Department of Plant Physiology, Zentrum für Molekularbiologie der Pflanzen, Tübingen University, 72076 Tübingen, Germany.
Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany.
Development. 2021 Dec 1;148(23). doi: 10.1242/dev.199820. Epub 2021 Dec 8.
Hydrophobic cell wall depositions in roots play a key role in plant development and interaction with the soil environment, as they generate barriers that regulate bidirectional nutrient flux. Techniques to label the respective polymers are emerging, but are efficient only in thin roots or sections. Moreover, simultaneous imaging of the barrier constituents lignin and suberin remains problematic owing to their similar chemical compositions. Here, we describe a staining method compatible with single- and multiphoton confocal microscopy that allows for concurrent visualization of primary cell walls and distinct secondary depositions in one workflow. This protocol permits efficient separation of suberin- and lignin-specific signals with high resolution, enabling precise dissection of barrier constituents. Our approach is compatible with imaging of fluorescent proteins, and can thus complement genetic markers or aid the dissection of barriers in biotic root interactions. We further demonstrate applicability in deep root tissues of plant models and crops across phylogenetic lineages. Our optimized toolset will significantly advance our understanding of root barrier dynamics and function, and of their role in plant interactions with the rhizospheric environment.
根中疏水性细胞壁沉积物在植物发育和与土壤环境相互作用中起着关键作用，因为它们形成了调节养分双向流动的屏障。标记各自聚合物的技术正在出现，但仅在细根或切片中有效。此外，由于其化学成分相似，同时对木质素和栓质这两种屏障成分进行成像仍然存在问题。在这里，我们描述了一种与单光子和多光子共聚焦显微镜兼容的染色方法，该方法允许在一个工作流程中同时可视化初生细胞壁和不同的次生沉积物。该方案允许以高分辨率有效地分离出木质素和栓质特异性信号，从而能够精确剖析屏障成分。我们的方法与荧光蛋白的成像兼容，因此可以补充遗传标记，或有助于剖析生物根系相互作用中的屏障。我们进一步证明了它在植物模型和不同系统发育谱系作物的深层根组织中的适用性。我们优化的工具集将极大地促进我们对根屏障动态和功能的理解，以及它们在植物与根际环境相互作用中的作用的理解。
