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1.
Plant Physiol ; 195(2): 1561-1585, 2024 May 31.
Article in English | MEDLINE | ID: mdl-38318875

ABSTRACT

The inflorescence (spadix) of skunk cabbage (Symplocarpus renifolius) is strongly thermogenic and can regulate its temperature at around 23 °C even when the ambient temperature drops below freezing. To elucidate the mechanisms underlying developmentally controlled thermogenesis and thermoregulation in skunk cabbage, we conducted a comprehensive transcriptome and metabolome analysis across 3 developmental stages of spadix development. Our RNA-seq analysis revealed distinct groups of expressed genes, with selenium-binding protein 1/methanethiol oxidase (SBP1/MTO) exhibiting the highest levels in thermogenic florets. Notably, the expression of alternative oxidase (AOX) was consistently high from the prethermogenic stage through the thermogenic stage in the florets. Metabolome analysis showed that alterations in nucleotide levels correspond with the developmentally controlled and tissue-specific thermogenesis of skunk cabbage, evident by a substantial increase in AMP levels in thermogenic florets. Our study also reveals that hydrogen sulfide, a product of SBP1/MTO, inhibits cytochrome c oxidase (COX)-mediated mitochondrial respiration, while AOX-mediated respiration remains relatively unaffected. Specifically, at lower temperatures, the inhibitory effect of hydrogen sulfide on COX-mediated respiration increases, promoting a shift toward the dominance of AOX-mediated respiration. Finally, despite the differential regulation of genes and metabolites throughout spadix development, we observed a convergence of gene expression and metabolite accumulation patterns during thermogenesis. This synchrony may play a key role in developmentally regulated thermogenesis. Moreover, such convergence during the thermogenic stage in the spadix may provide a solid molecular basis for thermoregulation in skunk cabbage.


Subject(s)
Araceae , Gene Expression Regulation, Plant , Plant Proteins , Plant Proteins/metabolism , Plant Proteins/genetics , Araceae/genetics , Araceae/physiology , Araceae/metabolism , Oxidoreductases/metabolism , Oxidoreductases/genetics , Inflorescence/genetics , Transcriptome/genetics , Metabolome , Thermogenesis/genetics , Mitochondrial Proteins/metabolism , Mitochondrial Proteins/genetics
2.
Biochem J ; 477(17): 3417-3431, 2020 09 18.
Article in English | MEDLINE | ID: mdl-32856714

ABSTRACT

Cyanide-resistant alternative oxidase (AOX) is a nuclear-encoded quinol oxidase located in the inner mitochondrial membrane. Although the quality control of AOX proteins is expected to have a role in elevated respiration in mitochondria, it remains unclear whether thermogenic plants possess molecular mechanisms for the mitochondrial degradation of AOX. To better understand the mechanism of AOX turnover in mitochondria, we performed a series of in organello AOX degradation assays using mitochondria from various stages of the appendices of Arum maculatum. Our analyses clearly indicated that AOX proteins at certain stages in the appendices are degraded at 30°C, which is close to the maximum appendix temperature observed during thermogenesis. Interestingly, such temperature-dependent protease activities were specifically inhibited by E-64, a cysteine protease inhibitor. Moreover, purification and subsequent nano LC-MS/MS analyses of E-64-sensitive and DCG-04-labeled active mitochondrial protease revealed an ∼30 kDa protein with an identical partial peptide sequence to the cysteine protease 1-like protein from Phoenix dactylifera. Our data collectively suggest that AOX is a potential target for temperature-dependent E-64-sensitive cysteine protease in the appendices of A. maculatum. A possible retrograde signalling cascade mediated by specific degradation of AOX proteins and its physiological significance are discussed.


