Future Development Division, Advanced Engineering Services Co., Ltd., 1-6-1 Takezono, Tsukuba, Ibaraki 305-0032, Japan.
Faculty of Liberal Arts and Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan.
Life Sci Space Res (Amst). 2018 Aug;18:42-51. doi: 10.1016/j.lssr.2018.05.005. Epub 2018 May 21.
The mechanism by which gravity controls the polar transport of auxin, a plant hormone regulating multiple physiological processes in higher plants, remains unclear, although an important role of PIN proteins as efflux carriers/facilitators in polar auxin transport is suggested. We are going to study the effect of microgravity on the polar transport of auxin, focusing on the cellular localization of its efflux carrier, PsPIN1 in etiolated pea seedlings and ZmPIN1a in etiolated maize seedlings grown under microgravity conditions on the International Space Station (ISS) using immunohistochemical analyses according to space experimental plans (Ueda, 2016). To obtain adequate results regarding the cellular localization of functional proteins, prolonged chemical fixation processes as well as chemical fixatives should be well-matched to the properties of functional proteins as antigens since experimental analyses will be performed on the ground after keeping samples for a long duration on the ISS. As a result of ground verification, clear detection of the cellular localization of PsPIN1 and ZmPIN1a immunohistochemically was successful based on the results of several kinds of chemical fixation tested, even when etiolated pea and maize seedlings were fixed by immersion in chemical fixative for a long duration. The addition of 0.1% (w/v) Nonidet P-40 to chemical fixative composed of 50% (v/v) ethanol and 5% (v/v) acetic acid or that of 50% (v/v) methanol and 5% (v/v) acetic acid has led to a significant improvement in the immunohistochemical detection of PsPIN1 or ZmPIN1a. These chemical fixatives were also shown to be storage-stable for a long time before use. In this study, adequate chemical fixatives and fixation protocols were developed, which can be used to detect localization of PsPIN1 and ZmPIN1a proteins in young etiolated pea and maize seedlings, respectively, using anti PsPIN1 and ZmPIN1a antibodies. These protocols can be used in spaceflight experiments to investigate the effects of the microgravity environment on the ISS on PIN protein localization in pea and maize seedlings.
重力如何控制植物激素生长素的极性运输机制尚不清楚，尽管 PIN 蛋白作为生长素极性运输的外排载体/促进剂的重要作用已被提出。我们将根据空间实验计划（Ueda，2016），使用免疫组织化学分析方法，研究微重力对生长素极性运输的影响，重点研究其外排载体 PsPIN1 在黄化豌豆幼苗和 ZmPIN1a 在微重力条件下生长的黄化玉米幼苗中的细胞定位。为了获得有关功能蛋白细胞定位的充分结果，化学固定过程以及化学固定剂应与功能蛋白作为抗原的特性相匹配，因为在国际空间站（ISS）上长时间保存样品后，将在地面上进行实验分析。经过地面验证，根据几种化学固定方法的测试结果，成功地通过免疫组织化学方法清晰地检测到 PsPIN1 和 ZmPIN1a 的细胞定位，即使将黄化豌豆和玉米幼苗长时间浸泡在化学固定剂中进行固定也是如此。向由 50%（v/v）乙醇和 5%（v/v）乙酸组成的化学固定剂中添加 0.1%（w/v）非离子型去垢剂 P-40 或 50%（v/v）甲醇和 5%（v/v）乙酸可显著改善 PsPIN1 或 ZmPIN1a 的免疫组织化学检测。这些化学固定剂在使用前也表现出长时间的储存稳定性。在这项研究中，开发了足够的化学固定剂和固定方案，可分别使用抗 PsPIN1 和 ZmPIN1a 抗体检测年轻黄化豌豆和玉米幼苗中 PsPIN1 和 ZmPIN1a 蛋白的定位。这些方案可用于太空飞行实验，以研究微重力环境对 ISS 上豌豆和玉米幼苗中 PIN 蛋白定位的影响。
