The lnvolvement of Respiration in Free Radical Processes ... - NCBI

Plant Physiol. (1994) 104: 1333-1339

The lnvolvement of Respiration in Free Radical Processes during Loss of Desiccation Tolerance in Germinating Zea mays L.' An Electron Paramagnetic Resonance Study Olivier Leprince**, Neil M. Atherton, Roger Deltour, and Ceorge A. F. Hendry

Department of Botany, University of Liège, B22 Sart Tilman, B 4000 Liège, Belgium (O.L., R.D.); Department of Chemistry (N.M.A.) and Natural Environment Research Council, Unit of Comparative Plant Ecology, Department of Animal and Plant Sciences (G.A.F.H.), University of Sheffield, Sheffield S1 O 2TN, United Kingdom

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stages of germination, when embryos are able to survive considerable desiccation up to the point of radicle emergence. This desiccation tolerance progressively lessens as the seed germinates. There is increasing evidence that the development of intolerance is correlated with increased free radical attack and damage during water loss (Senaratna and McKersie, 1986; Senaratna et al., 1987; Leprince et al., 1990; McKersie, 1991; Hendry et al., 1992). Dried, desiccationintolerant tissues generally exhibit extensive peroxidative damage to cellular membranes, resulting in loss of semipermeability, changes in their microviscosity and lipid phase properties (Senaratna et al., 1985, 1987), increases in FFA (Senaratna et al., 1985, 1987), and accumulation of thiobarbituric acid-reactive substances associated with lipid peroxidation (Leprince et al., 1990; Hendry et al., 1992). As suggested by the extensive literature (refs. in Halliwell, 1987; McKersie, 1991; Salin, 1991; Bowler et al., 1992; Winston, 1992), the origin of such peroxidative damage occumng under stress conditions is likely to be the formation of transient but highly reactive, partially reduced or activated forms

When germinating Zea mays 1. seeds are rapidly desiccated, free radical-mediated lipid peroxidation and phospholipid deesterification is accompanied by a desiccation-induced buildup of a stable free radical associated with rapid loss of desiccation tolerance. Comparison of the eledron paramagnetic resonance and eledron nuclear double resonance properties of this radical with those of the radical in dried, desiccation-intolerant moss showed that the two were identical. At the subcellular level, the radical was associated with the hydrophilic fraction resulting from lipid extraction. lsolated mitochondria subjected to drying were also found to accumulate an identical radical in vitro. When increasing concentrations of cyanide were used, a significant positive correlation was shown between rates of respiration and the accumulation of the radical in desiccation-intolerant tissues. Another positive correlation was found when rates of O2uptake by radicles at different stages of germination were plotted against free radical content following desiccation. This indicates that free radical production is closely linked to respiration in a process likely to involve the desiccation-induced impairment of the mitochondrial eledron transport chain to form thermodynamically favorable conditions to induce accumulation of a stable free radical and peroxidized lipids. Modulation of respiration using a range of inhibitors resulted in broadly similar modulation of the buildup of the stable free radical. One site of radical generation was likely to be the NADH dehydrogenase of complex I and probably as a dired consequence of desiccation-impaired electron flow at or close to the ubiquinone

of

pool.

Most vascular plants can withstand little desiccation except during the later stages of seed development and the early This work has been supported by a Belgian 'Action de Recherches Concertées," grant 88/93-129. O.L. was supported by a research assistant fellowship from the Fonds National de Ia Recherche Scientifique and by a European Environmental Research Organization short-tem fellowship. G.A.F.H. acknowledges the support of the Natural Environment Research Council. * Present address: U.S.Department of Agriculture, Agricultura1 Research Service, National Seed Storage Laboratory, Fort Collins, CO 80521. * Corresponding- author; fax 1-303-221-1427.

o*.

