Effects of Methomyl and Helminthosporium maydis Toxin on Matrix Volume, Proton Motive Force, and NAD Accumulation in Maize (Zea mays L.) Mitochondria

Methomyl and Helminthosporium maydis race T toxin block oxidative phosphorylation in mitochondria isolated from maize plants with Texas male sterile cytoplasm (T) but not in mitochondria isolated from those with Normal cytoplasm (N) (Bednarski, Izawa, Scheffer 1977 Plant Physiol 59: 540-545). Moreover, they have been reported to cause specific swelling in T mitochondria (Miller, Koeppe 1971 Science 173: 67-69; Koeppe, Cox, Malone 1978 Science 201: 1227-1229). We could not detect, by direct volume measurements, any change induced by these compounds in the mitochondrial matrix space. We show here that the proton motive force, which in maize mitochondria is composed of a large transmembrane potential and of a low transmembrane pH difference, is absent in T mitochondria incubated in the presence of methomyl or of Helminthosporium maydis race T toxin, while it is unchanged in N mitochondria. Methomyl and Helminthosporium maydis race T toxin induce, independently of the collapse of the proton motive force, a release of the cofactors NAD and coenzyme A from the mitochondrial matrix space. In particular, we show that NAD is transported in maize mitochondria, and that this transport, which is not dependent on the proton motive force, is inhibited by methomyl or Helminthosporium maydis race T toxin.     

Induction of a Sensitive Response to Helminthosporium maydis Race T Toxin in Resistant Mitochondria of Corn (Zea mays L.) by Removal of the Outer Mitochondrial Membrane

Mitochondria isolated from Texas cytoplasmically male sterile (Tms) and normal (N) versions of corn (Zea mays L.) exhibit differential sensitivity to toxin(s) produced by Helminthosporium maydis race T, the causal organism of southern corn leaf blight. Malate dehydrogenase was inhibited by toxin(s) in intact Tms mitochondria but was unaffected in N mitochondria. Removal or rupture of the outer mitochondrial membrane resulted in retention of sensitivity of malate dehy-drogenase in Tms mitochondria to toxin(s), and induction of a sensitive response in normally toxin-insensitive N mitochondria. This suggests that a permeability difference in the respective outer membranes of N and Tms mitochondria may affect the passage of toxin(s) to a mitochondrial site of action. Mitochondrial bioassays indicate that more toxin was bound by Tms mitochondria than by N mitochondria; the greatest toxin binding was associated with the inner membrane of Tms mitochondria.     

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

Bipolaris maydis race T toxin (BmT), and its functional analog, methomyl, uncoupled Texas male-sterile (T) cytoplasm mitochondria by decreasing the resistance of the inner membrane to protons. However, unlike protonophoric or ionophoric agents, BmT toxin and methomyl induced irreversible swelling. Packed volume measurements showed that mitochondrial volume was irreversibly increased by methomyl and BmT toxin indicating that mitochondria no longer functioned as differentially permeable osmometers. The decreased resistance of inner mitochondrial membranes to protons and the loss of osmotic volume regulation suggests that methomyl and BmT toxin induced the formation of hydrophilic pores in T mitochondrial inner membranes.     

A Comparison of Mitochondria Isolated from Male-Sterile and Nonserile Cytoplasm Etiolated Corn Seedlings

Toxins from Helminthosporium maydis race T and Phyllosticta maydis have been found to affect the functional processes of corn mitochondria isolated from Texas male-sterile (T) cytoplasm, but not of mitochondria isolated from nonsterile (N) cytoplasm. The effects of chemicals known to induce responses similar to those of the toxin were compared on mitochondria isolated from T and N cytoplasm inbreds (W64A, Zea mays L.). Valinomycin, gramicidin, and decenylsuccinic acid (DSA) each caused more swelling (measured by transmission changes in %) of N mitochondria than of T mitochondira. The stimulation of exogenous NADH oxidation was the same for N and T mitochondria in the valinomycin, DSA, and Ca²⁺ plus phosphate treatments, was greater for T mitochondria than for N mitochondria in the gramicidin and DNP treatments, and was greater for N mitochondrai than for T mitochondira in the Ca⁺² minus phosphate treatment. Sodium azide inhibited NADH oxidation equally for N and T mitochondria. In addition, N and T mitochondria had similar respiration rates for various substrates and equal efficiencies of oxidative phosphorylation. In contrast to the specificity of toxins for T mitochondria, none of the treatment effects were specific for N or T mitochondria. The results indicate that mitochondria isolated from N and T cytoplasm generally respond similarly to various conditions, but that there can be quantitative differences in the response. The extent to which these differences represent cytoplasmically controlled modification of mitochondrial physiology or structure is not known.     

