Antifreeze proteins in winter rye

Six antifreeze proteins, which have the unique ability to adsorb onto the surface of ice and inhibit its growth, have been isolated from the apoplast of winter rye leaves where ice forms at subzero temperatures. The rye antifreeze proteins accumulate during cold acclimation and are similar to plant pathogenesis-related proteins, including two endoglucanase-like, two chitinase-like and two thaumatin-like proteins. Immunolocalization of the glucanase-like antifreeze proteins showed that they accumulate in mesophyll cell walls facing intercellular spaces, in pectinaceous regions between adjoining mestome sheath cells, in the secondary cell walls of xylem vessels and in epidermal cell walls. Because the rye antifreeze proteins are located in areas where they could be in contact with ice, they may function as a barrier to the propagation of ice or to inhibit the recrystallization of ice. Antifreeze proteins similar to pathogenesis-related proteins were also found to accumulate in closely-related plants within the Triticum group but not in freezing-tolerant dicotyledonous plants. In winter wheat, the accumulation of antifreeze proteins and the development of freezing tolerance are regulated by chromosome 5. Rye antifreeze proteins may have evolved from pathogenesis-related proteins, but they retain their catalytic activities and may play a dual role in increasing both freezing and disease resistance in overwintering plants.     

Ethylene Induces Antifreeze Activity in Winter Rye Leaves

Antifreeze activity is induced by cold temperatures in winter rye (Secale cereale) leaves. The activity arises from six antifreeze proteins that accumulate in the apoplast of winter rye leaves during cold acclimation. The individual antifreeze proteins are similar to pathogenesis-related proteins, including glucanases, chitinases, and thaumatin-like proteins. The objective of this study was to study the regulation of antifreeze activity in response to ethylene and salicyclic acid, which are known regulators of pathogenesis-related proteins induced by pathogens. Nonacclimated plants treated with salicylic acid accumulated apoplastic proteins with no antifreeze activity. In contrast, when nonacclimated plants were exposed to ethylene, both antifreeze activity and the concentration of apoplastic protein increased in rye leaves. Immunoblotting revealed that six of the seven accumulated apoplastic proteins consisted of two glucanases, two chitinases, and two thaumatin-like proteins. The ethylene-releasing agent ethephon and the ethylene precursor 1-aminocyclopropane-1-carboxylate also induced high levels of antifreeze activity at 20 degrees C, and this effect could be blocked by the ethylene inhibitor AgNO(3). When intact rye plants were exposed to 5 degrees C, endogenous ethylene production and antifreeze activity were detected within 12 and 48 h of exposure to cold, respectively. Rye plants exposed to drought produced both ethylene and antifreeze activity within 24 h. We conclude that ethylene is involved in regulating antifreeze activity in winter rye in response to cold and drought.     

Winter rye antifreeze activity increases in response to cold and drought, but not abscisic acid

Antifreeze activity increases in winter rye ( Secale cereale L.) during cold acclimation as the plants accumulate antifreeze proteins (AFPs) that are similar to glucanases, chitinases and thaumatin-like proteins (TLPs) in the leaf apoplast. In the present work, experiments were conducted to assess the role of drought and abscisic acid (ABA) in the regulation of antifreeze activity and accumulation of AFPs. Antifreeze activity was detected as early as 24 h of drought treatment at 20°C and increased as the level of apoplastic proteins increased. Apoplastic proteins accumulated rapidly under water stress and reached a level within 8 days that was equivalent to the level of apoplastic proteins accumulated when plants were acclimated to cold temperature for 7 weeks. These drought-induced apoplastic proteins had molecular masses ranging from 11 to 35 kDa and were identified as two glucanases, two chitinases, and two TLPs, by using antisera raised against cold-induced rye glucanase, chitinase, and TLP, respectively. Apoplastic extracts obtained from plants treated with ABA lacked the ability to modify the growth of ice crystals, even though ABA induced the accumulation of apoplastic proteins within 4 days to a level similar to that obtained when plants were either drought-stressed for 8 days or cold-acclimated for 7 weeks. These ABA-induced apoplastic proteins were identified immunologically as two glucanases and two TLPs. Moreover, the ABA biosynthesis inhibitor fluridone did not prevent the accumulation of AFPs in the leaves of cold-acclimated rye plants. Our results show that cold acclimation and drought both induce antifreeze activity in winter rye plants and that the pathway regulating AFP production is independent of ABA.     