Subject(s)
Arum/enzymology , Mitochondria/enzymology , Mitochondrial Proteins/metabolism , Oxidoreductases/metabolism , Plant Proteins/metabolism , Proteolysis , Signal Transduction , Arum/genetics , Mitochondria/genetics , Mitochondrial Proteins/genetics , Oxidoreductases/genetics , Plant Proteins/genetics
3.
J Biochem ; 165(1): 57-65, 2019 Jan 01.
Article in English | MEDLINE | ID: mdl-30289493

ABSTRACT

Thermogenesis in plants involves significant increases in their cyanide-resistant mitochondrial alternative oxidase (AOX) capacity. Because AOX is a non-proton-motive ubiquinol oxidase, the dramatic drop in free energy between ubiquinol and oxygen is dissipated as heat. In the thermogenic skunk cabbage (Symplocarpus renifolius), SrAOX is specifically expressed in the florets. Although SrAOX harbours conserved cysteine residues, the details of the mechanisms underlying its redox regulation are poorly understood. In our present study, the two mitochondrial thioredoxin o cDNAs SrTrxo1 and SrTrxo2, were isolated from the thermogenic florets of S. renifolius. The deduced amino acid sequences of the protein products revealed that SrTrxo2 specifically lacks the region corresponding to the α3-helix in SrTrxo1. Expression analysis of thermogenic and non-thermogenic S. renifolius tissues indicated that the SrTrxo1 and SrAOX transcripts are predominantly expressed together in thermogenic florets, whereas SrTrxo2 transcripts are almost undetectable in any tissue. Finally, functional in vitro analysis of recombinant SrTrxo1 and mitochondrial membrane fractions of thermogenic florets indicated its reducing activity on SrAOX proteins. Taken together, these results indicate that SrTrxo1 is likely to play a role in the redox regulation of SrAOX in S. renifolius thermogenic florets.


Subject(s)
Araceae/enzymology , Mitochondria/enzymology , Mitochondrial Proteins/metabolism , Oxidoreductases/metabolism , Plant Proteins/metabolism , Thermogenesis , Thioredoxins/metabolism , Amino Acid Sequence , Araceae/genetics , Araceae/physiology , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , DNA, Plant/genetics , DNA, Plant/isolation & purification , Flowers , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Mitochondrial Proteins/chemistry , Oxidation-Reduction , Oxidoreductases/chemistry , Plant Proteins/chemistry , RNA, Messenger/genetics , Sequence Homology, Amino Acid , Thioredoxins/genetics
4.
Funct Plant Biol ; 45(12): 1195-1204, 2018 11.
Article in English | MEDLINE | ID: mdl-32291010

ABSTRACT

Arum concinnatum Schott is a highly thermogenic species, with the temperature of the appendix exceeding ~10.9°C above the ambient temperature during thermogenesis, whereas the rates of respiration of the male florets in intact inflorescences peak at 0.92µmol s-1 g-1, which is the highest rate so far measured among the plants. Here, we attempt the ex situ exogenous induction of thermogenesis in whole inflorescences and in separate appendices of the spadix, and explore the thermogenic patterns under controlled laboratory conditions of light and temperature. Mature but unopened inflorescences and appendices showed thermogenic responses when treated with salicylic acid (SA), but not when treated with distilled water (control). With regard to light conditions, the responses revealed only one significant difference for inflorescences, which concerns the higher maximum temperature in the continuous light treatment compared with continuous dark. Along the ambient temperature gradient, at the lowest temperature edge individuals remained stable close to ambient temperature and to control. These findings suggest that, in general, ex situ exogenous induction of thermogenesis can be achieved in whole inflorescences and in separate appendices of spadix of A. concinnatum using SA. This study also indicates that SA acts independently of light conditions, while exogenous induction of thermogenesis takes place within an ambient temperature range.


