nitric oxide synthase-mediated phytoalexin accumulation in soybean cotyledons in response to the...

Nitric Oxide Synthase-Mediated PhytoalexinAccumulation in Soybean Cotyledons in Response to theDiaporthe phaseolorum f. sp. meridionalis Elicitor1

Luzia Valentina Modolo, Fernando Queiroz Cunha, Marcia Regina Braga, and Ione Salgado*

Departamento de Bioquımica, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP,13083–970, Brazil (L.V.M., I.S.); Departamento de Farmacologia, Faculdade de Medicina de Ribeirao Preto,Universidade de Sao Paulo, Ribeirao Preto, SP, 14049–900, Brazil (F.Q.C.); and Secao de Fisiologia eBioquımica de Plantas, Instituto de Botanica, Sao Paulo, 01061–970, SP, Brazil (M.R.B.)

Phytoalexin biosynthesis is part of the defense mechanism of soybean (Glycine max) plants against attack by the fungusDiaporthe phaseolorum f. sp. meridionalis (Dpm), the causal agent of stem canker disease. The treatment of soybean cotyledonswith Dpm elicitor or with sodium nitroprusside (SNP), a nitric oxide (NO) donor, resulted in a high accumulation ofphytoalexins. This response did not occur when SNP was replaced by ferricyanide, a structural analog of SNP devoid of theNO moiety. Phytoalexin accumulation induced by the fungal elicitor, but not by SNP, was prevented when cotyledons werepretreated with NO synthase (NOS) inhibitors. The Dpm elicitor also induced NOS activity in soybean tissues proximal tothe site of inoculation. The induced NOS activity was Ca21- and NADPH-dependent and was sensitive to the NOS inhibitorsNG-nitro-l-arginine methyl ester, aminoguanidine, and l-N6-(iminoethyl) lysine. NOS activity was not observed in SNP-elicited tissues. An antibody to brain NOS labeled a 166-kD protein in elicited and nonelicited cotyledons. Isoflavones(daidzein and genistein), pterocarpans (glyceollins), and flavones (apigenin and luteolin) were identified after exposure tothe elicitor or SNP, although the accumulation of glyceollins and apigenin was limited in SNP-elicited compared withfungal-elicited cotyledons. NOS activity preceded the accumulation of these flavonoids in tissues treated with the Dpmelicitor. The accumulation of these metabolites was faster in SNP-elicited than in fungal-elicited cotyledons. We concludethat the response of soybean cotyledons to Dpm elicitor involves NO formation via a constitutive NOS-like enzyme thattriggers the biosynthesis of antimicrobial flavonoids.

Plants respond to attack by pathogens by activatinga wide variety of protective mechanisms designed toprevent pathogen replication and spreading. Suchdefenses include rapid and localized cell death (hy-persensitive response, HR) and the accumulation ofantimicrobial compounds known as phytoalexins,which play an important role in many plant-pathogen incompatible interactions (Paxton, 1991).Glyceollins are the major phytoalexins produced insoybean (Glycine max) plants. The timing and magni-tude of glyceollin accumulation differ markedly incompatible and incompatible interactions, but areconsistent with their proposed role in race-specificresistance (Graham et al., 1990; Paxton, 1991; Gra-ham, 1995 and refs. therein).

Glyceollins are pterocarpans derived from the phe-nylpropanoid pathway and they occur as a series ofisomers (I–IV; Paxton, 1995). Phe ammonia-lyase(PAL), the first enzyme in this pathway, catalyzes thenonoxidative deamination of the amino acid Phe toproduce cinnamate, the first structure with a phenyl-

propanoid skeleton in this biosynthetic pathway(Paxton, 1991). Daidzein, a dihydroxylated isofla-vone, is the immediate precursor of the glyceollins(Bailey and Mansfield, 1982; Paxton, 1995). Genistein(trihydroxylated isoflavone) is another antimicrobialisoflavonoid that is accumulated in soybean tissuesduring incompatible reactions (Dixon et al., 1995;Dakora and Phillips, 1996).

Various substances can induce glyceollin produc-tion in soybean tissues, including fungal elicitors,oligogalacturonides, H2O2, and salicylic acid (Degouseeet al., 1994; Gomez et al., 1994; Graham, 1995; Knorzeret al., 1999). Recently, nitric oxide (NO) was shown toinduce the expression of genes related to phytoalexinbiosynthesis in soybean and tobacco (Nicotiana taba-cum) cells in culture (Delledonne et al., 1998; Durneret al., 1998). This nitrogen radical also induces phy-toalexin accumulation in potato (Solanum tuberosum)tuber tissues (Noritake et al., 1996), potentiates theinduction of HR in soybean cells by reactive oxygenintermediates (Delledonne et al., 1998), and inducesdeath with the hallmarks of apoptosis in Kalanchoedaigremontiana, Taxus brevifolia (Pedroso et al., 2000a,2000b), Arabidopsis (Clarke et al., 2000), and Citrusinensis (Saviani et al., 2002) cells. These observationssuggest the existence of an NO-mediated signalingpathway in plant defense responses to pathogens.