Helminthosporium maydis T Toxin Decreased Calcium Transport into Mitochondria of Susceptible Corn

The effects of purified Helminthosporium maydis T (HmT) toxin on active Ca²⁺ transport into isolated mitochondria and microsomal vesicles were compared for a susceptible (T) and a resistant (N) strain of corn (Zea mays). ATP, malate, NADH, or succinate could drive ⁴⁵Ca²⁺ transport into mitochondria of corn roots. Ca²⁺ uptake was dependent on the proton electrochemical gradient generated by the redox substrates or the reversible ATP synthetase, as oligomycin inhibited ATP-driven Ca²⁺ uptake while KCN inhibited transport driven by the redox substrates. Purified native HmT toxin completely inhibited Ca²⁺ transport into T mitochondria at 5 to 10 nanograms per milliliter while transport into N mitochondria was decreased slightly by 100 nanograms per milliliter toxin. Malate-driven Ca²⁺ transport in T mitochondria was frequently more inhibited by 5 nanograms per milliliter toxin than succinate or ATP-driven Ca²⁺ uptake. However, ATP-dependent Ca²⁺ uptake into microsomal vesicles from either N or T corn was not inhibited by 100 nanograms per milliliter toxin. Similarly, toxin had no effect on proton gradient formation ([¹⁴C]methylamine accumulation) in microsomal vesicles. These results show that mitochondrial and not microsomal membrane is a primary site of HmT toxin action. HmT toxin may inhibit formation of or dissipate the electrochemical proton gradient generated by substrate-driven electron transport or the mitochondrial ATPase, after interacting with a component(s) of the mitochondrial membrane in susceptible corn.     

Helminthosporium maydis Race T Toxin Induces Leakage of NAD from T Cytoplasm Corn Mitochondria

The mechanism by which Helminthosporium maydis race T toxin inhibits respiration dependent on NAD⁺-linked substrates in T cytoplasm corn mitochondria was investigated. The toxin did not cause leakage of the soluble matrix enzyme malate dehydrogenase from the mitochondria or inhibit malate dehydrogenase or isocitrate dehydrogenase directly. The toxin did increase the permeability of the inner membranes of T cytoplasm, but not N cytoplasm, mitochondria to NAD⁺. Added NAD⁺ partially or fully restored toxin-inhibited electron transport in T cytoplasm mitochondria. Thiamin pyrophosphate had a similar effect when malate was the substrate. It was concluded that the inhibition of respiration of NAD⁺-linked substrates by the toxin is due to depletion of the intramitochondrial pool of NAD⁺ and other coenzymes.     

Inhibition of Dark CO(2) Fixation and Photosynthesis in Leaf Discs of Corn Susceptible to the Host-specific Toxin Produced by Helminthosporium maydis, Race T

The host-specific toxin produced by Helminthosporium maydis, race T, causes 50% inhibition of dark fixation of ¹⁴CO₂ by leaf discs of susceptible (Texas male sterile) corn when it is diluted to approximately 1/10,000 of the volume of the original fungus culture filtrate. Dilutions of 1/10 or less are required for equivalent inhibition of discs prepared from resistant (N) corn. Root growth and photosynthesis were considerably less sensitive (dilution values 1/3000 and 1/1200, respectively), as was leakage of ¹⁴C induced by toxin from preloaded discs. Based on literature values for dilutions causing ion leakage or inhibition of mitochondrial oxidation, toxin dilutions several orders of magnitude greater bring about inhibition of dark CO₂ fixation. Preincubation of discs in light increased sensitivity of dark fixation to toxin and an effect of light on symptom development was shown. Phosphoenolypruvate carboxylase activity in extracts of roots or leaves was not affected by toxin nor was the enzyme level altered in excised leaves treated with toxin. Inhibition of dark fixation of CO₂ provides a bioassaay for race T toxin which is both reliable and rapid.     

