Cold-Induced Alterations in Plasma Membrane Proteins That are Specifically Related to the Development of Freezing Tolerance in Cold-Hardy Winter Wheat

The objective of this study was to identify plasma membraneproteins that are specifically induced by cold acclimation inwheat (Triticum aestivum L.). Two cultivars with a marked differencein the genetic ability to cold-acclimate, namely, spring wheat(cv. Chinese Spring) and winter wheat (cv. Norstar), were usedas the experimental material. After four weeks of growth ina cold chamber, the freezing tolerance in the shoots of winterwheat increased to –18°C, whereas it increased onlyto –8°C in the shoots of spring wheat. In the caseof roots from both cultivars, freezing tolerance increased onlyslightly after the growth in the cold environment. Cold acclimationinduced remarkable changes in the electrophoretic patterns ofplasma membrane proteins which depended on both the cultivarand the tissue examined. Levels of polypeptides with molecularmasses from 22 to 31 kDa decreased in both the root and shootplasma membranes from both cultivars. Among these polypeptides,levels of those of 28 and 26 kDa decreased abruptly after oneweek of cold acclimation. By contrast, levels of polypeptidesof 89, 83, 52, 23, 18 and 17 kDa increased specifically in theshoots of winter wheat. The increases in the levels of the 23-,18- and 17-kDa polypeptides were proportional to the developmentof freezing tolerance. Freeze-fracture electron microscopy ofplasma membranes from shoot cells revealed that the number ofintramembrane particles on the fracture faces decreased markedlyin winter wheat after cold acclimation, but to a lesser extentin spring wheat. These results suggest that the plasma membranesmight undergo molecular reorganization during cold acclimation. ¹Contribution no. 3709 from the Institute of Low TemperatureScience, Hokkaido University.     

Alterations of gene expression in potato (Solanum commersonii) during cold acclimation

Plantlets of Solanum commersonii stem-culture were acclimated at 5°C day/night temperature for 14 days. Cold hardiness increased from – 3.5°C to – 8.6°C. During the course of acclimation, the synthesis of polypeptides was investigated and poly (A⁺) RNA was isolated. Translation products of poly(A⁺) RNA in a rabbit rcticulocyte lysate system were then analyzed. During the 14 days of acclimation, 23 cold-induced polypeptides were identified. Most of them disappeared following 1 day of de-acclimation at a 20/15°C day/night regime. The synthesis of one group of polypeptides is prominent and stable throughout the acclimation period. The other group is transient. The most prominent and stable polypeptides have molecular weights of 21, 22, 31 and 83 kDa.
Acclimation alters translatable mRNA population during the development of cold hardiness. Two mRNAs encoding in vitro translation products at 26 and 27 kDa were identified during the course of acclimation. These proteins may play important roles in the overall programming for the development of cold hardiness in tuber-bearing S. commersonii.     

Polysome Metabolism during Cold Acclimation in Wheat

The content, composition and biological activity of polysomesfrom three wheat genotypes were studied during cold acclimation.The structural integrity of the different polysome populationswas not affected by the hardening temperature. Polysomes werealso found to accumulate at higher level in cold hardened seedlingssuggesting a high protein synthesis capacity during the acclimationperiod. The in vitro translation of polysome-bound mRNAs inthe wheat germ cell-free system showed a high translation potentialof polysomes from cold hardened seedlings compared to that ofcontrol. The electrophoretic analysis of the translation productsby two-dimensional SDS-PAGE revealed the induction of severalnew mRNAs in cold hardened wheat seedlings. The presence ofthese new messengers in the polysomal fraction suggests thatnew messages have already been processed, transported and preferentiallyselected for translation by the ribosomes. The most importantchange was the induction and pronounced synthesis of four peptides[one high mol wt peptide of 200 kDa (pI 6.5) and three smallerones of 58 (pI 7.0), 48 (pI 7.1) and 48 (pI 7.2) kDa respectively]in the freezing tolerant cultivar Norstar. These specific polypeptideswere absent in the freezing sensitive cultivar Glenlea suggestingthat their induction and expression was associated with thefreezing tolerance capacity. (Received January 19, 1990; Accepted August 24, 1990)     

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 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.     