Immunolocalization of Antifreeze Proteins in Winter Rye Leaves,Crowns, and Roots by Tissue Printing

During cold acclimation, antifreeze proteins (AFPs) that are similar to pathogenesis-related proteins accumulate in the apoplast of winter rye (Secale cereale L. cv Musketeer) leaves. AFPs have the ability to modify the growth of ice. To elucidate the role of AFPs in the freezing process, they were assayed and immunolocalized in winter rye leaves, crowns, and roots. Each of the total soluble protein extracts from cold-acclimated rye leaves, crowns, and roots exhibited antifreeze activity, whereas no antifreeze activity was observed in extracts from nonacclimated rye plants. Antibodies raised against three apoplastic rye AFPs, corresponding to a glucanase-like protein (GLP, 32 kD), a chitinase-like protein (CLP, 35 kD), and a thaumatin-like protein (TLP, 25 kD), were used in tissue printing to show that the AFPs are localized in the epidermis and in cells surrounding intercellular spaces in cold-acclimated plants. Although GLPs, CLPs, and TLPs were present in nonacclimated plants, they were found in different locations and did not exhibit antifreeze activity, which suggests that different isoforms of pathogenesis-related proteins are produced at low temperature. The location of rye AFPs may prevent secondary nucleation of cells by epiphytic ice or by ice propagating through the xylem. The distributions of pathogenesis-induced and cold-accumulated GLPs, CLPs, and TLPs are similar and may reflect the common pathways by which both pathogens and ice enter and propagate through plant tissues.     

Snow-Mold-Induced Apoplastic Proteins in Winter Rye Leaves Lack Antifreeze Activity

During cold acclimation, winter rye (Secale cereale L.) plants secrete antifreeze proteins that are similar to pathogenesis-related (PR) proteins. In this experiment, the secretion of PR proteins was induced at warm temperatures by infection with pink snow mold (Microdochium nivale), a pathogen of overwintering cereals. A comparison of cold-induced and pathogen-induced proteins showed that PR proteins accumulated in the leaf apoplast to a greater level in response to cold. The PR proteins induced by cold and by snow mold were similar when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examined by immunoblotting. Both groups of PR proteins contained glucanase-like, chitinase-like, and thaumatin-like proteins, and both groups exhibited similar levels of glucanase and chitinase activities. However, only the PR proteins induced by cold exhibited antifreeze activity. Our findings suggest that the cold-induced PR proteins may be isoforms that function as antifreeze proteins to modify the growth of ice during freezing while also providing resistance to the growth of low-temperature pathogens in advance of infection. Both functions of the cold-induced PR proteins may improve the survival of overwintering cereals.     

Proteins accumulate in the apoplast of winter rye leaves during cold acclimation

During cold acclimation, winter rye ( Secale cereale L.) plants develop the ability to tolerate freezing temperatures by forming ice in intercellular spaces and xylem vessels. In this study, proteins were extracted from the apoplast of rye leaves to determine their role in controlling extracellular ice formation. Several polypeptides in the 15 to 32 kDa range accumulated in the leaf apoplast during cold acclimation at 5°C and decreased during deacclimation at 20°C. A second group of polypeptides (63, 65 and 68 kDa) appeared only when the leaves were maximally frost tolerant. Ice nucleation activity, as well as the previously reported antifreeze activity, was higher in apoplastic extracts from cold-acclimated than from nonacclimated rye leaves. These results indicate that apoplastic proteins exert a direct influence on the growth of ice. In addition, freezing injury was greater in extracted cold-acclimated leaves than in unextracted cold-acclimated leaves, which suggests that the proteins present in the apoplast are an important component of the mechanism by which winter rye leaves tolerate ice formation     

Antifreeze protein produced endogenously in winter rye leaves

After cold acclimation, winter rye (Secale cereale L.) is able to withstand the formation of extracellular ice at freezing temperatures. We now show, for the first time, that cold-acclimated winter rye plants contain endogenously produced antifreeze protein. The protein was extracted from the apoplast of winter rye leaves, where ice forms during freezing. After partial purification, the protein was identified as antifreeze protein because it modified the normal growth pattern of ice crystals and depressed the freezing temperature of water noncolligatively.     

