What is critical for plant thermogenesis? Differences in mitochondrial activity and protein expression between thermogenic and non-thermogenic skunk cabbages

Thermogenesis during the blooming of inflorescence is found in several but not all aroids. To understand what is critical for thermogenesis, we investigated the difference between thermogenic and non-thermogenic skunk cabbages (Symplocarpus renifolius and Lysichiton camtschatcensis), which are closely related in morphology and phylogeny. Critical parameters of mitochondrial biogenesis, including density, respiratory activity, and protein expression were compared between these two species. Mitochondrial density, respiratory activity, and the amount of alternative oxidase (AOX) in L. camtschatcensis spadix mitochondria were lower than in S. renifolius spadix mitochondria, while the level of uncoupling protein (UCP) was higher. AOX and UCP mRNAs in L. camtschatcensis were constitutively expressed in various tissues, such as the spadix, the spathe, the stalk, and the leaves. cDNA encoding two putative thermogenic proteins, AOX and UCP were isolated from L. camtschatcensis, and their primary structure was analyzed by multiple alignment and phylogenetic tree reconstruction. AOX and UCP protein of two the skunk cabbage species are closely related in structure, compared with other isoforms in thermogenic plants. Our results suggest that mitochondrial density, respiratory activity, and protein expression, rather than the primary structure of AOX or UCP proteins, may play critical roles in thermogenesis in plants.     

Jack of one trade,master of none: host choice by Drosophila magnaquinaria

Summary Host selection by phytophagous insects is generally thought to be based on chemical or nutritional characteristics of the host. This is especially true for monophagous insects. However, many other factors may influence host choice. The present study examines host selection by Drosophila magnaquinaria, whose sole host is the yellow skunk cabbage, Lysichitum americanum. Utilization of skunk cabbage was tested relative to a set of alternative hosts. In the pre-alighting stage of host selection, skunk cabbage was found to be less attractive than tomato, cucumber, and commercial mushrooms. In pairwise oviposition tests, there were no differences among hosts. There were no differences in larval survivorship among skunk cabbage, tomato, cucumber, or Ramaria, and larvae developed into pupae earlier on tomato than on skunk cabbage. These results indicate that this monophagy is not based on characteristics of the host. We suggest that habitat selection is the more important factor in determining the association between D. magnaquinaria and skunk cabbage.     

Development of Phodopus sungorus brown preadipocytes in primary cell culture: effect of an atypical beta-adrenergic agonist, insulin, and triiodothyronine on differentiation, mitochondrial development, and expression of the uncoupling protein UCP

A new cellular model for the study of brown adipocyte development and differentiation in vitro is presented. Preadipocytes isolated from brown adipose tissue (BAT) of the djungarian dwarf hamster Phodopus sungorus are able to proliferate and differentiate in vitro into true brown adipocytes able to express the BAT marker protein the uncoupling protein (UCP). Whereas basal UCP expression is very low, its mRNA levels as well as the UCP detected by immunoblotting are highly increased by beta-adrenergic stimulation. The novel, atypical beta-adrenergic compound D7114 (ICI Pharmaceuticals, Macclesfield, Cheshire, England) was found to increase the number of adipocytes as well as UCP mRNA and UCP content of mitochondria, indicating the involvement of an atypical or beta 3 receptor. Insulin was found to play an important role in brown adipocyte differentiation and mitochondrial development, whereas T3 seemed to be implicated more directly in UCP expression. In a defined, serum-free medium a synergistic stimulatory action of insulin and T3 on UCP expression was found, which seems to involve a pathway different from that of beta-adrenergic UCP stimulation.     

Functional expression of plant alternative oxidase decreases antimycin A-induced reactive oxygen species production in human cells

Alternative oxidase (AOX) plays a pivotal role in cyanide-resistance respiration in the mitochondria of plants, fungi and some protists. Here we show that AOX from thermogenic skunk cabbage successfully conferred cyanide resistance to human cells. In galactose medium, HeLa cells with mitochondria-targeted AOX proteins were found to have significantly less reactive oxygen species production in response to antimycin-A exposure, a specific inhibitor of respiratory complex III. These results suggest that skunk cabbage AOX can be used to create an alternative respiration pathway, which might be important for therapy against various mitochondrial diseases.     

