Stress CSP310 Protein Uncouples Oxidative Phosphorylation Otherwise than Other Plant Uncoupling Proteins Do

The addition of cold shock CSP310 protein to mitochondria isolated from both monocotyledonous (rye, wheat, and maize) and dicotyledonous (pea) plants uncoupled oxidation from phosphorylation. This uncoupling was caused neither by the damage to mitochondrial membranes nor by the activation of alternative cyanide-resistant oxidase. As distinct from the classical plant uncoupling mitochondrial protein (PUMP), CSP310 uncoupling effect was insensitive to BSA. Therefore, we believe that the mechanism of CSP310 action differs from that of known plant uncoupling proteins.     

Fatty Acid Interaction with Mitochondrial Uncoupling Proteins

The phenomena of fatty acid interaction with mitochondrial integral membrane proteins, namelyuncoupling proteins (UCPs), are reviewed to emphasize the fatty acid cycling mechanism thathas been suggested to explain the UCP function. Fatty acid-induced uncoupling is suggestedto serve in bioenergetic systems, to set the optimum efficiency, and to tune the degree ofcoupling of oxidative phosphorylation. Fatty acid interaction with the classic uncouplingprotein (UCP1) from mitochondria of thermogenic brown adipose tissue (BAT) is well known.UCP1 is considered to mediate purine nucleotide-sensitive uniport of monovalent unipolaranions, including anionic fatty acids. The return of protonated fatty acid leads to H⁺ uniportand uncoupling. Experiments supporting this mechanism are also reviewed for plant uncouplingmitochondrial protein (PUMP) and ADP/ATP carrier. The fatty acid cycling mechanism ispredicted, as well for the recently discovered uncoupling proteins, UCP2 and UCP3.     

Occurrence of Plant-Uncoupling Mitochondrial Protein (PUMP) in Diverse Organs and Tissues of Several Plants

The presence of plant-uncoupling mitochondrial protein (PUMP), previously described by Vercesi et al. (1995), was screened in mitochondria of various organs or tissues of several plant species. This was done functionally, by monitoring purine nucleotide-sensitive linoleic acid-induced uncoupling, or by Western blots. The following findings were established: (1) PUMP was found in most of the higher plants tested; (2) since ATP inhibition of linoleic acid-induced membrane potential decrease varied, PUMP content might differ in different plant tissues, as observed with mitochondria from maize roots, maize seeds, spinach leaves, wheat shoots, carrot roots, cauliflower, broccoli, maize shoots, turnip root, and potato calli. Western blots also indicated PUMP presence in oat shoots, carnation petals, onion bulbs, red beet root, green cabbage, and Sedum leaves. (3) PUMP was not detected in mushrooms. We conclude that PUMP is likely present in the mitochondria of organs and tissues of all higher plants.     

Mitochondrial Proton Leak and the Uncoupling Proteins

An energetically significant leak of protons occurs across the mitochondrial inner membranesof eukaryotic cells. This seemingly wasteful proton leak accounts for at least 20% of thestandard metabolic rate of a rat. There is evidence that it makes a similar contribution tostandard metabolic rate in a lizard. Proton conductance of the mitochondrial inner membranecan be considered as having two components: a basal component present in all mitochondria,and an augmentative component, which may occur in tissues of mammals and perhaps ofsome other animals. The uncoupling protein of brown adipose tissue, UCP1, is a clear exampleof such an augmentative component. The newly discovered UCP1 homologs, UCP2, UCP3,and brain mitochondrial carrier protein 1 (BMCP1) may participate in the augmentativecomponent of proton leak. However, they do not appear to catalyze the basal leak, as this isobserved in mitochondria from cells which apparently lack these proteins. Whereas UCP1plays an important role in thermogenesis, the evidence that UCP2 and UCP3 do likewiseremains equivocal.     

Plant Uncoupling Mitochondrial Protein Activity in Mitochondria Isolated from Tomatoes at Different Stages of Ripening

In the present study we have observed a higher state of coupling in respiring mitochondriaisolated from green as compared to red tomatoes (Lycopersicon esculentum, Mill.). Greentomato mitochondria produced a membrane potential () high enough to phosphorylate ADP,whereas in red tomato mitochondria, BSA and ATP were required to restore to the levelof that obtained with green tomato mitochondria. This supports the notion that such uncouplingin red tomato mitochondria is mediated by a plant uncoupling mitochondrial protein (PUMP;cf. Vercesi et al., 1995). Nevertheless, mitochondria from both green and red tomatoes exhibitedan ATP-sensitive linoleic acid (LA)-induced decrease providing evidence that PUMP isalso present in green tomatoes. Indeed, proteoliposomes containing reconstituted green or redtomato PUMP showed LA uniport and LA-induced H⁺ transport. It is suggested that the higherconcentration of free fatty acids (PUMP substrates) in red tomatoes could explain the lowercoupling state in mitochondria isolated from these fruits.     

