Release of Reactive Oxygen ... - Plant Physiology · by plant cells (“oxidative burst”; Baker...

Release of Reactive Oxygen Intermediates (SuperoxideRadicals, Hydrogen Peroxide, and Hydroxyl Radicals) andPeroxidase in Germinating Radish Seeds Controlled byLight, Gibberellin, and Abscisic Acid1

Peter Schopfer*, Claudia Plachy, and Gitta Frahry

Institut fur Biologie II der Universitat, Schanzlestrasse 1, D–79104 Freiburg, Germany

Germination of radish (Raphanus sativus cv Eterna) seeds can be inhibited by far-red light (high-irradiance reaction ofphytochrome) or abscisic acid (ABA). Gibberellic acid (GA3) restores full germination under far-red light. This experimentalsystem was used to investigate the release of reactive oxygen intermediates (ROI) by seed coats and embryos duringgermination, utilizing the apoplastic oxidation of 29,79-dichlorofluorescin to fluorescent 29,79-dichlorofluorescein as an invivo assay. Germination in darkness is accompanied by a steep rise in ROI release originating from the seed coat (livingaleurone layer) as well as the embryo. At the same time as the inhibition of germination, far-red light and ABA inhibit ROIrelease in both seed parts and GA3 reverses this inhibition when initiating germination under far-red light. During the laterstage of germination the seed coat also releases peroxidase with a time course affected by far-red light, ABA, and GA3. Theparticipation of superoxide radicals, hydrogen peroxide, and hydroxyl radicals in ROI metabolism was demonstrated withspecific in vivo assays. ROI production by germinating seeds represents an active, developmentally controlled physiologicalfunction, presumably for protecting the emerging seedling against attack by pathogens.

Reactive oxygen intermediates (ROI) comprise su-peroxide radicals (Oz

22), hydrogen peroxide (H2O2),

and hydroxyl radicals (zOH) formally originatingfrom one-, two-, or three-electron transfers to dioxy-gen (O2). These incompletely reduced oxygen speciesare primarily known as toxic byproducts of variouscellular O2-consuming redox processes such as pho-tosynthetic or respiratory electron transport, whichare responsible for causing symptoms of oxidativedamage if ROI production exceeds the capacity ofROI-scavenging reactions. The toxicity of ROIstrongly depends on the presence of a Fenton catalystsuch as iron ions or peroxidase, giving rise to ex-tremely reactive zOH radicals in the presence of H2O2and Oz

22 (Halliwell and Gutteridge, 1989; Chen and

Schopfer, 1999). In recent years it has emerged thatthe joint exudation of ROI and peroxidase plays animportant role in the defense system of plants againstpathogenic organisms (Vera-Estrella et al., 1993;Scott-Craig et al., 1995; Bestwick et al., 1998). Elicitor-mediated signals generated during the early phase ofpathogen attack can induce rapid production of ROIby plant cells (“oxidative burst”; Baker and Orlandi,1995; Lamb and Dixon, 1997; Wojtaszek, 1997),closely resembling the pathogen-activated ROI pro-duction by the inducible NADPH oxidase redox sys-tem in the plasma membrane of mammalian phago-

cytes (Henderson and Chappell, 1996). There isincreasing evidence suggesting that Oz

22 and/or

H2O2 can serve as integrating elements in the signal-ing circuitry controlling pathogen resistance re-sponses, acquired immunity, and programmed celldeath in plants (Jabs, 1999; Grant and Loake, 2000). Ithas recently been shown that roots of intact plantsare capable of releasing H2O2 into the surroundingmedium, even in the absence of pathogen attack orother stress elicitors (Frahry and Schopfer, 1998a).

Whereas the enzymatic mechanism involved inthe inducible ROI production by phagocytic leuko-cytes is well known (Henderson and Chappell,1996), the functionally corresponding enzyme sys-tem in plants has not yet been elucidated (Bolwelland Wojtaszek, 1997). Besides an hypotheticalNAD(P)H oxidase related to the leukocyte enzyme(Doke et al., 1996; Tenhaken and Rubel, 1998; Ka-wasaki et al., 1999; Frahry and Schopfer, 2000), thereare several other ROI-producing oxidases localizedat the plasma membrane or in the wall of plant cellssuch as peroxidases, amine oxidases, and oxalateoxidase that are potential sources for apoplasticH2O2 (Bolwell and Wojtaszek, 1997). IntracellularH2O2-producing reactions located in chloroplasts,mitochondria, and peroxisomes could possibly con-tribute to ROI release since H2O2 easily permeatesthrough cell membranes (Bass et al., 1983; Allan andFluhr, 1997; del Rıo et al., 1998).

In recent years there has been a growing interest inthe functional significance of ROI in seed aging, seedgermination, and early seedling development. Mostprevious investigations with seeds or seedlings were

1 This work was supported by the Land Baden-Wurttemberg(doctoral fellowship to G.F.), and in part by DeutscheForschungsgemeinschaft.