Antifreeze proteins modify the freezing process in planta

During cold acclimation, winter rye (Secale cereale L. cv Musketeer) plants accumulate antifreeze proteins (AFPs) in the apoplast of leaves and crowns. The goal of this study was to determine whether these AFPs influence survival at subzero temperatures by modifying the freezing process or by acting as cryoprotectants. In order to inhibit the growth of ice, AFPs must be mobile so that they can bind to specific sites on the ice crystal lattice. Guttate obtained from cold-acclimated winter rye leaves exhibited antifreeze activity, indicating that the AFPs are free in solution. Infrared video thermography was used to observe freezing in winter rye leaves. In the absence of an ice nucleator, AFPs had no effect on the supercooling temperature of the leaves. However, in the presence of an ice nucleator, AFPs lowered the temperature at which the leaves froze by 0.3 degrees C to 1.2 degrees C. In vitro studies showed that apoplastic proteins extracted from cold-acclimated winter rye leaves inhibited the recrystallization of ice and also slowed the rate of migration of ice through solution-saturated filter paper. When we examined the possible role of winter rye AFPs in cryoprotection, we found that lactate dehydrogenase activity was higher after freezing in the presence of AFPs compared with buffer, but the same effect was obtained by adding bovine serum albumin. AFPs had no effect on unstacked thylakoid volume after freezing, but did inhibit stacking of the thylakoids, thus indicating a loss of thylakoid function. We conclude that rye AFPs have no specific cryoprotective activity; rather, they interact directly with ice in planta and reduce freezing injury by slowing the growth and recrystallization of ice.     

Antifreeze proteins are secreted by winter rye cells in suspension culture

During cold-acclimation, winter rye ( Secale cereale L) leaves secrete antifreeze proteins (AFPs) into the apoplast. The AFPs bind to ice and modify its growth, which is easily observed in vitro . However, it is not yet known whether in planta AFPs interact with ice or whether they exert cryoprotective effects. These experiments are difficult to conduct with intact plants, so the aim of this work was to determine whether AFPs are produced in response to cold temperature in cell culture and to examine their function by using suspension cells. We showed that suspension cells secreted three of the six known winter rye AFPs into the culture medium during acclimation at 4°C. These AFPs were not present in washed suspension cells, thus indicating that they are not firmly bound to the cell walls. In order to examine the function of extracellular AFPs, non-acclimated (NA) winter rye suspension cells and protoplasts isolated from NA winter rye leaves were then frozen and thawed in the presence of AFPs extracted from cold-acclimated winter rye leaves. The AFPs had no effect on the survival of NA protoplasts after freezing; however, they lowered the lethal temperature at which 50% of the cells are killed by freezing (LT₅₀) of NA suspension cells by 2.5°C. We conclude that low above-zero temperatures induce winter rye suspension cells to secrete AFPs free in solution where they can protect intact suspension cells, but not protoplasts, from freezing injury, presumably by interacting with extracellular ice.     

Molecular Basis of Disease Resistance Acquired through Cold Acclimation in Overwintering Plants

Plants require substantial resistance against freezing and pathogens for overwintering. These two traits are acquired through cold acclimation. In contrast to freezing tolerance, molecular basis of disease resistance acquired through cold acclimation is poorly understood. Recent studies have suggested that pathogenesis-related (PR) proteins that are secreted into the apoplast during cold acclimation are responsible for the disease resistance. Interestingly, some of the cold-induced PR proteins display both antifungal and antifreeze activities, suggesting a dual function in protecting plants from overwintering stresses. The signaling pathway for cold-induced disease resistance is currently unknown but can be independent of pathogen-induced defense mechanisms.     

