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

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Abstract

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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.

Abstract

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.

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Fig. 1
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taken from the spathe. c Protoplasts isolated from the male (upper), sterile (middle), and female part (lower). The value in parenthesis indicates the isolation efficiency of protoplasts in each part. Few protoplasts were isolated from the appendix. d Bright-field (DIC) and fluorescent microphotographs (GFP) were collected on the Zeiss AxioImager A2 microscope equipped with appropriate filter sets

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All data generated or analyzed during this study are included in this published article and its supplementary information files.

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Acknowledgements

We thank Yoko Katayama (University of Miyazaki) for their assistance in the cultivation of skunk cabbage plants. This work was supported by a Grant-in-Aid for Scientific Research (grant no. 20H02917 to Y.I.-I., and grant nos. 18H02169 and 20K21282 to T. I.), by the Takeda Science Foundation, and by the Sasakawa Scientific Research Grant from The Japan Science Society.

Funding

This work was supported by a Grant-in-Aid for Scientific Research (grant no. 20H02917 to Y.I.-I., and grant nos. 18H02169 and 20K21282 to T. I.), by the Takeda Science Foundation, and by the Sasakawa Scientific Research Grant from The Japan Science Society.

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Author notes

  1. Haruhiko Maekawa and Miyabi Otsubo contributed equally to this study.

Authors and Affiliations

  1. Department of Agricultural and Environmental Sciences, Faculty of Agriculture, University of Miyazaki, 1-1 Gakuenkibanadai-nishi, Miyazaki, 889-2192, Japan

    Haruhiko Maekawa, Miyabi Otsubo, Koichiro Mizoguchi, Daiki Koyamatsu, Takehito Inaba & Yasuko Ito-Inaba

  2. Department of Frontier Research and Development, Kazusa DNA Research Institute, 2-6-7 Kazusakamatari, Kisarazu, Chiba, 292-0818, Japan

    Mitsuhiko P. Sato

  3. Department of Biochemistry and Molecular Biology, Graduate School of Science and Engineering, Saitama University, Shimo-Okubo 255, Sakura-ku, Saitama, 338-8570, Japan

    Tomoko Takahashi

  4. Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, 980-8577, Japan

    Yasuko Ito-Inaba

Authors
  1. Haruhiko Maekawa
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  2. Miyabi Otsubo
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  3. Mitsuhiko P. Sato
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  7. Takehito Inaba
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Contributions

HM, MO, KM, DK, TI, and YII conducted the experiments. YII and TI analyzed the data. YII, MPS, and TT contributed to the experimental design. YII wrote the manuscript. All authors have read and approved the manuscript.

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Correspondence to Yasuko Ito-Inaba.

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Communicated by Kinya Toriyama.

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Maekawa, H., Otsubo, M., Sato, M.P. et al. Establishing an efficient protoplast transient expression system for investigation of floral thermogenesis in aroids. Plant Cell Rep 41, 263–275 (2022). https://doi.org/10.1007/s00299-021-02806-1

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