Gene expression and metabolite levels converge in the thermogenic spadix of skunk cabbage.

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.

Degradation of mitochondrial alternative oxidase in the appendices of Arum maculatum.

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.

Lipoprotein profile and lipid metabolism of PXB-cells®, human primary hepatocytes from liver-humanized mice: proposal of novel in vitro system for screening anti-lipidemic drugs.

We investigated lipid metabolism in PXB-cells, which are human primary hepatocytes isolated from liver-humanized mice, and HepG2 and HuH-7 human hepatoma cell lines. Lipoprotein levels were higher in PXB-cells than in the 2 other cell lines, and PXB-cells mainly released triglycerides and cholesterol as very low density lipoprotein (VLDL), similar to actual liver tissue, whereas the major lipoprotein released from the 2 hepatoma cell lines was LDL. RT-PCR analysis demonstrated that the gene expression levels of apolipoprotein B100 (ApoB100), the apolipoprotein of VLDL/LDL, were similar in PXB-cells and HepG2 cells, while the overexpression of ApoC2, ApoC3, and ApoE, which are components of VLDL, but not LDL, was observed in PXBcells. A protein immunoassay revealed that ApoB100 levels secreted from PXB-cells and HuH-7 cells were similar; however, ApoC3 levels were higher in PXB-cells than in the two other cell lines. We also examined the anti-lipidemic activities of fenofibrate using this assay system. Fenofibrate suppressed lipoprotein production from PXB-cells in a dose-dependent manner mainly by activating the ß-oxidation pathway. These results suggest that PXB-cells produce high levels of lipoproteins and are suitable for screening anti-lipidemic agents.

Thioredoxin o-mediated reduction of mitochondrial alternative oxidase in the thermogenic skunk cabbage Symplocarpus renifolius.

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.

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 (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.

Metabolic interplay between cytosolic phosphoenolpyruvate carboxylase and mitochondrial alternative oxidase in thermogenic skunk cabbage, Symplocarpus renifolius.

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.

The biochemical basis for thermoregulation in heat-producing flowers.

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.

Transcriptome analysis of thermogenic Arum concinnatum reveals the molecular components of floral scent production.

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.

The oxygen supply to thermogenic flowers.

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.