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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cryobehaviour of the plasma membrane of protoplasts during a freezing-thaw cycle
  5. Alterations in the plasma membrane lipid composition during cold acclimation
  6. Lipid composition and cryobehaviour of the plasma membrane
  7. Proteomic analysis of plasma membrane proteins during cold acclimation
  8. Function of plasma membrane proteins associated with cold acclimation
  9. Perspectives
  10. References

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.


Abbreviations – 
CA

cold-acclimated

EIL

expansion-induced lysis

FJL

fracture-jump lesion

HII

hexagonal II

LOR

loss of osmotic responsiveness

LT50

temperature at which 50% of protoplasts are killed after freezing

NA

nonacclimated.

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cryobehaviour of the plasma membrane of protoplasts during a freezing-thaw cycle
  5. Alterations in the plasma membrane lipid composition during cold acclimation
  6. Lipid composition and cryobehaviour of the plasma membrane
  7. Proteomic analysis of plasma membrane proteins during cold acclimation
  8. Function of plasma membrane proteins associated with cold acclimation
  9. Perspectives
  10. References

The ability of many temperate plants to tolerate low temperatures and freezing is an important and essential trait to insure their geological distribution and maintain productivity. Eventually, many temperate plants are able to increase their freezing tolerance when exposed to low but non-freezing temperatures in an adaptive process, which is known as cold acclimation (Nakashima and Yamaguchi-Shinozaki 2006, Van Buikirk and Thomashow 2006, Chinnusamy et al. 2006). Through altered gene expression during cold acclimation, there are many, seemingly disparate responses occurring in many different aspects, including alterations in membrane composition and accumulation of compatible solutes. Because there is a consensus that irreversible dysfunction of the plasma membrane as a consequence of freeze-induced dehydration is the primary cause of freezing injury (Steponkus 1984), it is considered that many changes occurring during cold acclimation contribute to the increase in the cryostability of the plasma membrane during a freeze-thaw excursion.

In addition, it is known that many factors in the cytosol and intracellular organelles affect characteristics of the plasma membrane during both cold acclimation and freezing. In general, increased concentrations in compatible solutes result in a decrease in the extent of freeze-induced osmotic dehydration from cells (Levitt 1980). Some compatible solutes have shown specific cryoprotective effects during freezing. Those include protection of membranes and proteins by sugars (Uemura and Steponkus 2003), scavenging active oxygen species by proline (Smirnoff and Cumbes 1989) and the promotion of recovery process in freezing-damaged cells by glycinebetaine (Chen and Murata 2002). Furthermore, the cold-induced protein COR15am, which is localized in chloroplast stroma, was shown to increase the freezing tolerance of Arabidopsis protoplasts due to an increase in stability of the plasma membrane during osmotic dehydration (Artus et al. 1996, Steponkus et al. 1998). These results clearly indicate that plasma membrane cryostability is affected by many factors both in the plasma membrane and in the cytosol and intracellular organelles.

Nevertheless, it is reasonable that the major determinants to keep the plasma membrane functional under freezing conditions are those existing within the plasma membrane itself. Unless there are alterations within the plasma membrane, it will not be possible to increase the cryostability of the plasma membrane so that freezing tolerance of cells is enhanced. There is a good amount of evidence indicating that the plasma membrane changes considerably during the cold acclimation process. This minireview will focus on the plasma membrane-associated alterations occurring during freezing or cold acclimation and discuss the significance of these changes in increasing freezing tolerance of plant cells.