1 This work was supported by Fundacao de Amparo a Pesquisado Estado de Sao Paulo.

* Corresponding author; e-mail [email protected]; fax 55–19–3788–6129.

Article, publication date, and citation information can be foundat www.plantphysiol.org/cgi/doi/10.1104/pp.005850.

1288 Plant Physiology, November 2002, Vol. 130, pp. 1288–1297, www.plantphysiol.org © 2002 American Society of Plant Biologists

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In mammalian cells, the main mechanism of NOgeneration is through the enzyme NO synthase (NOS),which metabolizes l-Arg to l-citrulline with the for-mation of NO (Pollock et al., 1991). Although no NOSgene has been found, various studies have suggestedthe occurrence of a NOS-like enzyme in plant cells.NOS-like activity, measured by l-citrulline formationfrom l-Arg and/or by its sensitivity to mammalianNOS inhibitors, has been detected in several plantspecies (Cueto et al., 1996; Ninnemann and Maier,1996; Delledonne et al., 1998; Durner et al., 1998; Bar-roso et al., 1999; Ribeiro et al., 1999). In addition, usingantibodies produced against mammalian NOS iso-forms, NOS-like proteins have been localized in thecytosol and nucleus of maize (Zea mays) root tips(Ribeiro et al., 1999), and in peroxisomes and chloro-plasts of pea (Pisum sativum) leaves (Barroso et al.,1999). Western-blot analysis showed that the NOSimmunoreactive proteins in maize and pea extractshad a molecular mass of approximately 166 and 130kD, respectively (Barroso et al., 1999; Ribeiro et al.,1999), which is in the same molecular mass range asdescribed for mammalian NOS (Pollock et al., 1991).The involvement of a NOS-like enzyme in plant de-fense responses to pathogens has also been suggested(Delledonne et al., 1998; Durner et al., 1998). Increasedlevels of NOS activity were observed in tobacco plantsresistant to tobacco mosaic virus (Durner et al., 1998)and Ralstonia solanacearum (Huang and Knopp, 1998).Consistent with these observations, NOS inhibitorscompromised the responses of Arabidopsis leaves toattack by Pseudomonas syringae (Delledonne et al.,1998).

Soybean stem-canker disease represents one of thegreatest limitations to the cultivation of this crop inBrazil. Intense efforts have been made to developsoybean cultivars resistant to the fungus Diaporthephaseolorum f. sp. meridionalis (Dpm), the causal agentof this disease. However, very little is known aboutthe metabolic alterations that confer resistance toDpm. One of the experimental approaches used bythe Agronomical Institute of Campinas in Brazil toselect for resistance to Dpm has been the observationof a red color developed in the stem of soybeanplants inoculated with Dpm. Resistant cultivars de-velop an intense reddish color at the site of fungalinoculation, whereas in susceptible cultivars thecolor develops later, when the disease has alreadymanifested itself (N.R. Braga, personal communica-tion). This color in soybean tissues results from theaccumulation of certain glyceollin precursors follow-ing exposure to various biotic and abiotic factors(Ingham et al., 1981; Zahringer et al., 1981), and itsintensity is proportional to the phytoalexin content(Ayers et al., 1976b). Glyceollin precursors that havea red coloration include glycinol and the isopreny-lated compounds glyceolidin I and II (Ingham et al.,1981; Zahringer et al., 1981).

Considering that phytoalexin production seems tobe involved in the defense mechanism of soybeanplants against attack by Dpm, and that NO mayparticipate in plant defense responses, we have ex-amined the involvement of an NOS-like enzyme inthe activation of phenylpropanoid biosynthesis insoybean cotyledons treated with Dpm elicitor. Wealso compared the time course of the effects of theDpm elicitor and an NO donor compound on theformation of phenylpropanoid intermediates in soy-bean cotyledons. In addition, the induction of NOS-like activity and the effect of NOS inhibitors on thisprotein were analyzed. Our results demonstrate theinvolvement of a constitutive Ca21-dependent NOS-like enzyme in the soybean defense response to Dpmelicitor.

RESULTS

Flavonoids Elicited in Response to Dpm and SodiumNitroprusside (SNP)

The effect of SNP, an NO donor, on phytoalexinaccumulation in soybean was compared with that in-duced by a crude Dpm extract. A soybean cultivarresistant to Dpm (IAC-18) was treated with SNP orDpm elicitor for different periods, using the cotyledonassay. After treatment, the diffusates were analyzedfor phytoalexin content by HPLC with detection at 286nm. As shown in Figure 1, when cotyledons wereelicited with Dpm extract, the isoflavones daidzeinand genistein were detected after 6 h of incubation,whereas with SNP, these metabolites accumulatedearlier, being detected just 3 h after the beginning oftreatment. Glyceollins, the daidzein-derived pterocar-pans, were detected only after a 12-h incubation withDpm elicitor, and their production increased up to20 h. For SNP, only daidzein and genistein were de-tected up to 12 h after stimulation, and glyceollinsappeared only 20 h after elicitation. The Dpm extractstimulated the accumulation of a greater variety ofmetabolites than did SNP, the greatest difference be-ing observed after 20 h of treatment (Fig. 1).