Many EPR studies have demonstrated the central role of free radical processes in a number of environmental stresses throughout biology. Studies involving spin trapping techniques to detect short-lived free radicals demonstrated the formation of activated O2in plants during drought or chilling stress (Price et al., 1989; Rosen and Halpem, 1990;Hodgson and Raison, 1991). Price et al. (1989)observed in wheat chloroplasts prepared from droughted leaves an increase of superoxide, trapped by Tiron as spin-adduct, accompanied by a 2.3-fold increase in iron, a loss of Chl, and an increase in peroxidized lipids. Superoxide that formed as a result of impairment of the chloroplast electron transport chains was ~

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Abbreviations:AA,, antimycin AB;ENDOR, electron-nuclear double resonance; EPR, electron paramagnetic resonance; FCCP, p trifluoromethoxy(carbony1cyanide)phenylhydrazone;FFA, free fatty acid; SHAM, salicylhydroxamic acid; TBA, thiobarbituric acid. ~.

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believed to be the precursor of the hydroxyl radical formed through the iron-catalyzed Fenton reaction thus initiating oxidative injury (Price and Hendry, 1991). Correlations between rates of light-dependent superoxide production and lipid peroxidation during photosynthesis have been described in a wide range of plant species and used as evidence of electron leakage from stress-impaired electron flow within the thylakoid membranes (Halliwell, 1987, and refs. therein; Hodgson and Raison, 1991; Salin, 1991; Seel et al., 1991). Using intact plant tissues, we have used another EPR approach to focus on the nature and content of more stable free radicals that build up in vivo as markers of past evidence of oxidative stress. In germinating maize (Zea mays L.) radicles, we have previously observed that severe desiccation induced a buildup of a stable, EPR-detected, organic radical associated with the loss of desiccation tolerance (Leprince et al., 1990). An apparently identical EPR signal has also been identified in the embryonic axes of desiccation-intolerant seeds of Quercus robur accumulating with loss of moisture and seed viability (Hendry et al., 1992) and in two species of mosses subjected to high light irradiance during desiccation, coinciding again with loss of Chl and an increase in lipid peroxidation (Seel et al., 1991). Stable free radicals have also been detected in other seed tissues, such as cotyledon and axes of soybean (Priestley et al., 1985) and endosperm and embryo of maize (Buchvarov and Gantcheff, 1984), with their concentrationsincreasing during natural and accelerated aging and loss of viability. How such a radical (or radicals) originates is, however, unknown. We have recently shown that changes in rates of respiration' appeared to be linked to the loss of desiccation tolerance (Leprince et al., 1992) in maize radicles. Puntarulo et al. (1991) showed an increase of H202and 02production by mitochondria during germination of soybean. Hence, mitochondrial respiration may be a source of reduced O2 and of the desiccation-induced buildup of the stable, free radical. The goal of this paper is to report the exploration of this possibility. The experimental data confirm that the most likely source of both transient and stable free radicals in genninating maize is the mitochondrion.

Plant Physiol. Vol. 104, 1994

(see further) were packed into 3-mm-diameter quartz tubes, and scan ranges of 10 milliTesla were used with 25 decibels of nommal power. Other parameters were adjusted as necessary to obtain the most resolved spectra. The secondderivative presentation was used to discriminate against a broad background signal. The relative free radical1 concentrations were estimated by the height (cm) of the secondderivative signal amplitude corrected for instrument gain and expressed on a dry weight basis. EPR experiments were performed three times with two subreplicates each scanned twice. Theg value was calculated using 1,l diphenyl-2-picrylhydrazyl ( g value = 2.0036). ENDOR spectroscopy, combining the sensitivity of EPR with the resolution of NMR, was conducted using 100 dried 72-h-old radicles on a Bruker spectrometer at 109 K as described by Seel et al. (1991). Preparation of Cell Extracts