Cryobehaviour of the plasma membrane of protoplasts during a freezing-thaw cycle

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cryobehaviour of the plasma membrane of protoplasts during a freezing-thaw cycle
  5. Alterations in the plasma membrane lipid composition during cold acclimation
  6. Lipid composition and cryobehaviour of the plasma membrane
  7. Proteomic analysis of plasma membrane proteins during cold acclimation
  8. Function of plasma membrane proteins associated with cold acclimation
  9. Perspectives
  10. References

Significant differences in cryobehaviour of the plant plasma membrane were observed with protoplasts isolated from leaves of various herbaceous plant species before and after cold acclimation. These differences led to the identification of three distinct freeze-induced membrane lesions (Fig. 1), the incidence of which depends on the stage of cold acclimation and the extent of freeze-induced dehydration (or the nadir temperatures at which the samples were frozen) (Steponkus et al. 1993, Uemura et al. 1995).

image

Figure 1. Freeze-induced lesions associated with the plasma membrane in isolated protoplasts. In nonacclimated protoplasts, freeze-induced dehydration results in the formation of endocytotic vesicles that are not continuous to the plasma membrane. Under mild but injurious dehydration conditions, protoplasts cannot expand during thawing due to the lack of the plasma membrane materials (A). With severe dehydration, protoplasts do not respond osmotically and the hexagonal II (HII) phase is observed with the plasma membrane and other endomembranes (B). In cold-acclimated protoplasts, freeze-induced dehydration results in exocytotic extrusions continuous to the plasma membrane. Therefore, no lysis occurred at any injurious temperature. Instead, freezing injury is manifested as the loss of osmotic responsiveness associated with the fracture-jump lesion not with the HII phase formation (C).

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Nonacclimated protoplasts with mild dehydration

When protoplasts isolated from leaves of nonacclimated seedlings (NA protoplasts) are subjected to freeze-induced dehydration, two different lesions associated with the plasma membrane are observed (Fig. 1). Under conditions of freezing to the temperature at which 50% of protoplasts are killed (LT50) or above, osmotic contraction of the protoplasts results in surplus of the plasma membrane. To maintain proper tension on the plasma membrane, we must remove the surplus from the plasma membrane and many small vesicles are formed within a cell. Although the formation of endocytotic vesicles of the plasma membrane is not injurious per se (Dowgert and Steponkus 1984), these vesicles are not able to be incorporated into the plasma membrane during thawing. Thus, the protoplasts lyse during osmotic expansion after thawing of the suspending medium. This form of injury is referred to as expansion-induced lysis (EIL).

NA protoplasts with severe dehydration

When protoplasts are subjected to severe dehydration at temperatures below the LT50, the volume of the protoplasts is reduced considerably (Fig. 1). As a result, the protoplasts lose osmotic response during thawing of the suspending medium. This form of injury is referred to as loss of osmotic responsiveness (LOR) and, in NA protoplasts, is a consequence of the freeze-induced formation of hexagonal II (HII) phase in regions where the plasma membrane is brought into close apposition with various endomembranes (most often the chloroplast envelope and tonoplast) as the cell loses considerable amounts of water to the external ice (Gordon-Kamm and Steponkus 1984a, Uemura et al. 1995, Webb et al. 1994).

Cold-acclimated protoplasts

Cold acclimation results in a dramatic change in the cryobehaviour of the plasma membrane. With protoplasts isolated from cold-acclimated seedlings (CA protoplasts), freeze-induced osmotic contraction results in the formation of exocytotic extrusions instead of endocytotic vesicles. Exocytotic extrusions are continuous to the plasma membrane, indicating that the surface area of the plasma membrane is conserved during freeze-induced osmotic dehydration (Gordon-Kamm and Steponkus 1984b). During osmotic expansion upon thawing, exocytotic extrusions are reversibly incorporated into the plasma membrane, and hence, EIL does not occur at any injurious temperature in CA protoplasts. Manifestation of the injury in CA protoplasts is LOR at any injurious temperature. Furthermore, freeze-induced formation of the HII phase is not observed at any injurious temperature; instead, the fracture-jump lesion (LOR-FJL), which is characterized by localized deviations of the fracture plane of the plasma membrane in freeze-fracture electron micrographs (Fujikawa and Steponkus 1990, Steponkus et al. 1993), is observed throughout the plasma membrane.