Figure 2 compares the time course for the produc-tion of genistein, daidzein, and glyceollins in Dpm-and SNP-elicited soybean cotyledons. In both treat-ments, maximal genistein production occurred after12 h and decreased at 20 h. In contrast, the accumu-lation of daidzein and glyceollins showed differentpatterns for the two treatments. Maximal daidzeinproduction occurred after 12 h of incubation with theDpm elicitor. After this period, daidzein decreasedand the glyceollins began to increase. The differencesin the proportion of these two compounds repre-sented the conversion of daidzein into glyceollinsduring prolonged incubations. Maximal productionof daidzein also occurred after a 12 h of incubationwith SNP, and the levels of this precursor were un-changed after 20 h. The levels of glyceollins also

Nitric Oxide Synthase-Mediated Phytoalexin Accumulation in Soybean

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remained low, indicating a limited conversion ofdaidzein into glyceollins.

The diffusates of Dpm- and SNP-elicited cotyle-dons were also analyzed for the presence of the fla-

vones apigenin and luteolin because a spectral anal-ysis showed a high A350, a wavelength typical ofthese compounds. Both elicitors induced maximalaccumulation of luteolin after 12 h, which then de-

Figure 1. Isoflavonoids and pterocarpans produced by soybean cotyledons in response to Dpm elicitor and SNP. Soybeancotyledons (cultivar IAC-18) were elicited with 50 mL of Dpm extract (equivalent to 20 mg of Glc) or SNP (10 mM). After theindicated times, the diffusates were analyzed by HPLC at 286 nm. Metabolites were identified by comparing their retentiontimes with those of standards. Dz, Daidzein; Gt, genistein; Gs, glyceollins.

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1290 Plant Physiol. Vol. 130, 2002

creased during further incubation (Fig. 3). The re-sponse to SNP preceded that of Dpm because luteolinwas already detected after 3 h of elicitation. Apigeninwas induced principally by Dpm and began to ap-pear after 12 h of elicitation, its content being higherthan that detected after 20 h of treatment with SNP.

Overall, the response of soybean cotyledons to SNPwas faster (flavonoid production was already de-tected after 3 h) and more intense (higher produc-tion) than that to Dpm. However, SNP was not asefficient as Dpm in stimulating the biosynthesis ofmetabolites such as glyceollins and apigenin.

NO Dose-Dependent Phytoalexin Accumulation

Phytoalexin production in Dpm-elicited cotyledonswas compared with that induced by different con-centrations of SNP. In these assays, cultivars suscep-

tible (IAC-14) and resistant (IAC-18) to Dpm wereused, and phytoalexin accumulation was estimatedas the overall production of phenolics at 286 nm. Asshown in Figure 4, phytoalexin production in soy-bean cotyledons induced by SNP was dose depen-dent and, at 10 mm, was similar to that induced bythe fungal elicitor. No phytoalexin production wasobserved when SNP was replaced by ferricyanide, astructural analog of SNP, which is not an NO donor(data not shown). These results suggest that the NOradical generated by SNP was the signaling moleculeinvolved in the stimulation of phytoalexin produc-tion in soybean cotyledons. There were no significantdifferences among the phytoalexin responses of sus-ceptible (IAC-14) and resistant (IAC-18) cultivars af-ter exposure to Dpm elicitor or SNP as estimated bythe A286 (Fig. 4) and by HPLC analysis (data notshown).

Figure 2. Quantification of isoflavonoids and pterocarpans pro-duced by soybean cotyledons (cultivar IAC-18) in response to Dpmelicitor (A) and SNP (B). The total amount of phytoalexins producedby 20 soybean cotyledons in response to Dpm extract (equivalent to20 mg of Glc) or SNP (10 mM) was determined from calibrationcurves of daidzein (Dz), genistein (Gt), and glyceollin (Gs) standards.The data represent the mean 6 SE of three independent experimentswith 20 cotyledons per treatment.

Figure 3. Quantification of the flavones produced by soybean coty-ledons (cultivar IAC-18) in response to Dpm elicitor (A) and SNP (B).The total amount of flavones produced by 20 soybean cotyledons inresponse to Dpm extract (equivalent to 20 mg of Glc) or SNP (10 mM)was determined from calibration curves of apigenin (Ap) and luteolin(Lt) standards. The data represent the mean 6 SE of three independentexperiments with 20 cotyledons per treatment.



