Dynamics and precision of thermoregulatory responses of eastern skunk cabbage Symplocarpus foetidus

Inflorescences of the arum lily Symplocarpus foetidus are thermogenic and thermoregulatory. The spadix increases respiratory heat production rate as ambient temperature decreases. This study examined the relationships between spadix temperature (T_s), respiration rate () and ambient temperature (T_a) at equilibrium and during transient responses to step changes in T_a. Intact inflorescences inside a miniature constant temperature cabinet in the field showed the most precise temperature regulation yet recorded; over a 37.4 °C range in T_a (?10.3 to 27.1 °C), T_s changed only 3.5 °C (22.7 to 26.2 °C). Regulated temperatures were not related to spadix size (1.9–7.3 g) or circadian cycle. Dynamic responses to step changes in T_a involved a phasic change in T_s, first in the same direction as T_a, then reversing at 38.3 min, and finally approaching equilibrium at 87.6 min, on average. Meanwhile changed in a monotonic curve toward equilibrium. Models revealed that the dynamics of temperature change were inconsistent with simply a physical lag in the system, but involved some form of biochemical regulation, possibly by changes in activity of a rate‐limiting functional protein.     

The gene expression landscape of thermogenic skunk cabbage suggests critical roles for mitochondrial and vacuolar metabolic pathways in the regulation of thermogenesis

Floral thermogenesis has been described in several plant species. Because of the lack of comprehensive gene expression profiles in thermogenic plants, the molecular mechanisms by which floral thermogenesis is regulated remain to be established. We examined the gene expression landscape of skunk cabbage (Symplocarpus renifolius) during thermogenic and post-thermogenic stages and identified expressed sequence tags from different developmental stages of the inflorescences using super serial analysis of gene expression (SuperSAGE). In-depth analysis suggested that cellular respiration and mitochondrial functions are significantly enhanced during the thermogenic stage. In contrast, genes involved in stress responses and protein degradation were significantly up-regulated during post-thermogenic stages. Quantitative comparisons indicated that the expression levels of genes involved in cellular respiration were higher in thermogenic spadices than in Arabidopsis inflorescences. Thermogenesis-associated genes seemed to be expressed abundantly in the peripheral tissues of the spadix. Our results suggest that cellular respiration and mitochondrial metabolism play key roles in heat production during floral thermogenesis. On the other hand, vacuolar cysteine protease and other degradative enzymes seem to accelerate senescence and terminate thermogenesis in the post-thermogenic stage.     

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.     

Synchronicity of thermogenic activity, alternative pathway respiratory flux, AOX protein content, and carbohydrates in receptacle tissues of sacred lotus during floral development

The relationships between heat production, alternative oxidase(AOX) pathway flux, AOX protein, and carbohydrates during floraldevelopment in Nelumbo nucifera (Gaertn.) were investigated.Three distinct physiological phases were identified: pre-thermogenic,thermogenic, and post-thermogenic. The shift to thermogenicactivity was associated with a rapid, 10-fold increase in AOXprotein. Similarly, a rapid decrease in AOX protein occurredpost-thermogenesis. This synchronicity between AOX protein andthermogenic activity contrasts with other thermogenic plantswhere AOX protein increases some days prior to heating. AOXprotein in thermogenic receptacles was significantly higherthan in post-thermogenic and leaf tissues. Stable oxygen isotopemeasurements confirmed that the increased respiratory flux supportingthermogenesis was largely via the AOX, with little or no contributionfrom the cytochrome oxidase pathway. During the thermogenicphase, no significant relationship was found between AOX proteincontent and either heating or AOX flux, suggesting that regulationis likely to be post-translational. Further, no evidence ofsubstrate limitation was found; starch accumulated during theearly stages of floral development, peaking in thermogenic receptacles,before declining by 89% in post-thermogenic receptacles. Whilstcoarse regulation of AOX flux occurs via protein synthesis,the ability to thermoregulate probably involves precise regulationof AOX protein, most probably by effectors such as -keto acids. Key words: Alternative oxidase, alternative pathway respiration, Nelumbo nucifera, plant thermogenesis, starch Received 11 November 2007; Accepted 28 November 2007     

The energy-conserving and energy-dissipating processes in mitochondria isolated from wild type and nonripening tomato fruits during development on the plant