Total lipids were extracted from 50 to 70 dried. desiccationintolerant radicles in the presence of butylated hydroxytoluene (0.05%, w/v) according to the procedure of Nichols (1963). Different lipid classes were separated by TLC with hexane:diethyl ether:fonnic acid (80:20:2, v/v) as the solvent system, following the method of Christie (1989), and were identified after iodine exposure in comparison with known standards. Polar lipids, FFA, and triacylglycerols were scrapetl from the plates and dissolved in chloroform before being subjected to EPR analysis, together with the pellet of cellular debris resulting from backwashings during the lipid extraction procedure. Nuclei isolation was performed at 4OC with 72-h-old fresh radicles according to the method of Greimers and Deltour (1981). Mitochondria-iich fractions were prepared by homogenizing, at 4OC, 72-h-old freshly excised radicles in 100 mM KH2P04buffer (pH 7.5), 250 m sorbitol, 0.2 m EDTA, and 0.1% BSA, followed by a 6000g centrifugation for 20 s. After the pellet was discarded, the supematant was centrifuged at 15,OOOg for 10 min, and the pellet was resuspended in 50 m KH2P04 buffer (pH 7.2), 250 mM sorbitol. Pellets of crude mitochondri'i, nuclei, and pelletable cell debris were rapidly dried over CaC12 for 24 h at 16OC and used for EPR analysis.

MATERIALS AND METHODS Plant Material, Cermination, and Desiccation Tolerance Test

Kemels of Zea mays L. cv Vanessa were allowed to germinate for 96 h in the dark at a constant 16OC as previously described (Leprince et al., 1992). Samples were rapidly dehydrated over dry CaC12 under vacuum (20 mm of Hg air pressure) for 24 h (Leprince, 1993), and desiccation tolerance was monitored by radicle emergence and growth following hydration of dried kemels. In these conditions, desiccation tolerance lasts for 24 h after initial imbibition and is lost after 72 h (Leprince et al., 1992). EPR and ENDOR Spectroscopy

EPR spectra were recorded on a Bruker ER 200D spectrometer at room temperature or at 77 K as described by Leprince et al. (1990). Ten to 20 intact dried radicles or cell extracts

FFA Analysis

The FFA were separated by TLC following the method of Christie (1989), visualized with iodine vapor, and quantified by densitometry using a subroutine-macrofunction from a VIDAS image analysis system (Kontron Elektronic) with linolenic acid as standard. The optical measurements were performed on six plates from three different lipid extractions. O2Uptake Measurements and lnhibition Study

After a 72-h imbibition, kemels were incubated for 2 h at 16OC in KH2P04buffer (pH 7.5) with various concentrations of respiration inhibitors and uncouplers freshly prepared in 50 md KH2P04buffer (pH 7.5). KCN was added at a final concentration of 1 PM to 1 mM; SHAM at 5 mM; SHAM plus KCN mixture at 0.5 mM KCN plus 5 mM SHAM, A A 3 at 50 p ~ rotenone ; at 5 PM in the presence of 0.05% of Tween 20

Respiration and Desiccation-lnduced Free Radical Processes

1335

(v/v) and FCCP at 0.1 mM, with the pH being maintained at 7.5 throughout. Concentrations of AA3 and rotenone corresponded to sublethal concentrations after a 2-h incubation detennined from viability tests using triphenyltetrazolium and indigo carmine biological stains (800th and Hendry, 1993). 0 2 uptake in three to five control or inhibitor-treated radicles was assessed polarographically at 16OC in a liquidphase Clark-type electrode in 50 mM KH2P04buffer (pH 7.5) in the absence or presence of appropriate inhibitors as previously described (Leprince et al., 1992). Experiments were performed three times with four subreplicates. Statistical variance analysis was carried out using the hierarchical classification of Kruskal and Wallis and standard regression analysis using Spearman’s rank correlation. Chemicals were obtained from Sigma Chemical or BDH (Poole, Dorset, UK).