Effect of NOS Inhibitors on Phytoalexin Production

When soybean cotyledons (IAC-14 and IAC-18 cul-tivars) were pretreated with the NOS inhibitors NG-nitro-l-Arg methyl ester (l-NAME) or aminoguani-dine (AMG) for 5 h and were subsequently elicitedwith the Dpm elicitor for 20 h, lower amounts ofphytoalexins were produced (50% inhibition) com-pared with controls not preincubated with the NOSinhibitors (Fig. 5). HPLC analysis showed that thedecrease in A286 was due mostly to a reduced contentof daidzein, genistein, and glyceollins (data notshown). l-NAME is a structural analog of l-Arg thatcompetitively inhibits inducible forms and irrevers-ibly inhibits constitutive isoforms of NOS in animals(Baylis et al., 1995). AMG, although not a structuralanalog of l-Arg, is an irreversible inhibitor of bothisoforms, but preferentially of the inducible form(Laszlo et al., 1995; Wolff and Lubeskie, 1995). A lowuptake of the inhibitors by the tissues, the absence ofthe inhibitors during elicitation, and a lower affinityof the putative plant NOS enzyme for these com-pounds compared with that of animals could explainthe incomplete inhibition of phytoalexin accumula-tion in Figure 5. In contrast, pretreating soybeancotyledons with the NOS inhibitors did not preventphytoalexin formation in response to SNP elicitation(Fig. 5). These results suggested that NO producedby a NOS-like enzyme could be involved in the sig-naling pathway leading to phytoalexin induction insoybean tissues in response to Dpm elicitor.

Induction of NOS Activity in Elicited Cotyledons

Soybean cotyledons (cultivar IAC-18) were elicitedwith the fungal elicitor or with SNP for different

periods, and the tissues were then prepared to deter-mine NOS activity using the Arg-citrulline assay, asdescribed in “Materials and Methods.” This is a morereliable method for measuring NOS activity than isNO quantification because NO can be generated inplants by routes other than NOS (see Wendehenne etal., 2001; Salgado et al., 2002). The difference betweenthe total l-[U-14C]citrulline formation and that inhib-ited by simultaneous incubation with l-NAME andAMG was used to estimate NOS activity. The timecourse for NOS activity showed that cotyledon elic-itation with the Dpm extract induced maximal en-zyme activity after 6 h of incubation, which corre-sponded to 3 pmol l-[U-14C]citrulline h21 mg21

when 1 mm l-[U-14C]Arg was used (Fig. 6). In aconverse manner, SNP did not evoke NOS activity incotyledon tissues (Fig. 6).

To further characterize the NOS activity induced insoybean cotyledon tissues, the Ca21 and NADPHdependence and the effect of l-N6-(iminoethyl) Lys(l-NIL) on l-[U-14C]citrulline formation after 6 h ofelicitation were examined using 10 mm l-[U-14C]Arg.l-NIL is a potent inhibitor of animal NOS, beingabout 28 times more selective for inducible NOS thanfor the constitutive isoform (Connor et al., 1995;Moore et al., 1996). As shown in Figure 7, l-[U-14C]citrulline formation in control and SNP-elicitedtissues was 4.5 and 4.0 pmol min21 mg21, respec-tively. These values did not change in the presence ofEGTA or l-NIL, and they were not affected whenNADPH was absent from the reaction mixture. Theseresults indicate that there was a basal l-[U-14C]citrulline incorporation in these conditions that

Figure 4. Dose-dependent induction of phytoalexin by SNP in soy-bean cotyledons. Soybean cotyledons from 7-d-old seedlings weretreated with 50 mL of SNP solution at the indicated concentrations.After 20 h in the dark, the diffusates were analyzed for their phytoa-lexin content (phenolics at 286 nm) as described in “Materials andMethods.” Control cotyledons were treated with 50 mL of deionizedwater or elicitor prepared from the fungus Dpm (equivalent to 13 mgof Glc). The bars represent the mean 6 SE of four to six replicates.

Figure 5. Effect of NOS inhibitors on phytoalexin production in-duced by Dpm elicitor or SNP. Prior to phytoalexin induction, soy-bean cotyledons were submerged in MES buffer (50 mM), L-NAME (3mM), or AMG (3 mM) for 5 h. The cotyledons were then dried on filterpaper and the exposed surface was treated with 50 mL of MES (50mM), Dpm elicitor (equivalent to 13 mg of Glc), or SNP (10 mM) for20 h. After incubation, the diffusates were analyzed for their phytoa-lexin content. The bars represent the mean 6 SE of two experimentseach done in triplicate.

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could not be attributed to NOS activity. On the otherhand, cytosolic fractions extracted from cotyledontissues previously elicited with a Dpm extract for 6 hshowed a higher rate of l-[U-14C]citrulline formation(11 pmol min21 mg21), which returned to basal levelsin the presence of l-NIL and EGTA, or in the absence

of NADPH (Fig. 7). Therefore, NOS activity wasl-NIL sensitive (7.7 pmol min21 mg21), Ca21 depen-dent (7.8 pmol min21 mg21), and NADPH dependent(5.8 pmol min21 mg21) under these conditions.