Subject(s)
Arum , Flowers , Male , Salicylic Acid , Temperature , Thermogenesis
5.
Plant Cell Environ ; 41(2): 367-373, 2018 02.
Article in English | MEDLINE | ID: mdl-29121698

ABSTRACT

The respiration rate of the thermogenic inflorescences of Japanese skunk cabbage Symplocarpus renifolius can reach 300 nmol s-1 g-1 , which is sufficient to raise spadix temperature (Ts ) up to 15 °C above ambient air temperature (Ta ). Respiration rate is inversely related to Ta , such that the Ts achieves a degree of independence from Ta , an effect known as temperature regulation. Here, we measure oxygen consumption rate (Mo2 ) in air (21% O2 in mainly N2 ) and in heliox (21% O2 in He) to investigate the diffusive conductance of the network of gas-filled spaces and the thermoregulatory response. When Ts was clamped at 15 °C, the temperature that produces maximal Mo2 in this species, exposure to high diffusivity heliox increased mean Mo2 significantly from 137 ± 17 to 202 ± 43 nmol s-1 g-1 FW, indicating that respiration in air is normally limited by diffusion in the gas phase and some mitochondria are unsaturated. When Ta was clamped at 15 °C and Ts was allowed to vary, exposure to heliox reduced Ts 1 °C and increased Mo2 significantly from 116 ± 10 to 137 ± 19 nmol s-1 g-1 , indicating that enhanced heat loss by conduction and convection can elicit the thermoregulatory response.


Subject(s)
Araceae/metabolism , Cell Respiration , Flowers/metabolism , Helium/metabolism , Oxygen/metabolism , Oxygen Consumption , Thermogenesis
6.
Biophys Physicobiol ; 15: 235-250, 2018.
Article in English | MEDLINE | ID: mdl-30713824

ABSTRACT

A wild plant called Skunk Cabbage is known to heat itself and keep its body warm before spring. We study its homothermal maintenance mechanism from a mesoscopic point of view. We take the increment process of the temperature time series and consider it as 'elastic' force that always tries to backlash its temperature to an intrinsic target temperature. We then propose a kind of extended Poisson distribution for the model of the 'elastic' force. The hypothesis testing result by Kolmogorov-Smirnov test suggests that the proposed distribution is a plausible candidate of the model for the 'elastic' force, on the temperature range in which the system is in equillibrium. In addition, it turns out that the parameters in the model captures well the linear behaviour of the expectations of the 'elastic' force at each of the present temperatures and similarly, the constancy of the variances of the force. Especially, the linearity of the expected increments over displacements of tempertures indicates that the backlash might be considered to be like the elastic force of a spring as described by Fuch's law.

7.
Plant Signal Behav ; 11(11): e1247138, 2016 11.
Article in English | MEDLINE | ID: mdl-27739913

ABSTRACT

Skunk cabbage (Symplocarpus renifolius) blooms in early spring and its inflorescence, referred to as the spadix, can produce enough heat to melt snow. Here, we investigated glycolytic carbon flow at the PEP branch-point in thermogenic spadices. Our analyses revealed that petals and pistils in thermogenic florets exhibited higher expression of SrPEPC and SrAOX transcripts than those of SrPK, SrPEPCK, and SrPEPtase. Moreover, enzymatic analyses showed high activities of PEPC in the extracts from thermogenic florets. Finally, mitochondria from thermogenic florets showed low respiratory activities when pyruvate was used as a substrate, although a significant malate-mediated cyanide-insensitive respiration was observed. Collectively, these results suggest that PEP metabolism, primarily catabolized by PEPC, plays a critical role in thermogenesis in S. renifolius.


Subject(s)
Araceae/enzymology , Cytosol/enzymology , Mitochondria/enzymology , Mitochondrial Proteins/metabolism , Oxidoreductases/metabolism , Phosphoenolpyruvate Carboxylase/metabolism , Plant Proteins/metabolism , Araceae/metabolism , Cytosol/metabolism , Mitochondrial Proteins/genetics , Oxidoreductases/genetics , Phosphoenolpyruvate Carboxylase/genetics , Plant Proteins/genetics , Temperature
8.
Sci Rep ; 6: 24830, 2016 Apr 20.
Article in English | MEDLINE | ID: mdl-27095582

