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     

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.     

Antifreeze proteins in winter rye leaves form oligomeric complexes

Antifreeze proteins (AFPs) similar to three pathogenesis-related proteins, a glucanase-like protein (GLP), a chitinase-like protein (CLP), and a thaumatin-like protein (TLP), accumulate during cold acclimation in winter rye (Secale cereale) leaves, where they are thought to modify the growth of intercellular ice during freezing. The objective of this study was to characterize the rye AFPs in their native forms, and our results show that these proteins form oligomeric complexes in vivo. Nine proteins were separated by native-polyacrylamide gel electrophoresis from apoplastic extracts of cold-acclimated winter rye leaves. Seven of these proteins exhibited multiple polypeptides when denatured and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After isolation of the individual proteins, six were shown by immunoblotting to contain various combinations of GLP, CLP, and TLP in addition to other unidentified proteins. Antisera produced against individual cold-induced winter rye GLP, CLP, and TLP all dramatically inhibited glucanase activity in apoplastic extracts from cold-acclimated winter rye leaves, and each antiserum precipitated all three proteins. These results indicate that each of the polypeptides may be exposed on the surface of the protein complexes. By forming oligomeric complexes, AFPs may form larger surfaces to interact with ice, or they may simply increase the mass of the protein bound to ice. In either case, the complexes of AFPs may inhibit ice growth and recrystallization more effectively than the individual polypeptides.     

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.     

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.     

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 protein accumulation in freezing-tolerant cereals

Freezing-tolerant plants withstand extracellular ice formation at subzero temperatures. Previous studies have shown that winter rye ( Secale cereale L.) accumulates proteins in the leaf apoplast during cold acclimation that have antifreeze properties and are similar to pathogenesis-related proteins. To determine whether the accumulation of these antifreeze proteins is common among herbaceous plants, we assayed antifreeze activity and total protein content in leaf apoplastic extracts from a number of species grown at low temperature, including both monocotyledons (winter and spring rye, winter and spring wheat, winter barley, spring oats, maize) and dicotyledons (spinach, winter and spring oilseed rape [canola], kale, tobacco). Apoplastic polypeptides were also separated by SDS-PAGE and immunoblotted to determine whether plants generally respond to low temperature by accumulating pathogenesis-related proteins. Our results showed that significant levels of antifreeze activity were present only in the apoplast of freezing-tolerant monocotyledons after cold acclimation at 5/2⁰C. Moreover, only a closely related group of plants, rye, wheat and barley, accumulated antifreeze proteins similar to pathogenesis-related proteins during cold acclimation. The results indicate that the accumulation of antifreeze proteins is a specific response that may be important in the freezing tolerance of some plants, rather than a general response of all plants to low temperature stress.     

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.     

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.     

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.     

Localization of Proteins Immunologically Related to Subunits of Stress 310-kD Protein in Winter Wheat Mitochondria

We studied the localization of polypeptides immunochemically related to subunits of cold-shock 310-kD protein from winter rye (Secale cerealeL.) in mitochondria and submitochondrial structures of winter wheat (Triticum aestivumL.) seedlings. Polypeptides were separated by SDS-PAGE and probed with the antibody against 310-kD protein from rye seedlings. Wheat mitochondria contained the following polypeptides cross-reacting with this antibody: 66, 60, 55, and 23 kD in the inner membrane; 60 and 58 kD in the outer membrane; and 66 and 55 kD in the matrix.     

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.     

Artificial antifreeze polypeptides: alpha-helical peptides with KAAK motifs have antifreeze and ice crystal morphology modifying properties.

