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ENVIRONMENTAL STRESS AND ADAPTATION

Calcium Interacts with Antifreeze Proteins and Chitinase from Cold-Acclimated Winter Rye¹

Departments of Biology (S.K., M.G., C.M., L.A.B.) and Chemical Engineering (M.S., C.M.), University of Waterloo, Waterloo, Ontario N2L 3G1, Canada; and Department of Food Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada (A.G.M.)

During cold acclimation, winter rye (Secale cereale) plants accumulate pathogenesis-related proteins that are also antifreeze proteins (AFPs) because they adsorb onto ice and inhibit its growth. Although they promote winter survival in planta, these dual-function AFPs proteins lose activity when stored at subzero temperatures in vitro, so we examined their stability in solutions containing CaCl₂, MgCl₂, or NaCl. Antifreeze activity was unaffected by salts before freezing, but decreased after freezing and thawing in CaCl₂ and was recovered by adding a chelator. Ca²⁺ enhanced chitinase activity 3- to 5-fold in unfrozen samples, although hydrolytic activity also decreased after freezing and thawing in CaCl₂. Native PAGE, circular dichroism, and Trp fluorescence experiments showed that the AFPs partially unfold after freezing and thawing, but they fold more compactly or aggregate in CaCl₂. Ruthenium red, which binds to Ca²⁺-binding sites, readily stained AFPs in the absence of Ca²⁺, but less stain was visible after freezing and thawing AFPs in CaCl₂. We conclude that the structure of AFPs changes during freezing and thawing, creating new Ca²⁺-binding sites. Once Ca²⁺ binds to those sites, antifreeze activity, chitinase activity and ruthenium red binding are all inhibited. Because free Ca²⁺ concentrations are typically low in the apoplast, antifreeze activity is probably stable to freezing and thawing in planta. Ca²⁺ may regulate chitinase activity if concentrations are increased locally by release from pectin or interaction with Ca²⁺-binding proteins. Furthermore, antifreeze activity can be easily maintained in vitro by including a chelator during frozen storage.

² These authors contributed equally to the paper.

³ Permanent address: Department of Food Science and Nutrition, Osaka Shoin Women's University, 2–26, Hishiyanishi 4, Higashi-Osaka, 577–8550, Japan.

⁴ Permanent address: Departamento de Botánica, Facultad de Ciencias Naturales y Oceanográficas, Universidad de Concepción, Casilla 160–C, Concepción, Chile.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.038158.

^* Corresponding author; e-mail griffith{at}uwaterloo.ca; fax 519–746–0614.

Received December 19, 2003; returned for revision February 9, 2004; accepted February 9, 2004.

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