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.     

Molecular identity of uncoupling proteins in thermogenic skunk cabbage

Thermogenic skunk cabbage has been reported to have two types of uncoupling protein (UCP), a typical 6-transmembrane (TM) SrUCPA and an atypical 5-TM SrUCPB. To verify further the role of SrUCPs in thermogenic skunk cabbage, we examined the molecular identity of SrUCPs in more detail. Both mRNA and genomic analyses supported the presence of SrUCPA, but not SrUCPB. Furthermore, SrUCP protein purified from spadix mitochondria was identified as SrUCPA by mass spectrometry. These results clearly indicate that SrUCPA is the major expressed UCP in skunk cabbage, and the presence of atypical SrUCPB is unlikely to be associated with thermogenesis of skunk cabbage.     

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.     

Changes in the composition of xylem sap during development of the spadix of skunk cabbage (Symplocarpus foetidus)

The spadix of skunk cabbage, Symplocarpus foetidus, is thermogenic and maintains an internal temperature of around 20 degrees C even when the ambient air temperature drops below freezing. This homeothermic heat production is observed only during the stigma stage, and thereafter ceases at the male stage when pollen is shed. To clarify the regulatory mechanism by which the stigma stage-specific heat production occurs in the spadix, sugars, organic acids, and amino acids in xylem sap were analyzed and compared with those of post-thermogenic plants. Interestingly, no significant difference was observed in the total volume of xylem sap per fresh weight of the spadix between thermogenic (31.2+/-24.7 microl h(-1) g(-1)) and post-thermogenic (50.5+/-30.4 microl h(-1) g(-1)) plants. However, concentrations of sugars (sucrose, glucose, and fructose), organic acids (malate and succinate), and amino acids (Asp, Asn, Glu, Gln, Gly, and Ala) in xylem sap decreased remarkably in post-thermogenic plants. Our results indicate that the composition of the xylem sap differs during the development of the spadix of S. foetidus.     

Changes in the Composition of Xylem Sap during Development of the Spadix of Skunk Cabbage (Symplocarpus foetidus)

The spadix of skunk cabbage, Symplocarpus foetidus, is thermogenic and maintains an internal temperature of around 20 °C even when the ambient air temperature drops below freezing. This homeothermic heat production is observed only during the stigma stage, and thereafter ceases at the male stage when pollen is shed. To clarify the regulatory mechanism by which the stigma stage-specific heat production occurs in the spadix, sugars, organic acids, and amino acids in xylem sap were analyzed and compared with those of post-thermogenic plants. Interestingly, no significant difference was observed in the total volume of xylem sap per fresh weight of the spadix between thermogenic (31.2±24.7 μl h^?1g^?1) and post-thermogenic (50.5±30.4 μl h^?1g^?1) plants. However, concentrations of sugars (sucrose, glucose, and fructose), organic acids (malate and succinate), and amino acids (Asp, Asn, Glu, Gln, Gly, and Ala) in xylem sap decreased remarkably in post-thermogenic plants. Our results indicate that the composition of the xylem sap differs during the development of the spadix of S. foetidus.     

Temperature-triggered periodical thermogenic oscillations in skunk cabbage (Symplocarpus foetidus)

The natural occurrence of temperature-triggered and light-independent thermogenic oscillation in the spadix of skunk cabbage, Symplocarpus foetidus, was discovered. The identified thermogenic oscillator had an accurate periodical cycle (ca. 60 min per cycle) that apparently responded to an increase or decrease in the spadix temperature with a threshold of less than 0.9 degrees C. Neither a constant ambient air temperature nor transient changes in the ambient air temperature within 10 min (19 degrees C --> 15 degrees C --> 19 degrees C) induced the temperature oscillation in the spadix. Moreover, the periodical cycles were independent of the weight of the spadix (2.5-9.2 g) and the amplitudes of the temperature oscillations were correlated with the magnitude of the changes in the spadix temperatures. These results imply that periodical temperature oscillations in the spadix of S. foetidus possess a quantitative regulatory process that involves a temperature sensation and subsequent heat production. Based on these results, we propose a time-dependent thermogenic oscillatory model that acts as a precise thermal regulator under dynamic environmental temperature changes.     

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.     

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.     

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     

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.     

