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Genes encoding chitinase-antifreeze proteins are regulated by cold and expressed by all cell types in winter rye shoots

Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada
Physiologia Plantarum (Impact Factor: 3.26). 08/2001; 112(3):359-371. DOI: 10.1034/j.1399-3054.2001.1120309.x
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ABSTRACT One group of antifreeze proteins (AFPs) is composed of two chitinases that accumulate in the apoplast of winter rye leaves during cold acclimation. In this study, the 28- and 35-kDa chitinase-AFPs were localized in nonacclimated and cold-acclimated rye leaves by immunoelectron microscopy with an antiserum produced against the purified winter rye 35-kDa chitinase-AFP. In cold-acclimated winter rye leaves, labelled chitinase-AFPs were abundant in the walls of epidermal, parenchymal sheath and mesophyll cells and xylem vessels, while less label was present in walls of vascular parenchyma cells. In contrast, chitinase labelling was essentially absent in the nonacclimated cells except in xylem vessels. As shown by RNA blotting, the transcripts of chitinase-AFPs accumulated to a high level in rye leaves during cold acclimation, to a lesser extent in crowns and were not detectable in roots. mRNA transcripts of the 28-kDa chitinase-AFP were localized in rye leaves by in situ hybridization. The chitinase-AFP transcripts were found in the same cell types as the protein itself. We conclude that all metabolically active cell types in cold-acclimated winter rye leaves and crowns are able to synthesize chitinase-AFPs and secrete them into adjacent cell walls, where they may interact with ice to delay its propagation through the plant and modify its growth.

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    • "Interestingly, recent studies support that cold signals are closely linked to pathogen resistance. A group of pathogenesis-related (PR) proteins, including endochitinases, b-1,3-glucanases, and thaumatin-like proteins, is synthesized after exposure to low temperatures in overwintering grasses (Hon et al., 1995; Pihakaski-Maunsbach et al., 2001). The PR proteins not only play a role in pathogen resistance but are also able to resist freezing (Snider et al., 2000). "
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    • "To become freezing-tolerant, overwintering cereals must first undergo a complex adjustment of plant morphology and metabolism known as cold acclimation . As winter rye (Secale cereale) plants acclimate to cold temperatures, they secrete pathogenesis-related (PR) proteins, including endochitinases, b-1,3-gluca- nases, and thaumatin-like proteins into intercellular spaces and the xylem (Hon et al., 1995; Antikainen et al., 1996; Pihakaski-Maunsbach et al., 1996, 2001). These apoplastic PR proteins are thought to provide a preemptive defense against psychrophilic fungi, such as snow molds (Snider et al., 2000), that are able to infect overwintering cereals at low temperatures and reduce their winter survival (Ergon et al., 1998). "
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    ABSTRACT: 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 Ca(2+)-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 Ca(2+)-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 Ca(2+)-binding proteins. Furthermore, antifreeze activity can be easily maintained in vitro by including a chelator during frozen storage.
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    • "Recent studies have shown that a class of pathogenesis-related (PR) proteins such as endo-chitinase and b-1,3-glucanase accumulated in cold-acclimated rye apoplast [11,16]. Cold-induced accumulation of this class of PR proteins has been correlated with enhanced resistance to pathogens [11] [16]. Interestingly, cold-induced PR proteins from rye showed antifreeze activity and are thought to be bifunctional in cold acclimation. "
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