Responses of the plasma membrane to low temperatures

The plasma membrane is considered to be the primary site of injury when plant cells are subjected to extracellular freezing. In order for plants or plant cells to acquire freezing tolerance, it is, thus, necessary that the plasma membrane increases its cryostability during freeze-thaw excursion. During cold acclimation both under natural and artificial conditions, there are compositional, structural and functional changes occurring in the plasma membrane, many, if not all, of which ultimately contribute to increased stability of the plasma membrane under freezing conditions. In addition, changes in the cytosol or intracellular compartments also affect the cryobehaviour of the plasma membrane during freeze-induced dehydration. Although many alterations occurring during cold acclimation influence the cryobehaviour of the plasma membrane comprehensively, recent advances in functional genomics approaches provide interesting information on the function of specific proteins for plasma membrane behaviour under freezing conditions.     

Arabidopsis dynamin‐related protein 1E in sphingolipid‐enriched plasma membrane domains is associated with the development of freezing tolerance

The freezing tolerance of Arabidopsis thaliana is enhanced by cold acclimation, resulting in changes in the compositions and function of the plasma membrane. Here, we show that a dynamin‐related protein 1E (DRP1E), which is thought to function in the vesicle trafficking pathway in cells, is related to an increase in freezing tolerance during cold acclimation. DRP1E accumulated in sphingolipid and sterol‐enriched plasma membrane domains after cold acclimation. Analysis of drp1e mutants clearly showed that DRP1E is required for full development of freezing tolerance after cold acclimation. DRP1E fused with green fluorescent protein was visible as small foci that overlapped with fluorescent dye‐labelled plasma membrane, providing evidence that DRP1E localizes non‐uniformly in specific areas of the plasma membrane. These results suggest that DRP1E accumulates in sphingolipid and sterol‐enriched plasma membrane domains and plays a role in freezing tolerance development during cold acclimation.     

Rapid degradation of starch in chloroplasts and concomitant accumulation of soluble sugars associated with ABA-induced freezing tolerance in the moss Physcomitrella patens

Abscisic acid (ABA) has been postulated to play a role in the development of freezing tolerance during the cold acclimation process in higher plants, but its role in cold tolerance in tower land plants has not been elucidated. The moss Physcomitrella patens rapidly developed freezing tolerance when its protonemata were grown in a medium containing ABA, with dramatic changes in the LT50 value from -2 degrees C to over -10 degrees C. We examined physiological and morphological alterations in protonema cells caused by ABA treatment to elucidate early cellular events responsible for rapid enhancement of freezing tolerance. Microscopic observations revealed that ABA treatment for 1 day resulted in a dramatic alteration in the appearance of intracellular organelles. ABA-treated cells had slender chloroplasts, with a reduced amount of starch grains, in comparison with those of non-treated cells. The ABA-treated cells also had several segmented vacuoles while many of non-treated cells had one central vacuole. When frozen to -4 degrees C, freezing injury-associated ultrastructural changes such as formation of aparticulate domains and fracture-jump lesions were frequently observed in the plasma membrane of non-treated protonema cells but not in that of ABA-treated cells. The ABA treatment increased the osmotic concentration of the protonema cells, in correlation with accumulation of free soluble sugars. These results suggest that ABA-induced accumulation of soluble sugars, associated with morphological changes in organelles, mitigated freezing-induced structural damage in the plasma membrane, eventually leading to enhancement of freezing tolerance in the protonema cells.     

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.     

Freeze/thaw-induced destabilization of the plasma membrane and the effects of cold acclimation

Disruption of the plasma membrane is a primary cause of freezing injury. In this review, the mechanisms of injury resulting from freeze-induced cell dehydration are presented, including destabilization of the plasma membrane resulting from (a) freeze/thaw-induced osmotic excursions and (b) lyotropic phase transitions in the plasma membrane lipids. Cold acclimation dramatically alters the behavior of the plasma membrane during a freeze/thaw cycle—increasing the tolerance to osmotic excursions and decreasing the propensity for dehydration-induced lamellar to hexagonal-II phase transitions. Evidence for a casual relationship between the increased cryostability of the plasma membrane and alterations in the lipid composition is reviewed.     