Alterations in the plasma membrane lipid composition during cold acclimation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cryobehaviour of the plasma membrane of protoplasts during a freezing-thaw cycle
  5. Alterations in the plasma membrane lipid composition during cold acclimation
  6. Lipid composition and cryobehaviour of the plasma membrane
  7. Proteomic analysis of plasma membrane proteins during cold acclimation
  8. Function of plasma membrane proteins associated with cold acclimation
  9. Perspectives
  10. References

There are many alterations in the plasma membrane composition responding to cold acclimation. After an aqueous two-phase partition protocol to isolate and purify plasma membrane fractions from plant materials was established, several research groups initiated projects to determine changes in the plasma membrane composition during cold acclimation.

Cold acclimation results in many changes in the lipid composition of the plasma membrane, suggesting that these changes are associated with the occurrence of specific freeze-induced lesions associated with the plasma membrane (Steponkus et al. 1993, Uemura and Steponkus 1999). The most notable changes in the lipid composition is an increase in the proportion of phospholipids, which is observed in a wide range of plant species from monocotyledonous to dicotyledonous plants and from herbaceous to woody plants (Table 1). The increase in phospholipids in the plasma membrane during cold acclimation occurs at the early stage of cold acclimation, whereas the decrease in cerebrosides occurs gradually throughout the cold acclimation process. In many plant species examined, the increase in phospholipid is primarily a result of an increase in the proportion of unsaturated molecular species of phosphatidylcholine and phosphatidylethanolamine, two major phospholipid classes in the plasma membrane. In addition, there is a decrease in the proportion of cerebrosides occurring over a wide range of plants.

Table 1.  Increase in the proportion of phospholipids in the plasma membrane during cold acclimation
 Protein (mmol mg−1)Total lipids (mol %) 
PlantsNACANACAReferences
Secale cereale1.041.2638.743.4Uemura and Yoshida (1984)
1.071.4638.743.4Lynch and Steponkus (1987)
  36.643.3Uemura and Steponkus (1994)
Avena sativa  28.939.5Uemura and Steponkus (1994)
Dactylis glomerata1.111.3850.253.1Yoshida and Uemura (1984)
Helianthus tuberosus1.151.5046.946.9Ishikawa and Yoshida (1985)
Arabidopsis thaliana  46.857.1Uemura et al. (1995)
0.941.1251.555.2Kawamura and Uemura (2002)
Morus bombycis1.021.7257.169.0Yoshida (1984)

Comprehensive lipid analyses of the plasma membrane before and after cold acclimation revealed that there is not a single molecular species that is unique to the plasma membrane isolated from either NA or CA leaves or unique to the plasma membrane isolated from a particular plant species. Instead, cold acclimation alters the relative proportions of virtually every molecular species in the plasma membrane, and the proportions of each of the lipid classes are widely different in plasma membranes of different plant species. Thus, if we assume that there are certain connections between the cryobehaviour and the lipid composition of the plasma membrane (see below), any differences in the cryobehaviour of the plasma membrane during a freeze-thaw cycle should be attributed to the altered lipid–lipid (and/or lipid–protein) interactions in the plasma membrane that resulted from the altered lipid composition rather than unique lipid species produced during cold acclimation.

Lipid composition and cryobehaviour of the plasma membrane

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cryobehaviour of the plasma membrane of protoplasts during a freezing-thaw cycle
  5. Alterations in the plasma membrane lipid composition during cold acclimation
  6. Lipid composition and cryobehaviour of the plasma membrane
  7. Proteomic analysis of plasma membrane proteins during cold acclimation
  8. Function of plasma membrane proteins associated with cold acclimation
  9. Perspectives
  10. References

Differential cryobehaviour of the plasma membrane before and after cold acclimation can be simulated with liposomes prepared with total lipid extracts of the plasma membrane isolated from NA and CA leaves. When subjected to freeze-induced or osmotically induced dehydration, liposomes with NA and CA plasma membrane lipids exhibit endoliposomal vesicles and exoliposomal extrusions, respectively (Steponkus and Lynch 1989). Therefore, the plasma membrane lipids seem to be responsible for the fate of the plasma membrane during freeze-induced dehydration.