The level of NOS activity (7.0 pmol min21 mg21)after Dpm elicitation was approximately equivalentto NO formation at a rate of 112 pmol min21 in eachcotyledon, considering that we used only tissue im-mediately adjacent to the site exposed to elicitor formeasuring NOS activity (16 mg per cotyledon). Thetotal amount of NO produced per cotyledon after 6 hof elicitation, taking into account the increment inNOS activity, would be around of 20 nmol, or at least10 nmol if one considers that 50% (w/v) of the totalprotein extracted was recovered in the soluble frac-tion used to measure NOS activity. This amount ofNO would be equivalent to that effectively releasedfrom SNP after the same period of elicitation, assum-ing that 0.5 mmol SNP was applied to each soybeancotyledon (50 mL of 10 mm SNP), low amounts of NOwould be released from SNP during this treatment,and, because of its chemical reactivity, only a smallfraction of this exogenously applied radical wouldreach its target (Wendehenne et al., 2001; Salgado etal., 2002). Thus, it seems reasonable to suppose thatin both treatments, the amount of NO reaching itsmolecular target for phytoalexin biosynthesis wouldbe of the same order of magnitude.

NOS Immunoreactivity in Soybean Tissues

In immunoblotting experiments with solubilizedpolypeptides from cotyledons, a band with an appar-ent molecular mass of approximately 166 kD cross-reacted with antibodies to mammalian neuronal NOS(Fig. 8). This 166-kD NOS-immunoreactive proteinhas been detected previously in maize tissues (Ri-beiro et al., 1999). Control immunoblots with brainhomogenates detected the usual 155-kD NOS (For-stermann et al., 1991), but with greater reactivitywhen compared with that of cotyledons. As shown inFigure 8, the NOS immunoreactive protein waspresent in nonelicited tissues, and its content did not

Figure 6. NOS activity in elicited soybean cotyledons (cultivar IAC-18). Tissues from soybean cotyledons elicited with Dpm elicitor(equivalent to 20 mg of Glc) or SNP (10 mM) for different times wereassayed for NOS activity as described in “Materials and Methods”using 1 mM L-[U-14C]Arg (350 mCi mmol21). NOS activity was deter-mined as the difference between the total L-[U-14C]citrulline produc-tion and that observed in the presence of the NOS inhibitors L-NAMEand AMG, both at 3 mM. The points represent the mean 6 SE of twoexperiments each done in triplicate. *P , 0.05 (by Student’s t test),compared with the control (time 0).

Figure 7. NOS activity of elicited soybean cotyledons is sensitive toL-NIL and is Ca21 and NADPH dependent. Tissues from soybean(cultivar IAC-18) cotyledons elicited with Dpm extract or SNP for 6 hwere assayed for NOS activity using 10 mM L-[U-14C]Arg (70 mCimmol21). L-[U-14C]citrulline formation was determined in completereaction medium (Control), and in the presence of 3 mM L-NIL(1L-NIL), 2 mM EGTA (-Ca21), and without NADPH (-NADPH). Thebars represent the mean 6 SE of two experiments each done intriplicate. *P , 0.05 (by Student’s t test) compared with the totalL-[U-14C]citrulline production obtained in the presence of all NOScofactors.

Figure 8. Immunoblot of proteins solubilized from soybean cotyle-dons probed with antibodies raised against brain NOS. Concentratedsolubilized proteins were separated by SDS-PAGE, transferred tonitrocellulose membranes, and probed with rabbit anti-brain NOSantibodies. Lanes, from left to right, contain proteins from the fol-lowing sources: (1) detergent extract from brain (20 mg) and 150 mgof solubilized soybean cotyledons that was not elicited (2; control) orwas elicited with SNP (3; 10 mM) or Dpm (4; equivalent to 20 mg ofGlc). The arrows indicate the positions of the protein standards(indicated in kilodaltons). The result shown is representative of threesimilar experiments.



change upon treatment with SNP or Dpm. Theseresults suggest that the putative NOS protein in-volved in phytoalexin accumulation in soybean-elicited cotyledons was a constitutive enzyme, andthey agree with the Ca21 dependence and slight in-crease in NOS activity seen after elicitation withDpm. The 166-kD protein detected in cotyledonscross-reacted very weakly with antibodies raisedagainst mouse macrophage NOS (result not shown).

DISCUSSION

The results of this study indicate the involvementof an NOS-like enzyme in the response of soybeancotyledons to the elicitor extracted from spores ofDpm, the causal agent of stem canker disease. Phy-toalexins accumulated in soybean cotyledons elicitedwith the Dpm extract and with the NO donor SNP.Dpm-induced phytoalexin production was inhibitedwhen cotyledons were pretreated with mammalianNOS inhibitors, whereas these same inhibitors didnot affect flavonoid accumulation when the cotyle-dons were elicited with SNP. Consistent with theseobservations, Dpm elicitation induced NOS activityin soybean tissues, but no such activity was observedin SNP-elicited tissues. In Dpm-elicited cotyledons,the induction of NOS activity preceded the activationof isoflavone (daidzein and genistein) biosynthesis.Therefore, maximal NOS activity occurred whenisoflavone production was still low. In contrast,when soybean cotyledons were elicited with SNP,isoflavones accumulated earlier. As observed forisoflavones, the time course of luteolin productionwas faster after elicitation with SNP than after treat-ment with Dpm. These results agree with the hypoth-esis that the Dpm elicitor activated the phenylpro-panoid pathway through an NOS-like enzyme. Thus,in elicitation with Dpm, phytoalexin accumulationoccurred only after NOS activation, whereas the timecourse for the production of these metabolites wasfaster when NO was provided directly.