Bioenergetics of tomato (Lycopersicon esculentum) development on the plant was followed from the early growing stage to senescence in wild type (climacteric) and nonripening mutant (nor, nonclimacteric) fruits. Fruit development was expressed in terms of evolution of chlorophyll a content allowing the assessment of a continuous time-course in both cultivars. Measured parameters: the cytochrome pathway-dependent respiration, i.e., the ATP synthesis-sustained respiration (energy-conserving), the uncoupling protein (UCP) activity-sustained respiration (energy-dissipating), the alternative oxidase(AOX)-mediated respiration (energy-dissipating), as well as the protein expression of UCP and AOX, and free fatty acid content exhibited different evolution patterns in the wild type and nor mutant that can be attributed to their climacteric/nonclimacteric properties, respectively. In the wild type, the climacteric respiratory burst observed in vitro depended totally on an increse in the cytochrome pathway activity sustained by ATP synthesis, while the second respiratory rise during the ripening stage was linked to a strong increase in AOX activity accompanied by an overexpression of AOX protein. In wild type mitochondria, the 10-M linoleic acid-stimulated UCP-activity-dependent respiration remained constant during the whole fruit development except in senescence where general respiratory decay was observed.     

Respiration of thermogenic inflorescences of skunk cabbage Symplocarpus renifolius in heliox

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 (T_s) up to 15 ° C above ambient air temperature (T_a). Respiration rate is inversely related to T_a, such that the T_s achieves a degree of independence from T_a, an effect known as temperature regulation. Here, we measure oxygen consumption rate (?o ₂) 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 T_s was clamped at 15 ° C, the temperature that produces maximal ?o ₂ in this species, exposure to high diffusivity heliox increased mean ?o ₂ 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 T_a was clamped at 15 ° C and T_s was allowed to vary, exposure to heliox reduced T_s 1 ° C and increased ?o ₂ 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.     

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.     

Evidence that fasting can induce a selective loss of uncoupling protein from brown adipose tissue mitochondria of mice

The effects of fasting and refeeding on the concentration of uncoupling protein in brown adipose tissue mitochondria have been investigated in mice. Fasting mice for 48 h led to a large decrease in the total cytochrome oxidase activity of the interscapular brown fat pad. Mitochondrial GDP binding and the specific mitochondrial concentration of uncoupling protein also fell on fasting. After 24 h refeeding both GDP binding and the mitochondrial concentration of uncoupling protein were normalized, but there was no alteration in the total tissue cytochrome oxidase activity. Fasting appears to induce a selective loss of uncoupling protein from brown adipose tissue mitochondria, which is rapidly reversible on refeeding.     

Overexpression of plant uncoupling mitochondrial protein in transgenic tobacco increases tolerance to oxidative stress

An Arabidopsis thaliana cDNA clone encoding a plant uncoupling mitochondrial protein (AtPUMP1) was overexpressed in transgenic tobacco plants. Analysis of the AtPUMP1 mRNA content in the transgenic lines, determined by Northernblot, revealed variable levels of transgene expression. Antibody probing ofWestern blots of mitochondrial proteins from three independent transgenic lines showed significant accumulation of AtPUMP1 in this organelle. Overproduction of AtPUMP1 in transgenic tobacco plants led to a significantincrease in tolerance to oxidative stress promoted by exogenous hydrogen peroxide as compared to wild-type control plants. These results provide thefirst biological evidence for a role of PUMP in protection of plant cells against oxidative stress damage.     

Immunoelectron microscopical identification of the uncoupling protein in brown adipose tissue mitochondria

The distribution of the uncoupling protein (UCP) in brown adipocyte mitochondria of the hibernant Muscardinus avellanarius was obtained by ultrastructural immunocytochemistry. In both cryosections and sections of Lowicryl-embedded material UCP was localized in the mitochondrial cristae of brown adipocytes, but not in liver mitochondria. It should now be possible to easily identify the morphology of cells committed to BAT differentiation in the tissue as well as in cell culture.     

Increased uncoupling protein-2 and -3 gene expressions in skeletal muscle of STZ-induced diabetic rats

Streptozotocin (STZ)-induced diabetic animals are vulnerable to cold stress. Uncoupling proteins (UCPs) play an important role in regulating thermogenesis. We investigated the gene expressions of UCPs in brown adipose tissue (BAT), white adipose tissue (WAT), liver and gastrocnemius muscle of STZ-diabetic rats using Northern blot. UCP-1, -2 and -3 mRNA expressions in BAT were all remarkably lower in STZ-diabetic rats than those in control rats. Both UCP-2 and -3 gene expressions in gastrocnemius muscle were substantially elevated in STZ-diabetic rats and insulin treatment restored UCP gene expressions to normal levels. These results suggest that in STZ-diabetic rats, the overexpression of UCP-2 and UCP-3 in skeletal muscle provides a defense against hypothermogenesis caused by decreased UCPs in BAT.     

