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.     

Expression of antifreeze proteins in transgenic plants

The quality of frozen fruits and vegetables can be compromised by the damaging effects of ice crystal growth within the frozen tissue. Antifreeze proteins in the blood of some polar fishes have been shown to inhibit ice recrystallization at low concentrations. In order to determine whether expression of genes of this type confers improved freezing properties to plant tissue, we have produced transgenic tobacco and tomato plants which express genes encoding antifreeze proteins. Theafa3 antifreeze gene was expressed at high steady-state mRNA levels in leaves from transformed plants, but we did not detect inhibition of ice recrystallization in tissue extracts. However, both mRNA and fusion proteins were detectable in transgenic tomato tissue containing a chimeric gene encoding a fusion protein between truncated staphylococcal protein A and antifreeze protein. Furthermore, ice recrystallization inhibition was detected in this transgenic tissue.     

An ice-binding protein from an Antarctic sea ice bacterium

An Antarctic sea ice bacterium of the Gram-negative genus Colwellia, strain SLW05, produces an extracellular substance that changes the morphology of growing ice. The active substance was identified as a approximately 25-kDa protein that was purified through its affinity for ice. The full gene sequence was determined and was found to encode a 253-amino acid protein with a calculated molecular mass of 26,350 Da. The predicted amino acid sequence is similar to predicted sequences of ice-binding proteins recently found in two species of sea ice diatoms and a species of snow mold. A recombinant ice-binding protein showed ice-binding activity and ice recrystallization inhibition activity. The protein is much smaller than bacterial ice-nucleating proteins and antifreeze proteins that have been previously described. The function of the protein is unknown but it may act as an ice recrystallization inhibitor to protect membranes in the frozen state.     

Enhanced survival of yeast expressing an antifreeze gene analogue after freezing.

Yeast, like most organisms, survives poorly under freezing conditions. It has been proposed that after rapid cooling yeast suffers a loss in viability from the recrystallization of intracellular ice. Antifreeze proteins found in the blood of certain polar fishes have been shown to be potent inhibitors of ice recrystallization at very low concentrations. We have examined the feasibility of protecting rapidly cooled yeast cells from freezing damage by inhibiting the recrystallization of intracellular ice through in vivo expression of an antifreeze analogue gene. A chemically synthesized gene encoding a protein similar to but differing from the antifreeze proteins of the fish Pseudopleuronectes americanus (winter flounder) was genetically fused to the 3' end of a truncated staphylococcal Protein A gene. When the fused gene was expressed in the budding yeast Saccharomyces cerevisiae, its cells were shown to produce a new chimeric protein that inhibited the recrystallization of ice in vitro. Yeast cells expressing the chimeric antifreeze protein showed a twofold increase in survival after rapid freezing (95 degrees C/min to -196 degrees C) and moderate rates of warming (26 to 64 degrees C/min) compared to cells lacking the chimeric protein.     

Antifreeze protein from freeze-tolerant grass has a beta-roll fold with an irregularly structured ice-binding site

The grass Lolium perenne produces an ice-binding protein (LpIBP) that helps this perennial tolerate freezing by inhibiting the recrystallization of ice. Ice-binding proteins (IBPs) are also produced by freeze-avoiding organisms to halt the growth of ice and are better known as antifreeze proteins (AFPs). To examine the structural basis for the different roles of these two IBP types, we have solved the first crystal structure of a plant IBP. The 118-residue LpIBP folds as a novel left-handed beta-roll with eight 14- or 15-residue coils and is stabilized by a small hydrophobic core and two internal Asn ladders. The ice-binding site (IBS) is formed by a flat beta-sheet on one surface of the beta-roll. We show that LpIBP binds to both the basal and primary-prism planes of ice, which is the hallmark of hyperactive AFPs. However, the antifreeze activity of LpIBP is less than 10% of that measured for those hyperactive AFPs with convergently evolved beta-solenoid structures. Whereas these hyperactive AFPs have two rows of aligned Thr residues on their IBS, the equivalent arrays in LpIBP are populated by a mixture of Thr, Ser and Val with several side-chain conformations. Substitution of Ser or Val for Thr on the IBS of a hyperactive AFP reduced its antifreeze activity. LpIBP may have evolved an IBS that has low antifreeze activity to avoid damage from rapid ice growth that occurs when temperatures exceed the capacity of AFPs to block ice growth while retaining the ability to inhibit ice recrystallization.     