* Corresponding author; e-mail schopfer @uni-freiburg.de; fax49 –761–203–2612.

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concerned with the destructive effects of ROI such aslipid peroxidation during oxidative stress (Hendry,1993; Leprince et al., 1995; Reuzeau and Cavalie,1995). Much less attention has been paid to thosephysiological conditions where ROI are actively pro-duced for serving a useful function within the seed orin its interaction with the environment. There are afew reports from work with soybean suggesting thatseed germination is accompanied by a generation ofROI in the embryo axis as well as in the seed coat,which can be measured with suitable indicator reac-tions in situ or in the surrounding medium (Boveriset al., 1984; Puntarulo et al., 1988; 1991; Simontacchiet al., 1993; Gidrol et al., 1994; Khan et al., 1996). Asseed germination represents a developmental periodmost sensitive for pathogen infection, it is conceiv-able that ROI release in this stage plays an importantrole in protecting the emerging embryo against inva-sion by parasitic organisms. With this idea in mindwe have studied the release of ROI and peroxidaseduring seed germination in radish (Raphanus sativuscv Eterna). This species has the advantage that culti-vars are available in which germination can be ex-perimentally controlled by light and hormones. Us-ing these tools for the experimental manipulation ofgermination, we determined the time courses of ROIrelease by embryos and seed coats. As peroxidasemay be involved in the transformation of H2O2 intothe much more toxic zOH radical (Chen and Schopfer,1999), we were also interested in the question ofwhether ROI release is accompanied by a release ofthis enzyme.

A second aim of this study was to identify theindividual ROI species produced by germinatingseeds. The separate estimation of H2O2, Oz

22, and

zOH in a physiological reaction is difficult and has sofar not been reported. The specific determination ofH2O2 is relatively simple and can be achieved, forinstance, with the fluorometric scopoletin oxidationassay (Andreae, 1955; Frahry and Schopfer, 1998a).After testing various procedures described in the lit-erature we found that a photometric assay based on

the reduction of newly introduced tetrazolium com-pounds (Na,39-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic-acid hydrate [XTT]; and Na,2-[4-iodophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H-tetrazolium[WST-1]) was suitable for the determination of Oz

22 in

vivo (Sutherland and Learmonth, 1997; Berridge andTan, 1998). It turned out that the degradation ofdeoxy-Rib or the hydroxylation of benzoate to fluores-cent products could be used as indicator reactions forthe determination of zOH (Gutteridge, 1987; Halliwellet al., 1988).

RESULTS

Effect of Far-Red Light, Abscisic Acid (ABA), andGibberellic Acid (GA3) on Germination

Background information on the germination be-havior of the seed material used in the followingexperiments is given in Figures 1 and 2. The seeds ofradish cv Eterna germinate in darkness at 25°C by95% within 30 h after sowing. Continuous irradiationwith far-red light (high irradiance reaction of phyto-chrome) or treatment with ABA in darkness preventgermination almost completely for more than 4 d. Wehave previously shown that light or ABA inhibitgermination in such seeds by lowering the expansiveforce exerted by the embryo on the seed coat to alevel below the critical level that must be exceededfor rupturing this constraining structure (Schopferand Plachy, 1985, 1993). Figure 1 shows that the lightinhibition of germination can be reversed by GA3 ina concentration-dependent manner, whereas the hor-mone has no effect on germination in darkness. Tet-cyclacis (100 mm), an inhibitor of gibberellin biosyn-thesis, blocks germination in darkness and thisinhibition can be reversed by GA3 (data not shown).The promotion of germination by GA3 in far-red lightis accompanied by a strong increase in the ability togerminate under osmotic stress and can therefore beattributed to an increase in the expansive force that

Figure 1. Time course of germination of radishseeds kept in darkness (white symbols) or continu-ous far-red light (black symbols) on water (H2O) orsolutions containing GA3 (10–160 mM) or ABA (10mM) from the time of sowing. GA3 had no effect ondark germination at any of the concentrations test-ed; therefore, only the data for the highest concen-tration are shown.

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allows the embryo to overcome the mechanical seedcoat restraint even in far-red light (Fig. 2). These dataare in agreement with the interpretation that endog-enous gibberellin formation is necessary for embryoexpansion, and thus germination, and that far-redlight affects germination by interfering with gibberel-lin synthesis in the embryo (Inoue, 1991; Bewley andBlack, 1994; Sanchez and de Miguel, 1997).