Dynamic compositional changes of detergent-resistant plasma membrane microdomains during plant cold acclimation

Plants increase their freezing tolerance upon exposure to low, non-freezing temperatures, which is known as cold acclimation. Cold acclimation results in a decrease in the proportion of sphingolipids in the plasma membrane in many plants including Arabidopsis thaliana. The decrease in sphingolipids has been considered to contribute to the increase in the cryostability of the plasma membrane through regulating membrane fluidity. Recently we have proposed a possibility of another important sphingolipid function associated with cold acclimation.¹ In animal cells, it has been known that the plasma membrane contains microdomains due to the characteristics of sphingolipids and sterols, and the sphingolipid- and sterol-enriched microdomains are thought to function as platforms for cell signaling, membrane trafficking and pathogen response. In our research on characterization of microdomain-associated lipids and proteins in Arabidopsis, a cold-acclimation-induced decrease in sphingolipids resulted in a decrease of microdomains in the plasma membrane and there were considerable changes in membrane transport-, cytoskeleton- and endocytosis-related proteins in the microdomains during cold acclimation. Based on these results, we discuss a functional relationship between the changes in microdomain components and plant cold acclimation.Key words: Arabidopsis, cold acclimation, detergent-resistant plasma membrane, plasma membrane lipid, plasma membrane protein, microdomain, proteome analysisIn fall or early winter, plants recognize the decrease in temperature and change cellular metabolism to survive against freezing stress. This phenomenon is termed as cold acclimation.² Because the plasma membrane is the critical site in cell survival during freezing, diverse cold-acclimation-induced changes are believed to ultimately protect the plasma membrane from the irreversible damage under freezing stress.³ One of the notable changes during cold acclimation is a decrease in sphingolipids, a characteristic plasma membrane lipid.⁴ Sphingolipids have melting temperatures higher than do phosphsolipids, major plasma membrane lipids. Thus, quantitative decreases in sphonglipids are considered to increase in membrane fluidity at low temperatures.⁴ Some 20 years ago, however, experimental results that sphinglipids form lipid microdomains in the plasma membrane were reported in mammalian and yeast cells.^5–7 Sphingolipids are heterogeneously distributed and self-associated with sterols and specific proteins in the plasma membrane. The sphingolipid/sterol-enriched microdomains in the plasma membrane are sometime called “membrane (lipid) raft” or “caveolae” in mammalian cells, and similar domains have been proposed later in plant cells.^8–11 The microdomains are biochemically isolated as low-density detergent-resistant plasma membrane (DRM) fractions and contain specific proteins associated with membrane trafficking, signal transduction, membrane transport, cytoskeleton interaction and pathogen infection.¹² Consequently, the microdomains are suspected to function as platform for assembly of these functional protein complexes and temporal interaction between protein-protein or protein-lipid.⁷ The microdomains change not only in domain size by coalescence of individual domains but also in protein and lipid compositions by physiological stimulus.^12–15We hypothesized that a decrease of sphingolipids in the plant plasma membrane during cold acclimation might not only increase membrane fluidity but also change microdomain formation and/or function. Our recent paper characterized cold-responsiveness of lipid and protein components in plant DRMs.¹ Arabidopsis thaliana is able to increase in freezing tolerance after few days of cold treatment [the temperature of 50% survival is −7°C before cold treatment at 2°C and decreases to −15°C after 7-d-treatment]. We first isolated plasma membrane-enriched fractions using aqueous two-phase partition system from Arabidopsis seedlings before and after cold acclimation. Next, plasma membrane fractions were subjected to 1% (w/v) Triton X-100 on ice for 30 min and then sucrose density gradient centrifugation. DRM fractions appeared as two white bands at about 40% (w/w) sucrose. DRMs in plants are generally recovered as heavier fractions than those in animals.^16–18 This is probably because the ratio of protein to lipid is greater in plants than in animals. Arabidopsis DRM fractions were enriched in sphingolipids (glucocerebrosides) and sterols (free sterols, acylated sterylglucosides and sterylglucosides).