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.

中文翻译：

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拟南芥/植物线粒体的组织特异性分离-IMTACT（在特定细胞类型中标记的线粒体的分离）。

植物细胞包含许多具有明确描述的代谢功能的小室。线粒体仅占总细胞量的很小一部分，但仍是呼吸作用的部位，因此对于植物生命中所有发育阶段的细胞都至关重要。这样，它们与其余细胞成分的分离是许多生化和生理学实验的基本要求。尽管存在从不同器官（即叶，根，块茎等）中分离植物线粒体的程序，但它们通常很繁琐，并且不能在组织水平（即韧皮部，叶肉或花粉）提供分辨率。在这里，我们提出了一种在拟南芥中开发的称为IMTACT（分离标记在特定细胞类型中的线粒体）的新方法拟南芥（Arabidopsis），涉及通过表达OM64（外膜64）基因的合成版本，结合BLRP和BirA生物素连接酶基因的转基因品系，以组织特异性方式对线粒体进行生物素化。组织特异性可通过细胞特异性启动子（例如CAB3和SUC2）实现）。粗提物中标记的线粒体被磁珠保留，从而可以从器官或特定组织中简单快速地分离出高纯度和完整的细胞器。例如，我们可以证明，来自叶肉细胞的线粒体群体的大小显着大于从叶伴侣细胞中分离的线粒体群体的大小。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和组织特异性启动子的OM64–BLRP单发构建体。