ABSTRACT

Thermoregulation (homeothermy) in animals involves a complex mechanism involving thermal receptors throughout the body and integration in the hypothalamus that controls shivering and non-shivering thermogenesis. The flowers of some ancient families of seed plants show a similar degree of physiological thermoregulation, but by a different mechanism. Here, we show that respiratory control in homeothermic spadices of skunk cabbage (Symplocarpus renifolius) is achieved by rate-determining biochemical reactions in which the overall thermodynamic activation energy exhibits a negative value. Moreover, NADPH production, catalyzed by mitochondrial isocitrate dehydrogenase in a chemically endothermic reaction, plays a role in the pre-equilibrium reaction. We propose that a law of chemical equilibrium known as Le Châtelier's principle governs the homeothermic control in skunk cabbage.


Subject(s)
Body Temperature Regulation , Flowers/chemistry , Flowers/physiology , Hot Temperature , Plant Physiological Phenomena , Algorithms , Cell Respiration , Mitochondria/metabolism , Models, Biological
9.
PLoS One ; 10(11): e0142917, 2015.
Article in English | MEDLINE | ID: mdl-26588091

ABSTRACT

We investigated the association between the expression of a gene encoding gustatory receptor (G10) and division of labor in the honey bee, Apis mellifera. Among 10 GR genes encoding proteins 15% ~ 99% amino acid identity in the honey bee, we found that AmGR10 with 99% identity is involved in nursing or brood care. Expression of AmGR10 was restricted to organs of the hypopharyngeal gland, brain, and ovary in the nurse bee phase. Members of an extended nursing caste under natural conditions continued to express this gene. RNAi knockdown of AmGR10 accelerated the transition to foraging. Our findings demonstrate that this one gene has profound effects on the division of labor associated with the development and physiology of honeybee society.


Subject(s)
Bees/genetics , Behavior, Animal , Gene Expression Regulation/genetics , Receptors, Cell Surface/biosynthesis , Animals , Bees/growth & development , Brain/metabolism , Drosophila Proteins/genetics , Molecular Sequence Data , Receptors, Cell Surface/genetics , Signal Transduction
10.
Sci Rep ; 5: 8753, 2015 Mar 04.
Article in English | MEDLINE | ID: mdl-25736477

ABSTRACT

Several plant species can generate enough heat to increase their internal floral temperature above ambient temperature. Among thermogenic plants, Arum concinnatum shows the highest respiration activity during thermogenesis. However, an overall understanding of the genes related to plant thermogenesis has not yet been achieved. In this study, we performed de novo transcriptome analysis of flower organs in A. concinnatum. The de novo transcriptome assembly represented, in total, 158,490 non-redundant transcripts, and 53,315 of those showed significant homology with known genes. To explore genes associated with thermogenesis, we filtered 1266 transcripts that showed a significant correlation between expression pattern and the temperature trend of each sample. We confirmed five putative alternative oxidase transcripts were included in filtered transcripts as expected. An enrichment analysis of the Gene Ontology terms for the filtered transcripts suggested over-representation of genes involved in 1-deoxy-D-xylulose-5-phosphate synthase (DXS) activity. The expression profiles of DXS transcripts in the methyl-D-erythritol 4-phosphate (MEP) pathway were significantly correlated with thermogenic levels. Our results suggest that the MEP pathway is the main biosynthesis route for producing scent monoterpenes. To our knowledge, this is the first report describing the candidate pathway and the key enzyme for floral scent production in thermogenic plants.


