Glyceollin: A Site-Specific Inhibitor of Electron Transport in Isolated Soybean Mitochondria

Abstract

The glyceollin inhibition of electron transport by isolated soybean and corn mitochondria was similar to that of rotenone, acting at site I between the internal NADH Glyceollin, a phytoalexin produced during the hypersensitive response in soybeans, is hypothesized to be responsible for the expression of cultivar-specific resistance of soybeans to Phytophthora megasperma var sojae (7,26), and its accumulation has been correlated with the incompatible response of soybeans to Meloidogyne incognita (5), and Pseudomonos glycinea (8). Glyceollin inhibits fungal and nematode growth in vitro at concentrations similar to localized concentrations found in vivo (6,26).Inhibition of nematode respiration and electron transport from several substrates to 02 in isolated soybean mitochondria by glyceollin has been reported by Kaplan, Keen, and Thomason (6), but the specific site ofinhibition by glyceollin was not determined. We thus began our study with the a priori assumption that glyceollin could play a primary role in plant-affected pathogen toxicity, and autophytotoxicity through effects on mitochondrial electron transport. The experiments reported here deal only with the autophytotoxicity of glyceollin on electron transport of mitochondria isolated from etiolated soybean hypocotyls and cotyledons and corn shoots. All studies were designed to determine the unique site of glyceollin effect on electron transport and utilized various substrates, electron acceptors, and known inhibitors. MATERIALS AND METHODSSoybean seedlings (Glycine max L., cv Amsoy 71) were germinated and grown in the dark at 28°C in moist vermiculite saturated Isolation of mitochondria from cotyledon tissue was the same, except that the cotyledons were ground through an Oster Automatic Juicer (model 362; Oster Co., Milwaukee, WI) and filtered through Miracloth (Chicopee Mills, Inc., Milltown, NJ) before the first centrifugation.Purification of washed mitochondria consisted of resuspending washed mitochondria in 1 ml of standard resuspension medium consisting of 0.3 M mannitoL 20 mm Hepes, 0.1 mm EDTA, and 0.1% BSA at pH 7.2. The resuspended mitochondria were then placed on top of a discontinuous sucrose gradient similar to that used by Douce et aL (1), and consisting of 1.45 M sucrose (4 ml) layered under 1.2 M sucrose (10 ml). Tubes were centrifuged for 45 min at 60,000g at 4°C in a Sorvall AH 627 swinging bucket rotor placed in a Sorvall OTD 65 centrifuge.After centrifugation, mitochondria were located at the boundary of the two sucrose layers and were removed with a curved tip pasteur pipette. The purified mitochondria were then diluted slowly over a period of 20 min with the slow addition of 0.1 M sucrose containing 10 mm KH2PO4, and 0.1% BSA until a sucrose concentration of 0.3 M (pH 7.2) was achieved. The diluted mitochondrial suspension in 0.3 M sucrose was then centrifuged at 14,600g for 8 min. After centrifugation, the purified mitochondria were resuspended in the standard 0.3 M mannitol resuspension medium. Mitochondrial protein was esti...

Isoflavonoid-inducible resistance to the phytoalexin glyceollin in soybean rhizobia

“…While some results suggest a nonspecific mode of action involving disruption of membrane integrity (44), other data support the hypothesis that glyceollin works via specific sensitive target enzymes such as ATPase (12) or NADH-ubiquinone-oxidoreductase (2). From unpublished data, we know that the NADH-ubiquinone-oxidoreductase of B. japonicum 110spc4 is highly sensitive to glyceollin, being strongly affected at concentrations as low as 2 ,uM.…”

Role of Flavonoid and Isoflavonoid Molecules in Symbiotic Functioning and Host-Plant Defence in the Leguminosae

“…Even the mode of action of phytoalexins on pathogens and/or symbionts remains undefined. In the case of the glyceollins, Role of Flavonoid and Isoflavonoid Moleculeshowever, the lethal effect of these phytoalexins on pathogens and symbionts is exerted via inhibition of plasma membrane and tonoplast H + -transporting ATPases [32], and/or NADH-ubiquinone-oxidoreductase [33].…”

RNAi Silencing of Genes for Elicitation or Biosynthesis of 5-Deoxyisoflavonoids Suppresses Race-Specific Resistance and Hypersensitive Cell Death in Phytophthora sojae Infected Tissues

“…Another possibility is that the more elaborate 5-deoxyisoflavonederived pterocarpans, such as glyceollin contribute to cell death. Indeed, preliminary studies suggest that the glyceollins target both the membrane ATPase that serves as a proton pump (Giannini et al, 1988) and mitochrondrial electron transport (Boydston et al, 1983). Given the fact that alkalinization of the apoplast (K 1 /H 1 exchange) and prolonged oxidative stress both are key events in the HR (Heath, 2000), it is possible that such events could contribute to the HR.…”