Stimulation of potato tuber respiration by cold stress is associated with an increased capacity of both plant uncoupling mitochondrial protein (PUMP) and alternative oxidase

The CO₂ evolution of intact potato tubers (Solanum tuberosum, L., var. Bintje) was analyzed during a 10-day period of their warm (25 ± 2°C) or cold (5 ± 1°C) storage, to evaluate cold-stress effects on expression and activities of plant uncoupling mitochondrial protein (PUMP) and alternative oxidase (AOX). CO₂ evolution rates were analyzed at 20°C, to reflect their possible capacities. The 20°C CO₂ production declined from 13 to 8 mg kg^–1 h^–1 after 2 days of warm storage and then (after 3 to 7 days) decreased from 8 to 6.5 mg kg^–1 h^–1. In contrast, 20°C CO₂ evolution did not change after the first day of cold storage, increased up to 14.5 mg kg^–1 h^–1 after 2 days, and decreased to about 12 mg kg^–1 h^–1 after 3 to 7 days of cold storage. Cold storage increased PUMP expression as detected by Western blots and led to elevated capacities of both PUMP (44%) and CN-resistant AOX (10 times), but not the cytochrome pathway. Since we found that cold storage led to about the same mitochondrial respiration of 40 nmol O₂ min^–1 mg^–1 attributable to each of the respective proteins, we conclude that both AOX and PUMP equally contribute to adaptation of potato tubers to cold.     

Seed dormancy and control of germination in Sisymbrella dentata (L.) O.E. Schulz (Brassicaceae)

• Seed germination responsiveness to environmental cues is crucial for plant species living in changeable habitats and can vary among populations within the same species as a result of adaptation or modulation to local climates. Here, we investigate the germination response to environmental cues of Sisymbrella dentata (L.) O.E. Schulz, an annual endemic to Sicily living in Mediterranean Temporary Ponds (MTP), a vulnerable ecosystem.
• Germination of the only two known populations, Gurrida and Pantano, was assessed over a broad range of conditions to understand the role of temperatures, nitrate, hormones (abscisic acid – ABA and gibberellins – GA) and after‐ripening in dormancy release in this species.
• Seed germination responsiveness varied between the two populations, with seeds from Gurrida germinating under a narrower range of conditions. Overall, this process in S. dentata consisted of testa and endosperm rupture as two sequential events, influenced by ABA and GA biosynthesis. Nitrate addition caused an earlier testa rupture, after‐ripening broadened the thermal conditions that allow germination, and alternating temperatures significantly promoted germination of non‐after‐ripened seeds.
• Primary dormancy in S. dentata seeds likely allows this plant to form a persistent seed bank that is responsive to specific environmental cues characteristic of MTP habitats.

     

Acute and chronic cold exposure differentially affects the browning of porcine white adipose tissue

Piglets are characteristically cold intolerant and thus susceptible to high mortality. However, browning of white adipose tissue (WAT) can induce non-shivering thermogenesis as a potential strategy to facilitate the animal’s response to cold. Whether cold exposure can induce browning of subcutaneous WAT (sWAT) in piglets in a similar manner as it can in humans remains largely unknown. In this study, piglets were exposed to acute cold (4°C, 10 h) or chronic cold exposure (8°C, 15 days), and the genes and proteins of uncoupling protein 1 (UCP1)-dependent and independent thermogenesis, mitochondrial biogenesis, lipogenic and lipolytic processes were analysed. Interestingly, acute cold exposure induced browning of porcine sWAT, smaller adipocytes and the upregulated expression of UCP1, PGC1α, PGC1β, C/EBPβ, Cidea, UCP3, CKMT1 and PM20D1. Conversely, chronic cold exposure impaired the browning process, reduced mitochondrial numbers and the expression of browning markers, including UCP1, PGC1α and PRDM16. The present study demonstrated that acute cold exposure (but not chronic cold exposure) induces porcine sWAT browning. Thus, browning of porcine sWAT could be a novel strategy to balance the body temperature of piglets, and thus could be protective against cold exposure.     

Reconstitution of novel mitochondrial uncoupling proteins UCP2 and UCP3

Reconstitution of novel mitochondrial uncoupling proteins, human UCP2 and UCP3, expressed in yeast, was performed to characterize fatty acid (FA)-induced H⁺ efflux in the resulted proteoliposomes. We now demonstrate for the first time that representatives of physiologically abundant long chain FAs, saturated or unsaturated, activate H⁺ translocation in UCP2- and UCP3-proteoliposomes. Efficiency of lauric, palmitic or linoleic acid was roughly the same, but oleic acid induced faster H⁺ uniport. We have confirmed that ATP and GTP inhibit such FA-induced H⁺ uniport mediated by UCP2 and UCP3. Coenzyme Q₁₀ did not further significantly activate the observed H⁺ efflux. In conclusion, careful instant reconstitution yields intact functional recombinant proteins, UCP2 and UCP3, the activity of which is comparable with UCP1.     