The decrease in the incidence of EIL during cold acclimation is a result of an increase in the proportion of unsaturated species of phosphatidylcholine in the plasma membrane. This is evidenced by the results of membrane-engineering studies in which NA protoplasts were fused with liposomes containing unsaturated species of phosphatidylcholine, resulting in an enrichment of unsaturated phosphatidylcholine species in the plasma membrane (in other words, the plasma membrane becomes similar to that of CA protoplasts), leading to increased survival of the protoplasts after a freeze/thaw cycle (Steponkus et al. 1988, Uemura and Steponkus 1989). In addition, during freeze-induced dehydration, the plasma membrane of NA protoplasts fused with the liposomes showed the formation of exocytotic extrusions instead of endocytotic vesicles. Collectively, the cryobehaviour and more importantly the functional stability of the plasma membrane is clearly affected by the lipid composition in the plasma membrane.

An increase in the proportion of unsaturated species of phosphatidylcholine in the plasma membrane is primarily responsible for a decreased propensity for freeze-induced formation of the HII phase after cold acclimation. Osmotic dehydration of liposomes prepared from NA plasma membrane lipids but not CA plasma membrane lipids results in the formation of the HII phase (Cudd and Steponkus 1988). In addition, artificial enrichment of the proportion of unsaturated species of phosphatidylcholine in the plasma membrane of NA protoplasts using the protoplast–liposome fusion technique decreases the participation of the plasma membrane in freeze-induced formation of the HII phase significantly (Sugawara and Steponkus 1990).

We believe that, under the in situ conditions, the decreased propensity of freeze-induced formation of HII phase after cold acclimation is not the result of the increased proportion of unsaturated species of phosphatidylcholine per se. Rather, it is because of the collective changes in the various lipid components in the plasma membrane occurring during cold acclimation. It is known from many biophysical studies that phase behaviour of the membrane is influenced by complex interactions of factors including the hydration characteristic of the membrane, hydration-dependent (i.e. lyotropic) and temperature-dependent (i.e. thermotropic) phase behaviour of mixtures of lipids in the membrane, and the intrinsic curvature of constituent monolayers (Steponkus et al. 1993). These complex interactions may answer an obvious question regarding the influence of the increased proportion of unsaturated species of phosphatidylethanolamine in the cryobehaviour of the plasma membrane. It was known that the unsaturated species of phosphatidylethanolamine are apparently prone to form the HII phase thermotropically and lyotropically. However, because of interactions with other lipids and proteins in the plasma membrane, the plasma membrane after cold acclimation diminishes a probability of the lamellar to HII phase transition upon freeze-induced dehydration.

The FJL is proposed to be the result of membrane fusion occurring between the plasma membrane and intracellular membranes as a consequence of membrane-membrane apposition upon severe freeze-induced dehydration (Siegel 1986a,1986b, 1986c, Steponkus et al. 1993). On the fusion event, an intermediate, which is thought to be a common intermediate in the lamellar to HII phase transition, is formed at a critical temperature and a certain hydration. Thus, as with the HII formation, the occurrence of the FJL is affected by lipid composition of the membrane, which influences the intrinsic curvature of the constituent monolayers. An increase in the proportion of phospholipids and a decrease in the proportion of cerebrosides results in an increase in membrane hydration and a decrease in intrinsic curvature of the constituent monolayers, which, in turn, decreases the temperature at which the intermediates are formed below the lamellar-to-HII phase transition temperature. Thus, after cold acclimation, no HII phase is observed, but the FJL becomes the primary ultrastructural change associated with the form of injury (i.e. LOR).

