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 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.

Figure 1.

Effect of cations and freeze-thaw (FT) cycles on antifreeze activity. A, Apoplastic proteins in solutions containing 1 to 200 mm CaCl₂ at pH 5.5 (0FT), were assayed for antifreeze activity, and then frozen and thawed three times (3FT) and reassayed. The absence of Ca²⁺ was achieved by dialyzing proteins in 5 mm EDTA. B, Apoplastic proteins were dialyzed in 20 or 200 mm CaCl₂, MgCl₂, or NaCl at pH 5.5, then frozen and thawed three times. C, To determine if the effect of Ca²⁺ was reversible, apoplastic proteins were frozen and thawed in the presence of 20 or 200 mm CaCl₂, then dialyzed in either 5 mm EGTA or 50 mm EDTA at pH 5.5. AE is the activity of the original apoplastic extract in 20 mm CaCl₂ and 20 mm ascorbate, pH 3. D, Apoplastic proteins were dialyzed in 5 mm EDTA, sodium succinate, or EGTA at pH 5.5, then frozen and thawed three times. Antifreeze activity was quantified by determining changes in ice morphology with dilution as described in “Materials and Methods”. Data are presented as means ± se, n = 3.

Figure 2.

Effects of cations and freeze-thaw (FT) cycles on chitinase activity. A, Apoplastic proteins were dialyzed in solutions containing 20 to 200 mm CaCl₂, MgCl₂, or NaCl at pH 5.5. Proteins were dialyzed in 5 mm EDTA to achieve 0 mm cation. B, Apoplastic proteins were dialyzed in solutions containing 20 to 200 mm CaCl₂, MgCl₂, or NaCl at pH 5.5 (0FT), then frozen and thawed three times (3FT) and reassayed. C, To determine if the effect of Ca²⁺ was reversible, apoplastic proteins frozen and thawed in the presence of 20 or 200 mm CaCl₂ were then dialyzed in 5 mm EGTA or 50 mm EDTA at pH 5.5. AE is the activity of the original apoplastic extract in 20 mm CaCl₂ and 20 mm ascorbate, pH 3. D, Apoplastic proteins were dialyzed in 5 mm EDTA, sodium succinate, or EGTA at pH 5.5, then frozen and thawed three times. In all experiments, each sample was diluted in a solution containing the substrate 4-MU(GlcNAc)₃ and chitinase activity was measured by the release of free 4-MU as described in “Materials and Methods”. The dependence of chitinase activity on cation concentration was calculated using the final cation concentration in the assay medium. Data are presented as means ± se, n = 3.

Figure 3.

Effect of Ca²⁺ on activity of a purified native chitinase (NP3). A, NP3 was eluted from a colloidal chitin-affinity column with 20 mm acetic acid, pH 3. B, The original apoplastic extract (AE) and the eluted fractions were examined by SDS-PAGE. The apparent molecular masses of Bio-Rad Precision Protein Standards are shown on the left and of the prominent polypeptides are shown on the right. C, Aliquots of NP3 were dialyzed in solutions containing up to 200 mm CaCl₂, and then chitinase activity was assayed as described in “Materials and Methods”. The dependence of chitinase activity on cation concentration was calculated using the final cation concentration in the assay medium. Data are presented as means ± sd, n = 3.

Figure 4.

Effect of cations and freeze-thaw cycles on the organization of apoplastic proteins as determined by native PAGE. Proteins were separated in 9% polyacrylamide gels and stained with Coomassie Blue. A, Apoplastic proteins were dialyzed in 0 to 200 mm CaCl₂, then 5 μg protein were loaded per lane. Nine native proteins (NPs) were visible in the original apoplastic extract, AE. B, Separation of apoplastic proteins (10 μg per lane) dialyzed in 5 mm EDTA, 20 mm CaCl₂, and 20 mm MgCl₂. C, Apoplastic proteins were dialyzed in 0 to 200 mm CaCl₂, frozen and thawed three times, then 10 μg protein were loaded per lane.

Figure 5.

