Cryoprotective activities of group 3 late embryogenesis abundant proteins from Chlorella vulgaris C-27

The nucleotide sequence of hiC12, isolated as a cDNA clone of hardening-induced Chlorella (hiC) genes, was identified. The clone encodes a late embryogenesis abundant (LEA) protein having six repeats of a 11-mer amino acid motif, although in a slightly imperfect form. To overexpress the hiC61) and hiC12 genes, their coding regions were PCR amplified and subcloned into a pGEX-1lambdaT vector. The HIC6 and HIC12 proteins were expressed as GST fusion proteins in E. coli, then purified. The two HIC proteins were found to be effective in protecting a freeze-labile enzyme, LDH, against freeze-inactivation. On a molar concentration basis, they were about 3.1 x 10(6) times more effective in protecting LDH than sucrose and as effective as BSA. Cryoprotection tests with five kinds of chain-shortened polypeptides, synthesized based on the 11-mer amino acid motif of the HIC6 protein showed that the cryoprotective activity decreased with a decrease in the repeating units of the 11-mer motif. In fact, cryoprotective activities of three kinds of single 11-mer amino acids were very low even at high concentrations. All the results suggested that the sufficiently repeated 11-mer motif is required for the cryoprotective activities of Chlorella LEA proteins.     

Sequence composition versus sequence order in the cryoprotective function of an intrinsically disordered stress‐response protein

Intrinsically disordered stress proteins have been shown to act as chaperones, protecting proteins from damage caused by stresses such as freezing and thawing. Dehydration proteins (dehydrins) are intrinsically disordered stress proteins that are found in almost all land plants. They consist of a variable number of the short, semi‐conserved, Y‐, S‐, and K‐segments, with longer stretches of poorly conserved sequences in between. Previous studies have provided conflicting views on the details of the dehydrin cryoprotective mechanism of enzymes. Experiments with polyethylene glycol (PEG) have shown that PEG cryoprotective efficiency is the same as dehydrins of the same hydrodynamic radius, suggesting that the protein's disordered and polar nature is important, rather than the specific order of the residues. To further elucidate the mechanism, we created scrambled variants of the wild grape dehydrins K₂ and YSK₂ and tested their ability to protect lactate dehydrogenase and yeast frataxin homolog‐1 from freeze/thaw damage. The results show that for preventing aggregation, it is the sequence composition and the size of the dehydrin that is the most important factor in protection, while for freeze/thaw damage causing loss of secondary structure, it is the sequence composition that is most significant.     

Antibodies against individual thylakoid membrane proteins as molecular probes to study chemical and mechanical freezing damage in vitro

The release of proteins and the loss of biochemical activities under mechanical and chemical stresses during freezing of isolated thylakoid membranes were investigated, using polyacrylamide gel electrophoresis, single radial immunodiffusion and the measurement of cyclic photophosphorylation. Antibodies against purified proteins derived from the stromal (coupling factor CF1, ferredoxin-NADP⁺ reductase) and the lumenal side (plastocyanin) of the membrane vesicles were used as probes. Low initial solute concentrations were employed to generate mechanical stress. Chemical stresses were manipulated by varying the molar ratios of cryotoxic to cryoprotective solutes at high initial solute concentrations. Constant low amounts of ferredoxin-NADP⁺ reductase were lost from the membranes during freezing, irrespective of the composition of the suspending media. Damage at high initial osmolalities was accompanied by the release of CF1, which was influenced by the ratio of potentially cryotoxic to cryoprotective solutes, as demanded by the colligative theory of membrane cryopreservation. CF1 release and loss of cyclic photophosphorylation were linearly correlated at different ratios of salt to sucrose. However, the correlation data revealed that CF1 release could account for only part of the observed cryoinjury. Plastocyanin release was predominant at low initial osmolalities and was not influenced by the chemical composition of the suspending media. This indicates mechanical damage by membrane rupture. Under these circumstances loss of plastocyanin and loss of cyclic photophosphorylation were linearly correlated. Loss of photophosphorylation could be prevented by the addition of up to 1.2 mg plastocyanin/ml prior to freezing. It could also be ameliorated to a large extent by raising the phenazine methosulfate concentration in the test assay from 30 to 230 μM. This indicates that the membranes are able to reseal after rupture, maintaining a proton gradient upon illumination and that it is the loss of plastocyanin from their lumen that inhibits cyclic photophosphorylation.     

