Cryopreservation of Plant Mitochondria as a Tool for Protein Import or in Organello Protein Synthesis Studies.

Abstract

Cryopreserved chloroplasts and thylakoids have recently been proven to be suitable for protein import and integration assays. The possibility of recovering intact plant mitochondria after storage would also facilitate a wide range of investigations that are currently underway on the molecular biology of these organelles, e.g. mitochondrial transcription, RNA editing, in organello protein synthesis, and protein or transfer RNA import. Therefore, we addressed the question whether cryopreservation of isolated plant mitochondria was also possible. Tobacco (Nicotiana tabacum) or broad bean (Vicia faba) mitochondria were quick frozen and stored in liquid nitrogen in the presence of various concentrations of ethylene glycol as a cryoprotectant. After thawing, up to 90% of the mitochondria stored in 5 to 10% ethylene glycol appeared to retain an intact outer membrane and normal oxidative phosphorylation activity. Their ultrastructural aspect, observed by electron microscopy, was similar to that of freshly prepared mitochondria. Furthermore, efficient in organello protein synthesis was carried out with mitochondria stored in the presence of 7.5% ethylene glycol. Finally, the precursor of the [beta] subunit of the mitochondrial F1-ATPase from Nicotiana plumbaginifolia was successfully translocated into V. faba cryopreserved mitochondria and processed. These data demonstrate that plant mitochondria cryopreserved under the conditions described here remain functional and can be used for a variety of physiological and biochemical studies.