Ion transport processes in corn mitochondria : I. Effect of the local anesthetic dibucaine.

The local anesthetic dibucaine inhibited respiration-dependent contraction mediated by the K(+)/H(+) antiport system of isolated corn mitochondria. Respiration declined concurrently. Nigericin, an exogenous K(+)/H(+) exchanger, restored ion efflux in dibucaine-blocked corn mitochondria. It was concluded that dibucaine inhibited ion efflux via blockage of the K(+)/H(+) antiport. Further experiments determined that dibucaine also inhibited proton influx facilitated by protonophores and by the ATPase complex during state III respiration. These results are discussed in relation to the mechanism by which dibucaine inhibits proton translocation across the inner mitochondrial membrane.

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.

Glyceollin: a site-specific inhibitor of electron transport in isolated soybean mitochondria.

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 dehydrogenase and coenzyme Q. Coupled state 3 malate oxidation was inhibited by glyceollin and rotenone with apparent K(i) values of about 15 and 5 micromolar, respectively. Carbonylcyanide m-chlorophenyl hydrazone uncoupled state 4 malate oxidation was also inhibited by glyceollin and rotenone, but uncoupled succinate and exogenous NADH state 4 oxidation was only slightly inhibited by both compounds. Glyceollin also inhibited ferricyanide reduction with malate as the electron donor, with an apparent K(i) of 5.4 micromolar, but failed to inhibit such reduction with succinate or externally added NADH as electron donors. Glyceollin did not inhibit state 4 oxidation of malate, succinate, or exogenous NADH. Glyceollin did not act as a classical uncoupler or as an inhibitor of oxidative phosphorylation.

Oxidation of proline by mitochondria isolated from water-stressed maize shoots.

Proline oxidation and coupled phosphorylation were measured in mitochondria after isolation from shoots of water-stressed, etiolated maize (Zea mays L.) seedlings. Both state III and state IV rates of proline oxidation decreased as a logarithmic function of increased seedling water stress between -5 and -10 bars. Proline oxidation rates decreased 62% (state III) and 58% (state IV) as seedling water potentials were decreased from -5 to -10 bars. By comparison, oxidation of succinate, exogenous NADH, or malate + pyruvate decreased only 10 to 15% in this stress range. These decreases were a linear function of increased stress and were comparable to oxidation rates of mitochondria subjected to varying in vitro osmotic potentials. Osmotically induced in vitro stress reduced proline oxidation rates linearly with more negative osmotic potentials, a decrease that was similar to the responses of the other substrates to more negative osmotic potentials. Some decrease in coupling, with all substrates as determined by ADP/O ratios, was observed under osmotic stress. Mitochondria were also isolated from shoot tissue that had been stressed and then rewatered. On a percentage basis, the recovery of proline oxidation was greater than that of the other substrates.The decreases in the proline oxidase activity of mitochondria after only slight stress indicate a mitochondrial sensitivity to water stress at significantly less negative water potentials than previously reported for measurements of maize membrane permeability and respiratory activity.

Mitochondrial heredity: a determinant in the toxic response of maize to the insecticide methomyl.

Mitochondria isolated from etiolated Texas male-sterile (TMS) cytoplasm maize (Zea mays L.) seedlings were adversely affected by methomyl (Lannate, 90 wettable powder), while those isolated from normal-fertile seedlings were not. In a manner analogous to that reported for Bipolaris (Helminthosporium) maydis (race T) toxin, experiments with TMS mitochondria showed that 1 to 3 millimolar methomyl inhibited the state 4 oxidation rate of combined malate and pyruvate while stimulating that of succinate or exogenous reduced nicotinamide adenine dinucleotide. Similar concentrations of methomyl effected an inhibition of phosphorylation, an increase in the percentage of transmittance of light through mitochondrial suspensions, and a decrease in the density of the mitochondrial matrix. Methomyl (15 millimolar) had little effect on the physiological activity or ultrastructure of isolated normal-fertile mitochondria. These observations provide the opportunity to specifically assess the homogeneity, or lack of it, of a cytoplasmic heritable characteristic in a widely divergent group of higher plants.