Effects of Purified Helminthosporium maydis Race T Toxin on the Structure and Function of Corn Mitochondria and Protoplasts

A toxin preparation from Helminthosporium maydis Race T containing several closely related molecules with apparently identical biological activities was highly active against mitochondria and protoplasts from Texas male-sterile (T) cytoplasm corn (T mitochondria and T protoplasts, respectively) but had no effect on their male-fertile (N) cytoplasm counterparts. The toxin preparation caused multiple changes in isolated T mitochondria, including uncoupling of oxidative phosphorylation, stimulation of succinate and NADH respiration, inhibition of malate respiration, increased swelling, loss of matrix density, and unfolding of the inner membrane. Only 6 to 7 nanograms toxin per milligram mitochondrial protein (1.8 nanogram per milliliter) were required to fully uncouple oxidative phosphorylation and to completely inhibit malate respiration in isolated T mitochondria. Similar low concentrations of toxin caused collapse of T protoplasts after several days of culture. Severe ultrastructural damage to mitochondria in T protoplasts was observed within 20 minutes; no changes in other cellular components were observed at this time. These observations on the cytoplasmic specificity, multiple effects, and high activity of the toxin at the mitochondrial and cellular levels highlight its biological significance and potential usefulness in determining the molecular basis of southern corn leaf blight disease.     

A Differential Effect of Race T Toxin on Dark and Photosynthetic CO(2) Fixation by Thin Leaf Slices from Susceptible Corn

The effect of purified host-specific toxin from Bipolaris (Helminthosporium) maydis, race T, on dark or light-dependent CO₂ fixation was studied with thin (1 × 8 mm) corn (Zea mays L.) leaf slices supplied H¹⁴CO₃⁻. At 5 to 30 nanograms per milliliter (5 nanomolar), toxin significantly inhibited (20 to 40%) dark CO₂ fixation in susceptible (T) corn slices after either dark or light preincubations of 10-20 minutes. The same concentrations were effective to the same degree on photosynthesis, but the effect differed in that significant inhibition occurred after 25 minutes and only with light preincubation. Light preincubation without toxin did not shorten the time required for inhibition of photosynthesis after addition of toxin. Once photosynthetic inhibition was entrained, it was not reversed by subsequent periods of darkness. The results suggest the possibility that race T toxin affects two separate metabolic sites, and the data are discussed in view of currently held concepts of toxin action in susceptible tissue.     

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

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. Succinate-dependent 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) corn 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.     

Loss of Sensitivity to Helminthosporium maydis Race T Toxin during Aging of Mitochondria Isolated from Texas Cytoplasm Corn

Helminthosporium maydis Race T toxin caused the expected changes in freshly isolated mitochondria from T cytoplasm corn, namely complete uncoupling of oxidative phosphorylation, pronounced stimulation of succinate and NADH respiration, complete inhibition of malate respiration, and increased mitochondrial swelling. In contrast, identical toxin treatments of the mitochondria after 12 hours aging on ice resulted in partial uncoupling, much lower stimulation of succinate and NADH respiration, no inhibition of malate respiration, and no mitochondrial swelling. Almost all of the toxin sensitivity was lost by 6 hours aging. At this stage, the mitochondria were 208× and 66× less sensitive to toxin-induced changes in coupling of malate respiration and state 4 malate respiration rates, respectively. Loss of toxin sensitivity did not occur when the mitochondria were aged under nitrogen or in the presence of 5 millimolar dithiothreitol. This suggested that the aging effect was due to oxidation, possibly of sulfhydryl groups in one or more mitochondrial membrane proteins.     

A Comparison of Purified Host Specific Toxin from Helminthosporium maydis, Race T, and Its Acetate Derivative on Oxidation by Mitochondria from Susceptible and Resistant Plants

NADH or succinate oxidation and malate oxidation were differentially affected in mitochondria from both susceptible and resistant corn by a purified and chemically characterized preparation of host-specific toxin from Bipolaris (Helminthosporium) maydis, race T. NADH and succinate oxidation by susceptible T corn mitochondria were stimulated 50 to 200% with apparent uncoupling from the cytochrome chain at approximately 10(-9)m toxin (5 to 20 ng/ml). Significant inhibition of malate oxidation was observed at slightly higher toxin concentrations, but oxidation was still coupled to ADP utilization. Inhibition of malate oxidation also was observed in N corn (resistant) and soybean mitochondria at approximately 1,000-fold greater concentrations, but stimulation of NADH and succinate oxidation was not found at any toxin concentration tested.A fully acetylated toxin derivative at approximately 1 microgram per milliliter also caused stimulation of NADH or succinate oxidation in T corn mitochondria, but not those of N corn or soybean mitochondria at 100 micrograms per milliliter. Malate oxidation was inhibited to the same extent by toxin acetate with mitochondria from T corn, N corn, and soybean. The blocking of hydroxyl groups in race T toxin by acetyl functions eliminated selectivity toward malate oxidation only. The data suggest that inhibition of malate oxidation is either a separate or secondary effect of selective action of toxin on T corn mitochondria, perhaps by interference with transport in or out of the matrix. Sensitivity of T, but not N, corn mitochondria to purified toxin decays within minutes after pellets are suspended in aqueous osmotica, with no obvious change in mitochondrial integrity. The action of race T toxin seems to involve a labile process, such as ion gradient(s), or an unstable structural conformation of T corn mitochondria.     

