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

• Published in: The Plant journal : for cell and molecular biology 2020-07, Vol.103 (1), p.459-473
• Main Authors: Boussardon, Clément, Przybyla‐Toscano, Jonathan, Carrie, Chris, Keech, Olivier
• Format: Article
• Language: English
• Subjects: Arabidopsis; Arabidopsis - cytology; Arabidopsis - metabolism; Arabidopsis - physiology; Arabidopsis Proteins; Arabidopsis Proteins - metabolism; Arabidopsis Proteins - physiology; Beads; Biotin; Biotinylation; Cell size; cell types; Developmental stages; Leaves; Life Sciences; magnetic beads; Mesophyll; Mitochondria; Mitochondria - metabolism; Mitochondria - physiology; Mitochondrial Membrane Transport Proteins; Mitochondrial Membrane Transport Proteins - metabolism; Mitochondrial Membrane Transport Proteins - physiology; Organ Specificity; Organelles; Organs; Plant cells; Plant Leaves; Plant Leaves - metabolism; Plant Leaves - physiology; Plant mitochondria; Plant Roots; Plant Roots - metabolism; Plant Roots - physiology; Plant Tubers; Plant Tubers - metabolism; Plant Tubers - physiology; Plants, Genetically Modified; Pollen; Promoters; purification method; technical advance; Tissues; Tubers
• ISSN: 0960-7412; 1365-313X; 1365-313X
• DOI: 10.1111/tpj.14723

Summary 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. Significance Statement We propose a new method for ‘transformable’ plant species that allows the user to rapidly isolate highly pure mitochondria from any tissues in any given genotype provided that a tissue‐specific promoter is used.