Functional analysis of skunk cabbage SfUCPB, a unique uncoupling protein lacking the fifth transmembrane domain, in yeast cells

Skunk cabbage, Symplocarpus foetidus, expresses two uncoupling proteins (UCPs), termed SfUCPA and SfUCPB, in the thermogenic organ spadix. SfUCPB exhibits unique structural features characterized by the absence of the putative fifth transmembrane domain (TM5) observed in SfUCPA, which is structurally similar to UCP1, and is abundantly expressed in the thermogenic spadix. Here, we conducted a series of comparative analyses of UCPs with six transmembrane domains, SfUCPA and rat UCP1, and TM5-deficient SfUCPB, using a heterologous yeast expression system. All UCPs were successfully expressed and targeted to the mitochondria, although the expression level of SfUCPB protein was approximately 10% of rat UCP1. The growth rate, mitochondrial membrane potential, and ATP content were significantly lower in cells expressing SfUCPB than in those expressing rat UCP1 and SfUCPA. These results suggest that SfUCPB, a novel TM5-deficient UCP, acts as an uncoupling protein in yeast cells.     

The Respiratory Chain of Plant Mitochondria. II. Oxidative Phosphorylation in Skunk Cabbage Mitochondria

Mitochondria were prepared from the spadices of skunk cabbage (Symplocarpus foetidus) whose respiratory rate with succinate and malate showed 15% to 30% sensitivity to cyanide inhibition, and which showed respiratory control by added ADP. The observed respiratory control ratios ranged from 1.1 to 1.4. The change in pH of the mitochondrial suspension was recorded simultaneously with oxygen uptake: alkalinization of the medium, expected for phosphorylation of ADP, coincided with the period of acceleration in oxygen uptake caused by addition of an ADP aliquot. The ADP/O ratios obtained were 1.3 for succinate and 1.9 for malate. In the presence of 0.3 mm cyanide, the ADP/O ratio for succinate was zero, while that for malate was 0.7. These results are consistent with the existence of an alternate oxidase which interacts with the flavoprotein and pyridine nucleotide components of the respiratory chain and which, in the presence of cyanide, allows the first phosphorylation site to function with an efficiency of about 70%. In the absence of respiratory inhibitors, the efficiency of each phosphorylation site is also about 70%. This result implies that diversion of reducing equivalents through the alternate oxidase, thereby bypassing the 2 phosphorylation sites associated with the cytochrome components of these mitochondria, occurs to a negligible extent during the oxidative phosphorylation of ADP or State 3.Addition of ADP or uncoupler to skunk cabbage mitochondria respiring in the controlled state or State 4, results in reduction of cytochrome c and the oxidation of the cytochromes b, ubiquinone and pyridine nucleotide. A site of interaction of ADP with the respiratory chain between cytochromes b and cytochrome c is thereby identified by means of the crossover theorem. Flavoprotein measured by fluorescence is also oxidized upon addition of ADP or uncoupler, but flavoprotein measured by optical absorbance changes becomes more reduced under these conditions. Depletion of the mitochondria by pretreatment with ADP and uncoupler prevents reduction of most of the fluorescent flavoprotein by succinate. These results indicate that skunk cabbage mitochondria contain both high and low potential flavo-proteins characterized by different fluorescence/absorbance ratios similar to those demonstrated to be part of the respiratory chain in mitochondria from animal tissues.     

Determination of molecular mass of the aroid alternative oxidase by radiation-inactivation analysis.