A field study of seed germination in the endangered Trillium reliquum Freeman (Trilliaceae)

Previous studies examining the seeds of most Trillium species have reported double dormancy, a type of seed dormancy where two cold periods and one warm period are needed for complete germination. In the present paper, we describe a field study examining the federally endangered Trillium reliquum Freeman (Trilliaceae) in which moderate to high numbers of seeds germinated after one winter following seed production. Sixteen baskets with seeds were placed in four T. reliquum populations (four baskets in each population) in Georgia, USA, in June 2005. In spring 2006, all seed baskets contained seedlings. Germination percentages ranged from 33.3 to 83.3% across sites with a mean of 56.9 ± 3.9%. Trillium reliquum had higher germination percentages compared with other field‐based germination studies with other Trillium species. Our findings will inform future demographic studies of T. reliquum and suggest that double dormancy in seeds may not be as widespread as previously reported within the genus Trillium.     

茶树叶片类脱水素蛋白的Western-blot分析

为了解茶树脱水素种类与功能,采用Western-blot技术,研究了不同季节及越冬过程中茶树叶片脱水素蛋白家族的表达模式。结果显示:(1)茶树叶片总蛋白提取采用酚-甲醇/醋酸铵沉淀法,用时短、蛋白浓度高、SDSPAGE电泳条带清晰,背景干净,满足茶树Western-blot技术要求。(2)在不同季节及越冬期中发现14~95kD共9种不同分子量的茶树类脱水素蛋白,其中95、65、48、37、34和14kD等6种蛋白表达量较为稳定,季节与越冬期变化不明显;58kD脱水素仅在冬季表达,越冬期不断上升,2月份增加到最高,表达丰度高;28kD脱水素蛋白在冬季表达量高,越冬期与茶树抗寒力变化规律一致;21kD脱水素在夏季和越冬期后期有较高的表达。研究表明,这3种脱水素可能在茶树抗逆中起着重要作用。     

活性氮、活性氧及植物激素在种子休眠解除中的作用及相互关系研究进展

休眠是植物种子对环境变化的适应机制,其机理至今未完全清楚阐明。前期对种子休眠机制的研究主要集中在激素调节上,近期的研究结果表明,一氧化氮（nitric oxide,NO）参与打破种子的休眠,并与其所引起的种子中活性氧的变化有关。本文简要综述活性氮（reactive nitrogen species,RNS）、活性氧（reactive oxygen species,R0s）和植物激素在种子休眠解除中的作用及相互关系研究进展。     

Characterization of the structure and DNA complexity of mung bean mitochondrial nucleoids

Electron microscopic images of mitochondrial nucleoids isolated from mung bean seedlings revealed a relatively homogeneous population of particles, each consisting of a chromatin-like structure associated with a membrane component. Association of F-actin with mitochondrial nucleoids was also observed. The mitochondrial nucleoid structure identified in situ showed heterogeneous genomic organization. After pulsed-field gel electrophoresis (PFGE), a large proportion of the mitochondrial nucleoid DNA remained in the well, whereas the rest migrated as a 50–200 kb smear zone. This PFGE migration pattern was not affected by high salt, topoisomerase I or latrunculin B treatments; however, the mobility of a fraction of the fastmoving DNA decreased conspicuously following an in-gel ethidium-enhanced UV-irradiation treatment, suggesting that molecules with intricately compact structures were present in the 50-200 kb region. Approximately 70% of the mitochondrial nucleoid DNA molecules examined via electron microscopy were open circles, supercoils, complex forms, and linear molecules with interspersed sigma-shaped structures and/or loops. Increased sensitivity of mtDNA to DNase I was found after mitochondrial nucleoids were pretreated with high salt. This result indicates that some loosely bound or peripheral DNA binding proteins protected the mtDNA from DNase I degradation.     