RESULTS Characterization of the EPR Response in Desiccation-lntolerant Radicles The EPR response detected at room temperature in intact dried maize radicles of cv Vanessa (Fig. la) was identical with that previously reported in dried material from cv Baron (Leprince et al., 1990). The radical spectrum was a single lhe, slightly broadened on the high-field side; the breadth between points of maximum width was 1.11milliTesla and the g value at maximum absorption was 2.0045. This EPR signal was compared with that ,present in green, desiccated tissues of Dicranella palustris, a desiccation-intolerant moss (Seel et al., 1991). Comparisons were made by recording the spectra of maize and moss separately and by recording the spectra of a maize and moss mixture, which gave approximately equal spectral intensities of each of the two components. The EPR spectrum of the mixture was indistinguishable from that of either component (Fig. 1). Further evidence for the identity and similarity of the radical in each tissue was provided by a comparison of the ENDOR responses (Fig. 2). The ENDOR response in the proton region was a characteristic matix ENDOR signal, giving no immediate indication of proton hyperfine coupling that might otherwise have assisted in assigning a structure to the radical. The spectra did show a dip at the free proton

Figure 1. Comparison of second-derivative EPR spectra of intact, dried, desiccation-intolerantmaterial. a, Radicles (72 h old) of maize (cv Vanessa); b, photosynthetic tissues of the D. palustrk moss (examined by Seel et al., 1991); c, maize radicles and moss packed in the same EPR tube. mT, MilliTesla.

I 12

I 14

I 16

L 18 M H z

Figure 2. Comparison of first-derivative ENDOR spectra of desiccation-intolerant maize radicles (a) and desiccated D. palustris moss (b). The arrowhead indicates a significant notch at the free proton frequency.

frequency that arises because excitation of NMR transitions in nuclei with zero hyperfine coupling does not relieve saturation of the EPR transition. The depth of the dip is particularly sensitive to microwave power; both shape and pattern of response to the physical and instrumental settings are determined by spin diffusion. The important point in the present context is that the matrix ENDOR responses for the maize and moss samples show the same dependence on these parameters. This would be expected for chemically identical species in comparable physical environments. The line shape was independent of temperature down to 105 K, indicating that the radicals were strongly immobilized at room temperature and that g anisotropy was small. This supports the earlier argument (Seel et al., 1991) that the species is not a peroxy radical. The term “carbon centered” was used earlier to describe the radical in moss (Seel et al., 1991), but further, recent experiments indicate that this is a quinone (Atherton et al., 1993). Attempts were made to locate this free radical at the subcellular level. Severa1 cellular fractions were prepared from dried desiccation-intolerant radicles including total lipid, FFA, triacylglycerol, and polar lipid fractions, powdered pellets of defatted cellular debris resulting from backwashings during the lipid extraction procedure, dried crude mitochondria, nuclei, and cellular debris extracted from fresh desiccation-intolerant radicles (Table I). Although an accurate quantification was not possible, the strongest EPR responses were detected in the defatted cell extracts and in the dried mitochondria pellet. A weak EPR signal was found in the cellular debris but was not detected in other cellular fractions. This suggests that the free radical may be located in and/or generated in vitro by drylng mitochondria. A summary of the desiccation-induced buildup of the stable radical, lipid peroxidation, and accumulation of FFA in genninating tissues is presented in Table 11. Desiccation-tolerant radicles exhib-

Leprince et al.

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Table 1. Estimation of free radical accumulation detected by EPR in

various cellular extracts prepared from desiccation-intolerant germinating maize radicles O, Little or no detectable response, +++, strong response. Free Radical Accumulation

Cellular Extracts

Total lipids" Polar lipidsa

O O O O

Neutra1 lipids: FFA" Triacy lglycerol" Defatted cellular debris"

coefficient (r = 0.968). A similar high correlation ( r = 0.980) was obtained in a second experiment in which the rates of O2 uptake by radicles in various stages of germiriation were plotted against the free radical content from desiccated radicles of ecpivalent stages (Fig. 3B). Both regressions indicate a correlation between respiration and desiccation-induced buildup of the stable, free radical. Identification of the Sites of Electron Leakage by lmpaired Electron Transport Chains and Their Linkage to Free Radical Accumulation

+++ O +++

Nucleib Mitochondriab Cellular debrisb a Sample prepared from dried material. first from fresh material and then dried.


