Study of hydrophobic proteins and protein complexes involved in cryopreservation of banana (Musa spp.) meristems
Vertommen, Annelies; Carpentier, S.C; Remmerie, N; Witters, E; Swennen, R.; Panis, B (2008)
Agrifood Research Working papersMTT:n selvityksiä
Cryopreservation (or conservation at ultra-low temperatures (-196 °C)) of meristems is the best method to preserve the banana (Musa spp.) diversity safely. A prerequisite for successful application of this technique is the avoidance of irreversible cell membrane damage caused by the formation of intracellular ice crystals. Ice crystallization can only be prevented through a reduction of the cellular water content (= dehydration) to the strict minimum. Acclimation is often essential to survive such a low water content (1). Membrane proteins likely play an important role in the acquisition of dehydration tolerance. As such, a study of the change in the membrane proteome of banana meristems of a dehydration tolerant and sensitive variety will expand the current knowledge of the physiology underlying cryo- and dehydration tolerance. This information will be used to improve the efficiency of current cryopreservation protocols. One approach to study the meristem proteome is by its separation through two-dimensional electrophoresis (2DE) (2). However, highly hydrophobic membrane proteins largely escape from "classical" 2DE analysis because of their low abundance and their limited solubility in neutral detergent/urea lysis buffers (3). Low abundance can be solved by enriching fractionation steps. Physical, as well as chemical methods have been described. For banana meristems, fractionation was executed by differential centrifugation in order to obtain a microsomal fraction. However, further chemical enrichment of this fraction was needed. Seigneurin-Berny et al. (1999) developed a simple technique to extract highly hydrophobic proteins from chloroplast membranes (4). The method is based on the differential solubilization of membrane proteins in chloroform/methanol mixtures. We optimized this extraction method for banana meristems by determining the ideal proportion of chloroform/methanol to be used as well as by testing alternative precipitation methods and different acrylamide concentrations. Subsequently, the optimised method was applied to search for differential proteins of a dehydration tolerant and sensitive banana variety. An alternative technique to study hydrophobic proteins, which also gives information about the organization of protein complexes and/or protein-protein interactions, is Blue native PAGE (BN-PAGE). This technique, originally developed by Schägger and von Jagow (5) allows separation of protein complexes and hydrophobic proteins in the mass range of 10 kDa to 1 MDa. This technique comprises (i) the use of mild, neutral detergents for solubilisation and (ii) the application of Coomassie Brilliant Blue G 250 to give a negative charge to proteins and protein complexes. This will allow separation according to molecular mass. The technique was optimized and applied to study protein complexes present in the microsomal fraction of banana meristems. Preliminary results are presented.
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