Antifreeze proteins in overwintering plants: a tale of two activities

Antifreeze proteins are found in a wide range of overwintering plants where they inhibit the growth and recrystallization of ice that forms in intercellular spaces. Unlike antifreeze proteins found in fish and insects, plant antifreeze proteins have multiple, hydrophilic ice-binding domains. Surprisingly, antifreeze proteins from plants are homologous to pathogenesis-related proteins and also provide protection against psychrophilic pathogens. In winter rye (Secale cereale), antifreeze proteins accumulate in response to cold, short daylength, dehydration and ethylene, but not pathogens. Transferring single genes encoding antifreeze proteins to freezing-sensitive plants lowered their freezing temperatures by approximately 1 degrees C. Genes encoding dual-function plant antifreeze proteins are excellent models for use in evolutionary studies to determine how genes acquire new expression patterns and how proteins acquire new activities.     

Calcium interacts with antifreeze proteins and chitinase from cold-acclimated winter rye

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.     

Wintersweet accumulates apoplastic chitinase with a dual role in petals

Most monocotyledons like cereals accumulate antifreeze proteins in the apoplast during cold acclimation, but it is still uncertain whether dicotyledons do. Here we report the isolation and characterisation of a 33-kD apoplastic chitinase extracted from the corolla of wintersweet (Chinmonanthus praecox communis L.), which was purified using successive column chromatography on Phenyl-Sepharose, DEAE-Sepharose, and CM-Sepharose. Antifreezing activity of chitinase was confirmed in terms of the formation of bipyramidal ice crystals and high thermal-hysteresis values. Interestingly, chitinase was also found to affect germination of fungal spores of four major plant pathogens. From these data, we hypothesize that, under natural conditions, wintersweet as one of the overwintering dicotyledons also accumulates apoplastic antifreeze proteins like monocotyledons. To our knowledge, this is the first report on the isolation of dicotyledon apoplastic chitinase with high-level antifreeze and antifungal activities.     

Sucrose metabolism in spring and winter wheat in response to high irradiance, cold stress and cold acclimation

Effects of low‐temperature stress, cold acclimation and growth at high irradiance in a spring (Triticum aestivum L. cv. Katepwa) and a winter wheat (Triticum aestivum L. cv. Monopol) were examined in leaves and crowns with respect to the sucrose utilisation and carbon allocation. Light‐saturated and carbon dioxide (CO₂)‐saturated rates of CO₂ assimilation were decreased by 50% in cold‐stressed spring and winter wheat cultivars. Cold‐ or high light‐acclimated Katepwa spring wheat maintained light‐saturated rates of CO₂ assimilation comparable to those of control spring wheat. In contrast, cold‐ or high light‐acclimated winter wheat maintained higher light and CO₂‐saturated rates of CO₂ assimilation than non‐acclimated controls. In leaves, during either cold stress, cold acclimation or acclimation to high irradiance, the sucrose/starch ratio increased by 5‐ to 10‐fold and neutral invertase activity increased by 2‐ to 2.5‐fold in both the spring and the winter wheat. In contrast, Monopol winter wheat, but not Katepwa spring wheat, exhibited a 3‐fold increase in leaf sucrose phosphate synthase (SPS) activity, a 4‐fold increase in sucrose:sucrose fructosyl transferase activity and a 6.6‐fold increase in acid invertase upon cold acclimation. Although leaves of cold‐stressed and high light‐grown spring and winter wheat showed 2.3‐ to 7‐fold higher sucrose levels than controls, these plants exhibited a limited capacity to adjust either sucrose phosphate synthase or sucrose synthase activity (SS[s]). In addition, the acclimation to high light resulted in a 23–31% lower starch abundance and no changes at the level of fructan accumulation in leaves of either winter or spring wheat when compared with controls. However, high light‐acclimated winter wheat exhibited a 1.8‐fold higher neutral invertase activity and high light‐acclimated spring wheat exhibited an induction of SS(d) activity when compared with controls. Crowns of Monopol showed higher fructan accumulation than Katepwa upon cold and high light acclimation. We suggest that the differential adjustment of CO₂‐saturated rates of CO₂ assimilation upon cold acclimation in Monopol winter wheat, as compared with Katepwa spring wheat, is associated with the increased capacity of Monopol for sucrose utilisation through the biosynthesis of fructans in the leaves and subsequent export to the crowns. In contrast, the differential adjustment of CO₂‐saturated rates of CO₂ assimilation upon high light acclimation of Monopol appears to be associated with both increased fructan and starch accumulation in the crowns.     