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

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) as monitored by the absorbance change of a cationic dye, safranine. The relative membrane potential (Δψ) could also be detected indirectly as ⁴⁵Ca²⁺ uptake. Hmt toxin added to T mitochondria dissipated the steady state Δψ similar to addition of a protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Toxin analogs (Cpd XIII: C₄₁H₆₈O₁₂ and Cpd IV: C₂₅H₄₄O₆), reduced native toxin (RT2C: C₄₁H₈₄O₁₃) and Pm toxin (band A: C₃₃H₆₀O₈, produced by the fungus, Phyllosticta maydis) were effective in dissipating Δψ 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 Δψ formed in N mitochondria. The striking similarities of the HmT toxin (band 1: C₄₁H₆₈O₁₃) and Cpd XIII on T mitochondrial activities provide strong evidence supporting the correctness of the polyketol structure assigned to the native toxin. Since the Δψ 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.     

Mitochondrial DNA variation in maize plants regenerated during tissue culture selection

Summary Plants resistant to Helminthosporium maydis race T were obtained following selection for H. maydis pathotoxin resistance in tissue cultures of susceptible, Texas male-sterile (T) cytoplasm maize. The selected lines transmitted H. maydis resistance to their sexual progeny as an extranuclear trait. Of 167 resistant, regenerated plants, 97 were male fertile and 70 were classified male sterile for reasons that included abnormal plant, tassel, anther or pollen development. No progeny were obtained from these male-sterile, resistant plants. Male fertility and resistance to the Phyllosticta maydis pathotoxin that specifically affects T cytoplasm maize were co-transmitted with H. maydis resistance to progeny of male-fertile, resistant plants. These three traits previously were associated only with the normal (N) male-fertile cytoplasm condition in maize. Three generations of progeny testing provided no indication that the cytoplasmic association of male sterility and toxin susceptibility had been broken by this selection and regeneration procedure. Restriction endonuclease analysis of mitochondrial DNA (mtDNA) revealed that three selected, resistant lines had distinct mtDNA organization that distinguished them from each other, from T and from N cytoplasm maize. Restriction patterns of the selected resistant lines were similar to those from T cytoplasm mtDNA; these patterns had not been observed in any previous analyses of various sources of T cytoplasm. The mtDNA analyses indicated that the male-fertile, toxin-resistant lines did not originate from selection of N mitochondrial genomes coexisting previously with T genomes in the T cytoplasm line used for selection.Scientific Journal Series Article no. 11,185 of the Minnesota Agricultural Experiment Station and no. 2295 of the Florida Agricultural Experiment Station. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee of warrantly of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable     

Reduction of Adenosine Triphosphate Levels in Susceptible Maize Mesophyll Protoplasts by Helminthosporium maydis Race T Toxin

Helminthosporium maydis race T (HMT) toxin caused a reduction in the steady-state ATP levels when leaf mesophyll protoplasts isolated from maize containing Texas male-sterile (T) but not male-fertile (N) cytoplasm were incubated in the dark. At a toxin concentration 10 times the mean effectived dose for inhibition of root growth, the ATP levels began to fall in 30 to 90 seconds, fell by 50% in about 4 minutes, and reached 23% of the original levels in 100 minutes. This is faster than any previously observed response of whole cells or tissues to HMT toxin. In protoplasts incubated in the light, ATP levels were 25% higher than in the dark and were either unaffected or only slightly diminished by toxin. 3-(3,4-Dichlorophenyl)-1, 1-dimethylurea (DCMU), an inhibitor of photosynthetic electron transport, overcame the effect of light on both toxin-treated and control protoplasts. Oligomycin, an inhibitor of mitochondrial ATP synthesis, mimicked the effects of toxin in the dark, in the light, and in the light plus DCMU, but it was not specific for T cytoplasm. During the first 24 hours of culture, ATP levels in control protoplasts increased in both the light and dark. In the dark, ATP was not detectable after 24-hour incubation in the presence of toxin, whereas in the light a substantial amount of ATP remained. Our results are compatible with the hypothesis that mitochondria in vivo are inhibited by HMT toxin. Other responses of cells and tissues to toxin can be explained in terms of reduced ATP levels.     