Ice-active proteins from the Antarctic nematode Panagrolaimus davidi

The Antarctic nematode Panagrolaimus davidi has an ice-active protein that shows recrystallization inhibition but no thermal hysteresis. It belongs to a class of ice-active proteins found in a variety of freezing-tolerant organisms that display insignificant levels of thermal hysteresis in the context of the environmental temperatures to which they are exposed. The recrystallization inhibition activity of the P. davidi ice-active protein is present at low concentrations, is relatively heat stable, is affected more by acid than by alkaline pH, is not calcium dependant and is not affected by reagents that target carbohydrate residues or sulphydryl linkages. A hexagonal ice crystal growth form also indicates the presence of an ice-active protein. This protein could have important functions in the survival of intracellular freezing by this organism by controlling the stability of ice after its formation.     

A theoretical model of a plant antifreeze protein from Lolium perenne.

Antifreeze proteins (AFPs), found in certain organisms enduring freezing environments, have the ability to inhibit damaging ice crystal growth. Recently, the repetitive primary sequence of the AFP of perennial ryegrass, Lolium perenne, was reported. This macromolecular antifreeze has high ice recrystallization inhibition activity but relatively low thermal hysteresis activity. We present here a theoretical three-dimensional model of this 118-residue plant protein based on a beta-roll domain with eight loops of 14-15 amino acids. The fold is supported by a conserved valine hydrophobic core and internal asparagine ladders at either end of the roll. Our model, which is the first proposed for a plant AFP, displays two putative, opposite-facing, ice-binding sites with surface complementarity to the prism face of ice. The juxtaposition of the two imperfect ice-binding surfaces suggests an explanation for the protein's inferior thermal hysteresis but superior ice recrystallization inhibition activity and activity when compared with fish and insect AFPs.     

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.     

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.     

Inhibition of recrystallization in ice by chimeric proteins containing antifreeze domains

Using synthetic DNA, we assembled a gene encoding a protein identical in sequence to one of the antifreeze proteins produced by the fish Pseudopleuronectes americanus (winter flounder). To address the relationship between structure and function, we also assembled genes encoding proteins varying in sequence and length. The synthetic genes were cloned into a bacterial expression vector to generate translational fusions to the 3' end of a truncated staphylococcal protein A gene; the chimeric proteins encoded by these fusions, varying only in their antifreeze domains, were isolated from Escherichia coli. The antifreeze domains conferred the ability to inhibit ice recrystallization, which is characteristic of naturally occurring antifreeze proteins, on the chimeric proteins. The chimeric proteins varied in their effectiveness of inhibiting ice recrystallization according to the number of 11-amino acid repeats present in the antifreeze moiety. A protein with only two repeats lacked activity, while the inhibitory activity increased progressively for proteins containing three, four, and five repeats. Some activity was lost upon removal of either the salt bridge or the carboxyl-terminal arginine, but surprisingly, not when both features were absent together.     

甲虫抗冻蛋白热滞活性的二维吸附结合模型

甲虫抗冻蛋白是一种具有规则结构的昆虫抗冻蛋白。在相同浓度条件下,甲虫抗冻蛋白比鱼类抗冻蛋白有更高的热滞活性,目前已成为人们重点研究的一类抗冻蛋白。根据甲虫抗冻蛋白的结构特点及其在冰晶表面的吸附模式,应用二维吸附结合模型计算分析了具有6￣11个β-螺旋(β-helix)结构片段的甲虫抗冻蛋白变体分子,得到了它们的热滞活性随溶液浓度变化的规律,特别是热滞活性与甲虫抗冻蛋白的β-螺旋结构片段数的关系。结果显示,抗冻蛋白在冰晶表面的覆盖度是一个影响其热滞活性的重要因素。     