The functional molecular mass of the cyanide-resistant salicylhydroxamate-sensitive duroquinol oxidase activity from Sympocarpus foetidus (skunk cabbage) and Sauromatum guttatum spadix mitochondria was determined by radiation-inactivation analysis. The functional molecular mass for the oxidase activity was found to be 26,700 Da for skunk cabbage and 29,700 Da for Sauromatum guttatum mitochondria frozen at -70 degrees C. Irradiation of dried mitochondrial samples resulted in a larger target size of 38,000 Da, and in some cases, a stimulation of activity at low dose of radiation. The functional molecular mass of cytochrome c oxidase activity from skunk-cabbage and bovine heart mitochondria was also investigated. Dried and frozen mitochondrial samples from both species yielded similar target sizes, in the range 70,900-73,400 Da. Purified bovine heart cytochrome c oxidase was also irradiated, and yielded a functional molecular mass of 66,400 Da. The target size of cytochrome c oxidase agrees with literature values insofar as the target size is considerably smaller than the molecular mass of the entire complex.     

Trypanocidal CoQ analogues: their effect on other mitochondrial systems

1. A comparative study of compounds which inhibit the respiration of the infective form of the protozoan parasite Trypanosoma brucei brucei, such as salicylhydroxamic acid, other substituted benzhydroxamic acid, esters of 2,3- and 3,4-dihydroxybenzoic acid and structurally related compounds, showed that they have a remarkable degree of selectivity for the trypanosome as compared to rat liver mitochondria even though they are putative CoQ analogues and both respiratory systems are dependent on CoQ. 2. The minimal inhibition of mammalian mitochondrial function could not be assigned to inhibition of ubiquinone function in these mitochondria. 3. CoQ-reducing mitochondrial dehydrogenases from rat liver, trypanosomes and skunk cabbage (Symplocarpus foetidus) were insensitive to these inhibitors. 4. The alternative oxidase of skunk cabbage mitochondria was sensitive to a spectrum of trypanosome respiration inhibitors suggesting a similarity to the oxidase of the trypanosome although differing degrees of sensitivity and differing responses to alterations in the molecular structure of the inhibitors indicate that the milieu of the active sites are dissimilar.     

Modified glutamine catabolism in macrophages of Ucp2 knock-out mice

Uncoupling protein 2 (UCP2) belongs to a family of transporters of the mitochondrial inner membrane and is reported to uncouple respiration from ATP synthesis. Our observation that the amino acid glutamine specifically induces UCP2 protein expression prompted us to investigate metabolic consequences of a UCP2 knockdown (Ucp2-KO) when glutamine is offered as a substrate. We found that Ucp2-KO macrophages incubated in the presence of glutamine exhibit a lower ammonium release, a decreased respiratory rate, and an intracellular accumulation of aspartate. Therefore, we conclude that UCP2 expression is required for efficient oxidation of glutamine in macrophages. This role of UCP2 in glutamine metabolism appears independent from the uncoupling activity of UCP2.     

Partial purification of the cyanide-resistant alternative oxidase of skunk cabbage (Symplocarpus foetidus) mitochondria.

A partial purification of the cyanide-resistant, alternative oxidase from skunk cabbage (Symplocarpus foetidus L.) spadix mitochondria is described. Skunk cabbage mitochondria were solubilized in N,N-bis-(3-D-glucon-amido-propyl)deoxycholamide and the alternative oxidase was purified using a batch DEAE-cellulose treatment, followed by precipitation with Extracti-Gel and chromatography on Sephadex G-200. Following pooling and concentrating of the most active fractions from the gel filtration column, a 20- to 30-fold purification of the alternative oxidase was obtained, with no evidence of contamination by cytochrome c oxidase (complex IV) or cytochrome c reductase (complex III). Polyacrylamide gel electrophoresis of the partially purified oxidase showed major polypeptides at 36 and 29 kD, both of which react with monoclonal antibodies raised against the Sauromatum guttatum alternative oxidase. The purified oxidase fraction showed no absorbance in the visible spectral region, and addition of sodium borohydride induced no absorbance changes in the ultraviolet region. The purified alternative oxidase catalyzed the four-electron reduction of oxygen to water in the absence of citrate, but catalyzed an apparent two-electron reduction of oxygen to hydrogen peroxide in the presence of 0.7 M citrate.     

