The process of cold acclimation is not well understood, but evidence is accruing for a genetic role in this process. This project will focus on one aspect of gene regulation during cold acclimation, and the relationship of increased protein expression in the adjustment of metabolism to low temperature in plants. A member of a class of proteins that becomes more abundant in plants and is therefore upregulated in during exposure to low temperature will be characterized in terms of structural organization of the protein(s) and gene(s), and regulation in response to low temperature and other related environmental stresses. Previous work has demonstrated that the synthesis of these proteins in leaf tissue is closely associated with cold acclimation (cold acclimation protein CAP). Using preparative electrophoresis techniques, sufficient amounts of one of these proteins have been obtained (CAP79) to determine amino acid sequences of CNBr fragments using a gas-phase sequenator to separate phenylthiohydantoin-amino acid derivatives. The amino acid sequences, show that CAP79 is a 70kDa heat shock cognate. The sequence information was also used in the synthesis of oligonucleotides to probe a lambda Zap II cDNA library and to amplify specific genomic sequences by polymerase chain reaction (PCR). Polyclonal antisera to this group of proteins have been modified. Using these antibodies inframe cDNAs will be isolated the identity of clones selected by oligos from the expression library will be confirmed. The cDNAs will be characterized, and used to isolate additional genomic sequences, with the intent to characterize their structure and regulation by environmental factors that influence cold tolerance levels. Isolation of all parts of these genes including the regulatory regions is the goal. Cell fractionation and immunocytochemical staining studies are planned to determine the cellular localization of this low temperature upregulated protein(s). Experiments are described that may suggest associative interactions of CAP79 with other cellular components, and potential for repair of cryoinjury to cold sensitive proteins.//