Cryoprotective effect of the serine-rich repetitive sequence in silk protein sericin

The silk proteins, fibroin and sericin, are produced in the silk gland of Bombyx mori, and hydrophilic sericin envelops fibroin with successive sticky layers in the formation of a cocoon. To study the biological functions of sericin, we focused on the serine-rich sericin peptide consisting of 38 amino acids, which is a highly conserved and internally repetitive sequence of a sericin protein. The corresponding gene was chemically synthesized, and the PCR-amplified gene was ligated to oligomerize sericin peptide and fused at the amino terminus to a His-tagged and proteolytic cleavage sequence in an inducible expression vector. When the dimers of sericin peptides were overexpressed in Escherichia coli, the transformants showed a prominent increase in cell viability after freezing in medium. Further, the purified dimeric sericin peptide from E. coli was found to be effective in protecting lactate dehydrogenase from denaturation caused by freeze-thaw. Both of these protective effects against freezing stress in cells and proteins were also observed with sericin hydrolysate. These results indicate that this unique sericin peptide, like sericin, has a high cryoprotective activity and will be valuable as a new biomaterial for industrial use.     

The effect of cryogenic storage on human erythrocyte membrane proteins as determined by polyacrylamide-gel electrophoresis

A study was conducted to determine the effects of freezing on the major membrane proteins of isolated human erythrocyte membranes. Membranes in low or normal ionic strength medium were frozen at slow or fast freezing rates. The membrane protein composition and elution of proteins from the membranes were studied utilizing polyacrylamide-gel electrophoresis in a sodium dodecyl sulfate or an acetic acid-urea-phenol solvent system. Neither a change in the composition of the membrane proteins nor any elution of membrane protein during freezing and thawing was observed. The data indicate that any human erythrocyte membrane damage during freezing and thawing was not related to a change in major membrane protein composition. Human red cell membranes were stable at ?80 or ?196 °C in the absence of a cryoprotective agent.     

植物的低温蛋白

综述了与植物耐冻性有关的一些植物内源蛋白质或多肽 ,包括低温防护蛋白、抗冻蛋白、植物脱水素、膜关联耐冻性多肽蛋白质。结果表明 ,植物的耐冻性与其低温蛋白 (cold induced proteins)有着密切的关系 ,并指出了抗冻蛋白行使功能的两种可能的作用方式。同时 ,耐冻性与除低温外的其它环境胁迫因子的植物抗性如抗干旱、抗病虫、高盐耐性、乙烯耐性等密切相关     

Changes in protein metabolism during the acquisition of tolerance to cryopreservation of carrot somatic embryos

High concentrations of sucrose are often used to cryopreserve regenerable plant cell cultures in liquid nitrogen. A 21-h pretreatment of carrot somatic embryos in medium containing 0.4 M sucrose allows 80 % of them to germinate after freezing. Substitution of sucrose by polyethylene glycol 6000 led to lower germination rates. However, a high level of freezing tolerance was restored by addition of 1 μM abscisic acid in the pretreatment medium. Using these different media, both total water soluble protein, using SDS-PAGE, and boiling-stable protein, using 2-D electrophoresis, were studied in relation to acquisition of cryopreservation tolerance. Only boiling-stable protein patterns showed some changes: five polypeptides accumulated in 0.4 M sucrose-pretreated embryos or in embryos pretreated by media containing abscisic acid. This accumulation was not detected with polyethylene glycol 6000 used as sole cryoprotectant. Although over-accumulation of polypeptides was highest with media containing ABA, the best germination rates were linked to pretreatment with 0.4 M sucrose. The addition of okadaic acid in 0.4 M sucrose medium led to embryo death after freezing, confirming the existence of a message leading to metabolic changes and acquisition of cryotolerance. Water-soluble proteins obtained from 0.4 M sucrose-pretreated embryos appeared more active than those extracted from control embryos in protecting in vitro a freeze-labile enzyme. Boiling-stable proteins, corresponding to a part of total proteins, were more active than total proteins. These results suggest that these polypeptides may be involved in a mechanism of protection needed for cell survival during freezing stress.     

