Antifreeze Proteins in Winter Rye Are Similar to Pathogenesis-Related Proteins

Abstract

The ability to control extracellular ice formation during freezing is critical to the survival of freezing-tolerant plants. Antifreeze proteins, which are proteins that have the ability to retard ice crystal growth, were recently identified as the most abundant apoplastic proteins in cold-acclimated winter rye (Secale cereale 1.) leaves. In the experiments reported here, amino-terminal sequence comparisons, immuno-cross-reactions, and enzyme activity assays all indicated that these antifreeze proteins are similar to members of three classes of pathogenesis-related proteins, namely, endochitinases, endo-p-l,3-glucanases, and thaumatin-like proteins. Apoplastic endochitinases and endo-p-l,3-glucanases that were induced by pathogens in freezing-sensitive tobacco did not exhibit antifreeze activity. Our findings suggest that subtle structural differences may have evolved in the pathogenesis-related proteins that accumulate at cold temperatures in winter rye to confer upon these proteins the ability to bind to ice.

Immunolocalization of Antifreeze Proteins in Winter Rye Leaves, Crowns, and Roots by Tissue Printing

“…Anti-GLP antiserum raised against the 32-kD polypeptide, used in a dilution of 1:10,000, recognized a polypeptide with a molecular mass of 32 kD (Fig. 2B), which was identified as a GLP by Hon et al (1995). With a dilution of 1:500, the anti-GLP antiserum also recognized a 35-kD polypeptide (Fig.…”

“…With a dilution of 1:500, the anti-GLP antiserum also recognized a 35-kD polypeptide (Fig. 3D, lane 4) also known to be a GLP (Hon et al, 1995). Anti-CLP antiserum recognized only one 35-kD polypeptide at a dilution of 1:1,000 ( Fig.…”

“…Briefly, apoplastic extracts from CA rye leaves were dialyzed against 20 m M NH,HCO,, pH 8.0, and stirred with colloidal chitin. The colloidal chitin was washed with 20 mM NH,HCO,, pH 5.2, to remove nonspecifically bound proteins before CLP (35 kD) was eluted with 20 mM acetic acid, pH 3.0 (Hon et al, 1995).…”

“…Magnification bar = 17 /urn. TLPs (Hon et al, 1995). Antisera were raised against one of each type of AFP, namely the 32-kD GLP, 35-kD CLP, and 25-kD TLP ( Fig.…”

“…These proteins were identified as GLPs, CLPs, and TLPs (Hon et al, 1995). Similar proteins, endo-P-1,3-glucanases, endochitinases, and TLPs, are known to be related to the mechanism for plant disease resistance (Carr and Klessig, 1989;Bol et al, 1990;Collinge et al, 1993;Stintzi et al, 1993) and are therefore classified as PR proteins.…”