Influence of Helminthosporium maydis, Race T, Toxin on Potassium Uptake in Maize Roots

The effect of a toxin extract of Helminthosporium maydis, race T on K⁺ (⁸⁶Rb) uptake by excised root segments of normal (N) and Texas cytoplasmic male-sterile (T) versions of corn inbred W64A was investigated. The uptake of K⁺ was inhibited in both N and T roots by the toxin. This was true for both basal (freshly excised) and augmented (pretreated with aeration) K⁺ uptake. Augmented uptake was more toxin-sensitive than basal uptake (irrespective of cytoplasm type), and the augmented uptake in T roots was seven to eight times more toxin-sensitive than in N roots.     

The binding of host-selective toxin analogs to mitochondria from normal and ;texas' male sterile cytoplasm maize

Tritium-labeled toxin analogs were prepared by reduction with NaB³H₄ of either the toxin from Helminthosporium maydis race T or a toxin component from Phyllosticta maydis. These reduced analogs had high radiochemical specific activities, high biological activities, and plant specificities identical to the native toxins. A filtration assay was developed to test the binding of these labeled analogs to isolated mitochondria. Binding was not energy dependent nor was there measurable matrical uptake. The analogs were shown to be lipophilic, a characteristic which gave rise to considerable nondisplaceable binding. Under conditions limiting nondisplaceable binding, the displaceable binding was shown to be linear with respect to toxin concentration and unsaturable. No significant differences were observed in the binding characteristics between the mitochondria from normal and male-sterile (Texas) cytoplasm maize. The findings suggest that, at physiologically relevant concentrations, these toxin analogs permeate the membranes of susceptible and resistant mitochondria alike. The lack of demonstrable specific binding does not rule out the involvement of a classical receptor site but does indicate that other kinds of molecular interactions may be involved in the mechanisms for toxicity and specificity.     

Cytoplasm-specific Effects of Helminthosporium maydis Race T Toxin on Survival of Corn Mesophyll Protoplasts

High yields of mesophyll protoplasts were obtained from leaves of corn (Zea mays L., inbred W64A). Many protoplasts survived a week in the dark in a simple osmoticum. Culture filtrate from Helminthosporium maydis race T at dilutions of 1:10,000 to 1:20,000 destroyed protoplasts with Texas male-sterile (T) cytoplasm. Substantial damage to protoplasts with nonmale-sterile (N) cytoplasm occurred only at a 1:20 dilution. High concentrations of partially purified H. maydis race T (HMT) toxin (32.5-130 μg dry weight/ml) did not reduce survival of protoplasts with N cytoplasm or C or S male-sterile cytoplasms after 6 days of exposure. Protoplasts with T or TRf (fertility restored) cytoplasm collapsed within 1 to 3 days after treatment with 0.13 μg of HMT toxin/ml, which was one-fifth the level causing 50% inhibition of T cytoplasm seedling root growth. Protoplasts with T cytoplasm which were washed after 30 minutes or more of exposure to HMT toxin also collapsed within a few days. Cultured W64A T protoplasts and freshly isolated protoplasts from inbreds C103 and Mo17 with T cytoplasm were less sensitive to HMT toxin than freshly isolated W64A T protoplasts. Toxin-treated protoplasts survived longer in the light than in the dark. The sensitivity and specificity of the system described will facilitate physiological, ultrastructural, and genetic studies of toxin action.     

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.     