Freezing of isolated thylakoid membranes in complex media. VIII. Differential cryoprotection by sucrose, proline and glycerol

The cryoprotective efficiency of sucrose, proline and glycerol for chloroplast membranes isolated from spinach leaves ( Spinacia oleracea L. cv. Monatol) was determined after freeze-thaw treatment in media containing the predominant inorganic electrolytes of the chloroplast stroma. In most cases, the protective capacity of equimolar concentrations of the cryoprotectants followed the order sucrose > proline > glycerol. The lower the freezing temperature the less cryoprotectant was necessary for comparable preservation of the capacity of photosynthetic electron transport. Likewise, the cryoprotective efficiency of sucrose for cyclic photophosphorylation and light-induced proton gradient increased with decreasing freezing temperature. In contrast, while proline effectively stabilized these membrane reactions at mild and moderate freezing temperatures, it was much less efficient at more severe freezing stress. Cryoprotection of photophosphorylation and proton gradient formation at given initial concentrations of glycerol was largely independent of the freezing temperature. While dissociation of the peripheral part of chloroplast coupling factor (CF₁) during freeze-thaw treatment cannot be prevented in the presence of lower initial concentrations of proline and glycerol and. at mild freezing temperatures, of sucrose, the latter may stabilize this protein complex at least under more severe freezing conditions. The differences in the cryoprotective efficiency of the solutes are discussed relative to their non-ideal activity-concentration profiles, solution properties and penetration behaviour across the thylakoid membrane.     

Effects of freezing on the release and inactivation of chloroplast coupling factor 1

The three high-molecular-weight subunits of chloroplast coupling factor (CF₁) are the primary proteins released from pyrophosphate-washed thylakoids exposed to freezing. Identical subunit profiles are found in the supernatant proteins of thylakoids exposed to different intensities of freezing stress by the inclusion of sugars with varying degrees of cryoprotective efficiency. Isolated CF₁ is inactivated by freezing in the presence of NaCl, glucose, and sucrose but raffinose can protect against loss of enzymatic activity during freezing. The low specific activity of the supernatant proteins released from the thylakoid and the inability to recover the Ca²⁺-dependent ATPase activity lost from the membrane suggest that inactivation accompanies release of CF₁ during freezing.     

Frost tolerance and biochemical changes during hardening and dehardening in contrasting white clover populations

Successful winter survival of perennial plants, like white clover, is dependent on proper timing of both hardening and dehardening. The purpose of this study was to investigate the regulation of these processes in two cultivars (AberCrest and AberHerald) and two Norwegian ecotypes (Særheim collected at 58°46′N lat. and Bodø at 67°20′N lat.) of white clover (Trifolium repens L.). For hardening and dehardening, plants were exposed to controlled temperature conditions and frost hardiness of stolons was tested by programmed freezing at the rate of 3°C per hour. In addition, stolons were analysed for starch, soluble sugars and soluble amino acids. Cultivars AberCrest and AberHerald, selected for growth at low temperature and winter hardiness in the United Kingdom, were significantly less hardy than the Norwegian populations. After six weeks of hardening (2 weeks at 6°C and 4 weeks at 0.5°C), estimated LT₅₀ values were ?13.8, ?13.0, ?17.8 and ?20.3°C for AberCrest, AberHerald, Saerheim and Bodø, respectively. The rate of dehardening increased with increasing temperature. At low temperature (6°C), the northern ecotype from Bodø was more resistant to dehardening than AberHerald. However, at 18°C the absolute rate of dehardening (°C day^?1) was twice as high in Bodø as in AberHerald plants. Stolon elongation during dehardening was initiated at lower temperatures in AberHerald than in plants of the Bodø ecotype. The content of total soluble sugars, sucrose and the amino acids proline and arginine were significantly higher in hardy plants of Bodø than in those of AberHerald. Sucrose levels decreased during dehardening and correlations between sucrose content and LT₅₀ during this process were statistically highly significant for both Bodø and AberHerald. The least hardy populations of white clover were characterized by thick stolons, long internodes and large leaves.     