Release of ROI during Germination

The oxidative burst can be very sensitively demon-strated with the ROI probe, 29,79-dichlorofluorescin(DCFH) that is oxidized by H2O2 to highly fluores-cent 29,79dichlorofluorescein (DCF) in a peroxidase-dependent reaction (Keston and Brandt, 1965). Al-though DCFH does not penetrate the plasmamembrane, it can be introduced into the cell in theform of non-polar, unreactive DCFH-diacetate that issubsequently deacetylated by endogenous esterase.The polar, reactive DCFH liberated intracellularly, aswell as the DCF produced in the reaction with ROI,remain trapped in the cytoplasm (Bass et al., 1983).The reaction can be inhibited by catalase and zOHscavengers, indicating that H2O2 and zOH can partic-ipate in the oxidation of DCFH (Bass et al., 1983;Cathcart et al., 1983; Scott et al., 1988; Simontacchi etal., 1993). We have adapted this procedure for the invivo measurement of extraprotoplasmic ROI by pre-paring the substrate DCFH from DCFH-diacetatewith esterase in the reaction mixture before addingthe plant material to be analyzed. In this way ROI-dependent formation of the fluorochrome could beconfined to the external space and quantitativelymeasured in the incubation medium.

Embryos and coats of germinating radish seedsproduce a rapid increase in fluorescence in the sur-rounding incubation medium containing DCFH andsolidified with agar (Fig. 3). The reaction can beobserved after less than 2 min in the root hair regionof the radicle of young seedlings after liberation fromthe seed coat. A somewhat longer incubation time isneeded to demonstrate a similar reaction in the seed-ling shoot, in the isolated seed coat, or in less ad-vanced stages of germination. The ROI release can bequantitatively determined by measuring the increasein DCF fluorescence in a liquid incubation medium(Fig. 4). As the linearity of the reaction holds for atleast 30 min, an end-point determination after anincubation period of 15 min could conveniently beused in the standard assay for measuring ROI releasein the following experiments.

Germination of radish seeds under the experimen-tal conditions shown in Figure 1 is accompanied bythe release of ROI originating from the embryo aswell as from the seed coat. The data shown in Figure

Figure 2. Effect of osmotic constraint on the germination of radishseeds kept in darkness (white symbols) or continuous far-red light(black symbols) in the absence or presence of 160 mM GA3 onosmotic test solutions adjusted to negative water potentials (-c0)indicated on the abscissa. Final germination percentages were de-termined 72 h after sowing.

Figure 3. Demonstration of ROI release by germinating seeds andyoung seedlings of radish. The yellow-green fluorescence indicatesthe conversion of DCFH into DCF in the surrounding of ROI-producing tissues. The pictures show a young seedling, an emptyseed coat, and a germinating seed with protruding radicle 2 (top) or30 (bottom) min after embedding in DCFH-containing agar medium.From a seed population germinated for 48 h in darkness.

Release of Reactive Oxygen Intermediates and Peroxidase in Radish

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5 can be summarized as follows: ROI release can bedetected in the seed coat already 6 to 12 h aftersowing, i.e. 6 h before the onset of visible germina-tion in the seed population. In contrast, the embryoacquires the ability to produce ROI only about 12 hlater, i.e. after the onset radicle growth. There is asteep rise in ROI release in the coat and embryo ofseeds germinating in darkness. Far-red light andABA inhibit this rise almost completely in both seedparts; and GA3 reverses the inhibitory effect of far-red light in both seed parts, but has little effect dur-ing germination in darkness. This set of data showsthat seed coat and embryo are capable of a “burst” ofROI production, whereby the seed coat covers theearly phase and the embryo the later phase of thegermination process. In both seed parts the effects offar-red light, ABA, and GA3 on ROI release are tem-porally correlated with the effects of these factors ongermination.

Release of Peroxidase during Germination

The coats of radish seeds secrete peroxidase duringthe later stage of germination and the initial seedlingdevelopment. Time courses indicating the rate ofperoxidase release by isolated seed coats incubatedunder the experimental conditions of Figure 1 areshown in Figure 6. These data can be summarized asfollows: Peroxidase release starts between 24 and36 h after sowing and reaches a maximal rate 24 to48 h later, i.e. after the young plant has slipped outof the coat and has entered the seedling stage; per-oxidase release by isolated seed coats can be almostcompletely prevented by ABA, but only partly in-hibited by far-red light; and application of GA3promotes peroxidase release in far-red light as wellas in darkness. Moreover, GA3 shifts the period ofmaximal release on the time axis. This effect is mostpronounced in darkness. Thus, although peroxidaserelease is promoted by conditions promoting germi-

nation, it is only loosely correlated with ROI pro-duction.

Identification of Oz2

2, H2O2, and zOH as ROIProduced during Germination

The activated NADPH oxidase system in theplasma membrane of leukocytes reduces O2 to Oz

22,

which can subsequently dismutate to H2O2 1 O2spontaneously or in a reaction catalyzed by superox-ide dismutase (Henderson and Chappell, 1996). Inaddition, zOH can be generated under these conditionif Fe ions or peroxidase catalyze the further reductionof H2O2 to zOH utilizing Oz

22 as an electron donor

(Halliwell and Gutteridge, 1989; Chen and Schopfer,1999). If the “oxidative burst” of germinating radishseeds involves a similar sequence of reactions, itshould be possible to identify Oz

22, H2O2, and zOH in

the incubation medium. Table I shows that the DCFHoxidation by seed coats can be inhibited by a generalantioxidant (ascorbate) as well as by scavengers ofOz

22 (superoxide dismutase), H2O2 (catalase), and

zOH (benzoate, adenine, mannitol, formate, and thio-urea), but not by urea that demonstrates no scaven-ger activity (Halliwell and Gutteridge, 1989). Thesedata suggest that Oz

22, H2O2, as well as zOH are

involved in ROI production in this material.For identifying Oz

22, H2O2, and zOH in the “oxida-

tive burst” of germinating radish seeds we employedassay reactions that are specific for one of these ROI.