Use of ruthenium red to observe binding of Ca²⁺ to apoplastic proteins extracted from NA and CA winter rye leaves. A, Apoplastic proteins (50 or 100 μg per sample) were blotted onto nitrocellulose and stained with ruthenium red. The ability of cations to displace ruthenium red was determined after washing blots with 100 mm solutions of CaCl₂, MgCl₂, NaCl, or KCl. Unstained proteins appear yellow-gray in color and stained proteins appear red. B, Apoplastic proteins (100 μg) from CA winter rye leaves were incubated with Pronase E for 2 h, and then blotted and stained with ruthenium red. Antifreeze activity was assayed by observing the shape of growing ice crystals. The c-axis of the ice crystals is parallel to the plane of the page for the control sample and normal to the plane of the page for the Pronase E sample. C, Effect of freeze-thaw cycles on ruthenium red binding to apoplastic proteins from CA winter rye leaves (WR). Apoplastic proteins (100 μg) were dialyzed in Tris-EDTA, subjected to three freeze-thaw cycles (F-T) in the presence of 40 mm CaCl₂, MgCl₂, or NaCl, and then blotted and stained with ruthenium red. Unfrozen and freeze-thawed controls in Tris-EDTA buffer were also included. Ovalbumin (100 μg) was the positive control for a Ca²⁺-binding protein.

Figure 6.

Effect of cations and freeze-thaw cycles on the structure of apoplastic proteins determined by CD. Apoplastic proteins (1 mg mL⁻¹) were dialyzed in 5 mm EDTA (pH 5.5), 20 mm CaCl₂, or 20 mm MgCl₂. Half the samples were stored at 4°C as unfrozen controls, and the remaining samples were subjected to three freeze-thaw cycles, then they were maintained at 4°C until analysis. Treatments are labeled by the number of freeze-thaw cycles followed by the salt, e.g. 0EDTA indicates unfrozen, control samples dialyzed in EDTA, and 3EDTA indicates that samples dialyzed in EDTA were subjected to 3 freeze-thaw cycles. A, Near-UV CD spectra. B, Near-UV CD difference spectra calculated by subtracting the 0EDTA control from the other spectra shown in graph A. In order to examine secondary structure in far-UV, samples were diluted to 50 μg apoplastic protein mL⁻¹. C, Far-UV CD spectra. D, Far-UV CD difference spectra calculated by subtracting the 0EDTA control from the other spectra shown in graph C.

Figure 7.

Hypothetical model summarizing changes observed in native apoplastic proteins exposed to Ca²⁺ and repeated freeze-thaw cycles. For clarity, only one native protein (NP3) is shown and is modeled with the simplest stoichiometry of one glucanase (G), one chitinase (Cht) and one thaumatin-like protein (TLP). NP3 itself has antifreeze activity (Yu and Griffith, 1999), and each of the three components exhibits antifreeze activity when separated as individual polypeptides (Hon et al., 1994, 1995). When maintained in the unfrozen state in the absence of Ca²⁺, NP3 exhibits both antifreeze and chitinase activities. When frozen and thawed, NP3 adopts a different structure and still exhibits both antifreeze and chitinase activities. When Ca²⁺ interacts with the native, unfrozen protein, the structure of NP3 becomes more compact, and the complex exhibits enhanced chitinase activity with no change in antifreeze activity. However, when NP3 is frozen and thawed repeatedly in the presence of Ca²⁺, NP3 binds more Ca²⁺ and both antifreeze and chitinase activities decrease.

Figure 8.

Antifreeze activity in apoplastic extracts determined by observing the morphology of ice crystals grown in solution. A, Circular ice crystals represent no antifreeze activity; B, hexagonal ice crystals represent low activity; C, hexagonal ice crystals with increased growth along the c axis represent moderate activity; and D, hexagonal bipyramids represent high antifreeze activity. In A and B, the basal plane of each ice crystals is parallel to the plane of the page. In C and D, the basal plane of each ice crystal is normal to the plane of the page. The magnification bar represents 10 μm.