Cryoprotectants lead to phenotypic adaptation to freeze-thaw stress in Lactobacillus delbrueckii ssp. bulgaricus CIP 101027T

This study was conducted to investigate the ability of cryoprotective chemicals to induce phenotypic cryoadaptation in Lactobacillus delbrueckii ssp. bulgaricus CIP 101027T. Tolerance to negative temperature stress (freezing at -20 degrees C and thawing at 37 degrees C) was induced by pretreatment with Me(2)SO, glycerol, lactose, sucrose, and trehalose. Interestingly, Me(2)SO has a significantly greater cryoprotective effect than glycerol. Furthermore, lactose, sucrose, and trehalose, often referred to as osmotica, were shown to have greater cryoadaptive than cryoprotective properties. These results suggest that bacteria such as L. delbrueckii ssp. bulgaricus could be phenotypically adapted to freezing and thawing by an osmotic stress applied prior to freeze-thaw stress.     

Isolation of freeze-tolerant laboratory strains of Saccharomyces cerevisiae from proline-analogue-resistant mutants

Since some amino acids, polyols and sugars in cells are thought to be osmoprotectants, we expected that several amino acids might also contribute to enhancing freeze tolerance in yeast cells. In fact, proline and charged amino acids such as glutamate, arginine and lysine showed a marked cryoprotective activity nearly equivalent to that of glycerol or trehalose, both known as major cryoprotectants for Saccharomyces cerevisiae. To investigate the cryoprotective effect of proline on the freezing stress of yeast, we isolated proline-analogue-resistant mutants derived from a proline-non-utilizing strain of S. cerevisiae. When cultured in liquid minimal medium, many mutants showed a prominent increase, two- to approximately tenfold, in cell viability compared to the parent after freezing in the medium at −20 °C for 1 week. Some of the freeze-tolerant mutants were found to accumulate a higher amount of proline, as well as of glutamate and arginine which are involved in proline metabolism. It was also observed that proline-non-utilizer and the freeze-tolerant mutants were able to grow against osmotic stress. These results suggest that the increased flux in the meta-bolic pathway of specific amino acids such as proline is effective for breeding novel freeze-tolerant yeasts. Received: 6 November 1996 / Accepted: 7 December 1996     

Effective cryoprotection of thylakoid membranes by ATP

Freezing of isolated spinach thylakoids in the presence of NaCl uncoupled photophosphorylation from electron flow and increased the permeability of the membranes to protons. Addition of ATP prior to freezing diminished membrane inactivation. On a molar basis, ATP was at least 100 times more effective in protecting thylakoids from freezing damage than low-molecularweight carbohydrates such as sucrose and glucose. The cryoprotective effectiveness of ATP was increased by Mg²⁺. In the absence of carbohydrates, preservation of thylakoids during freezing in 100 mM NaCl was saturated at about 1–2 mM ATP, but under these conditions membranes were not fully protected. However, in the presence of small amounts of sugars which did not significantly prevent thylakoid inactivation during freezing, ATP concentrations considerably lower than 0.5 mM caused nearly complete membrane protection. Neither ADP nor AMP could substitute for ATP. These findings indicate that cryoprotection by ATP cannot be explained by a colligative mechanism. It is suggested that ATP acts on the chloroplast coupling factor, either by modifying its conformation or by preventing its release from the membranes. The results are discussed in regard to freezing injury and resistance in vivo.Abbreviations CF₁ chloroplast coupling factor - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - PMS phenazine methosulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propandiol     

A cold-regulated Arabidopsis gene encodes a polypeptide having potent cryoprotective activity.