Oxidation of proline by plant mitochondria.

Mitochondria isolated from etiolated shoots of corn (Zea mays), wheat (Triticum aestivum), barley (Hordeum vulgare), soybean (Glycine max L. Merr.), and mung bean (Phaseolus aureus) exhibited a proline-dependent O(2) uptake subject to respiratory control. ADP/O ratios with proline as substrate were intermediate between ratios obtained with exogenous NADH and malate + pyruvate as substrates. Isotope studies showed proline metabolism to be dependent on O(2), but not NAD. The major ninhydrin-positive product formed via Delta(1)-pyrroline-5-carboxylic acid was glutamate. Mitochondria were capable of further metabolism of glutamate, as radioactive CO(2), organic acids, and aspartate were recovered after [(14)C]proline feeding experiments. These results demonstrate the mitochondrial association and O(2) dependence of plant proline metabolism.

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.

Kaempferol inhibitions of corn mitochondrial phosphorylation.

Kaempferol (3, 5, 7, 4'tetrahydroxyflavone) inhibited the rate of state 3 substrate oxidation, but not the state 4 rate. This, along with the kaempferol inhibition of substrate-driven calcium-phosphate deposition, provided evidence that kaempferol was acting specifically on the phosphorylation mechanism and not on electron transfer. Kaempferol, however, did not inhibit ATP-driven contraction while oligomycin did. Comparisons of kaempferol with mersalyl indicated that kaempferol did not inhibit phosphorylation by blocking phosphate transport. Both kaempferol and 2,4-dinitrophenol inhibited calcium-phosphate transport, but kaempferol did not stimulate respiration to the extent that 2,4-dinitrophenol did under acceptorless conditions. Kaempferol had no effect on NADH-driven contraction in a potassium chloride reaction medium. The site of kaempferol effect is thus seen to be unique from oligomycin and more like aurovertin, likely acting before the formation of the phosphorylated high energy intermediate, but not as an uncoupler in the traditional 2,4-dinitrophenol mode.

Ultrastructural evidence for calcium phosphate deposition by isolated corn shoot mitochondria.

This study demonstrates that isolated corn (Zea mays L. Wf9 xM14) shoot mitochondria will accumulate calcium phosphate crystals in vitro that are comparable to those observed in both in vivo and in vitro animal mitochondria. The intensity of deposition, as observed by electron microscopy, increased with incubation time, substrate concentration, and concentration of calcium and inorganic phosphate. Calcium phosphate deposits were initially observed as amorphous, nearly spherical areas that lacked sharp or clear edges, but which were crystalline in nature under heavier loading conditions and longer incubation times.

Corn Mitochondrial Swelling and Contraction-an Alternate Interpretation.

Mitochondria isolated from 3-day-old etiolated corn shoots (Zea mays L.) can be categorized into three separate groups, each group characteristic of the cell type from which the mitochondria were isolated. Phloem sieve tubes and some adjacent parenchyma cells contain mitochondria that have few cristae and little amorphous matrix. Mitochondria from meristematic and undifferentiated cells have more cristae and matrix. Vaculate and differentiated cells have mitochondria with well-developed cristae and abundant matrix. Each mitochondrial type exhibits typical in vitro spontaneous swelling and substrate-induced contraction responses. characterized by change or lack of change in cristae size and in density of amorphous material. For the second and third types of mitochondria, swelling and contraction are characterized by a change in degree of cristae size and in matrix density. The first type undergoes few changes upon swelling or contraction. Radical changes of the inner membrane, withdrawal and infolding, are associated with cell differentiation and not with swelling and contraction of isolated corn shoot mitochondria.

Localization of lead accumulated by corn plants.