Phytoalexin production in several defense re-sponses against attack by pathogens is regulated atthe level of the enzymes PAL, chalcone synthase(CHS), and chalcone isomerase (Dixon and Paiva,1995). The present observations that isoflavones(daidzein and genistein), pterocarpans (glyceollins),and flavones (apigenin and luteolin) accumulated inDpm-elicited cotyledons via NOS activity suggestthat the NO produced endogenously in this interac-tion could stimulate phytoalexin biosynthesis by reg-ulating the expression of the initial enzymes of thephenylpropanoid pathway. Delledonne et al. (1998)reported that NG-nitro-l-Arg, a structural analog ofl-Arg, prevented the accumulation of PAL tran-scripts in soybean cell cultures inoculated with anavirulent strain of Pseudomonas syringae. This treat-ment also inhibited the transcription of CHS, the firstenzyme of the phenylpropanoid pathway branch.

These authors also showed that SNP induced theexpression of the PAL and CHS genes in soybean cellsin culture. NO donors such as S-nitrosoglutathioneand S-nitroso-N-acetylpenicillamine, and the injec-tion of mammalian NOS in tobacco seedlings, wereable to induce the expression of PAL genes (Durner etal., 1998).

Our results also show that although NO was effec-tive in stimulating the initial steps of the phenylpro-panoid pathway, this radical was apparently not theonly signaling molecule engaged in the late conver-sion of daidzein into glyceollins. As shown in Figures1 and 2, the conversion of daidzein into glyceollinswas more synchronized in Dpm- than in SNP-elicitedtissues, despite the high effectiveness of the NO do-nor in eliciting the accumulation of the precursordaidzein. In soybean, as in a number of other hostspecies, there is good evidence that reactive oxygenspecies (ROS) play a major role in the initiation of theresistance against pathogens (Bolwell and Wojtaszek,1997; Knorzer et al., 1999). For instance, hydrogenperoxide (H2O2) is an effective inducer of phytoa-lexin accumulation in soybean hypocotyls (Degouseeet al., 1994; Gomez et al., 1994) and in suspensioncultures of cells (Apostol et al., 1989). Furthermore, asynergistic interaction between NO and H2O2 hasbeen reported for the induction of cell death in soy-bean cell cultures (Delledonne et al., 2001). In prelim-inary experiments, we observed no such synergisticinteraction during phytoalexin induction in cotyle-dons of the soybean cultivar IAC-18. H2O2, tested inthe concentration range of 2 mm to 1 m, was ineffec-tive in inducing phytoalexin production. Moreover,H2O2 dose dependently reduced the phytoalexin ac-cumulation induced by SNP (L.V. Modolo, M.R.Braga, and I. Salgado, unpublished data). In accor-dance with these preliminary observations, Delle-donne et al. (1998) and Durner et al. (1998) found thatalthough NO acted synergistically with ROS to po-tentiate cell death, it also acted independently of ROSto induce the expression of defense-related genes,including the PAL gene. Previous studies haveshown that the oxidative burst and the loss of cellviability are not directly linked to phytoalexin induc-tion in soybean, cotton (Gossypium hirsutum) , andcarrot (Daucus carota) cells (Davis et al., 1993; Koch etal., 1998). A detailed analysis of the oxidative burst insoybean also showed that although H2O2 plays acentral role as an “orchestrator” of the HR, it was notvery effective in inducing the accumulation of tran-scripts for PAL and CHS (Tenhaken et al., 1995). Incontrast, Degousee et al. (1994) investigated the rela-tionship between oxidative processes and phytoa-lexin biosynthesis and demonstrated that H2O2 leadto significant glyceollin elicitation in soybean hypo-cotyls and radicles. Together, these results and thoseof our preliminary experiments indicate that the un-coupling of HR from NO-mediated phytoalexin in-duction by Dpm elicitor in cotyledons of the soybean

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cultivar IAC-18 is not unlikely. Thus, in our system,H2O2 is apparently not the signal triggered by theDpm elicitor that acts synergistically with NO toactivate specific defense genes involved in glyceollinsynthesis. On the other hand, glyceollin accumula-tion in soybean tissues is affected by genotype, age,and the developmental state of specific organs, and isunder strong regulation by several endogenous con-ditions (Abbasi and Graham, 2001; Abbasi et al., 2001and refs. therein). Thus, the elucidation of the signal-ing molecules, which act in concert with NO duringelicitation by Dpm, requires further investigation.