We previously showed that cor15, a cold-regulated gene from Arabidopsis thaliana, encodes a 15 kDa polypeptide designated COR15. Here we report that COR15 has potent cryoprotective activity in an in vitro cryoprotection assay. Specifically, COR15 was very effective in protecting the cold-labile enzyme lactate dehydrogenase against freeze-inactivation; on a concentration basis, it was about 10(6) times more effective than sucrose and 10(2)-10(3) times more effective than other proteins including bovine serum albumin. The possible role of cor15 in cold acclimation is discussed.     

Salt treatment induces frost hardiness in leaves and isolated thylakoids from spinach

Frost hardiness of spinach (Spinacia oleracea L.) leaves was increased by high concentrations of NaCl in the hydroponic culture medium. Freezing damage was determined by measurement of slow chlorophyll fluorescence quenching after freezing of leaves. Both the osmolality of the leaf sap and forst hardiness of the leaves were linearly correlated with the salt concentration in the hydroponic culture medium. Freezing damage occurred, irrespective of the extent of frost hardening, when dehydration of cells during extracellular ice formation decreased cellular volume to approximately 14% of the volume of unfrozen cells. The resistance of isolated, washed thylakoids against mechanical and chemical damage by freezing was investigated. Chemical damage by freezing caused by salt accumulation was measured as release of chloroplast coupling factor (CF1; EC 3.6.1.3), and mechanical damage was measured as release of the lumenal protein plastocyanin from the membranes during an in-vitro freeze-thaw cycle. Isolated thylakoids from salt-treated frost-hardy spinach and those from plants hardened under natural conditions did not exhibit improved tolerance against chemical freezing stress exerted by high salt concentrations. They were, however, more hardy than thylakoids from unhardened control leaves against mechanical damage by freezing.Abbreviation CF1 peripheral part of chloroplast coupling factor ATPase     

On the mechanisms of frost injury and frost hardening of spruce chloroplasts

Hill reaction and noncyclic photophosphorylation of isolated class C chloroplasts of spruce (Picea abies (L.) Karst.), as well as ¹⁴CO₂ fixation by whole needles at constant laboratory conditions proceeded at high rates during spring and early summer, declined during late summer and autumn by about 60%, remained at this level during winter, and recovered quickly in early spring. During summer, the whole needles proved to be frost labile, since after exposure to-20°C and careful thawing, fast chlorophyll degradation occurred. In addition, only photosynthetically inactive chloroplasts could be isolated from those precooled needles. On the contrary, during winter the photochemical activities of plastids from freshly harvested needles did not differ from those of artificially frozen-thawed needles. When isolated spruce chloroplasts were exposed to the same subfreezing temperatures as the whole needles, no influence of freezing on the photochemical activities was observed, irrespective of whether the plastids were isolated from frost sensitive or frost hardened needles. It is concluded that frost damage to spruce chloroplasts is due to an attack of membrane toxic compounds or lytic enzymes which were liberated upon freezing from more labile compartments. Frost hardening of the chloroplasts, as determined by the stability of chlorophyll after exposure of the needles to low temperatures, as well as by the isolation of photosynthetically active chloroplasts from such precooled needles, appeared to depend at least on 2 processes: (i) an alteration of the composition of the photosynthetically active membranes and (ii) and additional stabilization of these membranes by protecting substances. The first process was indicated by a large increase (decrease) of the capability of isolated chloroplasts for PMS-mediated photophosphorylation which accompanied natural or artificial frost hardening (dehardening). Production of cryoprotecting compounds was suggested by a significant higher stability against NaCl observed with class C chloroplasts isolated from frost hardened needles as compared to that of plastids from frost labile material. The decrease of the capability for both, the ferricyanide dependent photoreactions of the plastids and the CO₂ fixation by whole needles, which was observed during the frost hardening phase, cannot be due to freezing injuries; it rather appears to be a consequence of the frost hardening process.     