Reversible Effects of Toxin from Helminthosporium maydis Race T on Oxidative Phosphorylation by Mitochondria from Maize

Host-selective toxin from Helminthosporium maydis race T inhibited oxidative phosphorylation (AT³²P formation) and stimulated ATPase activity by mitochondria from male-sterile (T) but not from normal (N) cytoplasm maize (Zea mays L.). Toxin increased the rate of NADH oxidation, but succinate oxidation was slightly, and malate-pyruvate oxidation was strongly inhibited as the associated ATP formation was abolished. There was a 1-minute lag before toxin gave maximal stimulation of NADH oxidation; the responses to 2,4-dinitrophenol and valinomycin were immediate. There was also a delay in the effect of toxin on ATP formation. T mitochondria were more sensitive than were N mitochondria to uncoupling by nigericin plus K⁺; there was no evidence, however, that the action of toxin is related to that of nigericin or other ionophores. With NADH as the substrate, the degree of uncoupling increased with increases in toxin concentration up to a saturating level; kinetics of the response suggested reversibility. T mitochondria exposed to toxin for 5 minutes regained normal rates of respiration and of ATP formation when they were washed with toxin-free medium, showing that the uncoupling effect is reversible. Evidently HM-T toxin does not bind firmly to its site(s) of action, in contrast to reports for another hostselective toxin.     

The sterols of normal and male-sterile maize tassels during development

The steroids of normal and male-sterile (Texas type) genotypes of maize were investigated during tassel development. A bioassay for estrogen activity of the normal meiotic and postmeiotic tassels was negative, indicating estrogen activity (estrone equivalent) much less than one ng/g of plant tissue. The sterols found were cholesterol, campesterol, stigmasterol, sitosterol, and probably isofucosterol, stigmast-7-enol, and 24-methylenecholesterol. In the premeiotic, meiotic, and postmeiotic stages of both genotypes between 300 and 400 μg of C₂₈ and C₂₉ free sterols per g tassels (wet wt) were found, the proportions of the sterols being ca 45% sitosterol, 30% stigmasterol, and 13% campesterol, with less than 5% each of the remaining sterols. In all three stages before saponification more free sterols were found in the normal than in the male-sterile tassels. The differences were significant at the 95% level in the meiotic and post-meiotic stages. The amounts of these sterols derived from esters decreased from approximately 140 μg/g in the premeiotic stage to 50 μg/g in the meiotic stage, and to an undetectable amount in the postmeiotic stage. After application of cholesterol-[4-¹⁴C] to the normal and male-sterile maize leaves for 3 days at meiosis, the label was found in the free sterols and steryl esters of the leaves but only in the free sterols of the tassels.     

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.     

Expression in yeast of the T-urf13 protein from Texas male-sterile maize mitochondria confers sensitivity to methomyl and to Texas-cytoplasm-specific fungal toxins.

The mitochondrial gene T-urf13 from maize (Zea mays L.) with Texas male-sterile (T) cytoplasm codes for a unique 13 kd polypeptide, T-URF13, which is implicated in cytoplasmic male sterility and sensitivity to the insecticide methomyl and to host-specific fungal toxins produced by Helminthosporium maydis race T (HmT toxin) and Phyllosticta maydis (Pm toxin). A chimeric gene coding for T-URF13 fused to the mitochondrial targeting peptide from the Neurospora crassa ATP synthase subunit 9 precursor was constructed. Expression of this gene in the yeast Saccharomyces cerevisiae yielded a polypeptide that was translocated into the membrane fraction of mitochondria and processed to give a protein the same size as maize T-URF13. Methomyl, HmT toxin and Pm toxin inhibited growth of yeast cells expressing the gene fusion on medium containing glycerol as sole carbon source and stimulated respiration with NADH as substrate by isolated mitochondria from these cells. These effects were not observed in yeast cells expressing T-URF13 without a targeting peptide. The results show that T-URF13 is sufficient to confer sensitivity to methomyl and the fungal toxins in a heterologous eukaryotic system, and suggest that mitochondrial localization of T-URF13 is critical for these functions.     

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.     

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.     