+

Samples prepared

To explore the relationship between desiccation-impaired respiratory electron transport chains and free radical accumulation and to identify possible sites respon:sible for its formation, the EPR responses of 72-h-old radicle.; were monitored, a fter desiccation, in the presence of variou:$respiratory inhibitors known to modify activated O2production in animal and plant mitochondria (Rich and Bonner, 1978; Turrens and Boveris, 1980; Nohl and Jordan, 1986). The effects of inhibitors were confirmed polarographically in 72-h -old treated radicles (Table 111). In the presence of the uncoiipler FCCP, O2 consumption rates were enhanced by 1.3-foltl after treatment, indicating that mitochondrial respiration in control radicles operates with a limiting control of atlenylates as previously observed (Lambers, 1985, and refs. therein). The EPR responses of inhibitor-treated and then desiccated, intolerant radicles were of three different types: (a) Rotenone and FCCP induced an increase in EPR amplitudes of 47 and 10% over control values, respectively; (b) AA, affected respiration rates but did not alter significantly the free radical production in desiccated material; and (c) KCN, SHAM, and the combination of both brought about a reduction in the stable free radical content to 63,51, and 47%, of the untreated controls, respectively, accompanied by a significíintreduction in O2uptake by 69 to 32% of the rates of the uninhibited controls. Results of this experiment also confinned the correlations between O2 uptake rates and promotion of radical oxidative processes observed in Figure 3. 1

ited little peroxidative damage (measured by accumulation of TBA-reactive substances and accumulation of FFA), in marked contrast to desiccation-intolerant material. The desiccation-induced EPR response increased in parallel with the loss of desiccation tolerance and lipid peroxidation. O2Consumption Correlated with Desiccation-lnduced Buildup of Free Radicals

Two independent experiments were developed to establish a correlation between O2consumption by radicles and subsequent free radical accumulation after desiccation. The first consisted of measuring the inhibition of O2 uptake by KCN (1 ~ L Mto 1 m)and the EPR responses after KCN addition and desiccation. Addition of KCN in 72-h-old radicles reduced both O2 consumption and EPR signal amplitude in treated, and then desiccated, material (Fig. 3A). Maximum inhibition of O2 uptake and maximum decrease in the amplitude of the desiccation-induced EPR response were obtained with 0.5 and 1 mM KCN, respectively. The plot of O2 consumption rates versus free radical content in treated radicles following desiccation gave a high positive correlation

Table II. Evidence for the involvement of desiccation-induced free radical processes during the loss of desiccation tolerance, namely, lipid peroxidation (measured as the accumulation of TBA-reactive substances against malonyldialdehyde as standard [mean f SE, n = 5-61), phospholipid de-esterification (measured by the accumulation of the FFA fractions from JLC plates [mean f SE, n = 6]), and buildup of a stable organic radical (estimated by the amplitude height of the EPR signal corrected on the dry weight basis [mean f SE, n = 4-8, except for footnote cl)

Control

% of regermination

95 92 86 39 1 O ~

~

a

Free Radical Accumulation

Phospholipid De-Esterification

Lipid Peroxidation' Desiccation Tolerance Desiccated

nmol of malonyldialdehyde mg-' protein

Coritrol

Desiccated

Control

cm g-' dry wt

FFA, % of polar Iipids

b

b

b

b

b

0.1 66 f 0.064 0.085 & 0.019 0.085 f 0.019 0.015 & 0.014 0.024 & 0.064

0.1 92 f 0.022 O. 152 f 0.029 0.606 f 0.022 0.630 f 0.016 0.921 f 0.087

b

b

b

3.8 r t 1.4

3.7 f 1.5

Data adapted from Leprince et al. (1990).

Not determined.

44c

b

b

b

8.9 :t 5.6

24.2 f 4.5

72

b

b

b

~~

Deterrnined from one replicate.