Efficiency of cold hardiness induction by desiccation stress in four winter cereals

A number of defined desiccation treatments without low temperature exposure were able to induce freezing tolerance in 20 cultivars of winter cereals. A maximal degree of freezing tolerance was induced in epicotyls at 24°C in 24 hours at 40% relative humidity in rye and wheat, 7 days at 54% RH in barley, and 4 days at 70% RH in oats. Freezing tolerance was not correlated to water content of the plants after desiccation treatment but was related to the genetic capacity of the cultivars to frost harden. Levels of freezing tolerance induced by desiccation were similar to those induced by cold acclimation in rye and wheat, but considerably less in barley and oats. This is associated with a more rapid desiccation injury in barley and oats, precluding the completion of the hardening process.     

Characterization of antifreeze activity in Antarctic plants

Deschampsia antarctica and Colobanthus quitensis are the only vascular plants to have colonized the Maritime Antarctic, which is characterized by its permanently low temperature and frequent summer frosts. To understand how the plants survive freezing temperatures year-round, antifreeze activity was assayed in apoplastic extracts obtained from both non-acclimated and cold-acclimated Antarctic plants. By observing the shape of ice crystals grown in dilution series of the extracts, it was found that D. antarctica had antifreeze activity, but C. quitensis did not. D. antarctica exhibited antifreeze activity in the non-acclimated state and this activity increased after cold acclimation. The antifreeze activity in D. antarctica was labile to proteolysis and high temperature, active over a wide pH range, and associated with molecules greater than 10 kDa in molecular weight. These results show that D. antarctica produces antifreeze proteins that are secreted into the apoplast. When examined by SDS-PAGE, the apoplastic extracts from cold-acclimated D. antarctica exhibited 13 polypeptides. It is concluded that D. antarctica accumulates AFPs as part of its mechanism of freezing tolerance. Moreover, this is the first plant in which antifreeze activity has been observed to be constitutive.     

Immunogold localization of glucanase-like antifreeze protein in cold acclimated winter rye

Summary Apoplastic antifreeze proteins (AFPs) accumulate in winter rye (Secale cereale L. cv. Musketeer) leaves during cold acclimation. Two of the rye AFPs with molecular masses of 32 and 35 kDa are similar in their amino acid sequences and epitopes to -1, 3-endoglucanase. Localization of these AFPs, which we refer to as glucanase-like proteins (GLPs), was carried out with antiserum raised against the 32 kDa AFP. Specimens from leaves and roots of non-acclimated (NA) plants and cold acclimated (CA) plants were prepared by freeze-substitution for high resolution immunoelectron microscopy. In CA leaves, high levels of GLPs were observed in cell walls of mesophyll cells adjacent to intercellular spaces and in secondary thickenings of xylem vessels. Taken together with the absence of GLPs in vacuoles, these results confirm the apoplastic accumulation of AFPs in CA winter rye. Within the cells of CA leaves, GLPs were localized in cisternae of the rough endoplasmic reticulum, the Golgi apparatus and the plasma membrane, which indicates that GLPs are secreted via an exocytic bulk-flow pathway. The occurrence of high levels of GLPs in CA leaves, their low presence in NA leaves and the lack of GLPs in roots all suggest that there is a correlation between increased accumulation of GLPs and increased freezing tolerance of these plant materials. Furthermore, the localization of GLPs in the immediate vicinity of pathways for free water within the tissues supports the view that these proteins have an important role in the crystallization and/or recrystallization of water when the leaves of CA winter rye are exposed to freezing temperatures.Abbreviations AFP antifreeze protein - BSA bovine serum albumin - CA cold acclimated - GAR goat antirabbit antiserum conjugated with colloidal gold - GLP glucanase-like protein - NA non-acclimated - PBS phosphate buffered saline - PR pathogenesis related     

Cold-active winter rye glucanases with ice-binding capacity

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 beta-1,3-glucanases and an acidic beta-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 degrees 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 beta-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 beta-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.