Selective Inhibition of K+, Na+, Cl?, and PO43? Uptake in Zea mays L. by Bipolaris (Helminthosporium) maydis Race T Pathotoxin: Evidence for a Plasmalemma Target Site? 1

Pathotoxin preparations were obtained from either axenic culture filtrate of race T of Bipolaris maydis (Nisikado) Shoemaker (new culture media and toxin purification procedures are described) or extracts of maize leaves infected with the fungus. The toxins (10(-6) to 10(-8)m) caused inhibition of [(86)Rb]K(+) uptake in leaf discs and apical root segments of Zea mays L. cv W64A Texas (Tcms) and normal (N) cytoplasms. Significant inhibition was measurable as early as 5 min after adding toxin. In Tcms per cent inhibition was increased by increasing toxin concentration and time in toxin, by using solution at pH 5 rather than pH 7, by decreasing external KCl concentration over the range 50 to 0.1 mm (in the presence of 0.5 mm CaSO(4)), or by exposing leaf discs to light rather than dark during the uptake period in toxin. Root uptake of (22)Na(+) and (36)Cl(-) was inhibited to a lesser extent than K(+). Inhibition of (32)PO(4) (3-) uptake occurred after 40 min when cyclosis had ceased.When combined with data in the literature, our data indicate that the plasmalemma is the probable primary site of toxin action in N and Tcms maize. Comparison of the effects of toxin on K(+) uptake in N and Tcms maize suggests the existence of more than one mode of toxin action: a weak disruptive effect in N and Tcms, and in addition, specific membrane sites in Tcms involved in monovalent ion uptake.Six genotypes in N or Tcms cytoplasm which exhibited different degrees of disease susceptibility in the field showed a corresponding gradation of susceptibility to the toxin when a K(+) uptake bioassay was used. This correlation is strong evidence that the sites of toxin action affecting K(+) transport have characteristics closely related to cellular factors regulating susceptibility to fungal attack.     

Host-Specific Effects of Toxin from the Rough Lemon Pathotype of Alternaria alternata on Mitochondria

Host-specific toxin from the rough lemon (Citrus jambhiri Lush) pathotype of Alternaria alternata (ACR toxin) was tested for effects on mitochondria isolated from several citrus species. The toxin caused uncoupling of oxidative phosphorylation and changes in membrane potential in mitochondria from leaves of the susceptible host (rough lemon); the effects differed from those of carbonylcyanide-m-chlorophenylhydrazone, a typical protonophore. ACR toxin also inhibited malate oxidation, apparently because of lack of NAD⁺ in the matrix. In contrast, the toxin had no effect on mitochondria from citrus species (Dancy tangerine and Emperor mandarin [Citrus reticulata Blanco], and grapefruit [Citrus paradisi Macf.]) that are not hosts of the fungus. The effects of the toxin on mitochondria from rough lemon are similar to the effects of a host-specific toxin from Helminthosporium maydis (HMT) on mitochondria from T-cytoplasm maize. Both ACR and HMT toxins are highly selective for the respective host plants. HMT toxin and methomyl had no effect (toxic or protective) on the activity of ACR toxin against mitochondria from rough lemon.     

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     

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.     

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.     

Identification of Cytoplasmic Male Sterility in Chinese Radish Following PCR Analysis of Mitochondrial DNA

In order to understand the molecular characteristics of the Chinese radish, the mitochondrial DNA structure and sequence were analyzed in Chinese wild radish and cultivated varieties. A total of four male-sterile lines, four maintainer lines, 17 cultivars, and a single Chinese wild radish were used, along with 25 male-sterile individuals and 159 fertile plants. We found that the cytoplasm of Chinese radishes could be classified into two types: the normal type and the Ogura type. The Ogura-type cytoplasm was detected in 25 male-sterile plants. The 159 fertile plants had normal cytoplasm. Both the Ogura cytoplasm and the normal cytoplasm were detected in the male-sterile ??RA??. The orf138 gene in mitochondrial DNA was detected in cultivated Chinese radish cultivars but not in the wild radish. The Chinese radish orf138 nucleotide sequence was determined in four male-sterile lines and displayed complete homology to the known orf138 type A nucleotide sequence. Three types of mitochondrial orfB (type 1, type 2 and type 3) were found in Chinese radishes. Type 1 was only present in the male-sterile lines. Chinese cultivated radishes were divided into type 2 and type 3, while the Chinese wild radish only had type 3 cytoplasm.     