Subject(s)
Arum/genetics , Flowers/genetics , Gene Expression Profiling/methods , Gene Expression Regulation, Plant , Arum/metabolism , Biosynthetic Pathways/genetics , Energy Metabolism/genetics , Flowers/chemistry , Flowers/metabolism , Gene Ontology , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Odorants/analysis , Oxidoreductases/genetics , Oxidoreductases/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Temperature , Terpenes/metabolism , Transferases/genetics , Transferases/metabolism
11.
Plant Cell Environ ; 38(4): 827-37, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25256124

ABSTRACT

Thermogenic flowers produce heat by intense respiration, and the rates of O2 consumption (Mo2) in some species can exceed those of all other tissues of plants and most animals. By exposing intact flowers to a range of O2 pressures (Po2) and measuring Mo2, we demonstrate that the highest respiration rates exceed the capacity of the O2 diffusive pathway and become diffusion limited in atmospheric air. The male florets on the inflorescence of Arum concinnatum have the highest known mass-specific Mo2 and can be severely diffusion limited. Intact spadices of Japanese skunk cabbage Symplocarpus renifolius are diffusion limited in air only when Mo2 is maximal, but not at lower levels. True flowers of the sacred lotus Nelumbo nucifera and the appendix of Arum concinnatum are never diffusion limited in air. Mo2 - Po2 curves are evaluated quantitatively with the 'Regulation Index', a new tool to measure dependence of Mo2 on ambient Po2 , as well as the conventional 'Critical Po2 '. The study also includes measurements of Po2 within thermogenic tissues with O2-sensitive fibre optics, and reveals that the diffusion pathway is complicated and that O2 can be provided not only from the surface of the tissues but also from the pith of the flower's peduncle.


Subject(s)
Araceae/physiology , Arum/physiology , Flowers/physiology , Nelumbo/physiology , Oxygen/metabolism , Plant Transpiration/physiology , Cell Respiration , Diffusion , Hot Temperature , Inflorescence/physiology , Oxygen/analysis , Regression Analysis , Thermogenesis/physiology
12.
Genome Biol ; 14(5): R41, 2013 May 10.
Article in English | MEDLINE | ID: mdl-23663246

ABSTRACT

BACKGROUND: Sacred lotus is a basal eudicot with agricultural, medicinal, cultural and religious importance. It was domesticated in Asia about 7,000 years ago, and cultivated for its rhizomes and seeds as a food crop. It is particularly noted for its 1,300-year seed longevity and exceptional water repellency, known as the lotus effect. The latter property is due to the nanoscopic closely packed protuberances of its self-cleaning leaf surface, which have been adapted for the manufacture of a self-cleaning industrial paint, Lotusan. RESULTS: The genome of the China Antique variety of the sacred lotus was sequenced with Illumina and 454 technologies, at respective depths of 101× and 5.2×. The final assembly has a contig N50 of 38.8 kbp and a scaffold N50 of 3.4 Mbp, and covers 86.5% of the estimated 929 Mbp total genome size. The genome notably lacks the paleo-triplication observed in other eudicots, but reveals a lineage-specific duplication. The genome has evidence of slow evolution, with a 30% slower nucleotide mutation rate than observed in grape. Comparisons of the available sequenced genomes suggest a minimum gene set for vascular plants of 4,223 genes. Strikingly, the sacred lotus has 16 COG2132 multi-copper oxidase family proteins with root-specific expression; these are involved in root meristem phosphate starvation, reflecting adaptation to limited nutrient availability in an aquatic environment. CONCLUSIONS: The slow nucleotide substitution rate makes the sacred lotus a better resource than the current standard, grape, for reconstructing the pan-eudicot genome, and should therefore accelerate comparative analysis between eudicots and monocots.


Subject(s)
Genome, Plant , Nelumbo/genetics , Adaptation, Biological , Amino Acid Substitution , Evolution, Molecular , Molecular Sequence Data , Mutation Rate , Nelumbo/classification , Nelumbo/physiology , Phylogeny , Vitis/genetics
13.
Annu Rev Plant Biol ; 64: 637-63, 2013.
Article in English | MEDLINE | ID: mdl-23638828

