Simple preparation of plant epidermal tissue for laser microdissection and downstream quantitative proteome and carbohydrate analysis.
Simple preparation of plant epidermal tissue for laser microdissection and downstream quantitative proteome and carbohydrate analysis.
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DOI：
10.3389/fpls.2015.00194
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发表时间：
2015
影响因子：
5.6
通讯作者：
Voigt CA
中科院分区：
文献类型：
--
作者：
Falter C;Ellinger D;von Hülsen B;Heim R;Voigt CA
表皮细胞向外的细胞壁和相关的质膜代表了植物防御入侵病原体的第一层。细胞壁的修饰和防御结构的形成是植物防御的决定性因素。精确分离这些应力诱导的结构将允许对调节机制和细胞壁适应进行具体分析。然而，还没有提供从模式植物拟南芥（Arabidopsis thaliana）大规模制备表皮组织的方法，该方法将允许对完整的激光显微切割的表皮防御结构进行蛋白质组和细胞壁分析。我们发展了胶带-液体盖玻片技术（ACT），用于A.也可用于草叶。该方法与随后的染色技术兼容，以可视化应力相关的细胞壁结构，这些结构通过激光显微切割（LM）结合激光压力分解从表皮组织层中精确分离。我们成功地证明，这些特定的表皮组织样品可用于定量下游蛋白质组和细胞壁分析。ACT的发展，简单的叶表皮制备和兼容性，LM和下游定量分析开辟了新的可能性，在精确检查的压力和病原体相关的细胞壁结构在表皮细胞。由于所开发的组织处理技术同样适用于A. thaliana，包括与白粉病的相互作用的完善的模型病理系统，可以进行研究，以确定植物-微生物相互作用的主要调节机制，并将其潜在地推广到作物育种中。
The outwardly directed cell wall and associated plasma membrane of epidermal cells represent the first layers of plant defense against intruding pathogens. Cell wall modifications and the formation of defense structures at sites of attempted pathogen penetration are decisive for plant defense. A precise isolation of these stress-induced structures would allow a specific analysis of regulatory mechanism and cell wall adaption. However, methods for large-scale epidermal tissue preparation from the model plant Arabidopsis thaliana, which would allow proteome and cell wall analysis of complete, laser-microdissected epidermal defense structures, have not been provided. We developed the adhesive tape – liquid cover glass technique (ACT) for simple leaf epidermis preparation from A. thaliana, which is also applicable on grass leaves. This method is compatible with subsequent staining techniques to visualize stress-related cell wall structures, which were precisely isolated from the epidermal tissue layer by laser microdissection (LM) coupled to laser pressure catapulting. We successfully demonstrated that these specific epidermal tissue samples could be used for quantitative downstream proteome and cell wall analysis. The development of the ACT for simple leaf epidermis preparation and the compatibility to LM and downstream quantitative analysis opens new possibilities in the precise examination of stress- and pathogen-related cell wall structures in epidermal cells. Because the developed tissue processing is also applicable on A. thaliana, well-established, model pathosystems that include the interaction with powdery mildews can be studied to determine principal regulatory mechanisms in plant–microbe interaction with their potential outreach into crop breeding.
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