Figure 4. Time course of DCFH oxidation by radish seed coats.Three coats from 48-h dark-germinated seeds were incubated in 1.5mL of DCFH assay medium in darkness.

Figure 5. Time course of ROI release by embryos and coats of radishseeds kept in darkness (left) or continuous far-red light (right) onwater (H2O) or solutions containing GA3 (160 mM) or ABA (10 mM)from the time of sowing. Embryos and seed coats were separatedimmediately before the assay.

Schopfer et al.




First, the production of Oz22 by embryos and seed

coats was demonstrated using the reduction of thetetrazolium salts XTT or WST-1 as indicator reactions(Fig. 7). Inhibitor data documenting the specificity ofthe assay and characterizing the underlying Oz

22-

generating reaction are compiled in Table II. TheOz

22 production by seed coats could be inhibited by

superoxide dismutase and its low molecular weightmimic Mn-desferal (Rabinowitch et al., 1987; Able etal., 1998). The difference in effectiveness betweenthese two Oz

22-eliminating agents may be due to the

restricted permeability of the enzyme through thecell wall (Able et al., 1998). Of diagnostic significanceis the finding that Oz

22 production could be inhibited

by DPI, an inhibitor of the leukocyte NADPH oxidase(Henderson and Chappell, 1996), but not by the per-oxidase inhibitors KCN and NaN3. It should be notedin this context that inhibition by DPI alone can pro-vide no unequivocal evidence for the involvement ofthe oxidase since DPI inactivates also hemoproteinssuch as peroxidase (Frahry and Schopfer, 1998b).Catalase as well as zOH-scavenging substances suchas mannitol or formate had no significant inhibitoryeffect on Oz

22 formation by seed coats. Qualitatively

similar results were obtained in corresponding ex-periments with embryos, although the effectivenessof inhibitors was generally lower than in seed coats(data not shown). Taken together, these data showthat seed coats and embryos do produce Oz

22 during

germination. Moreover, differential sensitivity to di-agnostic inhibitors indicates that a cyanide-resistantenzyme, functionally resembling the leukocyteNADPH oxidase, rather than a peroxidase-type en-zyme, is responsible for this performance (Frahryand Schopfer, 2000).

Oz22 rapidly dismutates to O2 and H2O2, a rela-

tively stable ROI generally representing the domi-

nant product of “oxidative burst” reactions in plantsand animals. H2O2 can be specifically determined bythe peroxidase-dependent oxidation of phenolic sub-stances such as scopoletin. Using this sensitive flu-orometric assay, Figure 8 shows the time course ofH2O2 production by radish seed coats and embryosafter 48 h of germination in darkness. The reactioncould be inhibited by H2O2 scavengers (catalase andKI), ascorbate, and DPI, but not by zOH scavengers(mannitol and formate) and agents catalyzing thedismutation of Oz

22 (superoxide dismutase and Mn-

desferal; Table III). Again, qualitatively similar re-sults were obtained with embryos (data not shown).The results confirm the supposition that H2O2 is in-volved in the “oxidative burst” accompanying thegermination of radish seeds. Inhibitor evidence is

Figure 6. Time course of peroxidase release bycoats of radish seeds kept in darkness (whitesymbols) or continuous far-red light (black sym-bols) on water (H2O) or solutions containingGA3 (160 mM) or ABA (10 mM) from the time ofsowing. Coats were isolated from the seeds after24 h and further kept under the respective con-ditions. Activity of secreted peroxidase was de-termined in 2 mL of buffer in which seed coatshad been incubated for 20 min.

Table I. Effect of ROI scavengers on ROI release by radish seedcoats

The ROI-dependent oxidation of DCFH by three coats from 48-hgerminated seeds during a 15-min incubation period was determinedin the presence of various inhibitors. The seed coats were preincu-bated in the inhibitor solution for 15 min before the addition ofDCFH. Urea served as a negative control.

InhibitorROI

Release

%None 100 6 2Catalase (100 mg mL21) 53 6 3Superoxide dismutase (100 mg mL21) 57 6 2Na-ascorbate (10 mM) 43 6 2Na-benzoate (10 mM) 55 6 3Adenine (5 mM) 53 6 4Mannitol (10 mM) 43 6 3Na-formate (10 mM) 47 6 4Thiourea (10 mM) 34 6 2Urea (100 mM) 95 6 3





consistent with the concept that the H2O2 originatesfrom the dismutation of apoplastic Oz

22.

