Cotyledon Mitochondria Research Articles

Actin in Mung Bean Mitochondria and Implications for Its Function

Here, a large fraction of plant mitochondrial actin was found to be resistant to protease and high-salt treatments, suggesting it was protected by mitochondrial membranes. A portion of this actin became sensitive to protease or high-salt treatment after removal of the mitochondrial outer membrane, indicating that some actin is located inside the mitochondrial outer membrane. The import of an actin-green fluorescent protein (GFP) fusion protein into the mitochondria in a transgenic plant, actin:GFP, was visualized in living cells and demonstrated by flow cytometry and immunoblot analyses. Polymerized actin was found in mitochondria of actin:GFP plants and in mung bean (Vigna radiata). Notably, actin associated with mitochondria purified from early-developing cotyledons during seed germination was sensitive to high-salt and protease treatments. With cotyledon ageing, mitochondrial actin became more resistant to both treatments. The progressive import of actin into cotyledon mitochondria appeared to occur in concert with the conversion of quiescent mitochondria into active forms during seed germination. The binding of actin to mitochondrial DNA (mtDNA) was demonstrated by liquid chromatography-tandem mass spectrometry analysis. Porin and ADP/ATP carrier proteins were also found in mtDNA-protein complexes. Treatment with an actin depolymerization reagent reduced the mitochondrial membrane potential and triggered the release of cytochrome C. The potential function of mitochondrial actin and a possible actin import pathway are discussed.

Expression and kinetics of the mitochondrial alternative oxidase in nitrogen‐fixing nodules of soybean roots

ABSTRACTMitochondria from nitrogen‐fixing soybean nodules had previously been found to display very low rates of cyanide‐insensitive O2 consumption and to contain little protein immunoreactive with alternative oxidase (AOX) antibodies (A. Reams et al. 1992, Plant Physiology 99, pp. 712–717). Following the recent identification of novel regulatory mechanisms for AOX, we have re‐investigated AOX activity in nodule mitochondria. When AOX is fully activated by adding pyruvate and a reductant, significant AOX activity can be measured in nodule mitochondria when substrates that cause the ubiquinone (Q) pool to become > 90% reduced are provided. These nodule mitochondria contain ∼10% of the immunoreactive protein observed in soybean root or cotyledon mitochondria. Furthermore, transcripts from all three members of the soybean AOX multigene family were significantly less abundant in total RNA from nodules than from other tissues. The ratio of transcripts from the three AOX genes varied between the outer cortex and the inner infected region of the nodule. Measurements of Q redox state (Qr/Qt) versus O2 consumption showed that much higher levels of Q reduction were required for AOX to be active in mitochondria from nodules than in mitochondria from roots and cotyledons, and that root and nodule mitochondria were more dependent on added pyruvate for maximum AOX activity. Kinetic modelling suggests that these differences were simply the result of differences in the relative amount of active AOX protein, rather than differences in Q content or the AOX isoform expressed, in the different tissues.

Nitric oxide inhibits the cytochrome oxidase but not the alternative oxidase of plant mitochondria

Oxygen consumption via the cytochrome pathway in isolated soybean ( Glycine max [L.] Merr.) cotyledon mitochondria was inhibited by nitric oxide (NO) while respiration via the cyanide-insensitive alternative oxidase was not significantly affected. Inhibition of cytochrome pathway activity was rapidly reversible upon depletion of the added NO. NO production was also detected in solutions of NaNO 2 plus ascorbate and the extent of cytochrome pathway inhibition was dependent on the NO 2 − concentration. Little inhibition of alternative pathway respiration was observed under similar conditions. The alternative oxidase may play a role in nitric oxide tolerance in higher plants and in organisms such as trypanosomes which contain a plant-like alternative oxidase.

Regulation of Alternative Oxidase Activity by Pyruvate in Soybean Mitochondria.

