Purification, Characterization and cDNA Cloning of the 200 kDa Protein Induced by Cold Acclimation in Wheat

We have purified to homogeneity the 200 kDa protein inducedspecifically by low temperature in wheat (Triticum aestivumL.). The boiling solubility of the protein has been used asa main step in the purification procedure. Amino acid compositionindicates that the 200 kDa has a compositional bias for glycine(11.4%), threonine (13.3%), and alanine (22.0%). Using oligonucleotideprobes, we have isolated a clone (pWcs200) from a cold-acclimatedwinter wheat cDNA library. Northern analysis demonstrated thatthe expression of the corresponding gene was specifically upregulatedby low temperature. Southern analysis showed that the gene organizationand the relative copy number were identical in two cultivarsdiffering in their capacity to develop freezing tolerance. Proteinsequence and immunological analyses indicate that this proteinshares similar features with the 50 kDa protein induced duringcold acclimation of wheat. The two proteins are boiling-soluble,and possess similar repeated elements. These elements may beimportant for the development of freezing tolerance. We haveshown that the 200 kDa protein is the largest member of a familyof immunologically-related cold-induced proteins in wheat. Expressionof pWcs200 in E. coli yielded a product of around 200 kDa, indicatingthat the clone contains most of the coding region for this protein. (Received August 18, 1992; Accepted October 14, 1992)