The question arises whether zOH can be detected asa product of apoplastic oxygen metabolism activatedduring seed germination. zOH is an extremely short-lived ROI that has not yet been observed directly inbiological systems. Several methods for the indirectestimation of zOH in chemical reactions have beendescribed that, however, have so far rarely been ap-plied to living plant materials (Babbs, et al., 1989;Kuchitsu et al., 1995; v. Tiedemann, 1997). After mod-ification and careful standardization, the deoxy-Ribdegradation assay (Halliwell et al., 1988) and the

benzoate hydroxylation assay (Gutteridge, 1987)proved to be suitable for detecting zOH productionby radish seed coats. Deoxy-Rib can be oxidativelycleaved by zOH to form malondialdehyde reactingwith thiobarbituric acid to give an adduct that can bemeasured fluorometrically. In an alternate manner,zOH can be reacted with benzoate, resulting in theproduction of fluorescent hydroxybenzoates, ofwhich 2-hydroxybenzoate is the dominantly fluoresc-ing species at pH , 7 (Baker and Gebicki, 1984;Candeias et al., 1994). The incubation of ROI-producing seed coats in a medium containing deoxy-Rib or benzoate resulted in an increase of fluores-cence at the expected wavelengths, which could beinhibited by the zOH scavengers thiourea and for-mate (Fig. 9).

Because of its sensitivity and simplicity, the fluoro-metric determination of benzoate hydroxylationproducts (presumably mainly 2-hydroxybenzoate)was chosen for investigating the time course of zOHproduction by seed coats and embryos after 48 h ofgermination in darkness (Fig. 10). The spectrum ofthe fluorescence increase determined in these exper-iments showed a peak at 407 nm and was qualita-tively indistinguishable from the spectra obtainedwith 2-hydroxybenzoate or 3-hydroxybenzoate (Fig.11). Under the conditions of our measurements onerelative fluorescence unit (407 nm) was equivalent toa 2-hydroxybezoate concentration of 15 nm or a3-hydroxybenzoate concentration of 0.9 mm. The flu-orescence increase produced by seed coats in benzo-ate medium could be inhibited by zOH scavengers(adenine, mannitol, formate, and thiourea), H2O2scavengers (catalase and KI), Oz

22 scavengers (super-

oxide dismutase and Mn-desferal), as well as by per-oxidase inhibitors (KCN and NaN3). Urea, employedas an inactive analog of thiourea, had no inhibitoryeffect even at high concentrations (Table IV). It ap-pears from these results that zOH can also be gener-

Figure 7. Time course of Oz2

2 release by embryos and coats ofgerminating radish seeds. Ten embryos or 20 coats from 48-h dark-germinated seeds were incubated in 1 mL of XTT (black symbols) orWST-1 (white symbols) assay medium in darkness. Both reagentsprovided similar results after 12 h of incubation. XTT was selected forelaborating detailed time courses of Oz

22 formation. Data are nor-

malized on a per embryo/seed coat basis.

Figure 8. Time course of H2O2 release by embryos and coats ofgerminating radish seeds. Five embryos or 10 coats from 48-h dark-germinated seeds were incubated in 3 mL of scopoletin assay me-dium in darkness.

Table II. Effect of ROI scavengers and related inhibitors on Oz2

2

release by radish seed coatsThe Oz

22-dependent reduction of XTT by 20 coats from 48-h

germinated seeds during a 3-h incubation period was determined inthe presence of various inhibitors.

InhibitorOz

22

Release

%None 100 6 5Superoxide dismutase (100 mg mL21) 59 6 8Mn-desferal (300 mM) 0 6 0Diphenyleneiodonium (DPI) (100 mM)a 48 6 14KCN (1 mM) 97 6 14NaN3 (1 mM) 89 6 7Catalase (100 mg mL21) 82 6 21Na-benzoate (10 mM) 82 6 21Adenine (5 mM) 123 6 15Mannitol (10 mM) 81 6 14Na-formate (10 mM) 153 6 24Thiourea (10 mM) 91 6 20Urea (100 mM) 115 6 14

a Including 10 mL mL21 dimethylsulfoxide (ineffective in controls).

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ated during the “oxidative burst” of germinating rad-ish seeds. Inhibitor evidence reveals that the zOHproduction in this system depends on the availabilityof Oz

22, H2O2, and active peroxidase.

DISCUSSION

Using a sensitive assay for ROI originally devel-oped for demonstrating the oxidative burst in phago-cytic blood cells, this study shows that a similar risein ROI release is initiated in germinating seedsshortly before the radicle protrudes through the seedcoat. Up to now, ROI production by seeds has almostexclusively been considered under the aspect of oxi-dative stress, causing detrimental effects withinseeds such as a loss of viability and germinability

(Hendry, 1993; Leprince et al., 1995; Reuzeau andCavalie, 1995). In contrast to this view it is the inten-tion of the present report to demonstrate that theproduction of ROI during seed germination in factrepresents an active, beneficial biological reactionthat is connected with high germination capacity andvigorous seedling development. This view is sup-ported by the finding that a strong rise in ROI releasetakes place in the healthy, actively germinating seed,which coincides with the period in which the ex-panding embryo ruptures the coat and becomes ex-posed to environmental factors such as pathogenicmicroorganisms. This temporal correlation holdswhen germination is suppressed or promoted byphotomorphogenetic light or hormones such as ABAand GA3, indicating that both events are under asimilar kind of developmental control.

