Nitric oxide synthase-mediated phytoalexin accumulation in soybean cotyledons in response to the Diaporthe phaseolorum f. sp. meridionalis elicitor

Abstract

Phytoalexin biosynthesis is part of the defense mechanism of soybean (Glycine max) plants against attack by the fungus Diaporthe phaseolorum f. sp. meridionalis (Dpm), the causal agent of stem canker disease. The treatment of soybean cotyledons with Dpm elicitor or with sodium nitroprusside (SNP), a nitric oxide (NO) donor, resulted in a high accumulation of phytoalexins. This response did not occur when SNP was replaced by ferricyanide, a structural analog of SNP devoid of the NO moiety. Phytoalexin accumulation induced by the fungal elicitor, but not by SNP, was prevented when cotyledons were pretreated with NO synthase (NOS) inhibitors. The Dpm elicitor also induced NOS activity in soybean tissues proximal to the site of inoculation. The induced NOS activity was Ca(2+)- and NADPH-dependent and was sensitive to the NOS inhibitors N(G)-nitro-L-arginine methyl ester, aminoguanidine, and L-N(6)-(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 to the elicitor or SNP, although the accumulation of glyceollins and apigenin was limited in SNP-elicited compared with fungal-elicited cotyledons. NOS activity preceded the accumulation of these flavonoids in tissues treated with the Dpm elicitor. The accumulation of these metabolites was faster in SNP-elicited than in fungal-elicited cotyledons. We conclude that the response of soybean cotyledons to Dpm elicitor involves NO formation via a constitutive NOS-like enzyme that triggers the biosynthesis of antimicrobial flavonoids.

Figure 1

Isoflavonoids and pterocarpans produced by soybean cotyledons in response to Dpm elicitor and SNP. Soybean cotyledons (cultivar IAC-18) were elicited with 50 μL of Dpm extract (equivalent to 20 μg of Glc) or SNP (10 mm). After the indicated times, the diffusates were analyzed by HPLC at 286 nm. Metabolites were identified by comparing their retention times with those of standards. Dz, Daidzein; Gt, genistein; Gs, glyceollins.

Figure 2

Quantification of isoflavonoids and pterocarpans produced by soybean cotyledons (cultivar IAC-18) in response to Dpm elicitor (A) and SNP (B). The total amount of phytoalexins produced by 20 soybean cotyledons in response to Dpm extract (equivalent to 20 μg of Glc) or SNP (10 mm) was determined from calibration curves of daidzein (Dz), genistein (Gt), and glyceollin (Gs) standards. The data represent the mean ± se of three independent experiments with 20 cotyledons per treatment.

Figure 3

Quantification of the flavones produced by soybean cotyledons (cultivar IAC-18) in response to Dpm elicitor (A) and SNP (B). The total amount of flavones produced by 20 soybean cotyledons in response to Dpm extract (equivalent to 20 μg of Glc) or SNP (10 mm) was determined from calibration curves of apigenin (Ap) and luteolin (Lt) standards. The data represent the mean ± se of three independent experiments with 20 cotyledons per treatment.

Figure 4

Dose-dependent induction of phytoalexin by SNP in soybean cotyledons. Soybean cotyledons from 7-d-old seedlings were treated with 50 μL of SNP solution at the indicated concentrations. After 20 h in the dark, the diffusates were analyzed for their phytoalexin content (phenolics at 286 nm) as described in “Materials and Methods.” Control cotyledons were treated with 50 μL of deionized water or elicitor prepared from the fungus Dpm (equivalent to 13 μg of Glc). The bars represent the mean ± se of four to six replicates.

Figure 5

Effect of NOS inhibitors on phytoalexin production induced by Dpm elicitor or SNP. Prior to phytoalexin induction, soybean cotyledons were submerged in MES buffer (50 mm), l-NAME (3 mm), or AMG (3 mm) for 5 h. The cotyledons were then dried on filter paper and the exposed surface was treated with 50 μL of MES (50 mm), Dpm elicitor (equivalent to 13 μg of Glc), or SNP (10 mm) for 20 h. After incubation, the diffusates were analyzed for their phytoalexin content. The bars represent the mean ± se of two experiments each done in triplicate.

Figure 6

NOS activity in elicited soybean cotyledons (cultivar IAC-18). Tissues from soybean cotyledons elicited with Dpm elicitor (equivalent to 20 μg of Glc) or SNP (10 mm) for different times were assayed for NOS activity as described in “Materials and Methods” using 1 μm l-[U-¹⁴C]Arg (350 μCi μmol⁻¹). NOS activity was determined as the difference between the total l-[U-¹⁴C]citrulline production and that observed in the presence of the NOS inhibitors l-NAME and AMG, both at 3 mm. The points represent the mean ± se of two experiments 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 to l-NIL and is Ca²⁺ and NADPH dependent. Tissues from soybean (cultivar IAC-18) cotyledons elicited with Dpm extract or SNP for 6 h were assayed for NOS activity using 10 μm l-[U-¹⁴C]Arg (70 μCi μmol⁻¹). l-[U-¹⁴C]citrulline formation was determined in complete reaction medium (Control), and in the presence of 3 mm l-NIL (+l-NIL), 2 mm EGTA (-Ca²⁺), and without NADPH (-NADPH). The bars represent the mean ± se of two experiments each done in triplicate. *P < 0.05 (by Student's t test) compared with the total l-[U-¹⁴C]citrulline production obtained in the presence of all NOS cofactors.

Figure 8

Immunoblot of proteins solubilized from soybean cotyledons probed with antibodies raised against brain NOS. Concentrated solubilized proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with rabbit anti-brain NOS antibodies. Lanes, from left to right, contain proteins from the following sources: (1) detergent extract from brain (20 μg) and 150 μg of solubilized soybean cotyledons that was not elicited (2; control) or was elicited with SNP (3; 10 mm) or Dpm (4; equivalent to 20 μg of Glc). The arrows indicate the positions of the protein standards (indicated in kilodaltons). The result shown is representative of three similar experiments.