Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Wenhui Lyu, Jennifer Selinski, Lu Li, David Day, Monika Murcha, James Whelan, Yan Wang

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16 Citations (Scopus)


Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH 2 ). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O 2 min -1 mg total mitochondrial protein -1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Original languageEnglish
Article numbere56627
JournalJournal of Visualized Experiments
Issue number131
Publication statusPublished - 5 Jan 2018


  • Arabidopsis thaliana
  • Biochemistry
  • Blue native-polyacrylamide gel electrophoresis (BN-PAGE)
  • Density gradients
  • Fractionation
  • Issue 131
  • Mitochondrial isolation
  • Purity
  • Respiratory measurements
  • Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
  • Western blotting


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