ABSTRACT

The alternative oxidase is a membrane-bound ubiquinol oxidase found in the majority of plants as well as many fungi and protists, including pathogenic organisms such as Trypanosoma brucei. It catalyzes a cyanide- and antimycin-A-resistant oxidation of ubiquinol and the reduction of oxygen to water, short-circuiting the mitochondrial electron-transport chain prior to proton translocation by complexes III and IV, thereby dramatically reducing ATP formation. In plants, it plays a key role in cellular metabolism, thermogenesis, and energy homeostasis and is generally considered to be a major stress-induced protein. We describe recent advances in our understanding of this protein's structure following the recent successful crystallization of the alternative oxidase from T. brucei. We focus on the nature of the active site and ubiquinol-binding channels and propose a mechanism for the reduction of oxygen to water based on these structural insights. We also consider the regulation of activity at the posttranslational and retrograde levels and highlight challenges for future research.


Subject(s)
Mitochondrial Proteins/chemistry , Oxidoreductases/chemistry , Plant Physiological Phenomena , Plant Proteins/chemistry , Plants/enzymology , Antimycin A , Catalytic Domain , Fungi/enzymology , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Oxidation-Reduction , Oxidoreductases/genetics , Oxidoreductases/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Plants/metabolism , Temperature
14.
Appl Biochem Biotechnol ; 170(5): 1229-40, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23653140

ABSTRACT

Alternative oxidase (AOX) is a nonproton motive quinol-oxygen oxidoreductase which is a component of the mitochondrial respiratory chain in higher plants. In this study, we have characterized the catalytic activity and regulatory behaviors of Arum concinnatum AOX isoforms, namely AcoAOX1a and AcoAOX1b, and their artificial mutants in HeLa cells. We demonstrated that substitution of the motif-like sequence ENV on the C-terminal half of AcoAOX1a for QDT diminishes its activity and proposed that the innate inactivity of AcoAOX1b in HeLa cells is, at least in part, attributable to its QDT motif. Furthermore, we show that introduction of F130L in the hydrophilic N-terminal extension of AcoAOX1a resulted in greater activity in the presence of pyruvate. This result indicates that functional significance of the N-terminal extension is not particular to the conventional regulatory cysteine. On the basis of these findings, we discuss new insights into the structural integrity of AOX in HeLa cells and the applicability of mammalian cells for functional analysis of this enzyme.


Subject(s)
Arum/chemistry , Arum/physiology , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/metabolism , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Plant Proteins/chemistry , Plant Proteins/metabolism , Protein Engineering/methods , Amino Acid Motifs , Amino Acid Substitution , Catalysis , Enzyme Activation , HeLa Cells , Humans , Mitochondrial Proteins/genetics , Oxidoreductases/genetics , Plant Proteins/genetics , Structure-Activity Relationship
16.
Biochem J ; 445(2): 237-46, 2012 Jul 15.
Article in English | MEDLINE | ID: mdl-22512685

ABSTRACT

Symplocarpus renifolius and Arum maculatum are known to produce significant heat during the course of their floral development, but they use different regulatory mechanisms, i.e. homoeothermic compared with transient thermogenesis. To further clarify the molecular basis of species-specific thermogenesis in plants, in the present study we have analysed the native structures and expression patterns of the mitochondrial respiratory components in S. renifolius and A. maculatum. Our comparative analysis using Blue native PAGE combined with nano LC (liquid chromatography)-MS/MS (tandem MS) has revealed that the constituents of the respiratory complexes in both plants were basically similar, but that several mitochondrial components appeared to be differently expressed in their thermogenic organs. Namely, complex II in S. renifolius was detected as a 340 kDa product, suggesting an oligomeric or supramolecular structure in vivo. Moreover, the expression of an external NAD(P)H dehydrogenase was found to be higher in A. maculatum than in S. renifolius, whereas an internal NAD(P)H dehydrogenase was expressed at a similar level in both species. Alternative oxidase was detected as smear-like signals that were elongated on the first dimension with a peak at around 200 kDa in both species. The significance and implication of these data are discussed in terms of thermoregulation in plants.