Low temperature stress modulated secretome analysis and purification of antifreeze protein from Hippophae rhamnoides, a Himalayan wonder plant

Plants' distribution and productivity are adversely affected by low temperature (LT) stress. LT induced proteins were analyzed by 2-DE-nano-LC-MS/MS in shoot secretome of Hippophae rhamnoides (seabuckthorn), a Himalayan wonder shrub. Seedlings were subjected to direct freezing stress (-5 °C), cold acclimation (CA), and subzero acclimation (SZA), and extracellular proteins (ECPs) were isolated using vacuum infiltration. Approximately 245 spots were reproducibly detected in 2-DE gels of LT treated secretome, out of which 61 were LT responsive. Functional categorization of 34 upregulated proteins showed 47% signaling, redox regulated, and defense associated proteins. LT induced secretome contained thaumatin like protein and Chitinase as putative antifreeze proteins (AFPs). Phase contrast microscopy with a nanoliter osmometer showed hexagonal ice crystals with 0.13 °C thermal hysteresis (TH), and splat assay showed 1.5-fold ice recrystallization inhibition (IRI), confirming antifreeze activity in LT induced secretome. A 41 kDa polygalacturonase inhibitor protein (PGIP), purified by ice adsorption chromatography (IAC), showed hexagonal ice crystals, a TH of 0.19 °C, and 9-fold IRI activity. Deglycosylated PGIP retained its AFP activity, suggesting that glycosylation is not required for AFP activity. This is the first report of LT modulated secretome analysis and purification of AFPs from seabuckthorn. Overall, these findings provide an insight in probable LT induced signaling in the secretome.     

The mechanism by which fish antifreeze proteins cause thermal hysteresis

Antifreeze proteins are characterised by their ability to prevent ice from growing upon cooling below the bulk melting point. This displacement of the freezing temperature of ice is limited and at a sufficiently low temperature a rapid ice growth takes place. The separation of the melting and freezing temperature is usually referred to as thermal hysteresis, and the temperature of ice growth is referred to as the hysteresis freezing point. The hysteresis is supposed to be the result of an adsorption of antifreeze proteins to the crystal surface. This causes the ice to grow as convex surface regions between adjacent adsorbed antifreeze proteins, thus lowering the temperature at which the crystal can visibly expand. The model requires that the antifreeze proteins are irreversibly adsorbed onto the ice surface within the hysteresis gap. This presupposition is apparently in conflict with several characteristic features of the phenomenon; the absence of superheating of ice in the presence of antifreeze proteins, the dependence of the hysteresis activity on the concentration of antifreeze proteins and the different capacities of different types of antifreeze proteins to cause thermal hysteresis at equimolar concentrations. In addition, there are structural obstacles that apparently would preclude irreversible adsorption of the antifreeze proteins to the ice surface; the bond strength necessary for irreversible adsorption and the absence of a clearly defined surface to which the antifreeze proteins may adsorb. This article deals with these apparent conflicts between the prevailing theory and the empirical observations. We first review the mechanism of thermal hysteresis with some modifications: we explain the hysteresis as a result of vapour pressure equilibrium between the ice surface and the ambient fluid fraction within the hysteresis gap due to a pressure build-up within the convex growth zones, and the ice growth as the result of an ice surface nucleation event at the hysteresis freezing point. We then go on to summarise the empirical data to show that the dependence of the hysteresis on the concentration of antifreeze proteins arises from an equilibrium exchange of antifreeze proteins between ice and solution at the melting point. This reversible association between antifreeze proteins and the ice is followed by an irreversible adsorption of the antifreeze proteins onto a newly formed crystal plane when the temperature is lowered below the melting point. The formation of the crystal plane is due to a solidification of the interfacial region, and the necessary bond strength is provided by the protein "freezing" to the surface. In essence: the antifreeze proteins are "melted off" the ice at the bulk melting point and "freeze" to the ice as the temperature is reduced to subfreezing temperatures. We explain the different hysteresis activities caused by different types of antifreeze proteins at equimolar concentrations as a consequence of their solubility features during the phase of reversible association between the proteins and the ice, i.e., at the melting point; a low water solubility results in a large fraction of the proteins being associated with the ice at the melting point. This leads to a greater density of irreversibly adsorbed antifreeze proteins at the ice surface when the temperature drops, and thus to a greater hysteresis activity. Reference is also made to observations on insect antifreeze proteins to emphasise the general validity of this approach.     

