Chitinase genes responsive to cold encode antifreeze proteins in winter cereals

Antifreeze proteins similar to two different chitinases accumulate during c old acclimation in winter rye (Secale cereale). To determine whether these cold-responsive chitinases require post-translational modification to bind to ice, cDNAs coding for two different full-length chitinases were isolated from a cDNA library produced from cold-acclimated winter rye leaves. CHT9 is a 1,193-bp clone that encodes a 31.7-kD class I chitinase and CHT46 is a 998-bp clone that codes for a 24.8-kD class II chitinase. Chitinase-antifr eeze proteins purified from the plant were similar in mass to the predicted mature products of CHT9 and CHT46, thus indicating that there was little c hemical modification of the amino acid sequences in planta. To confirm thes e results, the mature sequences of CHT9 and CHT46 were expressed in Escheri chia coli and the products of both cDNAs modified the growth of ice. Transc ripts of both genes accumulated late in cold acclimation in winter rye. Sou thern analysis of winter rye genomic DNA indicated the presence of a small gene family homologous to CHT46. In hexaploid wheat, CHT46 homologs marred to the homeologous group 1 chromosomes and were expressed in response to co ld and drought. We conclude that two novel cold-responsive genes encoding c hitinases with ice-binding activity may have arisen in winter rye and other cereals through gene duplication.