A generalized method for transfecting root epidermis uncovers endosomal dynamics in Arabidopsis root hairs
A generalized method for transfecting root epidermis uncovers endosomal dynamics in Arabidopsis root hairs
复制标题
DOI：
10.1111/j.1365-313x.2007.03139.x
复制
发表时间：
2007-07-01
期刊：
影响因子：
7.2
通讯作者：
Blatt, Michael Robert
中科院分区：
文献类型：
--
作者：
Campanoni, Prisca;Sutter, Jens Uwe;Blatt, Michael Robert
通过使用共聚焦显微镜以及瞬时基因表达，在分析许多结构蛋白的细胞功能方面的进展已经加速。在过去的几年中，已经开发了几种瞬时表达的方法，但是除了一些易处理的物种和组织之外，它们的应用已经看到了有限的成功。我们已经开发了一种简单而有效的方法来可视化荧光蛋白在拟南芥根表皮共培养的幼苗与发根农杆菌。该方法同样适用于其他物种，包括小蓟属的瞬时基因表达，并可以与支持分子和生化分析相结合。该方法有望在不干扰植物生长的情况下，为根毛中的膜动力学、细胞发育和极性生长的研究提供显着的优势。由于该方法专门针对根表皮，它也提供了一个强大的工具，以接近根际相互作用的问题，如离子运输和养分获取。作为原理的证明，我们用质膜的荧光标记物（NpPMA 2-GFP，Nicotiana plumbaginifolia L.）质膜H+-ATP酶2）、内质网（YFP-HDEL）和高尔基体（唾液酸转移酶-GFP）来追踪它们在生长的拟南芥根毛和表皮中的分布。结果表明，在拟南芥根毛中，高尔基体的运动比以前报道的烟草叶表皮细胞的运动快，这与顶端生长细胞的高分泌动力学相一致;它们在细胞质内显示出比在烟草叶表皮中发现的更弥散的内质网模式，他们证实了先前的发现，即在生长的根毛顶端，内质网呈极化分布。
Progress in analysing the cellular functions of many structural proteins has accelerated through the use of confocal microscopy together with transient gene expression. Several methods for transient expression have been developed in the past few years, but their application has seen limited success beyond a few tractable species and tissues. We have developed a simple and efficient method to visualize fluorescent proteins in Arabidopsis root epidermis using co-cultivation of seedlings with Agrobacterium rhizogenes. The method is equally suitable for transient gene expression in other species, including Thellungiella, and can be combined with supporting molecular and biochemical analyses. The method promises significant advantages for study of membrane dynamics, cellular development and polar growth in root hairs without interference in the development of the plant. Since the method targets specifically the root epidermis, it also offers a powerful tool to approach issues of root-rhizosphere interactions, such as ion transport and nutrient acquisition. As a proof of principle, we carried out transfections with fluorescent markers for the plasma membrane (NpPMA2-GFP, Nicotiana plumbaginifolia L. Plasma Membrane H+-ATPase 2), the endoplasmic reticulum (YFP-HDEL), and the Golgi apparatus (sialyl transferase-GFP) to trace their distribution in growing Arabidopsis root hairs and epidermis. The results demonstrate that, in Arabidopsis root hairs, movement of the Golgi is faster than previously reported for tobacco leaf epidermal cells, consistent with the high secretory dynamics of the tip growing cell; they show a pattern to the endoplasmic reticulum within the cytoplasm that is more diffuse than found in tobacco leaf epidermis, and they confirm previous findings of a polarized distribution of the endoplasmic reticulum at the tip of growing root hairs.