The NOS-like enzyme described here showed char-acteristics similar to most other such enzymes foundso far in plants (Wendehenne et al., 2001) because itwas Ca21 dependent, partially sensitive to inhibitorsof different mammalian NOS isoforms, and constitu-tively expressed. The specific activity of this putativeenzyme was in the same range (3–4 pmol l-citrullineh21 mg21) as that described for maize tissues usingthe same assay technique to measure NOS activity incrude soluble extracts (Ribeiro et al., 1999). When apotent NOS inhibitor was used to characterize thespecific NOS activity, the estimated values for soy-bean cotyledons increased by approximately one or-der of magnitude (to 5.8–7.8 pmol l-citrulline min21

mg21), reaching values similar to those found byCueto et al. (1996) in soluble extracts of lupin (Lupi-nus albus) roots (2.5 pmol l-citrulline min21 mg21) atan equivalent l-Arg concentration. If NO is producedby a particular cell type and/or organelle, the mea-sured NOS activity may be significantly higher thanthat estimated from crude soluble extracts, as re-ported for purified peroxisomes of pea leaves (5.6nmol l-citrulline min21 mg21; Barroso et al., 1999).The NOS-immunoreactive protein detected in soy-bean cotyledons of the cultivar IAC-18 had a molec-ular mass of 166 kD, similar to that described inmaize tissues (Ribeiro et al., 1999), but was morereactive with antibody to brain NOS. However, thecharacteristics of the soybean enzyme differed fromthose of the putative NOS protein detected in peaperoxisome, which has a molecular mass of 130 kDand is immunorelated with the murine inducibleNOS, despite being Ca21 dependent and constitu-tively expressed (Barroso et al., 1999).

Although soybean resistance to Dpm in the fieldappears to be related to phytoalexin production, inthis study, there were no differences in the phytoa-lexin responses between the resistant and susceptiblecultivars induced by Dpm elicitor or SNP. Theseexperiments were done using detached cotyledonskept under controlled conditions. Factors that couldinfluence the plant response to pathogens under fieldconditions, such as high temperature and humidity(Classen and Ward, 1985; Graham, 1995; Smith,1996), were not considered here. Furthermore, plantresistance to a pathogen is a multifactorial phenom-enon that includes an arsenal of defensive reactions

(Hahn et al., 1989). In view of this and the fact thatthe mechanisms involved in the resistance of soybeanplants to stem canker fungus are still poorly under-stood, other responses in addition to phytoalexinproduction could be involved in the resistance ofsoybean to attack by Dpm in the field.

Whereas the antimicrobial function of phytoalexinsis well documented, the role of flavones such as api-genin and luteolin in plant defense against pathogensremains to be established. The anti-inflammatory ac-tivity of these molecules in mammalian cells has beenattributed to their antioxidant property, as well as to aregulatory action on NO production through theircapacity to inhibit NOS expression (Kim et al., 1999).Thus, it seems reasonable to suppose a similar actionfor apigenin and luteolin in controlling the level of NOat the site of infection in soybeans. Preliminary resultsfrom our laboratory have shown that diffusates fromsoybean cotyledons treated with Dpm elicitor or SNPinhibited NO production in LPS- and/or IFN-g-activated macrophages (L.S. Scuro and I. Salgado, un-published data).

There is increasing evidence that NO may influ-ence various developmental processes and that it hasa role in plant defense responses to pathogens (Sal-gado et al., 2002). Our data strongly suggest thatNO-induced phytoalexin production in soybean cot-yledons is mediated by an NOS-like enzyme and thatthe plant response to attack by Dpm is likely toinvolve this signaling pathway.

MATERIAL AND METHODS

Plant Material

Soybean (Glycine max) seeds of the cultivars IAC-14 and IAC-18, suscep-tible and resistant to the fungus Diaporthe phaseolorum f. sp. meridionalis(Dpm), respectively, were provided by Dr. Nelson R. Braga (Instituto Ag-ronomico de Campinas, Campinas, Sao Paulo, Brazil). The cotyledons usedwere detached from 7- to 8-d-old seedlings grown in vermiculite at roomtemperature in a greenhouse.

Preparation of the Elicitor

Elicitor from Dpm (strain 8498; kindly supplied by Dr. Margarida F. Ito,Instituto Agronomico de Campinas) was obtained by autoclaving (121°C,1.5 atm, 30 min) aqueous spore suspensions of 30- to 40-d-old culturesgrown in potato-dextrose-agar (Merck, Darmstadt, Germany) media in thedark at room temperature. The autoclaved suspension was centrifuged at10,000g for 6 min, and the pellet was discarded. Total carbohydrates werequantified in the supernatant by the phenol-sulfuric procedure (Dubois etal., 1956) using Glc as standard.

Elicitation Assay

The production of phytoalexins was evaluated using the soybean coty-ledon assay (Ayers et al., 1976a). A small section (i.d. 5 1.0 cm) wasremoved from the adaxial surface of each cotyledon and the woundedsurface was treated with 50 mL of test solution, as specified in the figurelegends. The cotyledons were kept in a petri dish containing water-absorbedfilter paper in the dark at 26°C for 20 h, unless stated otherwise. Thecotyledons were washed with deionized water (1 mL per cotyledon), andphytoalexin production was estimated in the diffusates by spectrophotom-etry at 286 nm.



