CRYOPRESERVATION OF PLANT-MITOCHONDRIA AS A TOOL FOR PROTEIN IMPORT OR IN ORGANELLO PROTEIN-SYNTHESIS STUDIES

Cryopreserved chloroplasts and thylakoids have recently been proven to be suitable for protein import and integration assays. The possibilit y of recovering intact plant mitochondria after storage would also fac ilitate a wide range of investigations that are currently underway on the molecular biology of these organelles, e.g. mitochondrial transcri ption, RNA editing, in organello protein synthesis, and protein or tra nsfer RNA import. Therefore, we addressed the question whether cryopre servation of isolated plant mitochondria was also possible. Tobacco (N icotiana tabacum) or broad bean (Vicia faba) mitochondria were quick f rozen and stored in liquid nitrogen in the presence of various concent rations of ethylene glycol as a cryoprotectant. After thawing, up to 9 0% of the mitochondria stored in 5 to 10% ethylene glycol appeared to retain an intact outer membrane and normal oxidative phosphorylation a ctivity. Their ultrastructural aspect, observed by electron microscopy , was similar to that of freshly prepared mitochondria. Furthermore, e fficient in organello protein synthesis was carried out with mitochond ria stored in the presence of 7.5% ethylene glycol. Finally, the precu rsor of the beta subunit of the mitochondrial F-1-ATPase from Nicotian a plumbaginifolia was successfully translocated into V. faba cryoprese rved mitochondria and processed. These data demonstrate that plant mit ochondria cryopreserved under the conditions described here remain fun ctional and can be used for a variety of physiological and biochemical studies.