Type II fish antifreeze protein accumulation in transgenic tobacco does not confer frost resistance.

Type II fish antifreeze protein (AFP) is active in both freezing point depression and the inhibition of ice recrystallization. This extensively disulfide-bonded 14 kDa protein was targeted for accumulation in its pro- and mature forms in the cytosol and apoplast of transgenic tobacco plants. Type II AFP gene constructs under control of a duplicate cauliflower mosaic virus 35S promoter, both with and without a native plant transit peptide sequence, were introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. AFP did not accumulate in the cytosol of transgenic plants, but active AFP was present as 2% the total protein present in the apoplast. Plant-produced AFP was the same size as mature Type II AFP isolated from fish, and was comparable to wild-type AFP in thermal hysteresis activity and its effect on ice crystal morphology. Field trials conducted in late summer on R1 generation transgenic plants showed similar AFP accumulation in plants under field conditions at levels suitable for large-scale production: but no difference in frost resistance was observed between transgenic and wild-type plants during the onset of early fall frosts.

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.