Establishing an efficient protoplast transient expression system for investigation of floral thermogenesis in aroids.

KEY MESSAGE: Floral thermogenesis is an important reproductive strategy for attracting pollinators. We developed essential biological tools for studying floral thermogenesis using two species of thermogenic aroids, Symplocarpus renifolius and Alocasia odora. Aroids contain many species with intense heat-producing abilities in their inflorescences. Several genes have been proposed to be involved in thermogenesis of these species, but biological tools for gene functional analyses are lacking. In this study, we aimed to develop a protoplast-based transient expression (PTE) system for the study of thermogenic aroids. Initially, we focused on skunk cabbage (Symplocarpus renifolius) because of its ability to produce intense as well as durable heat. In this plant, leaf protoplasts were isolated from potted and shoot tip-cultured plants with high efficiency (ca. 1.0 × 105/g fresh weight), and more than half of these protoplasts were successfully transfected. Using this PTE system, we determined the protein localization of three mitochondrial energy-dissipating proteins, SrAOX, SrUCPA, and SrNDA1, fused to green fluorescent protein (GFP). These three GFP-fused proteins were localized in MitoTracker-stained mitochondria in leaf protoplasts, although the green fluorescent particles in protoplasts expressing SrUCPA-GFP were significantly enlarged. Finally, to assess whether the PTE system established in the leaves of S. renifolius is applicable for floral tissues of thermogenic aroids, inflorescences of S. renifolius and another thermogenic aroid (Alocasia odora) were used. Although protoplasts were successfully isolated from several tissues of the inflorescences, PTE systems worked well only for the protoplasts isolated from the female parts (slightly thermogenic or nonthermogenic) of A. odora inflorescences. Our developed system has a potential to be widely used in inflorescences as well as leaves in thermogenic aroids and therefore may be a useful biological tool for investigating floral thermogenesis.

Characterization of two PEBP genes, SrFT and SrMFT, in thermogenic skunk cabbage (Symplocarpus renifolius).

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.