Analysis of Glyceollins, Daidzein, and Genistein

Diffusates from the soybean cotyledon assay (20 cotyledons) were ex-tracted with ethyl acetate, as described by Keen (1978). Organic fractionswere evaporated to dryness and the residues were then solubilized inmethanol and analyzed by HPLC in a chromatograph (DX500; Dionex,Sunnyvale, CA) fitted with a diode array detector. The samples were run ona 4.6 mm 3 250 mm column (ODS C18; Zorbax, Chadds Ford, PA) with alinear gradient from 20% to 60% (w/v) acetonitrile in 0.1% (w/v) trifluoro-acetic acid (0.7 mL min21) according to Pelicice et al. (2000). Peak areaversus compound concentration was plotted for various concentrations ofavailable standards. Daidzein and genistein were identified by calibrationwith authentic standards (Sigma, St. Louis). Diffusates from the Williams 82soybean cultivar from Illinois Foundation Seeds (Champaign; kindly sup-plied by Dr. M.G. Hahn, University of Georgia, Athens) were used as theglyceollin standard. All compounds were monitored at 286 nm.

Analysis of Apigenin and Luteolin

Aliquots of the diffusates from the soybean cotyledon assay (20 cotyle-dons) were evaporated, resuspended in methanol, and analyzed by HPLC ina chromatograph (Shimadzu, Kyoto) fitted with a UV-VIS detector. Thesamples were analyzed using a 4.6 mm 3 250 mm column (CLS ODS C18;Shimadzu) with a mobile phase of methanol in 10% (v/v) formic acid, in amethod modified from Garcıa-Viguera et al. (1998). Elution was done at aflow rate of 1 mL min21 using a gradient that started with 50% (v/v)methanol and increased to 60% (v/v) at 10 min, followed by 100% (v/v)methanol for 3 min and then by holding at 50% (v/v) methanol for 4 min.Peak area versus compound concentration was plotted for various concen-trations of apigenin and luteolin standards (Sigma). The runs were moni-tored at 336 nm and 350 nm for apigenin and luteolin, respectively.

Determination of NOS Activity

The NOS activity of cotyledon tissues treated with 50 mL of test solutionwas determined by the citrulline assay method modified from Rees et al.(1995). The conversion of l-[U-14C]Arg to l-[U-14C]citrulline was deter-mined in samples with and without the NOS inhibitors l-NAME (Sigma),AMG (RBI, Natick, MA), or l-NIL (Calbiochem, La Jolla, CA). In brief, 1.0 gof tissue, together with 50 mg of polyvinylpolypyrrolidone, was homoge-nized in 1.0 mL of cooled extraction buffer (50 mm Tris, pH 7.4, containing320 mm Suc, 10 mg mL21 leupeptin, 10 mg mL21 soybean trypsin inhibitor,2 mg mL21 aprotinin, 1 mm phenylmethylsulfonyl fluoride, 10 mm dithio-threitol, and 10 mm reduced glutathione). The homogenate was centrifugedat 10,000g for 10 min at 4°C. Forty microliters of the supernatant was addedto 100 mL of the assay buffer [40 mm HEPES, pH 7.2, containing 10 mm FAD,10 mm FMN, 1 mm dithiothreitol, 1.25 mm CaCl2, 50 mm 6(R)-5,6,7,8-tetrahydrobiopterin, 10 mg mL21 calmodulin, 1 mm b-NADPH, and 1 mm(50 nCi) or 2 mm (100 nCi) l-[U-14C]Arg; Amersham Pharmacia Biotech,Buckinghamshire, UK]. After incubation for 30 min at room temperature,the reaction was stopped by adding 1 mL of Dowex-Ag 50W suspended in100 mm HEPES containing 10 mm EDTA (1:1.5, v/v). The resin was removedby centrifugation (10,000g for 10 min at 18°C). Four hundred microliters ofthis supernatant was added to 3 mL of scintillation liquid and was countedin a counter (LS 6000; Beckman, Fullerton, CA). Protein content was deter-mined by the Coomassie Blue-binding method (Bradford, 1976) using pro-tein reagent (Bio-Rad, Hercules, CA), and bovine serum albumin asstandard.

Western Blotting

Western blotting of polypeptides solubilized from cotyledon tissues wasdone as described by Ribeiro et al. (1999) using a polyclonal antibody tohuman neuronal NOS (2 mg mL21) raised in rabbits (BD TransductionLaboratories, Lexington, KY).

ACKNOWLEDGMENTS

We thank the Conselho Nacional de Desenvolvimento Cientıfico e Tec-nologico and the Coordenacao de Aperfeicoamento de Pessoal de Nıvel

Superior for research fellowships. Denis U. Lima, Fabiola L.A.C. Mestriner,and Elzira E. Saviani helped with the HPLC analysis, the determination ofNOS activity, and the immunoblots, respectively.

Received March 20, 2002; returned for revision May 27, 2002; acceptedAugust 21, 2002.

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