Subject(s)
Araceae/metabolism , Arum/metabolism , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Oxidoreductases/metabolism , Plant Proteins/metabolism , Thermogenesis , Amino Acid Sequence , Araceae/genetics , Arum/genetics , Blotting, Western , Electron Transport , Electrophoresis, Gel, Two-Dimensional , Flowers , Mitochondria/genetics , Mitochondrial Proteins/genetics , Molecular Sequence Data , NADPH Dehydrogenase/genetics , NADPH Dehydrogenase/metabolism , Oxidoreductases/genetics , Phylogeny , Plant Proteins/genetics , Sequence Homology, Amino Acid , Tandem Mass Spectrometry
17.
Plant Physiol ; 157(4): 1721-32, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21988877

ABSTRACT

Heat production in thermogenic plants has been attributed to a large increase in the expression of the alternative oxidase (AOX). AOX acts as an alternative terminal oxidase in the mitochondrial respiratory chain, where it reduces molecular oxygen to water. In contrast to the mitochondrial terminal oxidase, cytochrome c oxidase, AOX is nonprotonmotive and thus allows the dramatic drop in free energy between ubiquinol and oxygen to be dissipated as heat. Using reverse transcription-polymerase chain reaction-based cloning, we reveal that, although at least seven cDNAs for AOX exist (AmAOX1a, -1b, -1c, -1d, -1e, -1f, and -1g) in Arum maculatum, the organ and developmental regulation for each is distinct. In particular, the expression of AmAOX1e transcripts appears to predominate in thermogenic appendices among the seven AmAOXs. Interestingly, the amino acid sequence of AmAOX1e indicates that the ENV element found in almost all other AOX sequences, including AmAOX1a, -1b, -1c, -1d, and -1f, is substituted by QNT. The existence of a QNT motif in AmAOX1e was confirmed by nano-liquid chromatography-tandem mass spectrometry analysis of mitochondrial proteins from thermogenic appendices. Further functional analyses with mitochondria prepared using a yeast heterologous expression system demonstrated that AmAOX1e is insensitive to stimulation by pyruvate. These data suggest that a QNT type of pyruvate-insensitive AOX, AmAOX1e, plays a crucial role in stage- and organ-specific heat production in the appendices of A. maculatum.


Subject(s)
Arum/enzymology , Flowers/enzymology , Mitochondrial Proteins/metabolism , Oxidoreductases/metabolism , Plant Proteins/metabolism , Pyruvic Acid/pharmacology , Amino Acid Motifs , Amino Acid Sequence , Arum/drug effects , Arum/genetics , Base Sequence , Cell Respiration , Chromatography, Liquid , DNA, Complementary/genetics , DNA, Plant/chemistry , DNA, Plant/genetics , Flowers/genetics , Hot Temperature , Mitochondrial Proteins/drug effects , Mitochondrial Proteins/genetics , Molecular Sequence Data , Organ Specificity , Oxidoreductases/drug effects , Oxidoreductases/genetics , Phylogeny , Plant Proteins/drug effects , Plant Proteins/genetics , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Sequence Alignment , Sequence Analysis, DNA , Tandem Mass Spectrometry
18.
Planta ; 231(6): 1291-300, 2010 May.
Article in English | MEDLINE | ID: mdl-20221632