Light and electron microscopic studies of corn plants (Zea mays L.) exposed to Pb in hydroponic solution showed that the roots generally accumulated a surface Pb precipitate and slowly accumulated Pb crystals in the cell walls. The root surface precipitate formed without the apparent influence of any cell organelles. In contrast, Pb taken up by roots was concentrated in dictyosome vesicles. Dictyosome vesicles containing cell wall material fused with one another to encase the Pb deposit. This encased deposit which was surrounded by a membrane migrated toward the outside of the cell where the membrane surrounding the deposit fused with the plasmalemma. The material surrounding the deposit then fused with the cell wall. The result of this process was a concentration of Pb deposits in the cell wall outside the plasmalemma. Similar deposits were observed in stems and leaves suggesting that Pb was transported and deposited in a similar manner.

Oxidation of reduced nicotinamide adenine dinucleotide phosphate by isolated corn mitochondria.

Isolated corn (Zea mays L.) mitochondria were found to oxidize reduced nicotinamide adenine dinucleotide phosphate in a KCl reaction medium. This oxidation was dependent on the presence of calcium or phosphate or both. Strontium and manganese substituted for calcium, but magnesium or barium did not. The oxidation of NADPH produced contraction of mitochondria swollen in KCl. Further evidence that the oxidation of NADPH was coupled was observed in respiratory control and adenosine diphosphate-oxygen ratios that were comparable to those reported for reduced nicotinamide adenine dinucleotide. The pathways of electron flow from NADH and NADPH were compared through the addition of electron transport inhibitors. The only difference between the two dinucleotides was that amytal was found to inhibit almost totally the state 3 oxidation of NADPH, but had little effect on the state 3 oxidation of NADH. The hypothetical pathways for electron flow from NADPH are discussed, as are the possible sites of calcium and phosphate stimulation.

Some effects of decenylsuccinic Acid on isolated corn mitochondria.

The effects of decenylsuccinic acid on the swelling and respiratory capacities of mitochondria isolated from etiolated corn (Zea mays L., Wf9 x M14) shoots were studied. Decenylsuccinic acid (0.1 mM to 1.0 mM) inhibited the oxidation of succinate and malate-pyruvate, stimulated the oxidation of reduced nicotinamide adenine dinucleotide, and uncoupled phosphorylation. The swelling of isolated corn mitochondria, as determined by percentage of transmittance changes, was stimulated by decenylsuccinic acid in potassium chloride reaction media and in sucrose reaction media without bovine serum albumin. In a diaphorase (2, 6-dichlorophenolindophenol as acceptor) reaction with intact mitochondria, only the dehydrogenation rate of malate was reduced by the addition of decenylsuccinic acid. The dehydrogenation of reduced nicotinamide adenine dinucleotide or of succinate was either not affected or was stimulated depending on the diaphorase reaction medium. The oxygen uptake of mitochondria oxidizing N, N, N', N'-tetramethyl-p-phenylenediamine diHCl and ascorbate was inhibited at decenylsuccinic acid concentrations greater than 0.5 mM.The results presented lead to the hypothesis that the primary effect of decenylsuccinic acid on isolated corn mitochondria is on the physical properties of the membranes and that decenylsuccinic acid-affected stimulation or inhibition of respiration results from the physical disruption of the membrane. These results appear to be consistent with those previously reported in whole plant studies.

The effects of drought stress on respiration of isolated corn mitochondria.

Mitochondria were isolated from etiolated corn shoots (Zea mays L.) that were stressed to a measured water potential. The rates of mitochondrial respiration in state III, state IV, and without phosphate or ADP on a milligram protein basis decreased as water stress increased with succinate, malatepyruvate, or reduced nicotinamide adenine dinucleotide as substrates. Coupling (as determined by respiratory control and ADP/O ratios) did not decrease with increasing water stress. At water potentials greater than -35 bars all respiration had ceased.

Southern corn leaf blight: susceptible and resistant mitochondria.