Synthesis and uptake of cytoplasmically synthesized pyruvate, pi dikinase polypeptide by chloroplasts

Polyadenylated RNA was isolated from maize leaves and translated in vitro. In agreement with a previous report by others, we found among the translation products a 110-kilodalton pyruvate orthophosphate dikinase (PPDK) precursor that is about 16 kilodaltons larger than the polypeptide isolated from cells. This maize PPDK precursor polypeptide was taken up from the translation product mixture by intact spinach chloroplasts and yielded a mature PPDK polypeptide (94 kilodaltons). The uptake and processing support the proposal that the extra 16-kilodalton size of the polypeptide from in vitro translation of maize leaf mRNA represents a transit sequence which is cleaved after its entry into chloroplasts. Moreover, these results provide additional evidence that in vivo in maize leaf cells PPDK polypeptide is synthesized in the cytoplasm and is transported into the chloroplasts.

Location of PPDK in C₃ plant leaves was investigated by immunochemical analysis. Intact chloroplasts were isolated from leaves of spinach, wheat, and maize. A protein blot of stromal protein in each case gave rise to bands corresponding to authentic PPDK polypeptide. This result indicates that PPDK is present in chloroplasts of C₃ plant leaves as it is in the case of C₄ plants.

     

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.     

Depolarization of the Electrogenic Transmembrane Electropotential of Zea mays L. by Bipolaris (Helminthosporium) maydis Race T Toxin, Azide, Cyanide, Dodecyl Succinic Acid, or Cold Temperature

The transmembrane electrical potential of root cells of Zea mays L. cv. W64A in a modified 1× Higinbotham solution was partially depolarized by semipurified toxin obtained from Bipolaris (Helminthosporium) maydis race T. At a given toxin concentration depolarization of Texas cytoplasm cells was much greater than for normal cytoplasm cells. This observation correlated directly to the differential host susceptibility to the fungus. The time course and magnitude of depolarization were dependent on toxin concentration; at high concentration the electropotential difference change was rapid. Cortex cells depolarized more slowly than epidermal cells indicating that the toxin slowly permeated intercellular regions. Toxin concentrations which affected electropotential difference were of the same magnitude as those required to inhibit root growth, ion uptake, and mitochondrial processes.

Azide, cyanide, and cold temperature (5 C) gave the same partial depolarization as did the toxin. Dodecyl succinic acid caused complete depolarization. These and other data indicate that one of the primary actions of the toxin is to inhibit electrogenic ion pumps in the plasmalemma.

     

T-URF 13 Protein from Mitochondria of Texas Male-Sterile Maize (Zea mays L.) : Its Purification and Submitochondrial Localization, and Immunogold Labeling in Anther Tapetum during Microsporogenesis

The protein T-URF13 (URF13) is specific to mitochondria of maize (Zea mays L.) with Texas (T) male-sterile cytoplasm and has been implicated in causing male sterility and susceptibility to T-cytoplasm-specific fungal diseases. T-URF13 was purified from isolated mitochondria from maize (line B73) with T cytoplasm by gel filtration and a quasi two-dimensional polyacrylamide gel electrophoresis system. Antibodies to the purified and denatured protein were produced in rabbits. Anti-T-URF13 antiserum was used to show that T-URF13 is in the inner membrane of mitochondria and behaves as an integral membrane protein when mitochondria are fractionated with sodium carbonate or Triton X-114. The antiserum and protein A tagged with 20-nanometer-gold particles were used to localize T-URF13 in T mitochondria by electron microscopy of sections of isolated mitochondria from etiolated shoots and sections of roots and of tapetal cells at pre-and post-degeneration stages of microsporogenesis. The microscopic study confirms that T-URF13 is specifically localized in the mitochondrial membranes of all of the T mitochondria tested, notably those in the tapetum from the meiocyte stage to the late-microspore stage. No change in the amount of labeled T-URF13 protein in the mitochondria of aging tapetal cells was detected.