Cytotoxicity and genotoxicity of xenoclauxin and desacetyl duclauxin fromPenicillium Duclauxii (delacroix)

The duclauxin derivatives xenoclauxin and desacetylduclauxin were examined for their effects on the growth of L-1210 murine leukemia cells, on the induction of DNA repair in the rat and mouse hepatocyte primary culture (HPC/DNA repair test), and on oxidative phosphorylation in mitochondria from rat livers in comparison to duclauxin. Both derivatives inhibited the growth of L-1210 culture cells as strongly as duclauxin. Duclauxin derivatives were negative in the HPC/DNA repair test. Xenoclauxin exhibited a potent uncoupling effect accompanying a marked depression of state 3 respiration of mitochondria in a similar fashion to that of duclauxin. Desacetylduclauxin significantly inhibited the state 3 respiration without causing uncoupling of oxidative phosphorylation in mitochondria. These results strongly suggest that xenoclauxin and desacetylduclauxin fromPenicillium duclauxii are not genotoxic but are cytotoxic mainly due to their potent inhibition of ATP synthesis in mitochondria.Abbreviations DNP 2,4-dinitrophenol - ETP electron transport particles - HPC hepatocyte primary culture cells - RC respiratory control - TdR thymine deoxyribonucleotide - UDS unscheduled DNA synthesis     

Assembly-dependent translation of subunits 6 (Atp6) and 9 (Atp9) of ATP synthase in yeast mitochondria

The yeast mitochondrial ATP synthase is an assembly of 28 subunits of 17 types of which 3 (subunits 6, 8, and 9) are encoded by mitochondrial genes, while the 14 others have a nuclear genetic origin. Within the membrane domain (F_O) of this enzyme, the subunit 6 and a ring of 10 identical subunits 9 transport protons across the mitochondrial inner membrane coupled to ATP synthesis in the extra-membrane structure (F₁) of ATP synthase. As a result of their dual genetic origin, the ATP synthase subunits are synthesized in the cytosol and inside the mitochondrion. How they are produced in the proper stoichiometry from two different cellular compartments is still poorly understood. The experiments herein reported show that the rate of translation of the subunits 9 and 6 is enhanced in strains with mutations leading to specific defects in the assembly of these proteins. These translation modifications involve assembly intermediates interacting with subunits 6 and 9 within the final enzyme and cis-regulatory sequences that control gene expression in the organelle. In addition to enabling a balanced output of the ATP synthase subunits, these assembly-dependent feedback loops are presumably important to limit the accumulation of harmful assembly intermediates that have the potential to dissipate the mitochondrial membrane electrical potential and the main source of chemical energy of the cell.     

Uncoupling Protein 1 and Sarcolipin Are Required to Maintain Optimal Thermogenesis,and Loss of Both Systems Compromises Survival of Mice under Cold Stress

The importance of brown adipose tissue as a site of nonshivering thermogenesis has been well documented. Emerging studies suggest that skeletal muscle is also an important site of thermogenesis especially when brown adipose tissue function is lacking. We recently showed that sarcolipin (SLN), an uncoupler of the sarco(endo)plasmic reticulum Ca²⁺ ATPase (SERCA) pump, could contribute to heat production in skeletal muscle. In this study, we sought to understand how loss of UCP1 or SLN is compensated during cold exposure and whether they are both necessary for thermogenesis. Toward this goal, we generated a UCP1;SLN double knock-out (DKO) mouse model and challenged the single and DKO mice to acute and long-term cold exposures. Results from this study show that there is up-regulation of SLN expression in UCP1-KO mice, and loss of SLN is compensated by increased expression of UCP1 and browning of white adipose tissue. We found that the DKO mice were viable when reared at thermoneutrality. When challenged to acute cold, the DKO were extremely cold-sensitive and became hypothermic. Paradoxically, the DKO mice were able to survive gradual cold challenge, but these mice lost significant weight and depleted their fat stores, despite having higher caloric intake. These studies suggest that UCP1 and SLN are required to maintain optimal thermogenesis and that loss of both systems compromises survival of mice under cold stress.     

Uncoupling protein mRNA, mitochondrial GTP-binding, and T4 5′-deiodinase activity of brown adipose tissue in Daurian ground squirrel during hibernation and arousal

The mRNA level of uncoupling protein (UCP) specific for brown adipose tissue (BAT) in Daurian ground squirrel, was detected by using a [32P]-labeled oligonucleotide probe. The UCP concentration in mitochondria was indirectly determined by titration with its specific ligand [H3]-labeled GTP. Type II T4 5'-deiodinase of BAT was assayed concomitantly. We found two species of mRNA for UCP with lengths of about 1.9 and 1.5 kb, respectively, both occurring in almost the same concentration. UCP mRNA content was elevated significantly during hibernation, but the UCP concentration did not change compared with that of nonhibernating controls kept at room temperature. When hibernating squirrels were aroused, the UCP mRNA remained at the elevated level as during hibernation, but the UCP concentration increased in comparison with that of nonhibernating controls or during hibernating. Changes in T4 5'-deiodinase activity in BAT were similar to the variations of the UCP mRNA level. These results suggest that the activation of T4 5'-deiodinase in BAT may be an important factor for the up-regulation and maintenance of UCP mRNA content needed for the synthesis of sufficient UCP to acquire the thermogenic capacity for arousal from hibernation.