Hyperactive antifreeze protein from winter flounder is a very long rod-like dimer of alpha-helices

The winter flounder (Pseudopleuronectes americanus) produces short, monomeric alpha-helical antifreeze proteins (type I AFP), which adsorb to and inhibit the growth of ice crystals. These proteins alone are not sufficiently active to protect this fish against freezing at -1.9 degrees C, the freezing point of seawater. We have recently isolated a hyperactive antifreeze protein from the plasma of the flounder with activity 10-100-fold higher than type I AFP. It is comparable in activity to the AFPs produced by insects, and is capable of conferring freeze resistance to the flounder. This novel AFP has a molecular mass of 16,683 Da and a remarkable amino acid composition that is >60% alanine. CD spectra indicate that the protein is almost entirely alpha-helical at 4 degrees C but partially denatures at 20 degrees C, resulting in a species with a moderately reduced helix content that is stable at up to 50 degrees C. This transformation correlates with irreversible loss of activity. Analytical ultracentrifugation (sedimentation velocity and equilibrium) indicates that the predominant species in solution is dimeric (molecular weight, 32,275). Size-exclusion chromatography reveals a 2-fold higher apparent molecular weight suggesting that this molecule has an unusually large Stokes radius. The axial ratio of the dimer calculated from the sedimentation velocity data is 18:1, confirming that this protein has an extraordinarily long, rod-like structure, consistent with a novel dimeric alpha-helical arrangement. The structural model that best fits these data is one in which the approximately 195 amino acids of each monomer form one approximately 290-A long alpha-helix and associate via a unique dimerization motif that is distinct from that of the leucine zipper and any other coiled-coil.     

Multiple genes provide the basis for antifreeze protein diversity and dosage in the ocean pout, Macrozoarces americanus

The ocean pout (Macrozoarces americanus) produces a set of antifreeze proteins that depresses the freezing point of its blood by binding to, and inhibiting the growth of, ice crystals. The amino acid sequences of all the major components of the ocean pout antifreeze proteins, including the immunologically distinct QAE component, have been derived by Edman degradation. In addition, sequences of several minor components were deduced from DNA sequencing of cDNA and genomic clones. Fifty percent of the amino acids are perfectly conserved in all these proteins as well as in two homologous sequences from the distantly related wolffish. Several of the conserved residues are threonines and asparagines, amino acids that have been implicated in ice binding in the structurally unrelated antifreeze protein of the righteye flounders. Aside from minor differences in post-translational modifications, heterogeneity in antifreeze protein components stems from amino acid differences encoded by multiple genes. Based on genomic Southern blots and library cloning statistics there are 150 copies of the 0.7-kilobase-long antifreeze protein gene in the Newfoundland ocean pout, the majority of which are closely linked but irregularly spaced. A more southerly population of ocean pout from New Brunswick in which the circulating antifreeze protein levels are considerably lower has approximately one-quater as many antifreeze protein genes. Thus, there appears to be a correlation between gene dosage and antifreeze protein levels, and hence the ability to survive in ice-laden seawater. Southern blot comparison of the two populations indicates that the differences in gene dosage were not generated by a simple set of deletions/duplications. They are more likely to be the result of differential amplification.     