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     

Altered cardiac tissue gene expression during acute hypoxic exposure

Summary Anoxia has been shown to induce the expression of one or more stress proteins in mammalian cells and tissues. A less severe form of oxygen depletion, hypoxic hypoxia, occurs in response to hypobaric decompression which simulates high altitude conditions. Under these conditions mouse hearts accumulate mRNAs for at least two polypeptides at substantially elevated levels. The molecular weights of these proteins, 85 kDa and 95 kDa, are similar to those reported for other mammalian stress proteins or glucose-regulated proteins. Time course experiments suggest that mRNAs for these species increase continuously for up to 16 hours of treatment, while mRNA for 71 kDa and 79 kDa polypeptides are elevated early in the treatment, but later decrease to control values. Total heart mRNA template activity is also increased by the hypobaric treatment. These results demonstrate that mouse cardiac tissue is capable of mounting a cellular stress-like response when exposed to moderately stressful conditions. It also provides a model for studying the direct effects of acute hypoxic stress on cellular gene expression, and its relationship to physiological adaptation.     

Heat-Stable COR (Cold-Regulated) Proteins Associated with Freezing Tolerance in Spinach

While most soluble proteins are coagulated by heating at 100°Cfor 10 minutes, some highly hydrophilic COR (Cold-regulated)proteins remain soluble in aqueous solution (Lin et al. 1990).We report here changes in levels of heat-stable proteins andtheir mRNAs during cold acclimation of spinach (Spinacia oleraceaL.). We analyzed heat-stable proteins and the heat-stable translationproducts from poly(A)⁺RNA generated in a wheat germ system.Heat-stable COR proteins with molecular masses of 140 kDa and85 kDa (CORs 140 and 85), were detected in the leaves of cold-acclimatedplants. Increased levels of CORs 140 and 85 correlated withthe development of freezing tolerance during cold acclimation.Interestingly, CORs 140 and 85 accumulated specifically in theleaves and stems and not in the roots of the cold-acclimatedplants. Consistent with this observation, freezing tolerancewas also induced in leaves and stems, but not in roots. Thesedata strongly suggest that CORs 140 and 85 are closely associatedwith freezing tolerance. Accumulation of COR 85 was also inducedby exogenous ABA, drought, and wounding. The possible rolesof CORs 140 and 85 in plants acclimating to low temperatureis given attention. (Received June 11, 1992; Accepted September 1, 1992)     

Sugars regulate cold-induced gene expression and freezing-tolerance in barley cell cultures

The hypothesis that the extracellular concentration of sugars helps regulate the acclimation of plant cells to cold was tested in this work. Suspension cultures were used to control the concentration of sugars in the medium supplied to barley cell cultures (Hordeum vulgare L. cv. Igri), replacing the medium daily to help maintain the concentration. Freezing tolerance and the levels of mRNA expression of the stress-response genes blt4.9 (coding for a non- specific lipid transfer protein) and dhn1 (coding for a dehydrin) were measured. Similar levels of freezing-tolerance and gene expression were obtained in the experiments as occur during cold-acclimation in the crown of the whole plant. In the cell cultures, cold (6/2 degrees C) did not induce an increase in freezing tolerance or in the expression of detectable levels of blt4.9 or dhn1 mRNAs when only 1 g l-1 sucrose was supplied. However, the cells in this low sucrose medium in the cold were not sugar-starved, indicating that this did not explain the failure of the cells to acclimate when grown in the cold environment. Ten g l-1 sucrose supplied to cells grown in the warm (25 degrees C) induced acclimation to freezing and up-regulation of expression of blt4.9 and dhn1 mRNAs. Osmolality of the medium did not explain this. Thirty g l-1 sucrose induced yet higher levels of freezing tolerance and of blt4.9 and dhn1 mRNAs in cultures grown in either the cold or the warm environment. The results implicate sugars in the regulation of cold acclimation     

Drought acclimation confers cold tolerance in the soil collembolan Folsomia candida

It has been noted that both summer drought and sub-zero winter temperatures induce the synthesis of sugars and polyols in invertebrate tissues. This has led several authors to suggest that many of the adaptations, previously viewed as a response to cold, might be part of a more universal desiccation tolerance mechanism. Here we show that acclimation of the soil dwelling collembolan Folsomia candida to a sublethal desiccation stress confers tolerance to cold shock and a significant increase in the molar percent of membrane fatty acids with a mid-chain double bond. These changes in membrane fatty acids are interpreted as conferring a significant reduction in the transition temperature of cell membranes, as would be expected in acclimation to cold, and these changes are therefore interpreted as contributing to the cross-tolerance. Drought acclimation was also shown to trigger the synthesis of the 70kDa family of heat-shock proteins (Hsp70). This group of heat shock proteins is implicated in the reestablishment of the normal three-dimensional structure of partially unfolded proteins and therefore are also likely to contribute to the observed cross-tolerance. This study provides evidence that the stresses exerted by desiccation and cold at the cellular level have sufficient similarities to induce overlapping adaptations.     