Analogs of host-specific phytotoxin produced by Helminthosporium maydis,race T: I. Synthesis

Fourteen analogs of the host-specific corn phytotoxin (T toxin) obtained from cultures of the fungal plant pathogen, Helminthosporium maydis, race T, were synthesized. Addition of difunctional Grignard reagents to aldehyde intermediates resulted in shorter versions (C₁₅–C₂₆) of native toxin (C₃₅–C₄₅), containing the β-polyketol functions which appear to account for the specificity and very high toxicity (10^?8–10^?9M) of T toxin toward certain corn varieties.     

Evaluation of a Chemical Method for Assay of Helminthosporium maydis Race T Toxin

A chemical test reported by Karr et al. (Plant Physiol. 55:727) to assay for host-specific toxin produced by Helminthosporium maydis race T was evaluated. Preparations from culture filtrates of both race T isolates, containing host-specific toxin, and similar preparations from race O isolates, containing no detectable host-specific toxin, gave positive reactions in the chemical assay. Also, preparations containing active or inactive toxin gave equal responses in this test. The procedure does not provide a reliable method for assaying H. maydis race T toxin.     

Isolation and Partial Characterization of Four Host-specific Toxins of Helminthosporium maydis (Race T)

Helminthosporium maydis, race T, produces four host-specific toxins in culture. These have been designated toxins I, II, III, and IV. A method for isolation and purification of the four toxins is presented, and the criteria of purity of preparations of toxins I, II, and III are given. Toxins I and II are chemically similar and yield the same molecular ion when subjected to mass spectrometry, while toxin III appears to be a glycoside of a compound related to toxins I and II. Toxins I, II, and III can be biologically derived from ¹⁴C-mevalonic acid or ¹⁴C-acetate, permitting preparation of ¹⁴C-labeled toxins. Some chemical, spectral, and chromatographic properties of toxins I, II, and III are presented, and these data are discussed relative to the possible structure of the three compounds. In addition, four host-specific toxins have been isolated from corn infected with H. maydis (race T). These toxins are recovered in the same fractions as toxins I, II, III, and IV using the isolation procedure described here. Three of the toxins isolated from infected corn cannot be distinguished from toxins I, II, and III on the basis of infrared spectra or chromatographic mobility.     

Analogs of host-specific phytotoxin produced by Helminthosporium maydis,race T: II. Biological activities

Inhibition of dark CO₂ fixation by susceptible corn leaves was used to compare the relative toxicity of synthetic analogs with that of the host-specific phytotoxin produced by the fungal corn pathogen, Helminthosporium maydis, race T. Analogs with C₁₅, C₂₅, or C₂₆ chain lengths and 1,5-dioxo-3-hydroxy functions were only slightly less toxic (2–6 × 10^?7M) than native T toxin (C₃₅–C₄₅ chain lengths) or its individual components (3 × 10^?8M). Like native toxin, analogs were host-specific in that they did not inhibit dark CO₂ fixation in leaf tissue of resistant corn at concentrations 10²–10³ times greater than those effective with susceptible corn. These findings support the structures previously proposed for native T toxin.     

Mitochondrial Interaction with Helminthosporium maydis Race T Toxin: Blocking by Dicyclohexylcarbodiimide

Treatment of mitochondria isolated from Texas male sterile cytoplasmcorn (T mitochondria) with high concentrations of dicyclohexylcarbodiimide(DCCD) (140 nmol DCCD mg^–1 mitochondrial protein) completelyand immediately inhibited T mitochondrial swelling by Helminthosporiummaydis Race T toxin (HmT toxin). In order to obtain a specificinteraction between DCCD and the ATPase complex T mitochondriawere incubated with lower DCCD concentrations (1–5 nmolDCCD mg^–1 mitochondrial protein) for up to 8 h at 4 °C.After 8 h incubation in the presence of 3.75 nmol DCCD mg^–1mitochondrial protein, toxin-induced swelling was decreasedby 69%. Specificity of DCCD action upon the ATPase complex wasconfirmed by (1) SDS gel electrophoresis and fluorographic analysesof proteins from [¹⁴C]-DCCD-treated T mitochondria and immunoprecipitatesand (2) physiological experiments showing that DCCD exertednone of its other documented effects. These data suggest thatHmT toxin interacts with the ATPase complex of T mitochondriaeither at or near the DCCD-binding protein within the membranesector of the complex. Key words: Zea mays L., Helminthosporium maydis, Mitochondria