Effect of NAD on Malate Oxidation in Intact Plant Mitochondria

Potato tuber mitochondria oxidizing malate respond to NAD⁺ addition with increased oxidation rates, whereas mung bean hypocotyl mitochondria do not. This is traced to a low endogenous content of NAD⁺ in potato mitochondria, which prove to take up added NAD⁺. This mechanism concentrates NAD⁺ in the matrix space. Analyses for oxaloacetate and pyruvate (with pyruvate dehydrogenase blocked) are consistent with regulation of malate oxidation by the internal NAD⁺/NADH ratio.     

Platanetin: A Potent Natural Uncoupler and Inhibitor of the Exogenous NADH Dehydrogenase in Intact Plant Mitochondria

Platanetin is a 3,5,7,8-tetrahydroxy, 6-isoprenyl flavone isolated from the bud scales of the plane tree (Platanus acerifolia Willd.). Its effects on the oxidative activities of isolated potato and mung bean mitochondria have been studied. The most noticeable effect is the selective inhibitory effect of this compound on the activity of the external NADH dehydrogenase of the inner membrane. A 50% inhibition of the NADH oxidation rate is obtained at a 2 micromolar concentration. This activity is probably due to the flavonoid structure and the high lipophilicity of platanetin associated with the presence of the isoprenyl chain. Another important effect of platanetin is its uncoupling activity on oxidative phosphorylation. The presence of easily dissociable hydroxyl groups and the high lipophilicity of platanetin allow a potent H⁺ transfer through the mitochondrial inner membrane. This uncoupling activity is comparable to that of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Platanetin is therefore the most active natural uncoupler known at the present time (full uncoupling at 2 micromolar with succinate as substrate). At higher concentrations (10 micromolar and more), platanetin can transfer electrons from the mitochondrial inner membrane to O₂; the branching point of this KCN-salicylhydroxamic acid insensitive platanetin dependent oxidative pathway is located at the level of flavoproteins, no transfer occurring when succinate is the substrate. The redox properties of platanetin are in accord with such an activity.     

The Pre-Endosymbiont Hypothesis: A New Perspective on the Origin and Evolution of Mitochondria

Mitochondrial DNA (mtDNA) is unquestionably the remnant of an α-proteobacterial genome, yet only ∼10%–20% of mitochondrial proteins are demonstrably α-proteobacterial in origin (the “α-proteobacterial component,” or APC). The evolutionary ancestry of the non-α-proteobacterial component (NPC) is obscure and not adequately accounted for in current models of mitochondrial origin. I propose that in the host cell that accommodated an α-proteobacterial endosymbiont, much of the NPC was already present, in the form of a membrane-bound metabolic organelle (the premitochondrion) that compartmentalized many of the non-energy-generating functions of the contemporary mitochondrion. I suggest that this organelle also possessed a protein import system and various ion and small-molecule transporters. In such a scenario, an α-proteobacterial endosymbiont could have been converted relatively directly and rapidly into an energy-generating organelle that incorporated the extant metabolic functions of the premitochondrion. This model (the “pre-endosymbiont hypothesis”) effectively represents a synthesis of previous, contending mitochondrial origin hypotheses, with the bulk of the mitochondrial proteome (much of the NPC) having an endogenous origin and the minority component (the APC) having a xenogenous origin.Considering the central role played in all eukaryotic cells by mitochondria or mitochondrion-related organelles (MROs, such as hydrogenosomes and mitosomes) (Hjort et al. 2010; Shiflett and Johnson 2010; Müller et al. 2012), the question of the origin and subsequent evolution of the mitochondrion has long captivated and challenged biologists. In a recent article in this series (Gray 2012), I discussed in detail several aspects of mitochondrial evolution, focusing particularly on how well the accumulating molecular data can be accommodated in current models of mitochondrial origin. In this context, the origin and evolution of the mitochondrial proteome, as opposed to the origin and evolution of the mitochondrial genome, were examined from the perspective of comparative mitochondrial proteomics. Somewhat disconcertingly, as more data have become available, we find ourselves considerably less certain about key aspects of how mitochondria originated than we were (or thought we were) several decades ago.Here, I summarize key points discussed in more detail in the previous article before presenting a novel perspective on how the mitochondrion might have originated. The new model proposed here, which represents a synthesis of both endogenous (“origin from within”) and xenogenous (“origin from outside”) modes, is advanced in an attempt to account for the inability of a purely endosymbiotic model, whose strongest support has come from studies of the mitochondrial genome, to adequately accommodate data on the mitochondrial proteome.     