The regulation of alternative oxidase activity by the effector pyruvate was investigated in soybean (Glycine max L.) mitochondria using developmental changes in roots and cotyledons to vary the respiratory capacity of the mitochondria. Rates of cyanide-insensitive oxygen uptake by soybean root mitochondria declined with seedling age. Immunologically detectable protein levels increased slightly with age, and mitochondria from younger, more active roots had less of the protein in the reduced form. Addition of pyruvate stimulated cyanide-insensitive respiration in root mitochondria, up to the same rate, regardless of seedling age. This stimulation was reversed rapidly upon removal of pyruvate, either by pelleting mitochondria (with succinate as substrate) or by adding lactate dehydrogenase with NADH as substrate. In mitochondria from cotyledons of the same seedlings, cyanide-insensitive NADH oxidation was less dependent on added pyruvate, partly due to intramitochondrial generation of pyruvate from endogenous substrates. Cyanide-insensitive oxygen uptake with succinate as substrate was greater than that with NADH, in both root and cotyledon mitochondria, but this difference became much less when an increase in external pH was used to inhibit intramitochondrial pyruvate production via malic enzyme. Malic enzyme activity in root mitochondria declined with seedling age. The results indicate that the activity of the alternative oxidase in soybean mitochondria is very dependent on the presence of pyruvate: differences in the generation of intramitochondrial pyruvate can explain differences in alternative oxidase activity between tissues and substrates, and some of the changes that occur during seedling development.

Pyruvate metabolism in mitochondria from cucumber cotyledons during early seedling development

both lightand dark-grown cucumber cotyledons at a variety of ages oxidize pyruvate only slowly (7-16nmol 02minting-1 protein; Hill et al., 1992). However, an increase in total mitochondrial protein in cucumber cotyledons leads to an increase in the capacity for pyruvate oxidation during the photosynthetic phase of development (Hill et al., 1992). In this paper we investigate the metabolism of pyruvate by cucumber cotyledon mitochondria. A detailed investi gation of the control of succinate oxidation by cucumber cotyledon mitochondria has established that the major controlling steps during lipid mobilization are dicarb oxylate uptake and the adenine nucleotide translocator (Hill et al., 1993). Since the rate of succinate oxidation is considerably greater than that of pyruvate (Hill et al., 1992), and the former is not limited by complexes III and IV (Hill et al., 1993), it seems unlikely that pyruvate oxidation is limited by these steps. We therefore investi gated the possibility that mitochondrial pyruvate oxida tion by isolated cotyledon mitochondria is limited by pyruvate uptake into the mitochondria. We did this in two ways: first, we compared the rates of oxidation of externally supplied pyruvate to that of pyruvate supplied internally through the action of NAD-malic enzyme (EC 1.1.1.39); second, we assayed pyruvate uptake

Studies on energy status and mitochondrial respiration during growth and senescence of mung bean cotyledons

Several characteristics of mitochondrial respiration and energy status have been studied during growth and senescence of mung bean (Phaseolus radiatus L.) cotyledons. The results showed that mitochondrial oxygen consumption, respiratory control, ADP:O ratios, and energy charge changed in the cotyledons during germination and growth of the seedlings. The respiration rate of intact cotyledons approximately reflected the trend of the oxidative activities of the isolated mitochondria. An increase was observed in both whole cotyledon respiration and mitochondrial oxygen uptake at the onset of senescence of mung bean cotyledons (day 3 after germination), which thereafter declined gradually. The capacity and activity of the alternative pathway increased markedly in mitochondria isolated from senescent cotyledons. After the onset of senescence, the mung bean cotyledon mitochondria exhibited a decrease both in the respiratory control ratios and ADP:O ratios, and the cotyledons exhibited a gradual decline in energy charge. All these results showed an irreversible deterioration of energy conservation in mung bean cotyledons. The role(s) of the alternative pathway in senescent mung bean cotyledons is discussed.

