Department of Plant Physiology, Umeå Plant Science Centre, Umeå University, 90187, Umeå, Sweden.
Department Biologie I - Botanik, Ludwig-Maximilians-Universität München, Großhadernerstrasse 2-4, Planegg-Martinsried, 82152, Germany.
Plant J. 2020 Jul;103(1):459-473. doi: 10.1111/tpj.14723. Epub 2020 Mar 13.
Plant cells contain numerous subcompartments with clearly delineated metabolic functions. Mitochondria represent a very small fraction of the total cell volume and yet are the site of respiration and thus crucial for cells throughout all developmental stages of a plant's life. As such, their isolation from the rest of the cellular components is a basic requirement for numerous biochemical and physiological experiments. Although procedures exist to isolate plant mitochondria from different organs (i.e. leaves, roots, tubers, etc.), they are often tedious and do not provide resolution at the tissue level (i.e. phloem, mesophyll or pollen). Here, we present a novel method called IMTACT (isolation of mitochondria tagged in specific cell types), developed in Arabidopsis thaliana (Arabidopsis) that involves biotinylation of mitochondria in a tissue-specific manner using transgenic lines expressing a synthetic version of the OM64 (Outer Membrane 64) gene combined with BLRP and the BirA biotin ligase gene. Tissue specificity is achieved with cell-specific promoters (e.g. CAB3 and SUC2). Labeled mitochondria from crude extracts are retained by magnetic beads, allowing the simple and rapid isolation of highly pure and intact organelles from organs or specific tissues. For example, we could show that the mitochondrial population from mesophyll cells was significantly larger in size than the mitochondrial population isolated from leaf companion cells. To facilitate the applicability of this method in both wild-type and mutant Arabidopsis plants we generated a set of OM64-BLRP one-shot constructs with different selection markers and tissue-specific promoters.
植物细胞包含许多具有明确划分的代谢功能的亚区室。线粒体仅占细胞总体积的很小一部分，但却是呼吸作用的场所，因此对植物生命的所有发育阶段的细胞都至关重要。因此，从细胞的其他成分中分离出线粒体是进行许多生化和生理实验的基本要求。虽然有从不同器官（例如叶片、根、块茎等）分离植物线粒体的程序，但这些程序往往繁琐，并且不能在组织水平上提供分辨率（例如韧皮部、叶肉或花粉）。在这里，我们提出了一种称为 IMTACT（特定细胞类型中标记的线粒体的分离）的新方法，该方法在拟南芥（Arabidopsis）中开发，涉及使用表达 OM64（外膜 64）基因的合成版本的转基因系在线粒体中进行组织特异性生物素化与 BLRP 和 BirA 生物素连接酶基因。组织特异性是通过细胞特异性启动子（例如 CAB3 和 SUC2）实现的。来自粗提取物的标记线粒体被磁珠保留，允许从器官或特定组织中简单快速地分离高度纯净和完整的细胞器。例如，我们可以表明，来自叶肉细胞的线粒体群体的大小明显大于从叶片伴细胞中分离的线粒体群体。为了促进该方法在野生型和突变型拟南芥植物中的适用性，我们生成了一组具有不同选择标记物和组织特异性启动子的 OM64-BLRP 一次性构建体。