Mitochondria catalyze the reduction of NAD by reduced methylviologen

Mitochondria from beef heart and yeast catalyze the reduction of NAD to NADH at the expense of reduced methylviologen (MV+). Based on protein the specific activity of mitochondria for this reaction is about 10 20-times higher than the consumption of oxygen in the presence of succinate or NADH. In 2H2O buffer (4S)-[4-2H]NADH is formed in high enantiomeric excess if the reduced methylviologen is electrochemically regenerated.     

The Effect of Exogenous Nicotinamide Adenine Dinucleotide on the Oxidation of Nicotinamide Adenine Dinucleotide-linked Substrates by Isolated Plant Mitochondria

The oxidation of malate, citrate, and α-ketoglutarate by cauliflower (Brassica oleacea L.) bud mitochondria was inhibited by rotenone. This inhibition was relieved upon addition of NAD⁺ to the medium, and ADP/O values were lowered to less than 2 when both rotenone and NAD⁺ were present. Dinitrophenol did not affect the relief of rotenone inhibition by exogenous NAD⁺.     

A Translocation Hypothesis based on the Structure of Plant Cytoplasm

Two types of linear structures have been seen in plant cytoplasm,largely by phase-contrast microscopy. Microscopic fine threads,o.Iµ to Iµ in diameter, were revealed in hair cells,where they formed endoplasmic systems along streaming pathwaysin the parietal cytoplasm and in transvacuolar strands. Duringcirculation streaming, small plastids and mitochondria-likeparticles were observed moving along the fine threads. Similarfine threads, together with small plastids and mitochondria-likeparticles occurred in phloem exudate and transcellular microscopicstrands. The transcellular strands, Iµ to 7µ indiameter, were seen in sieve-tube elements, phloem parenchyma,border parenchyma, and cortical cells. The movement of small plastids across end walls in border-parenchymacells, the appearance of the same structures within strandsin phloem cells, and the undiminished occurrence of small plastidsin successive drops of phloem exudate are collectively takenas evidence for their participation in translocation. Particlemovement is thought to occur through transcellular strands inassociation with fine threads, and to be motivated by a transcellularform of protoplasmic streaming.     

Effects of Polyamines on the Oxidation of Exogenous NADH by Jerusalem Artichoke (Helianthus tuberosus) Mitochondria

The effect of polyamines (putrescine, spermine, and spermidine) on the oxidation of exogenous NADH by Jerusalem artichoke (Helianthus tuberosus L. cv. OB1) mitochondria, have been studied. Addition of spermine and/or spermidine to a suspension of mitochondria in a low-cation medium (2 millimolar-K⁺) caused a decrease in the apparent K_m and an increase in the apparent V_max for the oxidation of exogenous NADH. These polycations released by screening effect the mitochondrially induced quenching of 9-aminoacridine fluorescence, their efficiency being dependent on the valency of the cation (C⁴⁺ > C³⁺). Conversely, putrescine only slightly affected both kinetic parameters of exogenous NADH oxidation and the number of fixed charges on the membranes. Spermine and spermidine, but not putrescine, decreased the apparent K_m for Ca²⁺ from about 1 to about 0.2 micromolar, required to activate external NADH oxidation in a high-cation medium, containing physiological concentrations of Pi, Mg²⁺ and K⁺. The results are interpreted as evidence for a role of spermine and spermidine in the modulation of exogenous NADH oxidation by plant mitochondria in vivo.     

Modulation of the Access of Exogenous NAD(P)H to the Alternative Pathway in Potato Tuber Callus Mitochondria with Triton X-100

Alternative oxidase activity in potato tuber (Solanum tuberosum L. cv Bintje) callus mitochondria with exogenous NAD(P)H as substrate is inhibited by low concentrations of the detergent Triton X-100. Alternative oxidase activity with succinate or malate as substrate is not affected by these low concentrations of Triton X-100. Cytochrome pathway activity was not influenced under these conditions, neither with endogenous nor with exogenous substrate. Washing of Triton X-100-treated mitochondria did partially restore both uninhibited and CN-resistant NADH oxidation, indicating that under these conditions Triton X-100 does not permanently remove major components from the mitochondrial membrane. Apparently, it is possible to manipulate mitochondria in such a way that the access of exogenous NADH to the alternative pathway is blocked while access to the cytochrome pathway is uninhibited. It is suggested that membrane conditions have a regulatory function (possibly via influencing the diffusion path) in the oxidation of exogenous NADH via the alternative pathway.     