Cryoprotectin protects thylakoids during a freeze-thaw cycle by a mechanism involving stable membrane binding

Chloroplast thylakoid membranes of higher plants are damaged by freezing both in vivo and in vitro. The resulting inactivation of photosynthetic electron transport has been related to transient membrane rupture, leading to the loss of soluble electron transport proteins and osmotically active solutes from the thylakoid lumen. We have recently purified and sequenced a protein from cold acclimated cabbage, that protects thylakoids from this freeze-thaw damage. The protein belongs to the WAX9 family of nonspecific lipid transfer proteins, but has no detectable lipid transfer activity. Conversely, other transport-active lipid transfer proteins show no cryoprotective activity. We show here that cryoprotectin binds to thylakoid membranes. Both cryoprotective activity and membrane binding were inhibited in the presence of specific sugars, most effectively by Glc-6-S. The binding of cryoprotectin to thylakoids reduced the fluidity of the membrane lipids close to the membrane/solution interface, but not in the hydrophobic core region. Using immobilized liposomes we could show that cryoprotectin was able to bind to pure lipid membranes.     

THE RELATION OF COLD RESISTANCE TO THE STATUS OF PHOSPHORUS AND CERTAIN METABOLITES IN RED-OSIER DOGWOOD (CORNUS STOLONIFERA MICHX.)

The process of cold acclimation in the winter hardy woody shrubCornus stolonifera Michx. was studied in relation to changesin phosphorus status and certain metabolites. Plants were coldacclimated effectively under controlled conditions which combinedshortening photoperiods and freezing temperatures. Metabolicchanges under these conditions were similar to those which occurnaturally in autumn. There was an extremely close relationshipbetween cold resistance and the status of phosphorus; i.e. ashardiness increased inorganic P decreased and acid soluble boundP and total organic P increased. Starch decreased during acclimationwhile the simpler carbohydrates increased. These included glucose,sucrose, fructose, raffinose, melibiose, xylose, and stachyosein decreasing order of occurrence. Proteins increased whilefree amino acids generally decreased. The non-volatile organicacids in decreasing quantitative order included malic, succinic,quinic, tartaric, citric, and shikimic acids. Of these onlyshikimic acid increased during hardening. These biochemicalchanges are discussed in reference to current theories of coldacclimation in plants. (Received June 18, 1966; )     

Freezing tolerance of sea urchin embryo pigment cells

Various stresses, including exposure to cold or heat, can result in a sharp increase in pigmentation of sea urchin embryos and larvae. The differentiation of pigment cells is accompanied by active expression of genes involved in the biosynthesis of naphthoquinone pigments and appears to be a part of the defense system protecting sea urchins against harmful factors. To clarify numerous issues occurring at various time points after the cold injury, we studied the effect of shikimic acid, a precursor of naphthoquinone pigments, on cell viability and expression of some pigment genes such as the pks and sult before and after freezing the cultures of sea urchin embryo cells. The maximum level of the pks gene expression after a freezing–thawing cycle was found when sea urchin cells were frozen in the presence of trehalose alone. Despite naphthoquinone pigments have been reported to possess antioxidant and cryoprotectant properties, our data suggest that shikimic acid does not have any additional cryoprotective effect on freezing tolerance of sea urchin embryo pigment cells.     

Formation of Ice Microparticles in the Ovarian Fluid and Homogenates of Unfertilized Russian Sturgeon Eggs during Cooling to –196°C

Cryopreservation of fish and amphibian eggs is still an unsolved problem. The formation of ice crystals inside and outside cells acts as a main detrimental factor during a deep freezing of fish eggs, as well as crystal growth (recrystallization and repeated crystallization). Designing efficient cryoprotective media is necessary in order to avoid egg injury from freezing. Additional components that are present in a cryoprotective medium and reduce the thermomechanical stress and cracks of frozen tissues might increase oocyte survival after freezing–thawing. Natural components of eggs and the ovarian fluid are promising as such additives. The formation of ice microparticles was studied in thin layers (0.2 mm) of the ovarian fluid and components of Russian sturgeon egg homogenates upon their cooling to a liquid nitrogen temperature (–196°C). The processes of freezing, ice cracking, and microparticle formation were observed as the temperature was decreased gradually. The shape and size of ice microparticles were found to depend on the composition of the freezing solution. Certain fractions of egg homogenate were assumed to be suitable as components of a cryoprotective medium.