Proteomic analysis of plasma membrane proteins during cold acclimation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cryobehaviour of the plasma membrane of protoplasts during a freezing-thaw cycle
  5. Alterations in the plasma membrane lipid composition during cold acclimation
  6. Lipid composition and cryobehaviour of the plasma membrane
  7. Proteomic analysis of plasma membrane proteins during cold acclimation
  8. Function of plasma membrane proteins associated with cold acclimation
  9. Perspectives
  10. References

As described above, a number of studies were published on alterations in lipid composition and cryobehaviour of the plasma membrane during cold acclimation. However, detailed and comprehensive studies on plasma membrane proteins in the cold acclimation process are limited so far. Since the middle of the 1980s, semiquantitative protein changes in the plasma membrane purified with an aqueous two-phase partition system were reported using one- or two-dimensional polyacrylamide gel electrophoresis (Uemura and Yoshida 1984, Yoshida and Uemura 1984, Zhou et al. 1994). However, these studies have only compared separation patterns on the gels and not analysed comprehensively the amount and identity of cold-responsive proteins associated with the plasma membrane.

Technical advances in molecular biology allow us to identify plasma membrane proteins that change in association with cold acclimation in combination with gene expression analysis. For example, a cell wall-plasma membrane linker protein increases at the mRNA level in Brassica napus (Goodwin et al. 1996) and annexin increases at the protein level in wheat after cold acclimation (Breton et al. 2000). Although these studies provide information on alterations of specific plasma membrane-associated proteins upon cold acclimation, it is still necessary to collect comprehensive information on the plasma membrane changes in the cold acclimation process, which is necessary when we consider the role of plasma membrane proteins in the cryostability and function of the plasma membrane.

With matrix-assisted laser-desorption ionization time-of-flight mass spectrometry coupled with peptide mass-fingerprinting analysis, a group of plasma membrane proteins that respond to cold acclimation has been identified in Arabidopsis (Fig. 2 and Table 2, Kawamura and Uemura 2003). In total, 38 cold-responsive proteins were identified: 26 of them were recovered in a solubilized fraction with isoelectric focusing electrophoresis and thought to be hydrophilic, peripheral proteins, and 12 of them were in an insolubilized fraction and thought to be hydrophobic, integral proteins. Changes in the majority of the cold-responsive plasma membrane proteins occurred within a day of cold acclimation, suggesting that cells respond to low temperatures quite rapidly. These proteins include membrane repair proteins, osmotic stress-related proteins, proteolysis-associated proteins and many functionally unknown proteins. Thus, it is suggested that cold acclimation results in drastic changes in the plasma membrane that are associated with many physiological processes to adapt to environmentally adverse conditions.

image

Figure 2. Flowchart of protein identification with matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) MS. After proteins are separated by electrophoresis, specified protein spots are cut and digested into small peptide fragments using trypsin. The peptide fragments are analysed with MALDI-TOF MS and peptide mass fingerprint spectra are obtained. Mass of each peak in the spectrum is then analysed with protein sequence databases using a proper programme provided on the Internet.