¹ Figure 1 shows the protein and lipid amounts in DRM during cold acclimation. DRM protein recovery rate from the plasma membrane was less than 10% and cold treatment resulted in a gradual decrease of the recovery: the recovery rate of DRM lipids from the plasma membrane rapidly decreased by half only after 2 days of cold acclimation. These data suggest a decrease in the proportion of microdomains in the plasma membrane and temporal changes in proteins and lipids in DRM during cold acclimation.Open in a separate windowFigure 1Changes in the protein and lipid amount in DRM recovered from plasma membrane fractions during cold acclimation. NA, non-acclimated; CA 2, CA 4 and CA 7, cold-acclimated for 2, 4 and 7 days, respectively. (Modified from Minami et al.)We found that there were significant differences in lipid alterations in plasma membrane and DRM fractions in cold acclimation (Fig. 2). The amount of total lipids (per mg of protein) in the plasma membrane fraction greatly increased after cold acclimation but not in the DRM fraction. In the plasma membrane fraction, cold acclimation for 2 days resulted in an increase in the proportions of phospholipids and free sterols and a decrease in the proportion of sphingolipids. In contrast, in the DRM fractions, free sterols increased after 2 days of cold acclimation but the proportion of phospholipids and sphingolipids did not change significantly. These results suggest that the changes in lipid classes in DRM differ from the changes in the whole plasma membrane. Our lipid analysis suggests that the decrease in sphingolipids in the plasma membrane affects the quantitative decrease of microdomains in the plasma membrane during cold acclimation (see Fig. 1). However, the lipid changes in the whole plasma membrane are unlikely to affect proportional changes in DRM-localized lipids except for free sterols.Open in a separate windowFigure 2Lipid changes in DRM and plasma membrane fractions during cold acclimation. NA, non-acclimated; CA 2, CA 4 and CA 7, cold-acclimated for 2, 4 and 7 days, respectively. FS, free sterols; ASG, acylated sterylglucosides; SG, sterylglucosides; GlcCer, glucocerebrosides; PL, phospholipids. (Modified from Minami et al.¹)We demonstrated quantitative changes of DRM-localized proteins during cold acclimation using two-dimensional differential gel electrophoresis (2D-DIGE) and western blot analyses.¹ 2D-DIGE analysis showed that one-third of the DRM-localized proteins quantitatively changed during cold acclimation. Subsequent mass spectrometric analysis of DRM proteins revealed significant changes in various proteins including increases in aquaporin, P-type H⁺-ATPase and endocytosis-related proteins and decreases in cytoskeletal proteins (tubulins and actins) and V-type H⁺-ATPase subunits during cold acclimation. The changes were first detected after 2 days of cold acclimation. Based on these results of protein analyses, Figure 3 illustrates changes in distribution patterns of DRM-localized proteins in the plasma membrane during cold acclimation. Cold acclimation induces the decrease in the amount of DRM proteins and lipids in the plasma membrane (Fig. 1), suggesting that component in microdomains decreases in the plasma membrane during cold acclimation. Furthermore, the proportion of some functional proteins changes in DRM during cold acclimation. Qualitative and quantitative changes of DRM proteins during cold acclimation are possibly associated with the plasma membrane functions. Plant cells at low temperature suffer from changes in membrane fluidity and cytoplasmic pH.^19–21 Upon freezing occurs, plant cells are subjected to severe dehydration and deformation stresses induced by extracellular ice formation.²² To avoid the occurrence of damages from these stresses, plants change plasma membrane components during cold acclimation.²³ H⁺-ATPase or aquaporins are thought to function in regulation of cytoplasmic pH or water transfer across the plasma membrane, respectively.^24,25 Cytoskeleton regulates cell structure and intracellular vesicle-trafficking processes reconstruct plasma membrane itself. Thus, the quantitative changes of these proteins in microdomains are likely associated with protective functions against freezing stress in cold acclimation.Open in a separate windowFigure 3Our hypothesis on changes in microdomains during plant cold acclimation. Cold acclimation results in a decrease in microdomains in the plasma membrane (see Fig. 1) and differential changes in various protein compositions in microdomains. We categorized DRM proteins as (1) membrane transport, (2) vesicle trafficking, (3) cytoskeleton, (4) microdomain-associated proteins and (5) others (e.g., plasma membrane and cell-wall reconstruction). Aquaporin, P-type H⁺-ATPase (1) and endocytosis-related proteins (2) increased and cytoskeletal proteins (3) and V-type H⁺-ATPase subunits (1) decreased in DRM during cold acclimation.We clearly demonstrated that cold acclimation decreased the amount of DRM and changed both lipid and protein compositions in plant DRM. Our study represents a first step towards elucidation of functions of plant microdomains in cold acclimation, strongly suggesting that microdomains, which function as a platform of membrane transport, membrane trafficking and cytoskeleton interaction, are associated with plant cold acclimation. Changes in microdomain lipids may also affect the protein activities during cold acclimation because sterols or sphingolipids are known to regulate activities of membrane transport or endocytosis. Thus, we suspect that the quantitative changes in microdomain lipids and proteins may correlate with development of freezing tolerance during cold acclimation. The hypothesis that the changes in microdomain components are functionally associated with plant cold acclimation should be reinforced by various approaches such as genetics, biochemistry or physical chemistry.     

