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.

Apex structures enhance water drainage on leaves.

The rapid removal of rain droplets at the leaf apex is critical for leaves to avoid damage under rainfall conditions, but the general water drainage principle remains unclear. We demonstrate that the apex structure enhances water drainage on the leaf by employing a curvature-controlled mechanism that is based on shaping a balance between reduced capillarity and enhanced gravity components. The leaf apex shape changes from round to triangle to acuminate, and the leaf surface changes from flat to bent, resulting in the increase of the water drainage rate, high-dripping frequencies, and the reduction of retention volumes. For wet tropical plants, such as Alocasia macrorrhiza, Gaussian curvature reconfiguration at the drip tip leads to the capillarity transition from resistance to actuation, further enhancing water drainage to the largest degree possible. The phenomenon is distinct from the widely researched liquid motion control mechanisms, and it offers a specific parametric approach that can be applied to achieve the desired fluidic behavior in a well-controlled way.

Pollination mutualism between Alocasia macrorrhizos (Araceae) and two taxonomically undescribed Colocasiomyia species (Diptera: Drosophilidae) in Sabah, Borneo.

Two taxonomically undescribed Colocasiomyia species were discovered from inflorescences of Alocasia macrorrhizos in Kota Kinabalu City, Sabah, Borneo, Malaysia. The aims of this study were to investigate the reproductive ecology of the flies and the plant, ascertain the importance of the flies as pollinators and examine the intimate association between flowering events and life history of the flies. We conducted sampling, observations and field pollination experiments. The flies were attracted by the odour of female-phase inflorescences in the early morning on the first day of anthesis. They fed, mated and oviposited in the inflorescences for 1 day. On the second day, the flies, covered with pollen grains, left the male-phase inflorescences for the next female-phase inflorescences. The immature forms of both fly species hatched, developed and pupated within the infructescences without damaging the fruits, and developed adults emerged when the mature infructescences dehisced. The flowering events and fly behaviours were well synchronized. In field pollination experiments, inflorescences bagged with a fine mesh (insect exclusion) produced almost no fruits, whereas those bagged with a coarse mesh (bee exclusion) produced as many fruits as the open-pollinated controls. These results indicate that these flies are the most efficient and specialised pollinators for their host, A. macrorrhizos. These flies, in return, depend on A. macrorrhizos for food and habitat through most of their life cycle. This study provides a deeper insight into the less recognised, highly intimate pollination mutualism between Araceae plants and Colocasiomyia flies.

Applying two-photon excitation fluorescence lifetime imaging microscopy to study photosynthesis in plant leaves.

This study investigates to which extent two-photon excitation (TPE) fluorescence lifetime imaging microscopy can be applied to study picosecond fluorescence kinetics of individual chloroplasts in leaves. Using femtosecond 860 nm excitation pulses, fluorescence lifetimes can be measured in leaves of Arabidopsis thaliana and Alocasia wentii under excitation-annihilation free conditions, both for the F (0)- and the F (m)-state. The corresponding average lifetimes are approximately 250 ps and approximately 1.5 ns, respectively, similar to those of isolated chloroplasts. These values appear to be the same for chloroplasts in the top, middle, and bottom layer of the leaves. With the spatial resolution of approximately 500 nm in the focal (xy) plane and 2 microm in the z direction, it appears to be impossible to fully resolve the grana stacks and stroma lamellae, but variations in the fluorescence lifetimes, and thus of the composition on a pixel-to-pixel base can be observed.