Mitochondria isolated from etiolated shoots of blight-susceptible and blight-resistant corn plants were subjected, in various respiratory states, to the pathotoxin released by Helminthosporium maydis (race T). The addition of the pathotoxin to susceptible mitochondria caused respiratory rate and oxidative phosphorylation changes. The addition of pathotoxin to susceptible mitochondria suspended in a potassium chloride reaction medium induced an immediate and irreversible swelling regardless of the respiratory state of the mitochondria. This membrane swelling correlates with the observed respiratory and coupling effects of the pathotoxin. In all instances, mitochondria isolated from blightresistant corn failed to exhibit any of the above responses to the pathotoxin.

The effect of calcium and inhibitors on corn mitochondrial respiration.

The effects of KCN, antimycin A, malonate, rotenone, and amytal on the oxidation of malate, succinate, and extramitochondrial reduced nicotinamide adenosine dinucleotide (NADH) by corn mitochondria were studied. Potassium cyanide and antimycin A inhibited the oxidation of all three substrates. Rotenone and amytal inhibited only the oxidation of malate, and malonate inhibited only the oxidation of succinate. Rotenone, amytal, and malonate did not inhibit the oxidation of extramitochondrial NADH. The calcium stimulation of the oxidation of extramitochondrial NADH was prevented by KCN and antimycin A but not by amytal, rotenone, or malonate. It is suggested that corn mitochondria possess a flavoprotein specific for extramitochondrial NADH and that this flavoprotein is sensitive to divalent cations.

Effects of gramicidin on corn mitochondria.

The effects of gramicidin D, S, and J on corn mitochondria respiration and swelling were studied. Only gramicidin D was found to have any pronounced effect on mitochondrial swelling. In buffered KCl gramicidin D produced a rapid, respiration-independent swelling which was not reversed with respiratory inhibitors or substrate exhaustion. The respiration rate of exogenous reduced nicotinamide adenine dinucleotide was stimulated by all three gramicidins, but the effects on malate-pyruvate and succinate respiration depended on the type of gramicidin and the reaction media. The respiration effects of gramicidin D may be due to action at specific sites for each substrate.

Divalent cation stimulation of substrate oxidation by corn mitochondria.

The oxidation of reduced nicotinamide adenine dinucleotide, malate-pyruvate, and succinate by corn mitochondria in buffered 0.2 m KCl was determined as a function of divalent cations. Ni(2+), Mg(2+), Co(2+), Ca(2+), Mn(2+), Sr(2+), and Ba(2+) stimulated reduced nicotinamide adenine dinucleotide oxidation in the absence of inorganic phosphate, with Ca(2+) and Sr(2+) having the greatest effect. Malate-pyruvate and succinate oxidation was stimulated by Ca(2+), Ba(2+), and Sr(2+), but only in the presence of inorganic phosphate. Ca(2+), Sr(2+), and Ba(2+) produced a simulated state 4 to state 3 transition with all three substrates, but only with malate-pyruvate and succinate was there a return to state 4. The order of divalent cation effectiveness suggests that the rate of water substitution from the cation inner coordination hydration sphere may be a rate-limiting step in certain mitochondrial reactions involving electron transport and phosphorylation.

Lead effects on corn mitochondrial respiration.

Oxidation of exogenous nicotinamide-adenine dinucleotide and succinate by corn mitochondria was measured as a function of lead chloride concentration. Lead chloride (50 to 62 micromoles per liter) stimulated oxidation of exogenous reduced nicotinamide-adenine dinucleotide by 174 to 640 percent depending on the reaction mediums, whereas lead chloride (12.5 micromoles per liter) inhibited succinate oxidation by more than 80 percent. When inorganic phosphate was included in reaction mediums the subsequent addition of lead was without effect due to the low solubility of lead phosphate. If addition of lead was followed by addition of phosphate the inhibition of succinate oxidation by lead was released, but there was no reduction in the stimulation of oxidation of reduced nicotinamide-adenine dinucleotide by lead. The effects of lead on plant growth might be accentuated under conditions of phosphate deficiency.