Floral thermogenesis has been found in dozens of primitive seed plants and the reproductive organs in these plants produce heat during anthesis. Thus, characterization of the molecular mechanisms underlying flowering is required to fully understand the role of thermogenesis, but this aspect of thermogenic plant development is largely unknown. In this study, extensive database searches and cloning experiments suggest that thermogenic skunk cabbage (Symplocarpus renifolius), which is a member of the family Araceae, possesses two genes encoding phosphatidyl ethanolamine-binding proteins (PEBP), FLOWERING LOCUS T (SrFT) and MOTHER OF FT AND TFL1 (SrMFT). Functional analyses of SrFT and SrMFT in Arabidopsis indicate that SrFT promotes flowering, whereas SrMFT does not. In S. renifolius, the stage- and tissue-specific expression of SrFT was more evident than that of SrMFT. SrFT was highly expressed in flowers and leaves and was mainly localized in fibrovascular tissues. In addition, microarray analysis revealed that, within floral tissues, SrFT was co-regulated with the genes associated with cellular respiration and mitochondrial function, including ALTERNATIVE OXIDASE gene proposed to play a major role in floral thermogenesis. Taken together, these data suggest that, among the PEBP genes, SrFT plays a role in flowering and floral development in the thermogenic skunk cabbage.

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Floral thermogenesis in plants plays a significant role in their reproductive function. Thermogenic aroids constitute a large family in highly thermogenic angiosperms, many of which possess intense heat-producing abilities. Several genes have been proposed to be involved in floral thermogenesis of aroids, but the biological tools to identify the functions of those genes at cellular and molecular levels are lacking. Among the many thermogenic aroids, we focused on skunk cabbage (Symplocarpus renifolius) because of its ability to produce intense, durable heat and small aboveground parts compared with other thermogenic aroids. In this study, leaf protoplasts were isolated from potted and shoot tip-cultured skunk cabbage plants and used to develop transient assay systems. The isolation protocol included an additional, sucrose gradient centrifugation step, which yielded high-purity protoplasts from both types of plants. The isolation and transfection efficiency of the protoplasts exceeded 1.0 × 105/g fresh weight and 50%, respectively, in both potted and shoot tip-cultured plants. Using this protoplast-based transient expression (PTE) system, we determined the protein localization of three mitochondrial energy-dissipating proteins, SrAOX, SrUCPA, and SrNDA1, fused to green fluorescent protein (GFP). In skunk cabbage leaf protoplasts, these three GFP-fused proteins were localized in MitoTracker-stained mitochondria. However, the green fluorescent particles in protoplasts expressing SrUCPA-GFP were enlarged compared with those in protoplasts expressing SrAOX-GFP and SrNDA1-GFP. Our PTE system is a powerful tool for functional gene analysis not only in thermogenic aroids but also in non-thermogenic aroids.