Salt-induced enhancement of antifreeze protein activity: a salting-out effect

Antifreeze proteins are a structurally diverse group of proteins characterized by their unique ability to cause a separation of the melting- and growth-temperatures of ice. These proteins have evolved independently in different kinds of cold-adapted ectothermic animals, including insects and fish, where they protect against lethal freezing of the body fluids. There is a great variability in the capacity of different kinds of antifreeze proteins to evoke the antifreeze effect, but the basis of these differences is not well understood. This study reports on salt-induced enhancement of the antifreeze activity of an antifreeze protein from the longhorn beetle Rhagium inquisitor (L.). The results imply that antifreeze activity is predetermined by a steady-state distribution of the antifreeze protein between the solution and the ice surface region. The observed salt-induced enhancement of the antifreeze activity compares qualitatively and quantitatively with salt-induced lowering of protein solubility. Thus, salts apparently enhance antifreeze activity by evoking a solubility-induced shift in the distribution pattern of the antifreeze proteins in favour of the ice. These results indicate that the solubility of antifreeze proteins in the solution surrounding the ice crystal is a fundamental physiochemical property in relation to their antifreeze potency.     

Characterization of a family of ice-active proteins from the Ryegrass,Lolium perenne

Five genes coding for ice-active proteins were identified from an expressed sequence tag database of Lolium perenne cDNA libraries. Each of the five genes were characterized by the presence of an N-terminal signal peptide, a region enriched in hydrophilic amino acids and a leucine-rich region in four of the five genes that is homologous with the receptor domain of receptor-like protein kinases of plants. The C-terminal region of all five genes contains sequence homologous with Lolium and Triticum ice-active proteins. Of the four ice-active proteins (IAP1, IAP2, IAP3 and IAP5) cloned, three could be expressed in Escherichia coli and recovered in a functional form in order to study their ice activity. All three ice-active proteins had recrystallization inhibition activity but showed no detectable antifreeze or ice nucleation activity at the concentration tested. IAP2 and IAP5 formed distinct hexagonal-shaped crystals in the nanolitre osmometer as compared to the weakly hexagonal crystals produced by IAP3.     

Characterization of a family of ice-active proteins from the Ryegrass, Lolium perenne

Five genes coding for ice-active proteins were identified from an expressed sequence tag database of Lolium perenne cDNA libraries. Each of the five genes were characterized by the presence of an N-terminal signal peptide, a region enriched in hydrophilic amino acids and a leucine-rich region in four of the five genes that is homologous with the receptor domain of receptor-like protein kinases of plants. The C-terminal region of all five genes contains sequence homologous with Lolium and Triticum ice-active proteins. Of the four ice-active proteins (IAP1, IAP2, IAP3 and IAP5) cloned, three could be expressed in Escherichia coli and recovered in a functional form in order to study their ice activity. All three ice-active proteins had recrystallization inhibition activity but showed no detectable antifreeze or ice nucleation activity at the concentration tested. IAP2 and IAP5 formed distinct hexagonal-shaped crystals in the nanolitre osmometer as compared to the weakly hexagonal crystals produced by IAP3.     

Functional importance of short-range binding and long-range solvent interactions in helical antifreeze peptides

Short-range ice binding and long-range solvent perturbation both have been implicated in the activity of antifreeze proteins and antifreeze glycoproteins. We study these two mechanisms for activity of winter flounder antifreeze peptide. Four mutants are characterized by freezing point hysteresis (activity), circular dichroism (secondary structure), Förster resonance energy transfer (end-to-end rigidity), molecular dynamics simulation (structure), and terahertz spectroscopy (long-range solvent perturbation). Our results show that the short-range model is sufficient to explain the activity of our mutants, but the long-range model provides a necessary condition for activity: the most active peptides in our data set all have an extended dynamical hydration shell. It appears that antifreeze proteins and antifreeze glycoproteins have reached different evolutionary solutions to the antifreeze problem, utilizing either a few precisely positioned OH groups or a large quantity of OH groups for ice binding, assisted by long-range solvent perturbation.