Temporal cell wall changes during cold acclimation and deacclimation and their potential involvement in freezing tolerance and growth

Plants adapt to freezing stress through cold acclimation, which is induced by nonfreezing low temperatures and accompanied by growth arrest. A later increase in temperature after cold acclimation leads to rapid loss of freezing tolerance and growth resumption, a process called deacclimation. Appropriate regulation of the trade-off between freezing tolerance and growth is necessary for efficient plant development in a changing environment. The cell wall, which mainly consists of polysaccharide polymers, is involved in both freezing tolerance and growth. Still, it is unclear how the balance between freezing tolerance and growth is affected during cold acclimation and deacclimation by the changes in cell wall structure and what role is played by its monosaccharide composition. Therefore, to elucidate the regulatory mechanisms controlling freezing tolerance and growth during cold acclimation and deacclimation, we investigated cell wall changes in detail by sequential fractionation and monosaccharide composition analysis in the model plant Arabidopsis thaliana, for which a plethora of information and mutant lines are available. We found that arabinogalactan proteins and pectic galactan changed in close coordination with changes in freezing tolerance and growth during cold acclimation and deacclimation. On the other hand, arabinan and xyloglucan did not return to nonacclimation levels after deacclimation but stabilized at cold acclimation levels. This indicates that deacclimation does not completely restore cell wall composition to the nonacclimated state but rather changes it to a specific novel composition that is probably a consequence of the loss of freezing tolerance and provides conditions for growth resumption.     

Heterotetrameric composition of aquaporin-4 water channels.

Aquaporin (AQP) water channel proteins are tetrameric assemblies of individually active approximately 30 kDa subunits. AQP4 is the predominant water channel protein in brain, but immunoblotting of native tissues has previously yielded multiple poorly resolved bands. AQP4 is known to encode two distinct mRNAs with different translation initiating methionines, M1 or M23. Using SDS-PAGE urea gels and immunoblotting with anti-peptide antibodies, four polypeptides were identified in brain and multiple other rat tissues with the following levels of expression: 32 kDa > 34 kDa > 36 kDa > 38 kDa. The 34 and 38 kDa polypeptides react with an antibody specific for the N-terminus of the M1 isoform, and 32 and 36 kDa correspond to the shorter M23 isoform. Immunogold electron microscopic studies with rat cerebellum cryosections demonstrated that the 34 kDa polypeptide colocalizes in perivascular astrocyte endfeet where the 32 kDa polypeptide is abundantly expressed. Velocity sedimentation, cross-linking, and immunoprecipitation analyses of detergent-solubilized rat brain revealed that the 32 and 34 kDa polypeptides reside within heterotetramers. Immunoprecipitation of AQP4 expressed in Xenopus laevis oocytes demonstrated that heterotetramer formation reflects the relative expression levels of the 32 and 34 kDa polypeptides; however, tetramers containing different compositions of the two polypeptides exhibit similar water permeabilities. These studies demonstrate that AQP4 heterotetramers are formed from two overlapping polypeptides and indicate that the 22-amino acid sequence at the N-terminus of the 34 kDa polypeptide does not influence water permeability but may contribute to membrane trafficking or assembly of arrays.     

Suppression of phospholipase Dalpha1 induces freezing tolerance in Arabidopsis: response of cold-responsive genes and osmolyte accumulation

Phospholipase D (PLD; EC 3.1.4.4) plays an important role in membrane lipid hydrolysis and in mediation of plant responses to a wide range of stresses. PLDalpha1 abrogation through antisense suppression in Arabidopsis thaliana resulted in a significant increase in freezing tolerance of both non-acclimated and cold-acclimated plants. Although non-acclimated PLDalpha1-deficient plants did not show the activation of cold-responsive C-repeat/dehydration-responsive element binding factors (CBFs) and their target genes (COR47 and COR78), they did accumulate osmolytes to much higher levels than did the non-acclimated wild-type plants. However, a stronger expression of COR47 and COR78 in response to cold acclimation and to especially freezing was observed in PLDalpha1-deficient plants. Furthermore, a slower activation of CBF1 was observed in response to cold acclimation in these plants compared to the wild-type plants. Typically, cold acclimation resulted in a higher accumulation of osmolytes in PLDalpha1-deficient plants than in wild-type plants. Inhibition of PLD activity by using lysophosphatidylethanolamine (LPE) also increased freezing tolerance of Arabidopsis, albeit to a lesser extent than did the PLD antisense suppression. Exogenous LPE induced expression of COR15a and COR47 in the absence of cold stimulus. These results suggest that PLDalpha1 plays a key role in freezing tolerance of Arabidopsis by modulating the cold-responsive genes and accumulation of osmolytes.