulated Copyright Clearance Center: 0032-0889/94/104/1455/04. NADH oxidase (semidehydrosascorbate reductase) of soybean plasma membrane: role in acidification of cytoplasm? I’rotoplasma LITERATURE ClTED 133 196-197 Njus PM, Kelley PM, Harnadek GJ, Pacquing YV (1987) Mecha- Asard H, Horemans N, Caubergs RJ (1992) Transmembrane elec- nism of ascorbic acid regeneration by cytochrome b561. Ann NY tron transport in ascorbate-loaded plasma membrane vesicles from Acad Sci 493: 108-119 higher plants involves a b-type cytochrome. FEBS Lett 306: Penel C, Castillo FJ (1991) Peroxidases of plant plasma membranes, 143-146 apoplastic ascorbate, and relation of redox activities to plant pa- Asard H, Venken M, Caubergs RJ, Reijnders W, Oltmann FL, De thology. In FL Crane, DJ Morri., HE Low, eds, Oxidoreduction at Greef JA (1989) b-Type cytochromes in higher plant plasma the Plasma Membrane: Relation to Growth and Transport, Vol 2. membranes. Plant Physiol90: 1077-1083 CRC Press, Boca Raton, FL, pp 121-147 Askerlund P, Larsson C, Widell S (1988) Localization of donor and Skotland T, Ljones T (1980) Direct spectrophotometric detection of acceptor sites of NADH dehydrogenase activities using inside-out ascorbate free radical formed by dopamine P-monooxygenase and and right-side-out plasma membrane vesicles from plants. FEBS ascorbate reductase. Biochim Biophys Acta 630 30-35 Lett 239 23-28 Wakefield LL, Cass AEG, Radda GK (1986) Electron transfer across Askerlund P, Larsson C, Widell S (1989) Cytochromes of plant the chrornaffin granule membrane. J Biol Chem 261: 9?46-9752