Protoplast-Based Transient Expression Combined with Plant Cultivation Systems as a Valuable Tool for Floral Thermogenesis Studies in Aroids

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.

Rhythmic temperature control in a homeothermic plant: A field study of thermogenic Japanese skunk cabbage, Symplocarpus renifolius

The spadices of skunk cabbage (Symplocarpus renifolius) can generate heat and maintain an internal temperature of about 23°C, even when the ambient air temperature drops below freezing. In this study, we continuously measured the spadix temperatures of skunk cabbage together with ambient air temperatures during March 2019 in their natural habitat in northern Honshu on Japan’s main island. Using Fourier transform we showed that the temperature of the thermogenic spadix oscillates over a period of approximately 60 min. Our data suggest that Japanese skunk cabbage possesses a novel timekeeping mechanism that may be associated with a homeothermic control of this plant.

Comparison of Whole Plastome Sequences between Thermogenic Skunk Cabbage Symplocarpus renifolius and Nonthermogenic S. nipponicus (Orontioideae; Araceae) in East Asia

Symplocarpus, a skunk cabbage genus, includes two sister groups, which are drastically different in life history traits and thermogenesis, as follows: The nonthermogenic summer flowering S. nipponicus and thermogenic early spring flowering S. renifolius. Although the molecular basis of thermogenesis and complete chloroplast genome (plastome) of thermogenic S. renifolius have been well characterized, very little is known for that of S. nipponicus. We sequenced the complete plastomes of S. nipponicus sampled from Japan and Korea and compared them with that of S. renifolius sampled from Korea. The nonthermogenic S. nipponicus plastomes from Japan and Korea had 158,322 and 158,508 base pairs, respectively, which were slightly shorter than the thermogenic plastome of S. renifolius. No structural or content rearrangements between the species pairs were found. Six highly variable noncoding regions (psbC/trnS, petA/psbJ, trnS/trnG, trnC/petN, ycf4/cemA, and rpl3/rpl22) were identified between S. nipponicus and S. renifolius and 14 hot-spot regions were also identified at the subfamily level. We found a similar total number of SSR (simple sequence repeat) motifs in two accessions of S. nipponicus sampled from Japan and Korea. Phylogenetic analysis supported the basal position of subfamily Orontioideae and the monophyly of genus Symplocarpus, and also revealed an unexpected evolutionary relationship between S. nipponicus and S. renifolius.