Desiccated

2'75 f 6 325 & 34 353 f 12 525 k 5 558 f 24 621 f 2 3

-


-

r = 0.968

90

x

0

85

41 o

70

I

I

I

I

I

I

75

80

85

90

95

100

/

Free radical content , % o f control

200

250

300

550

400

450

500

550

Free radical content

600

650

-1

Relative unit. c m ( g DW)

Figure 3. A, The relation given by line regression between KCN-

resistant free radical accumulation in intact 72-h-old radicles following desiccation (expressed as a percentage of untreated and desiccated controls) and KCN-resistant respiration in intact radicles (expressed as a percentage of O2 uptake of untreated controls; mean k SE, n = 4). 6, The relation between desiccation-induced radical content and O2 uptake by intact radicles in various stages of germination (mean f SE, n = 3-5). Spearman’s correlation coefficients are also given. DW, Dry weight. DISCUSSION

Results of the EPR study confirm that desiccation of intolerant maize seedlings brought about a higher production and accumulation of a stable, organic free radical than in desiccation-tolerant material. This stable free radical does not derive from lipid or lipid-soluble substances of low mo1 wt, because it was detected exclusively in the pellet of defatted cell debris following low-speed centrifugation. Isolated mitochondria, however, showed a similar EPR signal following a drying treatment in vitro (Table I). It is known that, in chloroplasts and mitochondria, quinones readily form free radicals (Cadenas, 1989). Nohl and Jordan (1986) reported a g value for ubiquinone of 2.004, which is close to the stable radical reported in our study. The recent identification of the similar signal in desiccated m o s also with g = 2.0045 (Atherton et al., 1993) and its association with mitochondria suggests that, in maize, it is a quinone radical. In this respect, its unusually high stability is not fully understood in the biological context but might be explained by the presence of the unpaired electron highly delocalized and resonance stabilized by an unsaturated acyl chain or phenolic compound and/or by tightly binding to a specific protein. Such association could render the whole molecule rather hydrophilic

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and would explain why the quinone radical did not appear in the hydrophilic fraction. The stable free radical also appeared to be identical with those detected in a wide range of tissues of other plants subjected to stresses that are known to promote oxidative injury, such as water loss (axial tissues of acom seeds [Hendry et al., 19921 and droughted leaves [Runeckles and Vaartnov, 1992]), high irradiance and water stress in mosses (Seel et al., 1991), O2 enrichment of roots (Goodman et al., 1986), and postanoxia of rhizomes (Crawford, 1993). This common EPR response following stress in both photosynthetic and nonphotosynthetic tissue indicates that this buildup is due to the presence of a sink molecule that traps and stabilizes electrons generated by the impaired electron transport chains to form a long-lived free radical. Although the precise mechanism is unknown, it seems likely that this EPR signal is the result rather than the cause of cell injury. This is supported by the buildup of TBA reactive substances, markers of peroxidized lipids, and in FFA, a marker of de-esterified phospholipids under the same conditions of stress. The strong, positive correlations between O2 consumption and free radical point to a central, if not causal, role played by respiration. On the other hand, loss of desiccation tolerante in genninating maize radicles has been associated with diminishing protection to activated O2by superoxide dismutase, GSH reductase, and peroxidases (Leprince et al., 1992) and with the severe impairment and loss of activities of several mitochondrial enzyme complexes (Leprince, 1992). It is interesting that it has been reported that the suppression of activity of several enzymes from the mitochondrial electron transport chains following mitochondrial genome alteration induced an increase in superoxide yield (Bandy and Davidson, 1990; Salin, 1991).During germination of soybean seeds, an increase in H202 and 02production by mitochondria has been observed (Puntarulo et al., 1991). The EPR experiments of Goodman et al. (1986) on wheat roots showed that O2 depletion and enrichment directly modulated the organic free radical concentration. Therefore, it seems likely that the desiccation-induced EPR response in our material originates from the following electron transfer sequence: following lethal desiccation, specific enzyme complexes prematurely termi-

Table 111. Effect of respiration inhibitors and uncoupler on desiccation-induced free radical accumulation and O2 uptake by 72-h-old radicles that are desiccation intolerant Mean data are expressed as percentages of untreated controls. Actual values of controls are reported in parentheses (PM O2 [g dry weight minl-’ and cm of EPR peak amplitude [g dry weightl-’). Treatment