Ribonucleic Acid Synthesis in Plant Mitochondria: Effects of Some Inhibitors and Cyclic Nucleotides

Mitochondria isolated from 48-h germinating Vigna sinensis (L.) Savi can incorporate [³H]uridine into acid-insoluble material. The incorporation is highly sensitive to rifampicin and partially so to ethidium bromide, two specific inhibitors of template function. The inhibitory effect of rifampicin can be partly counteracted by cyclic 3′:5′-AMP but not by cyclic 3′:5′-GMP, if they are allowed to interact with the synthetic system before the treatment with rifampicin. This indicates that cyclic AMP and rifampicin compete for a common site on the RNA polymerase responsible for DNA-dependent RNA synthesis. Inhibition by ethidium bromide is unaffected by prior nucleotide interaction with the system.     

Effects of Calcium on NAD(H)-Glutamate Dehydrogenase from Turnip (Brassica rapa L.) Mitochondria

The effect of Ca²⁺ and ammonia on mitochondrial NADH-glutamatedehydrogenase (GDH: EC 1.4.1.2 [EC] ) isolated from turnip root (Brassicarapa L.) activity was examined. Increasing the ammonia [(NH₄)₂SO₄]concentration led to significant substrate inhibition whichcould be reversed by micromolar levels of Ca²⁺. The sensitivityof the enzyme to ammonia inhibition and its reversal by Ca²⁺was affected by proteolysis. After treatment with various proteases,lower concentrations of Ca²⁺ were capable of fully activatingthe enzyme or overcoming the inhibitory effects of high ammonium,compared to non-treated enzyme. However, the protease-treatedenzyme was still sensitive to ethylene glycol-bis(ß-aminoethylether) N,N,N',N'-tetraacetate (EGTA). In contrast, NADH-GDHactivity was inhibited approx. 30% by organic mercurials (200µm), but the residual activity was not affected by thesubsequent additions of EGTA. NADH-GDH activity could also bestimulated by additions of high concentrations of NaCl (300mM) in the absence of added Ca²⁺. These results suggest thathydrophobic and -SH groups may be involved in the regulationof mitochondrial NADH-GDH activity by Ca²⁺. ² Present address: CSIRO Division of Horticulture, Urrbrae,S.A. 5064, Australia (Received April 18, 1990; Accepted July 23, 1990)     

Plant Parenthood in Milkweeds: A Direct Test of the Pollen Donation Hypothesis

Abstract For hermaphroditic plant species whose fruit production is limited by maternal resources, the "pollen donation hypothesis" views large floral displays as an adaptation to enhance the probability of fathering seeds on other plants. This hypothesis has frequently been used to describe the evolution of large floral displays in milkweeds ( Asclepias ). Most tests of the pollen donation hypothesis, however, have used indirect measures, such as flower production or pollen removal, to estimate male reproductive success. To test the pollen donation hypothesis directly, we performed a paternity analysis and determined the number of seeds sired by individual genotypes in a natural population of poke milkweed, A. exaltata , in southwestern Virginia. Seeds sired (male success) and seeds produced (female success) were significantly correlated with flower number per plant (for male success: r = 0.32, P < 0.05; for female success: r = 0.66, P < 0.0001). Functional gender of plants that reproduced both as males and females (N = 17) was not correlated with flower number per plant ( r = 0.35, P>0.05), indicating that plants with large floral displays did not reproduce primarily as males. Percent fruit-set and seed number per fruit were higher in 1986, when levels of pollinarium removal also were higher. Furthermore, several umbels that experienced high pollinator activity selectively matured fruits that contained many seeds. We argue that the evolution of large floral displays in milkweeds is the result of selection to increase overall reproductive success rather than male reproductive success alone.     

The Microelectrophoretic Behaviour of Plant Mitochondria compared with Rat Mitochondria

‘Heavy’ mitochondrial preparations of bean, cauliflower,and rat liver have been found to give unimodal distributionfor electrophoretic mobility against number of particles. ThepH-mean mobility curves were similar in form and consistentwith the mitochondrial surfaces being lipoproteins. de Duve (1959) separated ‘heavy’ and ‘light’(lysosome-rich) mitochondrial fractions from rat liver. Microelectrophoreticstudies on similar ‘heavy’ and ‘light’mitochondrial preparations from rat liver have shown the latterto consist mostly of mitochondria with some faster-moving particlestentatively identified with de Duve's lysosomes. ‘Light’mitochondrial preparations of bean showed no evidence of particlesadditional to mitochondria.     