The metabolic activity of the seed coat in themature seed can be ascribed to the aleurone layer, aliving tissue covering the inner surface of the coatthat is released from the quiescent state togetherwith the germinating embryo (Bewley and Black,1994). Whereas the role of the aleurone layer as asecretory tissue for hydrolases, peroxidase, and sev-eral other enzymes in the caryopses of cereals is wellestablished (Jones and Jacobsen, 1991), very little isknown about the morphological counterpart in theseeds of dicotyledonous plants (Yaklich et al., 1992).In galactomannan-storing seeds of legumes such asTrigonella foenum-graecum the aleurone layer secretsgalactan- and mannan-degrading hydrolases for thebreakdown of these storage polysaccharides in thecell walls of the endosperm during germination (Be-wley and Black, 1994). In Brassicaceae that lack astorage endosperm in the mature seeds, the aleu-rone tissue can be developmentally derived during

Figure 9. Demonstration of zOH release by radishseed coats using the deoxy-Rib degradation assay(a) or benzoate hydroxylation assay (b). Twenty or10 coats, respectively, from 48-h dark-germinatedseeds were incubated for 6 h in darkness in 1.5 mLof buffer containing 20 mM deoxy-Rib or 2.5 mM

benzoate and zOH scavengers (thiourea, TU; for-mate, FA). Degradation of deoxy-Rib was deter-mined by the increase in fluorescence at 553 nmagainst a blank without seed coats. Formation ofhydroxybenzoates was determined by the increasein fluorescence at 407 nm against a blank withoutbenzoate. The background fluorescence due tobenzoate (see Fig. 11) was subtracted.

Table III. Effect of ROI scavengers and related inhibitors on H2O2

release by radish seed coatsThe H2O2-dependent oxidation of scopoletin by 10 coats from

48-h germinated seeds during a 1-h incubation period was deter-mined in the presence of various inhibitors.

InhibitorH2O2

Release

%None 100 6 3Catalase (100 mg mL21) 72 6 5KI (10 mM) 47 6 10Na-ascorbate (10 mM) 63 6 6DPI (100 mM)a 42 6 4Superoxide dismutase (100 mg mL21) 100 6 6Mn-desferal (300 mM) 95 6 15Mannitol (10 mM) 121 6 12Na-formate (10 mM) 92 6 5Urea (100 mM) 90 6 5

a Including 10 mL mL21 dimethylsulfoxide (ineffective in controls).





seed maturation from the inner integument (Berg-feld and Schopfer, 1986) or from the outer layer ofthe endosperm (Groot and Van Caeseele, 1993). Inmustard seed coats, the induction of metabolic ac-tivity, including a rapid breakdown of storage pro-tein and storage fat, starts within less than 24 h aftersowing in a similar fashion as in the cotyledons ofthe embryo and is subject to a similar control bylight (Bergfeld and Schopfer, 1986). Our experi-ments with the coats of radish seeds provide evi-dence that the aleurone layer in the seeds of dicot-yledonous plants without bulky endosperm canfunction as a secretory tissue during germination byreleasing ROI and peroxidase to the apoplasticspace, presumably as a constitutive defense reactionagainst infection by microorganisms. In view of therelative chemical inertness of Oz

22 and H2O2, the

effectiveness of this reaction may depend chiefly onthe peroxidase-mediated generation of zOH. More-over, in view of the emerging role of ROI in signaltransduction (Jabs et al., 1997; Alvarez et al., 1998;Chamnongpol et al., 1998), it appears possible thatthese oxygen species are involved in activating andcoordinating various additional defense reactionsthat give the embryo a pre-emptive advantage forsurvival in a hazardous environment.

The ROI assay based on DCFH oxidation providesan integral measure for H2O2, zOH, and Oz

22 (after

its dismutation to H2O2 and O2) and is, therefore,perfectly suited for an overall assessment of ROIproduction by plant tissues in vivo. However, thisstrength also represents a major drawback in so faras the assay can give no information on the biosyn-thetic relationships and interactions between thesemolecules. The inhibitor evidence obtained with

various reagents scavenging one or more of the ROIsuggest that all three types of ROI can be producedin germinating radish seeds (Table I). We havetested this supposition by applying appropriate as-say reactions for Oz

22, H2O2, and zOH with the result

that all three ROI can be identified in the incubationmedium of embryos and coats isolated from germi-nating radish seeds. This conclusion is of coursecritically dependent on the reliability of the assayreactions used. We have therefore applied variousdiagnostic radical scavengers and inhibitors to testthe specificity of these reactions (Tables II–IV).These tests confirm that the assays employed in thisstudy are suitable for selectively detecting Oz

22,

H2O2, and zOH in a mixture of all three ROI. More-over, the inhibitor data provide information on thebiosynthetic relationships between various ROI.This information can be summarized as follows:Oz

22 is formed by a cyanide-insensitive, DPI-

sensitive reaction, pointing to a NAD(P)H-oxidase-type enzyme in the plasma membrane; H2O2 origi-nates from the dismutation of Oz

22 as its formation

can be inhibited by inhibiting Oz22 formation with

DPI; and zOH is formed in a cyanide-sensitive reac-tion depending on the presence of Oz

22 and H2O2.

