Characterisation of a fall protein of sugar pine and detection of its homologue associated with frost hardiness of western white pine needles

A 19-kDa protein of sugar pine (named Pinl I; i.e., protein I of Pinuslambertiana Dougl.) was detected in increasing amounts in the fall. This protein was separated by SDS–PAGE and also by two-dimensional gel electrophoresis. Pinl I was composed of two acidic isoforms. This protein was subjected to N-terminal amino acid sequence analysis. The two isoforms had an identical N-terminal amino acid sequence. The N-terminal peptide was synthesized and purified, and the purity of the synthetic peptide was greater than 95%. The peptide was conjugated to a carrier protein, keyhole limpet hemocyanin (KLH). Rabbits were immunized with peptide–KLH and the antipeptide antibody was purified from the crude antisera by immunoaffinity chromatography. The antibody was shown to bind specifically to PinI I. This anti-Pin I I antibody was used in a Western immunoblot to detect the homologues of Pin1 1 in two other five-needled pines: western white pine (Pinusmonticola Dougl.; named Pinm III) and eastern white pine (Pinusstrobus L.). The antibody was also used to monitor the seasonal variation of Pinm III in western white pine needles. Pinm III was shown to be associated with overwintering of western white pine seedlings. A significant correlation was observed between the frost hardiness of western white pine foliage and the content of Pinm III.