Establishing an efficient protoplast transient expression system for investigation of floral thermogenesis in aroids
Establishing an efficient protoplast transient expression system for investigation of floral thermogenesis in aroids
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DOI：
10.1007/s00299-021-02806-1
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发表时间：
2021-10
影响因子：
6.2
通讯作者：
H. Maekawa;Miyabi Otsubo;Mitsuhiko P. Sato;Tomoko Takahashi;Koichiro Mizoguchi;Daiki Koyamatsu;
中科院分区：
文献类型：
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作者：
H. Maekawa;Miyabi Otsubo;Mitsuhiko P. Sato;Tomoko Takahashi;Koichiro Mizoguchi;Daiki Koyamatsu;
花的产热是吸引传粉者的重要生殖策略。我们开发了必要的生物学工具，研究花产热利用两种产热的天南星，Symplocarpus renifolius和海芋odora.AbstractAroids包含许多物种具有强烈的产热能力，在他们的花序。一些基因已被提出参与这些物种的产热，但缺乏基因功能分析的生物学工具。在这项研究中，我们的目的是建立一个基于原生质体的瞬时表达（PTE）系统的研究产热aroids。最初，我们专注于臭鼬甘蓝（Symplocarpus renifolius），因为它能够产生强烈而持久的热量。从盆栽植株和茎尖培养植株中高效分离出叶原生质体。1.0× 105/g鲜重），半数以上的原生质体成功转染。使用这个PTE系统，我们确定了三个线粒体能量耗散蛋白，SrAOX，SrUCPA和SrNDA 1，融合到绿色荧光蛋白（GFP）的蛋白定位。这三种GFP融合蛋白定位于叶原生质体中MitoTracker染色的线粒体中，尽管表达SrUCPA-GFP的原生质体中的绿色荧光颗粒显著增大。最后，评估PTE系统是否建立在S. renifolius适用于产热型天南星科植物的花组织，S. renifolius和另一种产热的海芋（Alocasia odora）。虽然成功地从花序的几个组织中分离出了原生质体，但PTE系统仅适用于从A.芳香花我们开发的系统有可能被广泛应用于花序以及产热的天南星属植物的叶片，因此可能是一个有用的生物学工具，研究花的产热。
Key messageFloral 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 renifoliusandAlocasia odora.AbstractAroids 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 ofS. renifoliusis applicable for floral tissues of thermogenic aroids, inflorescences ofS. renifoliusand 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) ofA. odorainflorescences. 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.