The Respiratory Chain of Plant Mitochondria: IV. Oxidation Rates of the Respiratory Carriers of Mung Bean Mitochondria in the Presence of Cyanide

The half-time for oxidation of cytochrome b(557) in mitochondria from etiolated mung bean (Phaseolus aureus) hypocotyls is 5.8 milliseconds at 24 Celsius in the absence or presence of 0.3 mm KCN, when the oxidation is carried out by injecting a small amount of oxygenated medium into a suspension of mitochondria made anaerobic in the presence of succinate plus malonate. Since oxygen is consumed by the alternate, cyanide-insensitive respiratory pathway of these mitochondria, cycles of oxidation and reduction can be obtained with the oxygen pulses when cyanide is present. Reduced cytochromes (a + a(3)) also become oxidized at nearly the uninhibited rate under these conditions, a(3) completely and a partially. The half-time for oxidation of c(547) is also unaffected by 0.3 mm KCN, but c(549) has a half-time equal to that of c(547) in the presence of KCN, compared to the shorter one observed in the absence of inhibitor. The maximum extent of oxidation of the cytochromes c is about 70% in the presence of 0.3 mm KCN; this oxidation is rapidly followed by an extensive reduction which is synchronous with the reduction of cytochrome a observed under the same conditions. In the presence of cyanide, it appears likely that the cytochromes c and b(557) are oxidized by cytochrome oxidase in oxygen pulse experiments, rather than by the alternate oxidase. The oxidation of cytochrome b(553) is partially inhibited by KCN, but complete oxidation is attained in the aerobic steady state with excess oxygen. If the oxygen pulse experiment is carried out in the presence of sufficient malonate so that entry of reducing equivalents into the respiratory chain occurs at a rate negligible compared to inter-carrier electron transport, the half-time for flavoprotein oxidation is unaffected by 0.3 mm KCN while that for ubiquinone oxidation is but 2-fold larger. The observed net oxidation rate of these two carriers in mung bean mitochondria is more sensitive to the entry rate of reducing equivalents, as set by succinate concentration and malonate to succinate ratio, then it is in skunk cabbage (Symplocarpus foetidus) mitochondria. These observations are interpreted in terms of a respiratory carrier Y, placed between flavoprotein plus ubiquinone and the cytochromes, which is the fork in the split respiratory pathway to the two terminal oxidases and which has lower electron transport capacity in mung bean mitochondria than in skunk cabbage mitochondria.     

Uncoupling protein 2 from carp and zebrafish, ectothermic vertebrates.

Uncoupling protein 1 (UCP1) is of demonstrated importance in mammalian thermogenesis, and early hypotheses regarding the functions of the newly discovered UCP homologues, UCP2, UCP3 and others, have focused largely on their potential roles in thermogenesis. Here we report the amino acid sequences of two new UCPs from ectothermic vertebrates. UCPs from two fish species, the zebrafish (Danio rerio) and carp (Cyprinus carpio), were identified in expressed sequence tag databases at the European Molecular Biology Laboratory. cDNAs from a C. carpio 'peritoneal exudate cell' cDNA library and from a D. rerio 'day 0 fin regeneration' cDNA library were obtained and fully sequenced. Each cDNA encodes a 310 amino acid protein with an average 82% sequence identity to mammalian UCP2s. The fish UCP2s are about 70% identical to mammalian UCP3s, and 60% identical to mammalian UCP1s. Carp and zebrafish are ectotherms--they do not raise their body temperatures above ambient by producing excess heat. The presence of UCP2 in these fish thus suggests the protein may have function(s) not related to thermogenesis.     