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Table 2.  Identification of cold-responsive proteins in the plasma membrane of Arabidopsis thaliana. a Percentage of coverage at 50 p.p.m. for the matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) peptides. b Accession number in NCBI database ( http://www.ncbi.nlm.nih.gov). cAGI code in MIPS Arabidopsis database (http://www.mips.gsf.de/proj/thal/db/index.html). dUpward or downward arrow shows the day when plasma membrane proteins increase or decrease during cold acclimation.
 Molecular weight (kDa)/pI     
Spot numberExperimentalTheoreticalCover percentage (50 p.p.m.a)Protein nameAccession numberbCodecIncrease or decreased
Solubilized fraction
 144.3/4.740.1/4.5823RAD23 homologue9758825At5g384701d new[UPWARDS ARROW]
 240.7/6.1Not identified2d[UPWARDS ARROW]
 340.0/4.829.5/5.1229Dehydrin ERD10556472At1g204501d new[UPWARDS ARROW]
 437.9/4.720.8/5.4135Dehydrin ERD1413265523At1g761801d new[DOWNWARDS ARROW]
 535.2/4.824.6/4.9956Tobacco DREPP-like1550738At4g202601d[DOWNWARDS ARROW]2d[UPWARDS ARROW]
 634.8/6.130.7/6.015Synexin-like2911047At4g341501d new[UPWARDS ARROW]
 734.6/5.835.8/8.519NAD-dependent malate dehydrogenase3929649At1g532401d[DOWNWARDS ARROW]
 832.3/5.726.1/6.0457V-ATPase subunit E3600058At4g111501d[DOWNWARDS ARROW]
 931.3/5.128.3/5.3674Carbonic anhydrase 211692920At5g147401d[UPWARDS ARROW]
 1031.3/5.228.3/5.3670Carbonic anhydrase 211692920At5g147401d[UPWARDS ARROW]
 1131.3/5.328.3/5.3677Carbonic anhydrase 211692920At5g147401d[UPWARDS ARROW]
 1230.4/5.128.3/5.3671Carbonic anhydrase 211692920At5g147401d[UPWARDS ARROW]
 1330.4/5.228.3/5.3628Carbonic anhydrase 211692920At5g147401d[UPWARDS ARROW]
 1428.7/5.128.2/5.717Ferritin 115241018At5g016001d[UPWARDS ARROW]3d[DOWNWARDS ARROW]
 1528.7/5.824.1/5.9359Glutatione S-transferase (GST6) (Atpm24)407090At4g025201d[DOWNWARDS ARROW]2d[UPWARDS ARROW]
 1628.7/5.924.1/5.954GST (Atpm24)407090At4g025201d[DOWNWARDS ARROW]2d[UPWARDS ARROW]
 1728.7/6.124.1/6.234GST (ERD13)3201613At2g308601d[DOWNWARDS ARROW]2d[UPWARDS ARROW]
 1828.0/5.226.8/8.950Chaperonin 20 (Cpn21)14587373At5g207202d[UPWARDS ARROW]
 1927.9/5.128.2/5.716Ferritin 115241018At5g016002d[UPWARDS ARROW]
 2027.6/5.824.1/6.133GST6)18407435At2g477301d[DOWNWARDS ARROW]2d[UPWARDS ARROW]
 2122.9/5.547.6/6.121Rubisco large subunit19444321d[UPWARDS ARROW]
 2222.7/5.721.4/5.9839Outer membrane lipoprotein-like9759532At5g580701d[UPWARDS ARROW]
 2321.8/5.147.6/6.118Rubisco large subunit19444321d[UPWARDS ARROW]
 2418.8/5.617.8/5.6632Nodulin VfNOD18-like7630019At3g539901d[UPWARDS ARROW]
 2518.8/6.117.8/6.4141Nodulin VfNOD18-like7670026At3g170202d[UPWARDS ARROW]
 2616.6/5.714.7/5.782Mature rubisco small subunit13926229At1g670901d[UPWARDS ARROW]
 2715.9/5.714.7/5.748Mature rubisco small subunit13926229At1g670901d[DOWNWARDS ARROW]
Insolubilized fraction
 1103.4121.6/8.7425Phospholipase Dd3805845At4g357901d[UPWARDS ARROW]
 283.765.6/9.1622Pollen-specific like4725941At4g124201d[UPWARDS ARROW]3d[DOWNWARDS ARROW]
 363.274.7/7.216Ribophorin II like16604454At4g211501d[UPWARDS ARROW]
 459.159.4/7.6542Synaptotagmin like15027959At2g209901d[UPWARDS ARROW]
 555.5Not identified1d[UPWARDS ARROW]
 653.751.6/9.322Unknown protein15241803At5g623901d[UPWARDS ARROW]2d[DOWNWARDS ARROW]
 753.450.7/4.740Tubulin β-2/β-3166898At5g626901d[UPWARDS ARROW]3d[DOWNWARDS ARROW]
 851.449.5/9.129Elongation factor 1-α18086389At1g079302d[DOWNWARDS ARROW]
 946.644.7/10.33060S ribosomal protein L4-like15242558At5g028701d[UPWARDS ARROW]3d[DOWNWARDS ARROW]
 1040.4Not identified1d[UPWARDS ARROW]3d[DOWNWARDS ARROW]
 1130.831.3/5.724HIR protein-like18395770At3g012901d[UPWARDS ARROW]3d[DOWNWARDS ARROW]
 1227.628.2/5.113Chlorophyll a/b binding18403549At2g344301d[UPWARDS ARROW]3d[DOWNWARDS ARROW]
 1327.0Not identified1d[UPWARDS ARROW]3d[DOWNWARDS ARROW]
 1426.528.2/5.723Ferritin 115241018At5g016001d[UPWARDS ARROW]3d[DOWNWARDS ARROW]
 1524.624.9/10.43760-s ribosomal protein L1015223382At1g269101d[UPWARDS ARROW]3d[DOWNWARDS ARROW]

