Mode of Methomyl and Bipolaris maydis (race T) Toxin in Uncoupling Texas Male-Sterile Cytoplasm Corn Mitochondria

The effect of Helminthosporium maydis race T toxin on electron transport in susceptible cytoplasmic male-sterile Texas corn (Zea mays L.) mitochondria was investigated, using dichlorophenol indophenol and ferricyanide as electron acceptors. Succinatedependent electron transport was stimulated by the toxin, consistent with the well described increase in membrane permeability induced by the toxin. Malate-dependent electron transport was inhibited. This inhibition of electron transport increased as a function of time of exposure to the toxin. Mitochondria from normal-fertile (N) com were not affected by the toxin. Both the inhibition of electron transport and the increase in ion permeability, such as dissipation of membrane potential and Ca2+ gradients, induced by the toxin in T corn was prevented by N,N'-dicyclohexylcarbodiimide, a hydrophobic carbodiimide. A water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, was ineffective in preventing dissipation of membrane potential by the toxin. These results suggest that the various toxin actions are mediated via interaction of the toxin with one target site, most probably a 13 kilodalton polypeptide unique to T mitochondria. N,N'-dicyclohexylcarbodiimide may confer protection by modifying an amino acid residue in a hydrophobic portion of the target site.Helminthosporium maydis race T toxin specifically targets mitochondria of the Texas (T) line ofcytoplasmic male-sterile corn (Zea mays L.), while having no effect on normal-fertile corn or other types of cytoplasmic male-sterile corn. HmT3 toxin sensitivity and cytoplasmic male-sterility are tightly linked and are probably the result of the same recombination event in the mitochondrial DNA (8)(9)(10) HmT toxin also inhibits malate-driven respiration as measured by oxygen consumption (6). This inhibition may be the result of a direct interaction of the toxin with a target site in the NADH-ubiquinone oxidoreductase (complex I) or due to leakage of a cofactor, NAD. Malate-driven respiration, unlike succinate-driven respiration, requires a soluble cofactor, NAD. NAD has been shown to leak slowly from toxin-treated mitochondria (3, 22). Thus, it is possible that inhibition of malate-dependent respiration is caused by toxin-induced increases in mitochondrial membrane permeability and subsequent NAD leakage. However, in one case, HmT toxin inhibition of malate-dependent electron transport was shown to be separate from the action ofthe toxin on membrane permeability (6,30), suggesting that the toxin could possibly interact with two target sites.To understand the nature and the location of the toxin target site(s), we have investigated the effect of HmT toxin on electron transport activities by functionally dissecting the electron transport chain using ferricyanide and DCPIP as electron acceptors. The rationale was that assaying for acceptor reduction would be a more direct measurement ofelectron transport from complex I or II than assaying for 02 reduction.We also studied the effect of DCCD on toxi...

Section: Electron Transport Measured By Ferricyanide and Dcpip Reductionsupporting

confidence: 51%

mentioning

confidence: 86%

Effects of Helminthosporium maydis Race T Toxin on Electron Transport in Susceptible Corn Mitochondria and Prevention of Toxin Actions by Dicyclohexylcarbodiimide

Holden

Sze²

1989

“…Contraction of mitochondria is an osmotic phenomenon that is dependent upon the integrity of the inner membrane. Experimental conditions that damage the osmotic integrity of the inner membrane will eliminate contraction with no possible reversal of swelling (14). As evidenced in Figure 1, mitochondria isolated from osmotic-stressed tissue contract upon the addition of NADH which indicates that mild osmotic stress did not damage the inner membrane of either root or shoot mitochondria.…”

Section: Methodsmentioning

confidence: 81%

Effect of Osmotic Stress on Ion Transport Processes and Phospholipid Composition of Wheat (Triticum aestivum L.) Mitochondria

Klein

Burke²,

Wilson³

1986

Self Cite

The effect of osmotic stress on wheat (Triticum aestivum L.) mitochondrial activity and phospholipid composition was investipted. Preliminary growth measurements showed that osmotic stress (-0.25 or -0.5 megapascal external water potential) inhibited the rate of shoot dry matter accumulation while root dry matter accumulation was less sensitive. We have determined that differences in sensitivity to osmotic stress existed between tissues at the mitochondrial level. Mitochondria isolated from roots or shoots of stressed seedlings showed respiratory control and ADP/O ratios similar to control seedlings which indicates that stressed mitochondria were well coupled. However, under passive swelling conditions in a KCI reaction mixture, the rate and extent of valinomycininduced swelling of shoot mitochondria were increased by osmotic stress while root mitochondria were largely unaffected. Active ion transport studies showed efflux transport by stressed-shoot mitochondria to be partially inhibited since mitochondrial contraction required the addition of N-ethylmaleimide or nigericin. Efflux ion transport by root mitochondria was not inhibited by osmotic stress which indicates that stressinduced changes in ion transport were largely limited to shoot mitochondria. Characterization of mitochondrial fatty acid and phospholipid composition showed an increase in the percentage of phosphatidylcholine in stressed shoot mitochondria compared to the control. Mitochondrial fatty acid composition was not markedly altered by stress. No significant changes in either the phospholipid or fatty acid composition of stressed root mitochondria were observed. Hence, these results suggest that a tissue-specific response to osmotic stress exists at the mitochondrial level.Physiological responses of plants to water deficits generally vary with the severity and duration of the stress. The most sensitive processes are altered by a very mild stress and these changes intensify while additional processes become affected in accordance to their sensitivity to the stress (2).Sensitivity to water stress depends on the tissue in question. Under mild water stress, shoot growth is restricted while root growth continues (28,31 (1,15,22, 27), most have been restricted to water stress effects on shoot mitochondria while effects on root mitochondria have been neglected. Studies (1, 27) indicate that mitochondria isolated from air-dried shoots oxidize substrates (proline and to a lesser extent NADH, malate, and succinate) at reduced rates and exhibit generally poor coupling. These results and the lack of osmotic contraction by stmssed mitochondria (22) may indicate the loss ofmembrane integrity due to nonuniform shrinkage of mitochondrial membranes during desiccation (18). It should be emphasized that internal water deficits developed rather rapidly during air drying and became severe in a matter of hours.Because of the paucity of literature in this area, we examined the effect of mild osmotic stress on root and shoot mitochondrial ion and electron tra...

