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.

Floral traits and pollination ecology of European Arum hybrids.

Hybridisation is common in plants and can affect the genetic diversity and ecology of sympatric parental populations. Hybrids may resemble the parental species in their ecology, leading to competition and/or gene introgression; alternatively, they may diverge from the parental phenotypes, possibly leading to the colonisation of new ecological niches and to speciation. Here, we describe inflorescence morphology, ploidy levels, pollinator attractive scents, and pollinator guilds of natural hybrids of Arum italicum and A. maculatum (Araceae) from a site with sympatric parental populations in southern France to determine how these traits affect the hybrid pollination ecology. Hybrids were characterised by inflorescences with a size and a number of flowers more similar to A. italicum than to A. maculatum. In most cases, hybrid stamens were purple, as in A. maculatum, and spadix appendices yellow, as in A. italicum. Hybrid floral scent was closer to that of A. italicum, but shared some compounds with A. maculatum and comprised unique compounds. Also, the pollinator guild of the hybrids was similar to that of A. italicum. Nevertheless, the hybrids attracted a high proportion of individuals of the main pollinator of A. maculatum. We discuss the effects of hybridisation in sympatric parental zones in which hybrids exhibit low levels of reproductive success, the establishment of reproductive barriers between parental species, the role of the composition of floral attractive scents in the differential attraction of pollinators and in the competition between hybrids and their parental species, and the potential of hybridisation to give rise to new independent lineages.

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.

Different molecular bases underlie the mitochondrial respiratory activity in the homoeothermic spadices of Symplocarpus renifolius and the transiently thermogenic appendices of Arum maculatum.

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.

Identification of a gene for pyruvate-insensitive mitochondrial alternative oxidase expressed in the thermogenic appendices in Arum maculatum.

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.

Ubiquitous expression of a gene encoding for uncoupling protein isolated from the thermogenic inflorescence of the dead horse arum Helicodiceros muscivorus.

Uncoupling proteins (UCPs) are a family of mitochondrial inner membrane proteins that have been implicated in heat production in mammalian cells. The inflorescences of several members of the arum lily family (Araceae) have also been shown to produce heat during flowering, but the involvement of UCP-mediated heat production in plants is not known. In this work a gene has been isolated termed HmUCPa that encodes for a putative uncoupling protein from Helicodiceros muscivorus, a highly thermogenic arum lily. RT-PCR analysis revealed that the expression of HmUCPa was ubiquitously found, both in thermogenic male florets and appendix, and the non-thermogenic female florets, spathe and club-shaped organs of the spadix. These results suggest that HmUCPa is not primarily involved in organ-specific heat production in H. muscivorus.