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ENVIRONMENTAL STRESS AND ADAPTATION TO STRESS

Cold-Active Winter Rye Glucanases with Ice-Binding Capacity^1,2

Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1

Extracellular pathogenesis-related proteins, including glucanases, are expressed at cold temperatures in winter rye (Secale cereale) and display antifreeze activity. We have characterized recombinant cold-induced glucanases from winter rye to further examine their roles and contributions to cold tolerance. Both basic -1,3-glucanases and an acidic -1,3;1,4-glucanase were expressed in Escherichia coli, purified, and assayed for their hydrolytic and antifreeze activities in vitro. All were found to be cold active and to retain partial hydrolytic activity at subzero temperatures (e.g. 14%–35% at –4°C). The two types of glucanases had antifreeze activity as measured by their ability to modify the growth of ice crystals. Structural models for the winter rye -1,3-glucanases were developed on which putative ice-binding surfaces (IBSs) were identified. Residues on the putative IBSs were charge conserved for each of the expressed glucanases, with the exception of one -1,3-glucanase recovered from nonacclimated winter rye in which a charged amino acid was present on the putative IBS. This protein also had a reduced antifreeze activity relative to the other expressed glucanases. These results support the hypothesis that winter rye glucanases have evolved to inhibit the formation of large, potentially fatal ice crystals, in addition to having enzymatic activity with a potential role in resisting infection by psychrophilic pathogens. Glucanases of winter rye provide an interesting example of protein evolution and adaptation aimed to combat cold and freezing conditions.

² This paper is dedicated to the memory of Marilyn Griffith.

³ Present address: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Mahmoud W.F. Yaish (myaish{at}uoguelph.ca).

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.081935.

^* Corresponding author; e-mail myaish{at}uoguelph.ca; fax 519–837–2075.

Received April 11, 2006; returned for revision June 23, 2006; accepted June 23, 2006.

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