Phytochrome‐mediated control of respiratory activities of mitochondria in cotyledons of dark‐grown cucumber seedlings

Mitochondria isolated from cotyledons of dark‐grown cucumber (Cucumber sativus L., cv. Shimotsuki‐Aonaga) seedlings after illumination with continuous far‐red light showed an increased capacity for oxidation of malate or α‐ketoglutarate, as compared with those from cotyledons of non‐illuminated seedlings. This increase is supposed to be caused by phytochrome action (high irradiance response). Exogenous NAD+ had no effect on the rate of the oxidation of α‐ketoglutarate or malate by mitochondria isolated from far‐red light‐treated cotyledons, but it enhanced the oxidation rate of mitochondria from control cotyledons to the level of mitochondria from light‐treated ones. The NAD (NAD++ NADH) content was higher in mitochondria isolated from continuously far‐red light‐treated cotyledons than in mitochondria from controls. The NAD content was also increased by the treatment with a red light pulse and this response was reversed by a subsequent far‐red light pulse. It is proposed that phytochrome controls respiratory activities of cucumber mitochondria by changing the size of the NAD pool in the mitochondria.

Phytochrome-mediated control of respiratory activities of mitochondria in cotyledons of dark-grown cucumber seedlings

Phytochrome-mediated control of respiratory activities of mitochondria in cotyledons of dark-grown cucumber seedlings

Appearance of alternative respiration in cucumber cotyledon mitochondria after treatment with cycloheximide

Appearance of alternative respiration in cucumber cotyledon mitochondria after treatment with cycloheximide

Adenylate control of respiration in plants: the contribution of rotenone‐insensitive electron transport to ADP‐limited oxygen consumption by soybean mitochondria

The aim was to test the hypothesis that rotenone‐insensive electron transport (bypass of complex I) may underlie rapid state 4 (ADP‐limited) mitochondrial respiration. A comparison of mitochondria from soybean (Glycine max L. cv. Bragg) cotyledons and nodules showed that ADP‐sufficient (state 3) malate plus pyruvate oxidation by mitochondria from 7‐day‐old cotyledons was inhibited 50% by rotenone and state 4 rates were rapid, whereas nodule mitochondria were 80% inhibited by rotenone and had slower state 4 rates of malate plus pyruvate oxidation. Respiration of malate alone (pH 7.6) by cotyledon mitochondria was slow, especially in the absence of ADP; subsequent addition of pyruvate dramatically increased state 4 oxygen uptake concomitant with a rapid rise in mitochondrial NADH (determined by fluorimetry). Rotenone had no effect on this increased rate of state 4 respiration. The rate of malate oxidation by nodule mitochondria was relatively rapid compared with cotyledon mitochondria. The addition of pyruvate in state 4 caused a slow increase in matrix NADH and only a slight stimulation of oxygen uptake. Rotenone inhibited state 4 malate plus pyruvate oxidation by 50% in these mitochondria. From a large number of cotyledon and nodule mitochondrial preparations, a close correlation was found between the rate of state 4 oxygen uptake and rotenone‐resistance. During cotyledon development increased rotenone‐resistance was associated with an increase in the alternative oxidase. Addition of pyruvate to cotyledon mitochondria, during state 4 oxidation of malate in the presence of antimycin A, significantly stimulated O2 uptake and also almost eliminated respiratory control. Such combined operation of the rotenone‐insensitive bypass and the alternative oxidase in vivo will significantly affect the extent to which adenylates control the rate of electron transport.

Effect of the Embryonic Axis on the Development of Glyoxysomes, Plastids and Mitochondria in Cotyledons of Germinated Cucumber Seeds

The effect of the presence of the embryonic axis was examined on the following parameters of activities of cellular organelles in dark-grown cucumber cotyledons: activities of malate synthase, isocitrate lyase and 3-hydroxy-acyl-CoA dehydrogenase with glyoxysomes, ribulose bis-phosphate carboxylase activity and rate of chlorophyll formation with plastids, and activities of some respiratory enzymes (succinate and malate dehydrogenase and cytochrome oxidase) and respiratory activities (O 2 uptake and its cyanide-sensitivity, and respiratory control) with mitochondria. The development of these parameters depends upon the presence of the axis, and benzyladenine can bring about a full replacement of the action of the axis on the development of all the parameters except the two, respiratory control and cyanide-sensitivity of mitochondrial respiration. As to these two parameters, benzyladenine showed little effect on their development. On the basis of these results, the role of the axis in the development of the organelles in cotyledons is discussed.

