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

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 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.

Chitinase Gene Expression in Response to Environmental Stresses inArabidopsis thaliana: Chitinase Inhibitor Allosamidin Enhances Stress Tolerance

“…Antifreeze activity has been found in class I and class II chitinases from winter rye (Secale cereale). [31][32][33][34] These classes of chitinases were also induced in salt-adapted cells of tobacco (Nicotiana tabacum) 14) and winged bean (Psophocarpus tetragonolobus).…”

Cellular Response of Pea Plants to Cadmium Toxicity: Cross Talk between Reactive Oxygen Species, Nitric Oxide, and Calcium

“…condition and the cross talk processes occurring in different stress conditions. Some PRs have been described to be up-regulated under abiotic stress (Tateishi et al, 2001;Stressmann et al, 2004), which suggests the existence of common effectors with biotic stress. The analysis of the expression of PRs and HSP71.2 (for heat shock protein 71.2) in pea plants treated with Cd was carried out by semiquantitative RT-PCR and is shown in Fig.…”

“…An induction of chitinase activity has been observed in pea plants by Cd (Metwally et al, 2003) and other heavy metals like lead and arsenic (Békésiová et al, 2008) and also by osmotic stress (Tateishi et al, 2001), low temperature (Stressmann et al, 2004), and wounding (Wu and Bradford, 2003). Chitinases are probably components of the general defense response program of cells, although they can also play unknown specific roles in heavy metal stress.…”

Characterization of Cold-Responsive Extracellular Chitinase in Bromegrass Cell Cultures and Its Relationship to Antifreeze Activity

“…However, BiCHT1 products expressed in both E. coli and bromegrass had chitinase activity but did not affect the growth of ice crystals. Stressmann et al (2004) reported that when winter rye apoplast crude extract containing chitinase/AFP was frozen in the presence of 20 to 200 mM of CaCl 2 , the antifreeze activity was reduced by 30% and was recovered by adding a chelator. This effect was specific to high concentrations (20-200 mM) of CaCl 2 , and antifreeze activity before freeze-thaw cycles was unaffected by the presence of CaCl 2 .…”