Properties of Higher Plant Mitochondria. III. Effects of Respiratory Inhibitors

The effects of representative respiratory inhibitors were investigated on the coupled respiration of mung bean mitochondria using succinate and l-malate as substrates. The inhibitors studied were: (I) malonate, (II) amytal and rotenone, (III) antimycin A and 2-n-nonyl-4-hydroxyquinoline N-oxide (NOQNO), and (IV) cyanide and azide.     

Biosynthesis of Cardiolipin in Plant Mitochondria

The properties of cardiolipin synthase were investigated in mitochondria and submitochondrial fractions from etiolated mung bean (Vigna radiata L.) seedlings. Direct evidence is presented that the enzyme utilizes CDP-diacylglycerol in addition to phosphatidylglycerol for the synthesis of cardiolipin. Cardiolipin synthase had an alkaline pH optimum of about 9 and required divalent cations for activity. Maximal activity was obtained in the presence of 16 mM MnCl2. The apparent Km values for CDP-diacylglycerol and phosphatidylglycerol were 0.8 and 50 [mu]M, respectively. Cardiolipin synthase was localized predominantly in the inner membrane of mung bean mitochondria and displayed a substrate species specificity. Highest activities were measured with the dioleoyl species of both CDP-diacylglycerol and phosphatidylglycerol, and somewhat lower activities were measured with mixed species of the two substrates containing a palmitoyl and an oleoyl group. On the other hand, the cardiolipin synthase hardly used the dipalmitoyl species and strongly discriminated against CDP-dipalmitoylglycerol from a mixture with CDP-dioleoylglycerol.     

NADP-Utilizing Enzymes in the Matrix of Plant Mitochondria

Purified potato tuber (Solanum tuberosum L. cv Bintie) mitochondria contain soluble, highly latent NAD⁺- and NADP⁺-isocitrate dehydrogenases, NAD⁺- and NADP⁺-malate dehydrogenases, as well as an NADPH-specific glutathione reductase (160, 25, 7200, 160, and 16 nanomoles NAD(P)H per minute and milligram protein, respectively). The two isocitrate dehydrogenase activities, but not the two malate dehydrogenase activities, could be separated by ammonium sulfate precipitation. Thus, the NADP⁺-isocitrate dehydrogenase activity is due to a separate matrix enzyme, whereas the NADP⁺-malate dehydrogenase activity is probably due to unspecificity of the NAD⁺-malate dehydrogenase. NADP⁺-specific isocitrate dehydrogenase had much lower K_ms for NADP⁺ and isocitrate (5.1 and 10.7 micromolar, respectively) than the NAD⁺-specific enzyme (101 micromolar for NAD⁺ and 184 micromolar for isocitrate). A broad activity optimum at pH 7.4 to 9.0 was found for the NADP⁺-specific isocitrate dehydrogenase whereas the NAD⁺-specific enzyme had a sharp optimum at pH 7.8. Externally added NADP⁺ stimulated both isocitrate and malate oxidation by intact mitochondria under conditions where external NADPH oxidation was inhibited. This shows that (a) NADP⁺ is taken up by the mitochondria across the inner membrane and into the matrix, and (b) NADP⁺-reducing activities of malate dehydrogenase and the NADP⁺-specific isocitrate dehydrogenase in the matrix can contribute to electron transport in intact plant mitochondria. The physiological relevance of mitochondrial NADP(H) and soluble NADP(H)-consuming enzymes is discussed in relation to other known mitochondrial NADP(H)-utilizing enzymes.     

Effects of Landscape Structure on Medicinal Plant Richness in Home Gardens: Evidence for the Environmental Scarcity Compensation Hypothesis

Our research involves of how Paraguayan migrants who are living in Misiones, Argentina, manage medicinal plants in home gardens, and how this practice can be related to the landscape. We examine the relationship between the richness of home garden medicinal plants and landscape variables (e.g., distance to the forest) by applying PLS analysis, which combines principal component analysis with linear regression. We surveyed 60 home gardens localized in a rural area, and we characterized the surrounding landscape with geospatial tools. Paraguayans’ home gardens are extremely diverse sites (total of 136 medicinal species), where both native (82) and introduced species (50) are managed. People who live close to the native forest or mixed use areas (e.g., farms, secondary vegetation) tend to possess less native plants in their gardens because they are available nearby. While gardeners, who live in proximity to tree crops (e.g., pine plantations), have reduced access to wild medicinal resources; therefore, their effort is concentrated on maintaining native plants. These results reflect a relationship between accessibility to medicinal plants in the landscape and the management practices in the home gardens, a neglected driver in explaining the richness and composition of the medicinal plants in home gardens so far. Thus, we contributed evidence in support of the environmental scarcity compensation hypothesis. Finally, our study supports the idea that home gardens appear to function as a springboard for plant domestication.     

