Accumulation of type I fish antifreeze protein in transgenic tobacco is cold-specific

Expression of fish antifreeze protein (AFP) genes in plants is a possible means of increasing their frost resistance and freeze tolerance. Initial work involved transfer into tobacco of an AFP gene from winter flounder which codes for the alanine-rich, alpha-helical Type I AFP. Plants were transformed with a gene construct in which the preproAFP cDNA was inserted between the cauliflower mosaic virus 19S RNA promoter and the nopaline synthetase polyadenylation site. Although transgenic plants produced AFP mRNA, no AFP was detected on western blots. Re-evaluation of AFP expression in these transgenic plants showed that AFP accumulated to detectable levels only after exposure of the plant to cold. Extracts of plants incubated at 4 degrees C for 24 h contained a protein which co-migrated with winter flounder proAFP and was cross-reactive to Type I AFP antisera. Two other minor protein bands of slightly higher apparent M(r) also cross-reacted with the antisera and are thought to represent processing intermediates. The proAFP was unique to the transgenic plants and was absent in extracts taken prior to cold exposure. AFP levels increased over the first 48 h of cold incubation then remained stable. Since the alpha-helix content of Type I AFP has been shown to decrease markedly at warmer temperatures, we postulate that Type I AFP stability in transgenic plants is dependent on its secondary structure.