Modified glutamine catabolism in macrophages of Ucp2 knock-out mice

Uncoupling protein 2 (UCP2) belongs to a family of transporters of the mitochondrial inner membrane and is reported to uncouple respiration from ATP synthesis. Our observation that the amino acid glutamine specifically induces UCP2 protein expression prompted us to investigate metabolic consequences of a UCP2 knockdown (Ucp2-KO) when glutamine is offered as a substrate. We found that Ucp2-KO macrophages incubated in the presence of glutamine exhibit a lower ammonium release, a decreased respiratory rate, and an intracellular accumulation of aspartate. Therefore, we conclude that UCP2 expression is required for efficient oxidation of glutamine in macrophages. This role of UCP2 in glutamine metabolism appears independent from the uncoupling activity of UCP2.     

Leptin selectively reduces white adipose tissue in mice via a UCP1-dependent mechanism in brown adipose tissue

We tested the hypothesis that leptin, in addition to reducing body fat by restraining food intake, reduces body fat through a peripheral mechanism requiring uncoupling protein 1 (UCP1). Leptin was administered to wild-type (WT) mice and mice with a targeted disruption of the UCP1 gene (UCP1 deficient), while vehicle-injected control animals of each genotype were pair-fed to each leptin-treated group. Leptin reduced the size of white adipose tissue (WAT) depots in WT mice but not in UCP1-deficient animals. This was accompanied by a threefold increase in the amount of UCP1 protein and mRNA in the brown adipose tissue (BAT) of WT mice. Leptin also increased UCP2 mRNA in WAT of both WT and UCP1-deficient mice but increased UCP2 and UCP3 mRNA only in BAT from UCP1-deficient mice. These results indicate that leptin reduces WAT through a peripheral mechanism requiring the presence of UCP1, with little or no involvement of UCP2 or UCP3.     

Cyanide-insensitive Respiration in Plant Mitochondria

Pathways of electron transport have been studied in mitochondria isolated from hypocotyls of etiolated mung bean seedlings and skunk cabbage spadices that show cyanide-resistant respiratory activity. The residual flux through cytochrome c oxidase is shown to be small in comparison with the flux through an unidentified alternative oxidase that is known to have a high affinity for oxygen. This alternative oxidase is not a cytochrome. Skunk cabbage and mung bean mitochondria contain cytochromes a and a₃ that have absorption peaks differing slightly from those of animal preparations. A slow oxidation-reduction of cytochrome a₃-CN has been demonstrated. Cytochromes b undergo oxidation and reduction in the presence of cyanide but play no essential role in the cyanide-resistant pathway. Antimycin inhibits to an extent similar to that of cyanide; the respiratory chain bifurcates on the substrate side of the antimycin-sensitive site. Evidence is presented for the selective inhibition by thiocyanate, α, α′-dipyridyl, and 8-hydroxyquinoline of the alternative oxidase pathway, which may therefore contain a non-heme iron protein.     

Absence of UCP3 in brown adipose tissue does not impair nonshivering thermogenesis

We report on a novel Djungarian hamster mutant lineage that exhibits a loss of uncoupling protein (UCP) 3 mRNA and protein in brown adipose tissue (BAT), whereas UCP3 expression in skeletal muscle is only mildly diminished. In response to 2 d of cold exposure, UCP3 mRNA was 4.5-fold elevated in BAT of wild-type hamsters but remained undetectable in mutant hamsters. Notably, in BAT of warm- and cold-exposed mutant hamsters, UCP1 and UCP2 mRNA levels were increased. The tissue specificity of UCP3 deficiency suggests that the underlying unknown mutation impairs a factor controlling UCP3 gene expression selectively in brown adipocytes. In wild-type but not mutant primary brown adipocytes, UCP3 gene expression was stimulated by treatment with peroxisome proliferator activated receptor (PPAR) ligands. This implies that the underlying mutation causing UCP3 deficiency is expressed within brown adipocytes and disrupts PPAR-dependent transactivation of the UCP3 gene. On the functional level, we found no direct phenotypic consequences of altered UCP expression in BAT. The absence of UCP3 in BAT of cold-acclimated mutant hamsters affected neither maximal nonshivering thermogenesis elicited by noradrenaline nor the uncoupled respiration of isolated mitochondria in the presence of oligomycin and in response to palmitate.