Tissue‐specific isolation of Arabidopsis/plant mitochondria – IMTACT (isolation of mitochondria tagged in specific cell types)

Abstract

SummaryPlant 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.

Tagging and catching: rapid isolation and efficient labeling of organelles using the covalent Spy-System in planta

“…Several plant organelle isolation protocols were published in the last years, mostly facilitating the isolation of one type of organelle via affinity-based purification methods [36,37,[39][40][41][42]. In these protocols, either epitope or protein Tags (e.g.…”

“…The addition of such small tags does not add new biological functions and in most cases fusion proteins will retain normal structure and function [ 39 , 63 ]. But in most of the reported isolation protocols the Tags are not only fused to a targeting sequence for organelle labelling but also to a fluorescent protein (GFP) to allow the localization and visualization of the constructs [ 37 , 39 – 42 ]. Nevertheless, antibodies are expensive and as above-mentioned, affinity-based non-covalent systems have limitations in their field of application.…”

“…beads) followed by washing steps, and deliver organelles with high purity that can be used directly for subsequent analysis. Several epitope-tagging protocols achieving organelle isolation via co-IP have been published, focusing exclusively on one type of organelle [36][37][38][39][40][41][42].…”

“…Split-isopeptide Catcher-Tag systems could be used as specific, covalent alternatives to the non-covalent, affinity-based organelle isolation techniques, such as the biotin-streptavidin based isolation of nuclei or mitochondria published as INTACT [37] and IMTACT method [42], the Strep-Tactin based isolation of tagged mitochondria [40], the immunogenic chloroplast isolation via YFP [36], or epitope-tagging approaches for mitochondria isolation [38,39,41].…”

“…The INTACT and IMPTACT method allow the isolation of nuclei or mitochondria via protein-protein interaction, with the help of biotinylated targeting proteins that bind to streptavidin coated beads when mixed [37,42]. To achieve biotinylation of targeting fusion constructs, a bacterial biotin ligase needs to be co-expressed.…”