AEROBIOLOGY OF SYMPLOCARPUS FOETIDUS: INTERACTIONS BETWEEN THE SPATHE AND SPADIX

The aerobiology of the skunk cabbage, Symplocarpus foetidus (l .) Nutt., is examined as a factor contributing to efficient pollination and temperature regulation around the spadix. Field measurements show that the compass orientation of the asymmetrical opening of the spathe is random, while wind tunnel studies reveal that similar patterns of airflow are generated around the spadix regardless of the orientation of the spathe opening to the direction of airflow. Temperature measurements within a model of the inflorescence reveal that airflow around the spathe effectively maintains heat generated by the spadix, even at airflow speeds of 1.5 m/s. These results are discussed as exaptations of the spathe for pollination and for temperature regulation in sub-freezing weather.     

Interaction of purified alternative oxidase from thermogenic Arum maculatum with pyruvate

Plant alternative oxidase (AOX) activity in isolated mitochondria is regulated by carboxylic acids, but reaction and regulatory mechanisms remain unclear. We show that activity of AOX protein purified from thermogenic Arum maculatum spadices is sensitive to pyruvate and glyoxylate but not succinate. Rapid, irreversible AOX inactivation occurs in the absence of pyruvate, whether or not duroquinol oxidation has been initiated, and is insensitive to duroquinone. Our data indicate that pyruvate stabilises an active conformation of AOX, increasing the population of active protein in a manner independent of reducing substrate and product, and are thus consistent with an exclusive effect of pyruvate on the enzyme’s apparent V_max.     

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.     

Expression of uncoupling protein and alternative oxidase depends on lipid or carbohydrate substrates in thermogenic plants

Thermogenesis, in which cellular respiratory activity is considerably stimulated, requires mitochondrial uncoupling protein (UCP) in mammals and an alternative oxidase (AOX) in plants. Here, we show that the genes for both proteins are expressed in thermogenic plants, but the type correlates with the respiratory substrate. A novel gene termed PsUCPa encoding a variant of UCP was specifically expressed in thermogenic flowers of Philodendron selloum, which uses lipids as substrates. However, a gene termed DvAOX encoding for AOX protein was expressed in thermogenic flowers of Dracunculus vulgaris, which presumably uses carbohydrates as substrates. These findings suggest that cellular metabolism is a major determinant in selective expression of appropriate thermogenic genes in plants.     

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.     

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.     

Transgenic tobacco (Nicotiana tabacum L.) plants with increased expression levels of mitochondrial NADP+-dependent isocitrate dehydrogenase: evidence implicating this enzyme in the redox activation of the alternative oxidase

Many metabolic reactions are coupled to NADPH in the mitochondrial matrix, including those involved in thiol group reduction. One enzyme linked to such processes is mitochondrial NADP+-dependent isocitrate dehydrogenase (mtICDH; EC 1.1.1.42), although the precise role of this enzyme is not yet known. Previous work has implicated mtICDH as part of a biochemical mechanism to reductively activate the alternative oxidase (AOX). We have partially purified mtICDH from tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) cell suspension cultures and localized this to a 46-kDa protein on SDS-PAGE, which was verified by peptide sequencing. In the inflorescence of the aroid Sauromatum guttatum Schott (voodoo lily), mtICDH appears to be developmentally regulated, presenting maximal specific activity during the thermogenic period of anthesis when the capacity for AOX respiration is also at its peak. Transgenic tobacco plants were generated that overexpress mtICDH and lines were obtained that demonstrated up to a 7-fold increase in mtICDH activity. In isolated mitochondria, this resulted in a measurable increase in the reductive activation of AOX in comparison with wild type. When examined in planta in response to citrate feeding, a strong conversion of AOX from its oxidized to its reduced form was observed in the transgenic line. These data support the hypothesis that mtICDH may be a regulatory switch involved in tricarboxylic acid cycle flux and the reductive modulation of AOX.     

In vivo redox state of the ubiquinone pool in the spadices of the thermogenic skunk cabbage, <Emphasis Type="Italic">Symplocarpus renifolius</Emphasis>

In vivo ubiquinone (UQ) reduction levels were determined in thermogenic stigma and post-thermogenic male stages of spadices of the skunk cabbage, Symplocarpus renifolius. In contrast to Arum maculatum, in which the UQ pool is almost fully reduced during thermogenesis, the reduction levels of UQ9 and UQ10 were not affected by the thermogenic status or developmental stage of individual S. renifolius spadices. Moreover, these levels were controlled within the ranges 40–75% and 35–60%, respectively. These results suggest that the reduction state of the UQ pool per se is not primarily involved in thermoregulation in S. renifolius.