Cryobehavior of the plasma membrane in protoplasts isolated from cold-acclimated Arabidopsis leaves is related to surface area regulation

Extracellular freezing in plants results in dehydration and mechanical stresses upon the plasma membrane. Plants that acquire enhanced freezing tolerance after cold acclimation can withstand these two physical stresses. To understand the tolerance to freeze-induced physical stresses, the cryobehavior of the plasma membrane was observed using protoplasts isolated from cold-acclimated Arabidopsis thaliana leaves with the combination of a lipophilic fluorescent dye FM 1-43 and cryomicroscopy. We found that many vesicular structures appeared in the cytoplasmic region near the plasma membrane just after extracellular freezing occurred. These structures, referred to as freeze-induced vesicular structures (FIVs), then developed horizontally near the plasma membrane during freezing. There was a strong correlation between the increase in individual FIV size and the decrease in the surface area of the protoplasts during freezing. Some FIVs fused with their neighbors as the temperature decreased. Occasionally, FIVs fused with the plasma membrane, which may be necessary to relax the stress upon the plasma membrane during freezing. Vesicular structures resembling FIVs were also induced when protoplasts were mechanically pressed between a coverslip and slide glass. Fewer FIVs formed when protoplasts were subjected to hyperosmotic solution, suggesting that FIV formation is associated with mechanical stress rather than dehydration. Collectively, these results suggest that cold-acclimated plant cells may balance membrane tension in the plasma membrane by regulating the surface area. This enables plant cells to withstand the direct mechanical stress imposed by extracellular freezing.     

Changes in leaf ultrastructure and carbohydrates inArabidopsis thaliana L. (Heyn) cv. Columbia during rapid cold acclimation

Summary We studied cell ultrastructure and carbohydrate levels in the leaf tissue ofArabidopsis thaliana L. (Heyn) cv. Columbia during rapid cold acclimation. Freezing tolerance of the leaves from 26 day old plants was determined after 48 h and 10 days at 4°C. Acclimation treatment of 48 h decreased the lethal freezing temperature from –5.7°C to –9.4°C. Freezing tolerance was not altered further by acclimation at 4 °C for 10 days. Ultrastructural changes in the parenchyma cells were evident after 6 to 24 h of cold acclimation. The plasma membrane showed signs of extensive turnover. Evidence of membrane invaginations and sequestering of membrane material was observed. In addition, numerous microvesicles, paramural bodies, and fragments of endoplasmic reticulum were noticed in the vicinity of plasma membrane. Modifications in the structure of cell membranes were evident after 5 days of exposure to low temperature. Small, darkly stained globules were seen on the plasma membrane, tonoplast, chloroplast envelope membrane, mitochondrion outer membrane, dictyosome cisternae membrane, and microvesicle membrane. As far as we are aware, this type of membrane modification has not been described previously in plant cells exposed to low temperature. We propose to call these structures membraglobuli. Acclimation treatment also increased the concentrations of soluble sugars and starch. These observations suggest that cold acclimation inA. thaliana induces changes in both plasma membrane properties and carbohydrate composition.     