“…Phyllosticta maydis) were effective in dissipating &4, and decreasing Ca2 uptake with the following order: Pm (100) >> HmT (23)(24)(25)(26)(27)(28)(29)(30) > Cpd XIII (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) >> RT2C (0.4-1.8) > Cpd IV (0.2-1.0). In contrast, the toxins and analogs had no effect on A4A formed in N mitochondria.…”

mentioning

confidence: 99%

“…The toxin exerts uncoupling effects and inhibits electron transport (6). For example, malate-dependent oxygen consumption in T mitochondria is inhibited, while the toxin stimulates respiration (02 consumption) and abolishes state 3/state 4 transitions when NADH or succinate (in nonsalt osmoticum) are substrates (10,11,18,23,27,28). Loss ofrespiratory control can be induced by compounds that uncouple electron transport from oxidative phosphorylation by increasing membrane permeability to protons.…”

mentioning

confidence: 99%

Dissipation of the Membrane Potential in Susceptible Corn Mitochondria by the Toxin of Helminthosporium maydis, Race T, and Toxin Analogs

Holden¹,

Sze²

1987

We have tested directly the effect of Helminthosporium maydis T (Hmt) toxin and various analogs on the membrane potential formed in mitochondria isolated from a Texas (T) cytoplasmic male-sterile and a normal (N) corn. ATP, malate or succinate generated a membrane potential (negative inside) C44H1013) and Pm toxin (band A: C331H600, produced by the fungus, Phyllosticta maydis) were effective in dissipating &4, and decreasing Ca2 uptake with the following order: Pm (100) >> HmT (23-30) > Cpd XIII (11-25) >> RT2C (0.4-1.8) > Cpd IV (0.2-1.0). In contrast, the toxins and analogs had no effect on A4A formed in N mitochondria. The striking similarities of the HmT toxin (band 1: C41H680,3) and Cpd XIII on T mitochondrial activities provide strong evidence supporting the correctness of the polyketol structure assigned to the native toxin. Since the A4, in energized mitochondria is caused mainly by the electrogenic extrusion of H', the results support the idea that HmT toxin increases membrane permeability of T mitochondria to H'. The host specificity of the toxin suggests that an interaction with unique target site(s) on the inner mitochondrial membrane of T corn causes H' leakage.We are interested in understanding the mode of toxin action. Most of the evidence indicates the primary target for HmT toxin action is the mitochondrion of T corn. The toxin exerts uncoupling effects and inhibits electron transport (6). For example, malate-dependent oxygen consumption in T mitochondria is inhibited, while the toxin stimulates respiration (02 consumption) and abolishes state 3/state 4 transitions when NADH or succinate (in nonsalt osmoticum) are substrates (10,11,18,23,27,28). Loss ofrespiratory control can be induced by compounds that uncouple electron transport from oxidative phosphorylation by increasing membrane permeability to protons. We have shown previously that HmT toxin decreases Ca2+ uptake into T mitochondria (17) and increases membrane permeability to Ca2+ (14). Since Ca2+ uptake into the mitochondria is thought to depend on the membrane potential (inside negative) (8,14,17, 26), we tested directly whether the toxin could dissipate the electrochemical gradient of H+. Using safranine as a membrane potential probe, we show that HmT toxin dissipated the membrane potential in mitochondria of susceptible, but not resistant, corn. Synthetic toxin analogs, reduced native toxin and Pm toxin (produced by the fungus Phyllosticta maydis) were also effective in dissipating the membrane potential in T mitochondria, though they differed in their relative potency. Preliminary results of this study have been presented (15). MATERIALS AND METHODSA toxin produced by the fungus, Helminthosporium maydis race T, causes symptoms of leaf blight in corn with Texas malesterile cytoplasm (cms-T3), while corn with N cytoplasm is insensitive to the toxin (6). Both toxin sensitivity and male sterility are maternally inherited, and mitochondrial genes are thought to be responsible for these traits (22).