Control Rotenone FCCP AA3 KCN SHAM KCN + SHAM

Free Radical Concentration

O2Consumption

% of control

% of control

1O 0 (550 f 35) 147 f 16 110 & 3.8 94.5 f 4.7 62.7 k 9.5 51.2 f 4.3 46.7 k 11.4

100 (3.64 +. 5.4) 85.6 f 5.4 130.1 +. 11.7 68.1 rt 8.4 57.6 f 2.8 69.0 +. 7.6 32.1 +. 7.8

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Leprince et al.

nate electron transport and offer, thermodynamically, conditions to reduce molecular 02,yielding activated O2 (Cadenas, 1989)and then a series of one (or two) electron transfers accumulating a stable free radical and peroxidative damage. With the development of the respiratory systems in germinating maize (Leprince et a]., 1992), there is certainly increased opportunity for electron leakage to molecular 01, which could explain the increase in the EPR response with the onset and progress of desiccation intolerance (Table 11). The sequence of events described above is further supported by the data conceming the different sensitivity of inhibitors of respiration and their effects on stable free radical accumulation (Table 111). In submitochondrial particles of severa1 plant tissues, activated O2 generation at the NADH dehydrogenase of complex I has been identified by its insensitivity to KCN and AA3, but also by its sensitivity to rotenone, whereas that at ubiquinone-Cyt b of complex 111 was known by its insensitivity to SHAM and AA3 and sensitivity to KCN (Rich and Bonner, 1978; Salin 1991). The enhancement of free radical production in the presence of rotenone suggests the contribution of the flavoprotein NADH dehydrogenase of complex I to the EPR signal. Turrens et al. (1991) experimented with rat and rabbit hearts subjected to reoxygenation after reperfusion and showed that NADH dehydrogenase was highly reduced and contributed to a reperfusion oxidative injury by transferring electrons to O?, thereby generating activated 02.In our plant material, however, the presence of antimycin, which would indirectly affect the redox potential of upstream complexes, had no effect on the free radical fonnation. We assume that the electron flow has been disrupted between the complex I and 111. We previously observed that both the alternative oxidase (Leprince et al., 1992) and the NADH-Cyt c reductase were extremely sensitive to desiccation, whereas the succinate dehydrogenase remained unaffected by a desiccation treatment (Leprince, 1992). The inhibition of the free radical formation due to KCN and the absence of synergistic effects of the KCN plus SHAM mixture suggest that Cyt c oxidase may be another site of electron leakage and also point to the ubiquinone-Cyt b complex as a site at which electron flow is impeded, leading to stable free radical accumulation. Puntarulo et al. (1988) also reported that activated 0 2 formation in soybean mitochondria was cyanide sensitive, but the question of whether Cyt c oxidase produces activated O2is still unresolved (Cadenas, 1989). The sensitivity to SHAM, which was not significantly different from that of KCN and KCN plus SHAM, remains unexplained. Because the altemative oxidase does not generate 02or H202,it is possible that SHAM modified the electron flow between ubiquinone and Cyt c oxidase, subsequently back-controlling the production of the stable radical upon desiccation. An additional source of desiccationinduced stable free radical might ultimately follow collapse of the mitochondrial membrane potential. This was apparently simulated in our material by the addition of FCCP, which enhanced both respiration rates and stable radical accumulation. We conclude that the primary source of electrons leading to formation and accumulation of the stable free radical in desiccation-intolerant maize seeds is the mi-


tochondrion and that one significant casualty of desiccation damage is the ubiquinone pool. ACKNOWLEDCMENTS

We wish to thank Dr. W. Seel who kindly supplied the dried moss material and Dr. B.D. McKersie for reviewing the manuscript. Received July 12, 1993; accepted October 1, 1993. Copyright Clearance Center: 0032-0889/94/104/1333/07. LITERATURE CITED

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