PDF(Pubmed)
Abstract：
Arabidopsis root is a classic model system in plant cell and molecular biology. The sensitivity of plant roots to local environmental perturbation challenges data reproducibility and incentivizes further optimization of imaging and phenotyping tools. Here we present RoPod, an easy-to-use toolkit for low-stress live time-lapse imaging of Arabidopsis roots. RoPod comprises a dedicated protocol for plant cultivation and a customizable 3D-printed vessel with integrated microscopy-grade glass that serves simultaneously as a growth and imaging chamber. RoPod reduces impact of sample handling, preserves live samples for prolonged imaging sessions, and facilitates application of treatments during image acquisition. We describe a protocol for RoPods fabrication and provide illustrative application pipelines for monitoring root hair growth and autophagic activity. Furthermore, we showcase how the use of RoPods advanced our understanding of plant autophagy, a major catabolic pathway and a key player in plant fitness. Specifically, we obtained fine time resolution for autophagy response to commonly used chemical modulators of the pathway and revealed previously overlooked cell type-specific changes in the autophagy response. These results will aid a deeper understanding of the physiological role of autophagy and provide valuable guidelines for choosing sampling time during end-point assays currently employed in plant autophagy research.
摘要：
拟南芥根是植物细胞和分子生物学中的经典模型系统。植物根对局部环境扰动的敏感性挑战了数据的可重复性，并激励了成像和表型工具的进一步优化。这里我们介绍RoPod,一个易于使用的工具包，用于拟南芥根的低应力实时延时成像。RoPod包含用于植物培养的专用协议和可定制的3D打印容器，具有集成的显微镜级玻璃，同时用作生长和成像室。RoPod减少了样品处理的影响，保存实时样本以进行长时间的成像，并促进图像采集期间的治疗应用。我们描述了RoPods制造的协议，并提供了用于监测根毛生长和自噬活性的说明性应用管道。此外，我们展示了使用RoPods如何提高我们对植物自噬的理解，植物健康的主要分解代谢途径和关键参与者。具体来说,我们获得了对该途径常用化学调节剂的自噬反应的良好时间分辨率,并揭示了以前被忽视的细胞类型特异性自噬反应变化.这些结果将有助于更深入地了解自噬的生理作用，并为目前在植物自噬研究中使用的终点测定期间选择采样时间提供有价值的指南。
