Cabbage cryoprotectin is a member of the nonspecific plant lipid transfer protein gene family

We have recently purified a protein (cryoprotectin) from the leaves of: col d-acclimated cabbage (Brassica oleracea) to electrophoretic homogeneity, wh ich protects thylakoids isolated from the leaves of nonacclimated spinach ( Spinacia oleracea) from freeze-thaw damage. Sequencing of cryoprotectin sho wed the presence of at least three isoforms of WAX9 proteins, which belong to the class of nonspecific lipid transfer proteins. Antibodies raised agai nst two synthetic peptides derived from the WAX9 proteins recognized a band of approximately 10 kD in western blots of crude cryoprotectin preparation s. This protein and the cryoprotective activity could be precipitated from solution by the antiserum. We show further that cryoprotectin is structural ly and functionally different from WAX9 isolated from the surface wax of ca bbage leaves. WAX9 has lipid transfer activity for phosphatidylcholine, but no cryoprotective activity. Cryoprotectin, on the other hand, has cryoprot ective, but no lipid transfer activity. The cryoprotective activity of cryo protectin was strictly dependent on Ca2+ and Mn2+ and could be inhibited by chelating agents, whereas the lipid transfer activity of WAX9 was higher i n the presence of ethylenediaminetetraacetate than in the presence of Ca2and Mn2+.