This points to a Fenton-type reaction catalyzed byapoplastic peroxidase. It has recently been shownthat peroxidase, transformed into the perferryl state(Compound III) by Oz

22, catalyzes the generation of

Figure 10. Time course of zOH release by embryos and coats ofgerminating radish seeds. Ten embryos or 10 coats from 48-h dark-germinated seeds were incubated in 1.5 mL of benzoate assay me-dium in darkness. Data are normalized on a per embryo/seed coatbasis.

Figure 11. Fluorescence emission spectra of benzoate (2.5 mM) andreaction product(s) of the benzoate hydroxylation assay. Radish seedcoats were incubated for 6 h in assay medium with or withoutbenzoate as in Figure 10 (a). The spectra of authentic2-hydroxybenzoate (2-HOB, 0.2 mM), 3-hydroxybenzoate (3-HOB,10 mM), and 4-hydroxybenzoate (4-HOB, 1 mM) are included forcomparison (b).

Schopfer et al.




zOH from H2O2 (Chen and Schopfer, 1999). Takentogether, this information can be qualitatively ac-commodated in a functional scheme (Fig. 12). Ad-ditional work is needed to further test this scheme,e.g. by investigating the quantitative relationshipsbetween the reactants involved.

MATERIALS AND METHODS

Plant Material and Culture Conditions

Seeds of radish (Raphanus sativus cv Eterna) were ob-tained from J. Wagner Markensaaten GmbH (Heidelberg,Germany). Batches of 50 seeds were sown on chromato-graphic paper soaked with distilled water or hormonesolutions (GA3 from Serva, Heidelberg, or ABA fromFluka, Buchs, Switzerland) and were incubated at 25°C indarkness or far-red light (standard far-red source, 3.5Wm22; Mohr, 1966) as described previously (Schopfer andPlachy, 1984). Hormone solutions were adjusted to pH 6.5to 6.7 with KOH. Germination in the presence of osmoticstress was determined by incubating seed batches on de-fined solutions of polyethylene glycol 6000 (Roth,Karlsruhe, Germany) as described previously (Schopferand Plachy, 1993). Germination percentage was deter-mined using protrusion of the radicle by more than 2 mmas a criterion (Schopfer and Plachy, 1984). For biochemicalassays the seed coats were removed from the seeds withfine forceps, carefully avoiding injuring the embryos. Allhandling was done under green safe light.

Determination of ROI Release

A working solution containing 50 mm DCFH-diacetate(Serva) in K-phosphate buffer (20 mm, pH 6.0) preparedfrom a 25 mm DCFH-diacetate solution in acetone was

incubated for 15 min at 25°C with 0.1 g L21 of esterase(from pig liver, Boehringer Mannheim, Germany) fordeacetylation. This solution was immediately used for ROIassays by incubating three to 15 embryos or seed coats in1.5 mL for 15 min at 25°C on a shaker. A 1-mL aliquot of thesolution was removed and the increase in fluorescence(excitation: 488 nm, emission: 525 nm) due to the oxidationof DCFH to DCF was measured within a few minutes usinga fluorescence spectrophotometer (LS-3B, Perkin-Elmer,Foster City, CA). Blanks without plant material were run inparallel and used for subtracting spontaneous fluorescencechanges. Oxidation of DCFH by H2O2 is dependent onperoxidase (Keston and Brandt, 1965). As the addition ofhorseradish peroxidase to assays with embryos and seedcoats had no effect on the measurements, endogenous per-oxidase activity was obviously not rate limiting in cata-lyzing the assay reaction. The fluorescence increase waskept in the linear range of the assay as judged from cali-bration curves with H2O2 and peroxidase. In this range thefluorescence increase was proportional to the number ofseed coats incubated in the assay solution. The fluorescenceincrease could be completely inhibited by KCN (200 mm) orby boiling the plant material before the assay. ROI scav-enging reagents were obtained from Boehringer Mannheim(catalase and superoxide dismutase) or from Sigma (De-isenhofen, Germany). Reagents were adjusted to pH 6.0with HCl or KOH if necessary.

ROI formation by embryos and seed coats was visual-ized by embedding them in agar medium (10 g L21) con-taining the above reagent solution. Pictures were takenwith an epifluorescence microscope (Stemi SV6, Zeiss,Oberkochen, Germany) with excitation filter BP 450–490and emission filter LP 520 using Ektachrome 64T film(Kodak, Rochester, NY).

