Regulation of accumulation of antifreeze proteins in winter rye (Secale cereale L.)

Abstract

Winter rye acquires the ability to tolerate freezing when the plant is exposed to nonfreezing temperature. During cold acclimation, winter rye accumulates antifreeze proteins (AFPs) in the leaf apoplast where ice forms. The individual polypeptides with antifreeze activity were characterized under denaturing conditions to be similar to three classes of pathogenesis-related proteins; glucanase-like (GLPs), chitinase-like (CLPs), and thaumatin-like proteins (TLPs). The objectives of this study were two-fold; first, to characterize the winter rye antifreeze proteins in their native forms (Chapter Two), and second, to identify both hormonal and environmental regulators of AFP accumulation (Chapter Three and Four).

In characterizing the native state of these AFPs by native polyacrylamide gel electrophoresis, gel-filtration chromatography, immunoblotting, and immunoprecipitation (Chapter Two), my evidence suggests that the cold-induced rye AFPs form oligomeric complexes containing various combinations of GLP, CLP, and TLP, in addition to other unidentified proteins in vivo. The highest antifreeze activity was found in the apoplastic extract that is a mixture of all AFPs, followed by the complex containing GLP, CLP, and TLP, and then complexes consisting of GLP and TLP or GLP and CLP. By forming oligomeric complexes, antifreeze proteins may form larger surfaces to interact with ice or they may simply increase the mass of the protein bound to ice. In either case, the complexes of antifreeze proteins may inhibit ice growth and recrystallization more effectively than the individual polypeptides.

The cold-responsive AFPs are shown, for the first time to my knowledge, to be induced by drought (Chapter Three) and ethylene (Chapter Four) at a temperature noninducive for cold acclimation, which suggests that drought and ethylene may regulate the accumulation of AFPs involved in freezing tolerance in winter rye. The accumulation of AFPs in response to low temperature is abscisic acid (ABA)-independent (Chapter Three) because exogenous ABA did not induce accumulation of AFPs at warm temperature and application of the ABA biosynthesis inhibitor fluridone failed to prevent the accumulation of AFPs in cold-acclimated plants. Although drought (Chapter Three), ethylene (Chapter Four), salicylic acid (Chapter Four), and the low temperature parasitic fungus snow mold all induce the accumulation of 8-1.3-glucanase, chitinase, and thaumatin-like proteins immunologically similar to the low temperature-induced PR proteins in winter rye, the fact that only drought-, ethylene- and low temperature-induced PR proteins have antifreeze activity suggests that different genes encoding PR proteins may be expressed in response to different stimuli. One set of PR proteins genes responds to pathogens and salicylic acid (SA), whereas the second set is induced by low temperature, drought, and/or ethylene.