Changes in Respiratory Processes in Soybean Cotyledons During Development and Senescence

Summary The respiration of cotyledons from light-grown soybean seedlings (Glycine max L., Amsoy) showed a sharp peak of activity after two weeks of growth, followed by a slighter increase during senescence. Cotyledon respiration was found to be strongly cyanide-resistant. Mitochondria isolated from cotyledons at various stages of development displayed a progressive improvement in structure. The rate of succinate oxidation closely paralleled that of respiration, being also highly cyanide-resistant. Cotyledon mitochondria also oxidized glycine. However this oxidation was remarkably cyanide- and antimycin-resistant.

Solubilization and Partial Purification of N,N′-Dicyclohexylcarbodiimide-Sensitive ATPase from Pea Cotyledon Mitochondria

The N,N'-dicyclohexylcarbodiimide (DCCD)-sensitive ATPase of pea (Pisum sativum L.) cotyledon mitochondria was solubilized from submitochondrial particle membranes with sodium cholate and ammonium sulfate. Ammonium sulfate precipitation of the enzyme resulted in an increase in specific activity. At between 38% and 45% saturated ammonium sulfate, 20% of the ATPase activity was precipitated, with a specific activity 4 to 5 times higher than that of the crude enzyme. The precipitate was highly sensitive to DCCD.The properties of the ammonium sulfate preparation were investigated. It contained levels of cytochrome and NADH dehydrogenase contamination comparable to those of the highly purified F(0)F(1) preparations from animal tissue. The high degree of purification was corroborated by sodium dodecyl sulfate electrophoresis.

Streptomycin Resistance in Tobacco: IV. Effects of the Drug on the Ultrastructure of Plastids and Mitochondria in Cotyledons of Germinating Seeds

Summary The effects of streptomycin on the fine structure of chloroplasts and mitochondria were studied in cotyledons of germinating seeds from St-S (streptomycin sensitive) tobacco seedlings. The drug caused a reduction in thylakoid formation in the chloroplasts; prevented the development of normal grana; drastically reduced the formation of starch grains and lipid droplets in the organelle; and caused changesin the external shape of the plastids. In the mitochondria it caused swelling of the cristae, somtimes elongation, and usually degeneration of the organelle. In the St-R701 (streptomycin resistant) mutant, the resistance was expressed mainly by the ability of the chloroplasts to form normal grana in the presence of the drug. The possible biochemical mode(s) of action of streptomycin are discused.

Biogenesis of Mitochondria in Imbibed Peanut Cotyledons: Influence of the Axis

Development of mitochondrial activities (state 3 respiration, respiratory control ratio, ADP/O ratio) in peanut cotyledons occurs over the first 5 d from the start of imbibition. Mitochondria in cotyledons with the axis attached develop better than in those from which the axis has been removed. Initially, mitochondria are deficient in cytochrome c, but after 2 d from the start of imbibition this deficiency is overcome. Mitochondrial development in attached cotyledons, as measured by state 3 respiration, respiratory control ratio, ADP/O ratio, and succinate dehydrogenase and cytochrome oxidase activities, is severely impaired by cycloheximide. This indicates that de novo synthesis of proteins is necessary for mitochondria and their enzymes to develop, a situation which is in sharp contrast to the situation in pea cotyledons. Electron microscope studies also show that there is an increase in the numbers of mitochondria in peanut cotyledons with time after the start of imbibition. Two patterns of mitochondrial development exist in legumes: in imbibed peanut cotyledons respiratory activities increase due to biogenesis of mitochondria, whereas in pea cotyledons the increases are due to improvement of pre-existing organelles.