Energy conservation and dissipation in mitochondria isolated from developing tomato fruit of ethylene-defective mutants failing normal ripening: the effect of ethephon,a chemical precursor of ethylene

Alternative oxidase (AOX) and uncoupling protein (UCP) are present simultaneously in tomato fruit mitochondria. In a previous work, it has been shown that protein expression and activity of these two energy-dissipating systems exhibit large variations during tomato fruit development and ripening on the vine. It has been suggested that AOX and UCP could be responsible for the respiration increase at the end of ripening and that the cytochrome pathway could be implicated in the climacteric respiratory burst before the onset of ripening. In this study, the use of tomato mutants that fail normal ripening because of deficiencies in ethylene perception or production as well as the treatment of one selected mutant with a chemical precursor of ethylene have revealed that the bioenergetics of tomato fruit development and ripening is under the control of this plant hormone. Indeed, the evolution pattern of bioenergetic features changes with the type of mutation and with the introduction of ethylene into an ethylene-synthesis-deficient tomato fruit mutant during its induced ripening.     

Dialectics in carrier research: The ADP/ATP carrier and the uncoupling protein

A concise review is given of the research in our laboratory on the ADP/ATP carrier (AAC) and the uncoupling protein (UCP). Although homologous proteins, their widely different functions and contrasts are stressed. The pioneer role of research on the AAC, not only for the mitochondrial but also for other carriers, and the present state of their structure-function relationship is reviewed. The function of UCP as a highly regulated H⁺ carrier is described in contrast to the largely unregulated ADP/ATP exchange in AAC. General principles of carrier catalysis as derived from studies on the AAC and UCP are elucidated.     

Effects of floral thermogenesis on pollen function in Asian skunk cabbage Symplocarpus renifolius

The effects of temperature on pollen germination and pollen tube growth rate were measured in vitro in thermogenic skunk cabbage, Symplocarpus renifolius Schott ex Tzvelev, and related to floral temperatures in the field. This species has physiologically thermoregulatory spadices that maintain temperatures near 23°C, even in sub-freezing air. Tests at 8, 13, 18, 23, 28 and 33°C showed sharp optima at 23°C for both variables, and practically no development at 8°C. Thermogenesis is therefore a requirement for fertilization in early spring. The narrow temperature tolerance is probably related to a long period of evolution in flowers that thermoregulate within a narrow range.     

Structure-function relationships of the alternative oxidase of plant mitochondria: A model of the active site

A major characteristic of plant mitochondria is the presence of a cyanide-insensitive alternative oxidase which catalyzes the reduction of oxygen to water. Current information on the properties of the oxidase is reviewed. Conserved amino acid motifs have been identified which suggest the presence of a hydroxo-bridged di-iron center in the active site of the alternative oxidase. On the basis of sequence comparison with other di-iron center proteins, a structural model for the active site of the alternative oxidase has been developed that has strong similarity to that of methane monoxygenase. Evidence is presented to suggest that the alternative oxidase of plant mitochondria is the newest member of the class II group of di-iron center proteins.     

The role of different plant seedling shoots mitochondrial uncoupling systems in thermogenesis during low-temperature stress

A difference was found between the temperature of control and heat-treated winter wheat and pea seedlings shoots during low temperature stress. Functioning of three thermogenic mitochondrial systems was established: (i) alternative cyanide-resistant oxidase, (ii) plant uncoupling mitochondrial protein and (iii) stress protein CSP 310 and these three caused the higher temperature of winter wheat control shoots. In peas only two thermogenic systems, the alternative cyanide-resistant oxidase and plant uncoupling mitochondrial proteins were found.     

Structural requirements for the perception of ambient temperature signals in homeothermic heat production of skunk cabbage (Symlocarpus foetidus)

The spadix of skunk cabbage, Symplocarpus foetidus, is capable of maintaining an internal temperature of around 20 degrees C even when the ambient temperature drops to around 0 degrees C. To determine the crucial structure that is required for detection of ambient temperature signals, detailed measurements of the temperatures of the spadix were made under field conditions. The spadix temperature was well regulated even when the spathe or the leaf of the plant was removed. Furthermore, maintenance of the temperature of the central stalk at either 10 or 20 degrees C had no effect on the thermoregulation when the ambient temperature increased from 10 to 25 degrees C or decreased from 20 to 8 degrees C. Therefore, it seemed that the heat production in the spadix required neither the spathe, the leaf, nor the central stalk for perception of the external temperature signals. Finally, analysis of sugar composition in xylem exudates showed that the concentrations of sucrose, glucose, and fructose, all of which are potential energy sources of thermogenesis, did not change significantly at different ambient temperatures. It is concluded that the spadix is a unique organ in which the perception of ambient temperature signals and heat production occurs in S. foetidus.