Accumulation of Small Heat-Shock Protein Homologs in the Endoplasmic Reticulum of Cortical Parenchyma Cells in Mulberry in Association with Seasonal Cold Acclimation

Cortical parenchyma cells of mulberry (Morus bombycis Koidz.) trees acquire extremely high freezing tolerance in winter as a result of seasonal cold acclimation. The amount of total proteins in endoplasmic reticulum (ER)-enriched fractions isolated from these cells increased in parallel with the process of cold acclimation. Protein compositions in the ER-enriched fraction also changed seasonally, with a prominent accumulation of 20-kD (WAP20) and 27-kD (WAP27) proteins in winter. The N-terminal amino acid sequence of WAP20 exhibited homology to ER-localized small heat-shock proteins (smHSPs), whereas that of WAP27 did not exhibit homology to any known proteins. Like other smHSPs, WAP20 formed a complex of high molecular mass in native-polyacrylamide gel electrophoresis. Furthermore, not only WAP20 but also 21-kD proteins reacted with antibodies against WAP20. Fractionation of the crude microsomes by isopycnic sucrose-gradient centrifugation revealed that both WAP27 and WAP20 were distributed on a density corresponding to the fractions with higher activity of ER marker enzyme, suggesting localization of these proteins in the ER. When ER-enriched fractions were treated with trypsin in the absence of detergent, WAP20 and WAP27 were undigested, suggesting localization of these proteins inside the ER vesicle. The accumulation of a large quantity of smHSPs in the ER in winter as a result of seasonal cold acclimation indicates that these proteins may play a significant role in the acquisition of freezing tolerance in cortical parenchyma cells of mulberry trees.     

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.     

Characterization of an 80-kDa dehydrin-like protein in barley responsive to cold acclimation

Barley ( Hordeum vulgare L.) exposed to low temperature increases its freezing tolerance. This increase has been associated with several metabolic changes caused by low temperature, including expression of dehydrins (DHN), a family of proteins induced by dehydration and cold acclimation. DHNs play an undetermined role in dehydration responses during freezing. We have studied the accumulation of an 80-kDa DHN-like protein (P-80) in barley under cold acclimation 6/4°C (day/night), postulating that it is localized in tissues where primary ice nucleation occurs. P-80 was absent in nonacclimated plants and was detectable after 48 h of cold acclimation, reaching a stable level after 6 days. P-80 decreased when plants were returned to 20–25°C. Drought, ABA and high temperature did not increase the levels of P-80, suggesting that its expression could be specifically regulated by cold. Immunolocalization by tissue printing and fresh cross sections of leaves showed the protein to be associated with vascular tissues and epidermis. The localization of P-80 is consistent with our hypothesis because vascular tissue and the epidermis are preferential ice nucleation zones during the onset of freezing. The differential accumulation of P-80 may have an adaptive value by participating in tolerance mechanisms during freeze-induced dehydration.     

Environmental regulation and physiological basis of freezing tolerance in woody plants

Cold acclimation of plants is a complex process involving a number of biochemical and physiological changes. The ability to cold acclimate is under genetic control. The development of freezing tolerance in woody plants is generally triggered by non-freezing low temperatures but can also be induced by mild drought or exogenous abscisic acid, as well as by short photoperiod. In nature, the extreme freezing tolerance of woody plants is achieved during sequential stages of cold acclimation the first of which is initiated by short photoperiods and non-freezing low temperatures, and the second by freezing temperatures. Although recent breakthroughs have increased our knowledge on the physiological molecular basis of freezing tolerance in herbaceous species, which acclimate primarily in response to non-freezing low temperatures, very little is known about cold acclimation of woody plants. This article attempts to review our current understanding of the physiological aspects that underline cold acclimation in woody plants.     

The role of antioxidant defense in freezing tolerance of resurrection plant Haberlea rhodopensis