Determination of Oz2

2 Release

Batches of 10 embryos or 20 seed coats were incubated in1 mL of K-phosphate buffer (20 mm, pH 6.0) containing 500mm XTT or WST-1 (Polyscience Europe, Eppelheim, Ger-many) in darkness at 25°C on a shaker (Sutherland and

Figure 12. Tentative qualitative scheme illustrating the biosyntheticrelationships between various ROI formed by germinating radishseeds.

Table IV. Effect of ROI scavengers and related inhibitors on zOHrelease by radish seed coats

The zOH-dependent hydroxylation of benzoate by 10 seed coatsfrom 48-h germinated seeds during a 3-h incubation period wasdetermined in the presence of various inhibitors. Urea served as anegative control.

InhibitorzOH

Release

%None 100 6 5Catalase (100 mg mL21) 45 6 15KI (10 mM) 7 6 21Superoxide dismutase (100 mg mL21) 30 6 18Mn-desferal (300 mM) 26 6 29KCN (1 mM) 29 6 23NaN3 (1 mM) 214 6 10Adenine (10 mM) 26 6 10Mannitol (50 mM) 54 6 22Mannitol (100 mM) 8 6 35Na-formate (10 mM) 81 6 8Na-formate (20 mM) 235 6 27Thiourea (50 mM) 19 6 14Urea (100 mM) 83 6 27





Learmonth, 1997; Berridge and Tan, 1998). The increase inA470 (XTT) or 440 nm (WST-1) in the incubation mediumwas measured with a Kontron Uvikon 940 spectrophotom-eter. Reagent blanks (without tissue) and tissue blanks(without reagent) run in parallel were used to correct forunspecific absorbance changes. DPI was obtained fromBiomol (Hamburg, Germany). Mn-desferal (green com-plex) was prepared from deferoxamine mesylate (Sigma)and MnO2 as described by Beyer and Fridovich (1989).

Determination of H2O2 Release

Batches of five embryos or 10 seed coats were preincu-bated for 30 min in 3 mL of K-phosphate buffer (20 mm, pH6.0) to remove pre-formed H2O2 and were then incubatedin 3 mL of the same buffer containing 5 mm scopoletin(Sigma) and 3 mg mL21 horseradish peroxidase (Boehr-inger Mannheim) in darkness at 25°C on a shaker (An-dreae, 1955). The decrease in fluorescence (excitation: 346nm, emission: 455 nm) in the incubation medium wasmeasured using reagent blanks as reference. Fluorescencewas transformed into molar H2O2 concentration using alinear calibration curve.

Determination of zOH Release

Batches of 10 embryos or 10 seed coats were incubated in1.5 mL of K-phosphate buffer (20 mm, pH 6.0) containing2.5 mm Na-benzoate (Sigma) in darkness at 25°C on ashaker (Gutteridge, 1987). After clarifying the incubationmedium by centrifugation, the increase in fluorescence(excitation: 305 nm, emission: 407 nm) was measured in thefluorescence spectrophotometer using a quartz cell. Blankswithout benzoate run in parallel were used to correct forunspecific fluorescence originating from substances elutedfrom the plant material. A305 in the incubation medium was#0.12 and was not significantly higher in blanks, excludingoptical artifacts in the fluorescence measurements.

In an alternate manner, zOH production was estimated asdescribe by Halliwell et al. (1988) by incubating 20 seedcoats in 1.5 mL of buffer containing 20 mm 2-deoxy-D-Rib(Sigma). The formation of the breakdown product malon-dialdehyde was determined by mixing 0.5 mL of centri-fuged incubation medium with 0.5 mL of 2-thiobarbituricacid (Serva; 10 g L21 in 50 mm NaOH) and 0.5 mL oftrichloroacetic acid (28 g L21). After heating in boilingwater for exactly 10 min, cooling in tap water, and clarify-ing by centrifugation, the reaction product was measuredfluorometrically (excitation: 532 nm, emission: 553 nm)against reagent blanks.

Determination of Peroxidase Release

Batches of 20 seed coats removed from seeds 24 h aftersowing were transferred to small Petri dishes containing 2mL of distilled water or hormone solutions at 25°C. Afterappropriate periods of time the seed coats were blottedwith paper towels and were incubated further in disheswith 2 mL of K-phosphate buffer (50 mm, pH 7.0; contain-

ing hormones in case of hormone treatments) for 20 min ona shaker. Seeds and seed coats were kept continuously infar-red light or darkness (interrupted for short periods ofhandling under dim, green safe light). Peroxidase activityin the incubation medium was determined photometricallyat 436 nm (25°C) using 0.4 mm guaiacol (Merck, Darmstadt,Germany) and 2 mm H2O2 as substrates in K-phosphatebuffer (50 mm, pH 7.0) in a total volume of 2.5 mL.

Statistics

Data points represent means of four to 12 independentexperiments. ses are indicated except where too small to beshown graphically.

Received May 30, 2000; returned for revision September 20,2000; accepted November 7, 2000.

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