Function of plasma membrane proteins associated with cold acclimation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cryobehaviour of the plasma membrane of protoplasts during a freezing-thaw cycle
  5. Alterations in the plasma membrane lipid composition during cold acclimation
  6. Lipid composition and cryobehaviour of the plasma membrane
  7. Proteomic analysis of plasma membrane proteins during cold acclimation
  8. Function of plasma membrane proteins associated with cold acclimation
  9. Perspectives
  10. References

Then, next question is, whether these cold-responsive plasma membrane proteins affect the cold acclimation process to increase freezing tolerance. To determine the roles of the cold-responsive plasma membrane proteins in the cryobehaviour and/or function of the plasma membrane under low temperatures, we determined the freezing tolerance of seedlings and the cryobehaviour of the plasma membranes of protoplasts from transgenic Arabidopsis plants that overexpress specific plasma membrane proteins. We found that two cold-responsive plasma membrane proteins seem to have a positive effect on freezing tolerance acquisition in association with changing the cryobehaviour of the plasma membrane (Tominaga et al. 2005).

Outer-membrane lipoprotein-like protein (lipocalin-like protein, AtLCN) is one of the proteins that increase substantially during cold acclimation (Kawamura and Uemura 2003). All of the lipocalin family proteins have a similar structure, which includes a conserved ligand-binding pocket (Åkerstrom et al. 2000). However, there are few reports for plants. A lipocalin-like protein in wheat (TaTIL) is induced upon low temperature treatment at both the mRNA and protein levels (Frenette-Charron et al. 2002). AtLCN mRNA was reported to increase at low temperature, which is consistent with our previous results at the protein level. Thus, cold-induced expression of a lipocalin-like protein occurs both in dicotyledonous and monocotyledonous plants, suggesting a cryoprotective role of the lipocalin-like protein under freeze-induced dehydration.

The freezing tolerance of transgenic plants overexpressing AtLCN is greater than that of wild-type plants without cold acclimation. When leaves are frozen to −5 to −7° C, electrolyte leakage from leaves is much less in the transgenic plants than in wild-type plants. However, there is no or little difference in electrolyte leakage at temperatures above or below this temperature ranges. Furthermore, survival of protoplasts isolated from leaves at −10° C is greater in the transgenic plants than in wild-type plants when frozen under near equilibrium freezing conditions at the cooling rate of 0.25 and 0.5° C min−1 (Fig. 3). Immunoelectron microscopic studies show the accumulation of AtLCN near (both inside and outside) or on the plasma membrane. Therefore, it is believed that AtLCN may play a role in acquisition of freezing tolerance associated with an increase in the cryostability of the plasma membrane.

image

Figure 3. Effect of overexpression of AtLCN or ERD14 on the survival of NA protoplasts isolated from Arabidopsis leaves after freezing to −10° C with a cryomicroscope. Two AtLCN and an ERD14-overexpressing lines are employed as experimental materials. Nonacclimated (NA) protoplasts are frozen at various cooling rates to −10° C and subsequently thawed at 1.0° C min−1. The number of survived protoplasts is counted and calculated percentage survival against the number of vital protoplasts before a freeze-thaw cycle. Results are means ± sd (n = 3).

