Graduate School of Science and Engineering, Teikyo University, 1-1 Toyosatodai, Utsunomiya, Tochigi, 320-8551, Japan.
Advanced Instrumental Analysis Center, Teikyo University, 1-1 Toyosatodai, Utsunomiya, Tochigi, 320-8551, Japan.
J Plant Res. 2022 Mar;135(2):377-386. doi: 10.1007/s10265-021-01360-x. Epub 2021 Nov 23.
Laser microdissection (LMD) is used for isolating specific regions or single cells from a wide variety of tissue samples under direct microscopic observation. The LMD method enables the harvest of the cells of interest in a region or specific cells for several analyses, such as DNA/RNA analysis, proteomics, metabolomics, and other molecular analyses. Currently, LMD is used to study various biological events at the tissue or cellular level; it has been used in a wide range of research fields. In this report, we describe techniques for isolating different tissues/specific cells from cryosections of incised Arabidopsis flowering stems by LMD for spatiotemporal quantitative plant hormone analysis. The endogenous indole-3-acetic acid levels in the epidermis/cortex, vascular bundles, and pith of Arabidopsis flowering stems were approximately 19.0 pg mm, 33.5 pg mm, and 3.32 pg mm, respectively, and these endogenous levels were altered spatiotemporally after incision. We also analyzed jasmonic acid from LMD-isolated cells and showed that the endogenous levels increased in the range of approximately 200-3,500 pg mm depending on the tissue and region at 1 h after incision and then decreased to less than 100 pg mm or undetectable levels at 24 h after incision. Quantitative analyses of phytohormones, including jasmonic acid-related molecules, gibberellin, abscisic acid, and cytokinins, could also be performed using the same cell samples. These results showed that spatiotemporal changes in plant hormones could be quantitatively and simultaneously analyzed by LMD-isolated cells from cryosections with positional information. The combination of quantitative analysis by liquid chromatography-mass spectrometry (LC-MS) and sampling by the LMD method provides a comprehensive and quantitative understanding of spatiotemporal changes in plant hormones in a region- and tissue-specific manner. Therefore, LMD-LC-MS methods will contribute to our understanding of the physiological events that control the process of plant growth and development.
激光显微切割（LMD）用于在直接显微镜观察下从各种组织样本中分离特定区域或单个细胞。LMD 方法能够在一个区域或特定细胞中收获感兴趣的细胞，以进行多种分析，例如 DNA/RNA 分析、蛋白质组学、代谢组学和其他分子分析。目前，LMD 用于研究组织或细胞水平的各种生物学事件；它已在广泛的研究领域中使用。在本报告中，我们描述了通过 LMD 从切割的拟南芥开花茎的冷冻切片中分离不同组织/特定细胞的技术，用于时空定量植物激素分析。拟南芥开花茎的表皮/皮层、维管束和髓中的内源性吲哚-3-乙酸水平分别约为 19.0 pg mm、33.5 pg mm 和 3.32 pg mm，这些内源性水平在切割后具有时空变化。我们还分析了从 LMD 分离的细胞中的茉莉酸，并表明内源性水平在切割后 1 小时根据组织和区域在大约 200-3500 pg mm 的范围内增加，然后降低至 100 pg mm 或以下或 24 小时后不可检测水平小时。使用相同的细胞样本还可以对植物激素（包括茉莉酸相关分子、赤霉素、脱落酸和细胞分裂素）进行定量分析。这些结果表明，通过 LMD 从冷冻切片中分离的细胞，具有位置信息，可以对植物激素的时空变化进行定量和同时分析。液相色谱-质谱联用（LC-MS）定量分析与 LMD 方法取样相结合，提供了一种全面和定量的区域和组织特异性的植物激素时空变化理解。因此，LMD-LC-MS 方法将有助于我们理解控制植物生长和发育过程的生理事件。