Antifreeze polypeptides from fish are generally thought to inhibit ice crystal growth by specific adsorption onto ice surfaces and preventing addition of water molecules to the ice lattice. Recent studies have suggested that this adsorption results from hydrogen bonding through the side chains of polar amino acids as well as hydrophobic interactions between the non-polar domains on the ice-binding side of antifreeze polypeptides and the clathrate-like surfaces of ice. In order to better understand the activity of one of the antifreeze polypeptide families, namely the alpha-helical type I antifreeze polypeptides, four alpha-helical peptides having sequences not directly analogous to those of known antifreeze polypeptides and containing only positively charged and non-polar side chains were synthesized. Two peptides with regularly spaced lysine residues, GAAKAAKAAAAAAAKAAKAAAAAAAKAAKAAGGY-NH2 and GAALKAAKAAAAAALKAAKAAAAAALKAAKAAGGY-NH2, showed antifreeze activity, albeit weaker than seen in natural antifreeze polypeptides, by the criteria of freezing point depression (thermal hysteresis) and ice crystal modification to a hexagonal trapezohedron. Peptides with irregular spacing of Lys residues were completely inactive. Up to now, lysine residues have not been generally associated with antifreeze activity, though they have been implicated in some antifreeze polypeptides. This work also shows that lysine residues in themselves, when properly positioned on an alpha-helical polyalanine scaffold, have all the requisite properties needed for such an activity.     

Cyanogen bromide cleavage at methionine residues of polypeptides containing disulfide bonds

A method for cleaving polypeptides at their methionine residues without affecting intramolecular disulfide bonds is described. This method may be applied for cleaving recombinant heterologous hybrid polypeptides with release of the interesting peptide. The method may also be applied to assign the correct positions of disulfide bonds in protein molecules.     

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.     

Unassembled polypeptides of the plastidic ribosomes in heat-treated 70S-ribosome-deficient rye leaves

The polypeptides of the subunits of 70S ribosomes isolated from rye (Secale cereale L.) leaf chloroplasts were analyzed by two-dimensional polyacrylamide gel electrophoresis. The 50S subunit contained approx. 33 polypeptides in the range of relative molecular mass (M_r) 13000–36000, the 30S subunit contained approx. 25 polypeptides in the range of M_r 13000–40500. Antisera raised against the individual isolated ribosomal subunits detected approx. 17 polypeptides of the 50S and 10 polypeptides of the 30S subunit in the immunoblotting assay. By immunoblotting with these antisera the major antigenic ribosomal polypeptides (r-proteins) of the chloroplasts were clearly and specifically visualized also in separations of leaf extracts or soluble chloroplast supernatants. In extracts from rye leaves grown at 32° C, a temperature which is non-permissive for 70S-ribosome formation, or in supernatants from ribosome-deficient isolated plastids, six plastidic r-proteins were visualized by immunoblotting with the anti-50S-serum and two to four plastidic r-proteins were detected by immunoblotting with the anti-30S-serum, while other r-proteins that reacted with our antisera were missing. Those plastidic r-proteins that were present in 70S-ribosome-deficient leaves must represent individual unassembled ribosomal polypeptides that were synthesized on cytoplasmic 80S ribosomes. For the biogenesis of chloroplast ribosomes the mechanism of coordinate regulation appear to be less strict than those known for the biogenesis of bacterial ribosomes, thus allowing a marked accumulation of several unassembled ribosomal polypeptides of cytoplasmic origin.Abbreviations L polypeptide of large ribosomal subunit - M_r relative molecular mass - r-protein ribosomal polypeptide - S polypeptide of small ribosomal subunit - SDS sodium dodecyl sulfate     

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.     

Purification and structure analysis of antifreeze proteins from Ammopiptanthus mongolicus

We purified many kinds of antifreeze proteins with high activity from the leaves of Ammopiptanthus mongolicus by several biochemical techniques. The antifreeze activities of these AFPs were measured by both osmometry and differential scanning calorimetry, and the inhibition of growth of ice crystals by the AFPs was obvious. Additionally, the antifreeze proteins were analyzed by sequencing, glycosylation reaction, mass spectroscopy, and circular dichroism spectroscopy. Both samples have some other unique structures different from those of fishes and of insects. It was suggested that plant AFPs might have a particular antifreeze mechanism in comparison with that of fish and insects.