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Preliminary experiments suggest that overexpression of AtLCN results in a decrease in the incidence of EIL but not in the incidence of LOR. As mentioned above, EIL is a consequence of the formation of endocytotic vesiculation during freeze-induced contraction of protoplasts. Thus, AtLCN seems somehow to change the cryobehaviour of the plasma membrane under freeze-induced osmotic dehydration such that the plasma membrane minimizes the probability of formation of endocytotic vesicles. In addition, because LOR associated with either the HII formation in NA protoplasts or the FJLs in CA protoplasts is a primary lesion when frozen to temperatures lower than the LT50, AtLCN may have an additional effect on the incidence of these freeze-induced forms of injury associated with the plasma membrane. There is a possibility that AtLCN may affect the cryostability of the plasma membrane through an interaction with lipid components in the plasma membrane. Further experiments are necessary to clarify the function of AtLCN in freezing tolerance.

Another cold-responsive plasma membrane protein, ERD 14 (early responsive to dehydration protein 14), seems to affect the cryobehaviour of the plasma membrane in a different way. Protoplasts isolated from leaves of transgenic lines overexpressing ERD14 show a survival rate greater than those from leaves of wild-type plants at cooling rates of 0.25–2.5° C (Fig. 3). At slower rates, injury is due to freeze-induced dehydration associated with the plasma membrane. By contrast, at faster rates, intracellular freezing possibly occurs due to either ice formation in the cytosol or ice penetration through the plasma membrane. In fact, the incidence of intracellular freezing decreases in protoplasts of the ERD14 transgenic plants. Although the mechanism of intracellular freezing remains to be determined, ERD14 may minimize the penetration of ice from outside the protoplasts by alterations of characteristics of the plasma membrane during freezing.

Perspectives

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cryobehaviour of the plasma membrane of protoplasts during a freezing-thaw cycle
  5. Alterations in the plasma membrane lipid composition during cold acclimation
  6. Lipid composition and cryobehaviour of the plasma membrane
  7. Proteomic analysis of plasma membrane proteins during cold acclimation
  8. Function of plasma membrane proteins associated with cold acclimation
  9. Perspectives
  10. References

Upon subjecting to low temperatures, many plants including Arabidopsis respond in a short time to increase the probability of survival under freezing conditions. Because the plasma membrane may exert the most significant effect on survival, alterations in the plasma membrane are thought to be of utmost importance as determinants of plant survival. Although there are many studies so farindicating that the plasma membrane after cold acclimation is literally different from that before cold acclimation, the specific roles of the differences in increased freezing tolerance still require further investigation. With various mutants and transgenic plants overexpressing or downregulating (or lacking) the plasma membrane components, it becomes possible to understand functional roles of the specific components associated with the cold acclimation process. Diverse approaches should be taken and down-stream components of specific cold-responsive regulons should be analysed to obtain comprehensive diagram of the molecular mechanisms for cold acclimation in plants.

Acknowledgements –  We thank Dr Michael Schläppi (Marquette University, USA) for his valuable comments and helpful discussion on this minireview. The authors are also grateful to members of our laboratory in Iwate University for stimulated discussion and technical assistance. Works in our laboratory in Iwate University have been supported by grants from the Ministry of Education, Science, Sports and Culture of Japan (Grants-in-Aid for the 21st Century Center of Excellence Program and for the Scientific Research, no. 17380062), from the Ministry of Agriculture, Forestry and Fisheries of Japan (the Bio-design project) and from the President and the Dean of the United Graduate School of Agriculture, Iwate University.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cryobehaviour of the plasma membrane of protoplasts during a freezing-thaw cycle
  5. Alterations in the plasma membrane lipid composition during cold acclimation
  6. Lipid composition and cryobehaviour of the plasma membrane
  7. Proteomic analysis of plasma membrane proteins during cold acclimation
  8. Function of plasma membrane proteins associated with cold acclimation
  9. Perspectives
  10. References
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