Properties of Higher Plant Mitochondria. II. Effects of DNP, m-Cl-CCP, and Oligomycin on Respiration of Mung Bean Mitochondria

Effects of inhibitors of phosphorylation on the oxidation of succinate and of l-malate were investigated with tightly coupled mitochondria isolated from mung bean hypocotyls. When mitochondria were incubated with 2,4-dinitrophenol, or carbonyl cyanide m-chlorophenylhydrazone prior to the addition of substrate, the uncoupling effects of these chemicals were relatively small. This is probably caused by relative lack in these mitochondria of endogenous substrates, ATP, and/or “high-energy intermediates”. The action of uncoupling agents is, therefore, revealed in a more striking manner when they are introduced during the second state 4. Of the 2 uncoupling agents tested, malate oxidation consistently required 1.5 to 2 times higher concentration of the agents for the half-maximal effects than succinate oxidation. From the comparison of the degree of uncoupling it is concluded that 2,4-dinitrophenol is a better uncoupler of succinate oxidation, whereas carbonyl cyanide m-chlorophenylhydrazone functions as a more complete uncoupler of malate oxidation.

Oligomycin does not inhibit state 4 rates, while the increment of respiration due to added ADP is completely inhibited by this antibiotic. Identical half-maximal effects are observed with the same concentration of oligomycin in both succinate and l-malate oxidation. The oligomycin effect depends on the mitochondrial concentration employed. The concentration of this chemical required for the half-maximal effect is 55 to 80 mμmoles per mg mitochondrial protein. It is suggested that this inhibitor of phosphorylation binds all of the phosphorylation sites regardless of whether the sites are functional or not.

     

Testing the Stress-Gradient Hypothesis at the Roof of the World: Effects of the Cushion Plant Thylacospermum caespitosum on Species Assemblages

Many cushion plants ameliorate the harsh environment they inhabit in alpine ecosystems and act as nurse plants, with significantly more species growing within their canopy than outside. These facilitative interactions seem to increase with the abiotic stress, thus supporting the stress-gradient hypothesis. We tested this prediction by exploring the association pattern of vascular plants with the dominant cushion plant Thylacospermum caespitosum (Caryophyllaceae) in the arid Trans-Himalaya, where vascular plants occur at one of the highest worldwide elevational limits. We compared plant composition between 1112 pair-plots placed both inside cushions and in surrounding open areas, in communities from cold steppes to subnival zones along two elevational gradients (East Karakoram: 4850–5250 m and Little Tibet: 5350–5850 m). We used PERMANOVA to assess differences in species composition, Friedman-based permutation tests to determine individual species habitat preferences, species-area curves to assess whether interactions are size-dependent and competitive intensity and importance indices to evaluate plant-plant interactions. No indications for net facilitation were found along the elevation gradients. The open areas were not only richer in species, but not a single species preferred to grow exclusively inside cushions, while 39–60% of 56 species detected had a significant preference for the habitat outside cushions. Across the entire elevation range of T. caespitosum, the number and abundance of species were greater outside cushions, suggesting that competitive rather than facilitative interactions prevail. This was supported by lower soil nutrient contents inside cushions, indicating a resource preemption, and little thermal amelioration at the extreme end of the elevational gradient. We attribute the negative associations to competition for limited resources, a strong environmental filter in arid high-mountain environment selecting the stress-tolerant species that do not rely on help from other plants during their life cycle and to the fact the cushions do not provide a better microhabitat to grow in.     

植物种子衰老与线粒体关系的研究进展

种子的衰老是一个复杂的从量变到质变的生物学过程。种子衰老与线粒体功能异常密切相关,衰老的线粒体学说认为,线粒体中活性氧的过量产生是种子衰老的主要原因。深入了解种子衰老过程中线粒体的变化对于揭示种子衰老机理和种子安全保存具有重要意义。本文主要介绍了当前有关种子衰老过程中线粒体结构、呼吸作用和抗氧化系统的研究现状,并对种子衰老与线粒体关系研究中存在的问题进行了讨论。