ABSTRACT

The flowers of several families of seed plants warm themselves when they bloom. In some species, thermogenesis is regulated, increasing the rate of respiration at lower ambient temperature (T (a)) to maintain a somewhat stable floral temperature (T (f)). The precision of this regulation is usually measured by plotting T (f) over T (a). However, such measurements are influenced by environmental conditions, including wind speed, humidity, radiation, etc. This study eliminates environmental effects by experimentally 'clamping' T (f) at constant, selected levels and then measuring stabilized respiration rate. Regulating flowers show decreasing respiration with rising T (f) (Q (10) < 1). Q (10) therefore becomes a measure of the biochemical 'precision' of temperature regulation: lower Q (10) values indicate greater sensitivity of respiration to T (f) and a narrower range of regulated temperatures. At the lower end of the regulated range, respiration is maximal, and further decreases in floral temperature cause heat production to diminish. Below a certain tissue temperature ('switching temperature'), heat loss always exceeds heat production, so thermoregulation becomes impossible. This study compared three species of thermoregulatory flowers with distinct values of precision and switching temperature. Precision was highest in Nelumbo nucifera (Q (10) = 0.16) moderate in Symplocarpus renifolius (Q (10) = 0.48) and low in Dracunculus vulgaris (Q (10) = 0.74). Switching temperatures were approximately 30, 15 and 20 degrees C, respectively. There were no relationships between precision, switching temperature or maximum respiration rate. High precision reveals a powerful inhibitory mechanism that overwhelms the tendency of temperature to increase respiration. Variability in the shape and position of the respiration-temperature curves must be accounted for in any explanation of the control of respiration in thermoregulatory flowers.


Subject(s)
Flowers/physiology , Temperature , Araceae/physiology , Cell Respiration/physiology , Flowers/cytology , Nelumbo/physiology
19.
Peptides ; 31(5): 827-33, 2010 May.
Article in English | MEDLINE | ID: mdl-20206219

ABSTRACT

A palmitoyl conjugate of an insect pentapeptide that occurs in diapausing insects causes a reversible cell-cycle arrest and suppresses mitochondrial respiration. This peptide compound also causes growth arrest in murine leukemic cell line expressing human gene Bcr/Abl and a farnesoyl peptide induces embryonic diapause in Bombyx mori. These results demonstrate that the insect peptide compounds can lead to the understanding of a common pathway in developmental arrest in animals and may provide a new peptidominetic analog in the development of biopharmaceuticals and pest management.


Subject(s)
Cell Proliferation/drug effects , Cell Respiration/drug effects , Insect Proteins/chemistry , Oligopeptides/pharmacology , Animals , Bombyx , Cell Cycle/drug effects , Cell Line , Cell Line, Tumor , Cells, Cultured , Drosophila , Female , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Mice , Microscopy, Phase-Contrast , Molecular Structure , Moths , Oligopeptides/chemical synthesis , Oligopeptides/chemistry , Rats
20.
Biochim Biophys Acta ; 1797(1): 20-8, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19643077

ABSTRACT

Alternative oxidase (AOX) is a quinol-oxygen oxidoreductase, which is known to possess a dicarboxylate diiron reaction center held in structurally postulated alpha-helical bundle. However, little is known about the structural or functional features of its N-terminal region in any organism, with the exception of a regulatory cysteine residue (CysI) in angiosperm plants. Here, we show that transcripts of two AOX1 isozymes (AcoAOX1a and AcoAOX1b) are coexpressed in thermogenic appendices of Arum concinnatum, while their enzymatic activities seem to be distinct. Namely, AcoAOX1a, an abundantly expressed transcript in vivo, shows an apparent cyanide-insensitive and n-propyl gallate-sensitive respiration during ectopic expression of the protein in HeLa cells, whereas AcoAOX1b exhibits a lower transcript expression, and appears to be totally inactive as AOX at the protein level. Our functional analyses further reveal that an E83K substitution in AcoAOX1b, which is located far upstream of CysI in the N-terminal region, is the cause of this loss of function. These results suggest the presence of a naturally occurring inactive AOX homologue in thermogenic plants. Accordingly, our results further imply that the N-terminal region of the AOX protein functionally contributes to the dynamic activities of respiratory control within the mitochondria.


Subject(s)
Arum/enzymology , HeLa Cells/enzymology , Oxidoreductases/metabolism , Catalysis , DNA Primers , DNA Probes , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Mitochondrial Proteins , Oxidoreductases/chemistry , Oxidoreductases/genetics , Oxygen Consumption , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Plant Proteins , RNA, Plant/genetics , Recombinant Proteins/metabolism , Ribonucleases , Thermodynamics , Transcription, Genetic , Transfection
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