ATPase Activity of Pea Cotyledon Submitochondrial Particles

Submitochondrial particles freshly prepared by sonication from pea cotyledon mitochondria showed low ATPase activity. Activity increased 20-fold on exposure to trypsin. The pea cotyledon submitochondrial particle ATPase was also activated by "aging" in vitro. At pH 7.0 addition of 1 millimolar ATP prevented the activation. ATPase of freshly prepared pea cotyledon submitochondrial particles had a substrate specificity similar to that of the soluble ATPase from pea cotyledon mitochondria, with GTPase > ATPase. "Aged" or trypsin-treated particles showed equal activity with the two substrates. NaCl and NaHCO(3), which stimulate the ATPase but not the GTPase activity of the soluble pea enzyme, were stimulatory to both the ATPase and GTPase activities of freshly prepared submitochondrial particles. However, they were stimulatory only to the ATPase activity of trypsin-treated or "aged" submitochondrial particles. In contrast, the ATPase activity of rat liver submitochondrial particles was stimulated by HCO(3) (-), but inhibited by Cl(-), indicating that Cl(-) stimulation is a distinguishing property of the pea mitochondrial ATPase complex.

Development of Mitochondrial Activity in Pea Cotyledons Following Imbibition; Influence of the Embryonic Axis

The development of mitochondria in cotyledons during the initial stages following imbibition and the subsequent degeneration were faster when the embryonic axis was attached. This was more clearly demonstrated when mitochondrial activity was determined using malate or ct-oxo glutarate as substrate rather than NADH or succinate. Cycloheximide did not inhibit the initial development of mitochondria in attached cotyledons, although it inhibited the incorporation of l4C-amino acid into protein. Abscisic acid inhibited almost completely the initial increase in mitochondrial activities of attached cotyledons. The activities of several enzymes in mitochondrial fractions were almost the same in attached and detached cotyledons during 4 d after imbibition. The degeneration of mitochondria was not accompanied by the loss of enzymes. It was inferred that the initial development and the subsequent degeneration of mitochondria in cotyledons during 4 d after imbibition was brought about by the structural improvement and disorganiz ation of mitochondria, respectively. The initial differences in the development of mitochondrial activities between attached and detached cotyledons were suggested to be attributable to differences in the development of the activities of electron transfer pathway from endogenous NADH to ubiquinone.

Changes in Mitochondrial Properties Associated with Chloroplast Development in Jack Bean (Canavalia ensiformis [L] DC.)

Procedures were developed to isolate actively respiring mitochondria from jack bean (Canavalia ensiformis [L.] DC.) leaves and cotyledons. Consistent respiratory control values of about 2 for succinate oxidation were obtained in preparations from etiolated cotyledons. Jack bean mitochondria oxidized malate with high respiratory control (2 to 5) and ADP/O (up to 2.8).Mitochondria purified from greening leaves and cotyledons exhibited rapid increases in respiratory control for up to 4 hours of light exposure followed by a decrease in activity. ADP/O values remained constant at about 1.2 while oxygen consumption per unit mitochondrial protein increased constantly during the first 14 hours of greening in preparations from cotyledons. The changes in mitochondrial respiratory activity during greening were correlated with the reversible appearance of mitochondria in the condensed conformation observed by electron microscopy. Both isolated mitochondrial pellets and cotyledon mitochondria in situ were shown to exhibit these changes in conformation during greening.

Relative activities of NAD- and NADP- isocritric dehydrogenases in bean mitochondria modified by glycerol or NADP.

Mitochondria from cotyledons of Vigna sesquipedalis (L.) Fruwirth (starchy seed) showed no NAD-isocitric dehydrogenase (NAD-IDH) activity by the methods which have been known to be useful for the detection of NAD-IDH in mitochondria of plants including castor bean and alaska pea. When the Vigna cotyledon mitochondria were treated with glycerol, NAD-IDH activity appeared and NADP-isocitric dehydrogenase (NADP-IDH) activity was inhibited. The inhibition of mitochondrial NADP-IDH by glycerol was overcome by the addition of excess NADP. On the other hand, NADP-IDH activity in the soluble fraction of cell components was only slightly inhibited by glycerol and no NAD-IDH activity was elicited. It was postulated that NADP-IDH in mitochondria is converted to NAD-IDH by glycerol and back to NADP-IDH with NADP by the alteration in the spatial configuration of the enzyme. However, there could be 2 proteins as the other possibility. The NADP-IDH in the soluble fraction which is not subject to such alteration is different from the mitochondrial NADP-IDH.