Haberlea rhodopensis Friv. is unique with its ability to survive two extreme environmental stresses—desiccation to air-dry state and subzero temperatures. In contrast to desiccation tolerance, the mechanisms of freezing tolerance of resurrection plants are scarcely investigated. In the present study, the role of antioxidant defense in the acquisition of cold acclimation and freezing tolerance in this resurrection plant was investigated comparing the results of two sets of experiments—short term freezing stress after cold acclimation in controlled conditions and long term freezing stress as a part of seasonal temperature fluctuations in an outdoor ex situ experiment. Significant enhancement in flavonoids and anthocyanin content was observed only as a result of freezing-induced desiccation. The total amount of polyphenols increased upon cold acclimation and it was similar to the control in post freezing stress and freezing-induced desiccation. The main role of phenylethanoid glucoside, myconoside and hispidulin 8-C-(2-O-syringoyl-b-glucopyranoside) in cold acclimation and freezing tolerance was elucidated. The treatments under controlled conditions in a growth chamber showed enhancement in antioxidant enzymes activity upon cold acclimation but it declined after subsequent exposure to −10 °C. Although it varied under ex situ conditions, the activity of antioxidant enzymes was high, indicating their important role in overcoming oxidative stress under all treatments. In addition, the activity of specific isoenzymes was upregulated as compared to the control plants, which could be more useful for stress counteraction compared to changes in the total enzyme activity, due to the action of these isoforms in the specific cellular compartments.Supplementary informationThe online version contains supplementary material available at 10.1007/s12298-021-00998-0.     

Early Developments in RNA, Protein, and Sugar Levels during Cold Stress in Winter Rye (Secale cereale) Leaves

Changes in freezing tolerance of winter rye (Secale cerealeL. cv. Voima) were determined for leaf tissues during a 1-weekcold stress, which was performed by transferring the 7-d-oldseedlings from a greenhouse (25°C, long day) to 3°Cand short day conditions. The development of cold hardeningwas shown by using an ion leakage test and by determining theamounts of carbohydrates, soluble proteins and RNA. The firstevidence of the development of freezing resistance was foundafter 1 d at low temperature, i.e. an LT₅₀ value increased from-5 to -7°C. Plants cold treated for 7 d reached an LT₅₀value of -9°C. This increase in freezing tolerance was foundto be associated with the increased levels of soluble carbohydrates,total RNA and soluble proteins. These metabolic changes indicatethe association with adjustment of growth and cell metabolismto low temperatures at the beginning of cold acclimation ofwinter rye.Copyright 1994, 1999 Academic Press Secale cereale L., winter rye, cold stress, proteins, RNA, sugars     

Membrane rigidification functions upstream of the MEKK1-MKK2-MPK4 cascade during cold acclimation in Arabidopsis thaliana

The MEKK1-MKK2-MPK4 cascade is activated during cold acclimation. However, little is known regarding the perception of low temperature. In this study, we demonstrate that treatment of Arabidopsis with a membrane rigidifier, DMSO, caused MPK4 activation concomitantly with MEKK1 and MKK2 phosphorylation, as well as the cold-inducible gene COR15a expression. These processes are similar to the effects of cold treatment, whereas benzyl alcohol (BA), a membrane fluidizer, prevented such cold-induced events. Moreover, the DMSO-treated seedlings acquired freezing tolerance without cold acclimation. In contrast, the BA-pretreated seedlings did not show freezing tolerance. These results suggest that membrane rigidification activates this MAPK cascade and contributes to the acquisition of freezing tolerance.     

Differential remodeling of the lipidome during cold acclimation in natural accessions of Arabidopsis thaliana

Freezing injury is a major factor limiting the geographical distribution of plant species and the growth and yield of crop plants. Plants from temperate climates are able to increase their freezing tolerance during exposure to low but non‐freezing temperatures in a process termed cold acclimation. Damage to cellular membranes is the major cause of freezing injury in plants, and membrane lipid composition is strongly modified during cold acclimation. Forward and reverse genetic approaches have been used to probe the role of specific lipid‐modifying enzymes in the freezing tolerance of plants. In the present paper we describe an alternative ecological genomics approach that relies on the natural genetic variation within a species. Arabidopsis thaliana has a wide geographical range throughout the Northern Hemisphere with significant natural variation in freezing tolerance that was used for a comparative analysis of the lipidomes of 15 Arabidopsis accessions using ultra‐performance liquid chromatography coupled to Fourier‐transform mass spectrometry, allowing the detection of 180 lipid species. After 14 days of cold acclimation at 4°C the plants from most accessions had accumulated massive amounts of storage lipids, with most of the changes in long‐chain unsaturated triacylglycerides, while the total amount of membrane lipids was only slightly changed. Nevertheless, major changes in the relative amounts of different membrane lipids were also evident. The relative abundance of several lipid species was highly correlated with the freezing tolerance of the accessions, allowing the identification of possible marker lipids for plant freezing tolerance.