Alterations in membrane transport properties by freezing injury in herbaceous plants:

Leakage of ions from a thawed tissue is a common phenomenon of freezing injury. This leakage is usually assumed to be due to loss of membrane semipermeability or membrane rupture by freezing injury. Freeze injured, yet living, onion (Allium cepa L.) epidermal cells were used to study alterations in cell membranes that result in leakage of ions. In spite of a large efflux of ions, freeze injured cells could be plasmolysed and they remained plasmolysed for several days just like the unfrozen control cells. Injured cells also exhibited protoplasmic streaming. Passive transport of KCl, urea and methyl urea across the cell membranes of injured and control cells was also studied. No difference could be detected for the transport rates of urea and methyl urea between control and injured cells. However, a dramatic increase in the transport rate of KCl was found for the injured cells. Depending upon the extent of initial freezing injury, an increase or a decrease in injury symptoms was found in the post-thaw period. During the progress of freezing injury, 10 days after thawing, a swelling of the protoplasm was seen in the irreversibly injured cells. In spite of this swelling, these cells could be plasmolysed. It appears that the high amount of K⁺ that leaks out into the extracellular water, due to freezing injury, causes protoplasmic swelling by replacing Ca²⁺ in the plasma membrane. We conclude that protoplasmic swelling is a sign of secondary injury. The results presented in this study show that membrane semipermeability is not completely lost and membrane rupture does not occur during the initial stage of freezing injury. In fact, the cells have the ability to repair damage depending upon the degree of injury. Our results show there are specific alterations in membrane semipermeability (e.g., transport of K⁺) which could be repaired completely depending on the degree of injury. These findings suggest that ion leakage due to freezing injury is due to alteration in the membrane proteins and not in the membrane lipids.     

Plasma Membrane ATPase Activity following Reversible and Irreversible Freezing Injury

Plasma membrane ATPase has been proposed as a site of functional alteration during early stages of freezing injury. To test this, plasma membrane was purified from Solanum leaflets by a single step partitioning of microsomes in a dextran-polyethylene glycol two phase system. Addition of lysolecithin in the ATPase assay produced up to 10-fold increase in ATPase activity. ATPase activity was specific for ATP with a K_m around 0.4 millimolar. Presence of the ATPase enzyme was identified by immunoblotting with oat ATPase antibodies. Using the phase partitioning method, plasma membrane was isolated from Solanum commersonii leaflets which had four different degrees of freezing damage, namely, slight (reversible), partial (partially reversible), substantial and total (irreversible). With slight (reversible) damage the plasma membrane ATPase specific activity increased 1.5- to 2-fold and its K_m was decreased by about 3-fold, whereas the specific activity of cytochrome c reductase and cytochrome c oxidase in the microsomes were not different from the control. However, with substantial (lethal, irreversible) damage, there was a loss of membrane protein, decrease in plasma membrane ATPase specific activity and decrease in K_m, while cytochrome c oxidase and cytochrome c reductase were unaffected. These results support the hypothesis that plasma membrane ATPase is altered by slight freeze-thaw stress.     

In Vivo Perturbation of Membrane-Associated Calcium by Freeze-Thaw Stress in Onion Bulb Cells : Simulation of This Perturbation in Extracellular KCl and Alleviation by Calcium

Incipient freeze-thaw stress in onion bulb scale tissue is known to cause enhanced efflux of K⁺, along with small but significant loss of cellular Ca²⁺. During the post-thaw period, irreversibly injured cells undergo a cytological aberration, namely, `protoplasmic swelling.' This cellular symptom is thought to be caused by replacement of Ca²⁺ from membrane by extracellular K⁺ and subsequent perturbation of K⁺ transport properties of plasma membrane. In the present study, onion (Allium cepa L. cv Sweet Sandwich) bulbs were slowly frozen to either −8.5°C or −11.5°C and thawed over ice. Inner epidermal peels from bulb scales were treated with fluorescein diacetate for assessing viability. In these cells, membrane-associated calcium was determined using chlorotetracycline fluorescence microscopy combined with image analysis. Increased freezing stress and tissue infiltration (visual water-soaking) were paralleled by increased ion leakage. Freezing injury (−11.5°C; irreversible) caused a specific and substantial loss of membrane-associated Ca²⁺ compared to control. Loss of membrane-associated Ca²⁺ caused by moderate stress (−8.5°C; reversible) was much less relative to −11.5°C treatment. Ion efflux and Ca²⁺-chlorotetracycline fluorescence showed a negative relationship. Extracellular KCl treatment simulated freeze-thaw stress by causing a similar loss of membrane-associated calcium. This loss was dramatically reduced by presence of extracellular CaCl₂. Our results suggest that the loss of membrane-associated Ca²⁺, in part, plays a role in initiation and progression of freezing injury.     

Effects of paclobutrazol and chilling temperatures on lipids, antioxidants and ATPase activity of plasma membrane isolated from green bell pepper fruits

The effects of paclobutrazol treatment on plasma membrane lipid composition and ATPase activity of bell pepper fruit ( Capsicum annuum ) subjected to chilling temperatures were assessed. Application of the growth regulator paclobutrazol affected plant growth and fruit morphology. The plants were more compact and the fruits were less elongated than control fruits. There was about 60% more plasma membrane on a fresh weight basis from treated fruits. At harvest there was no difference in sterol to phospholipid ratio, or in phospholipid fatty acid composition of control compared with paclobutrazol treated fruit. However, plasma membrane ATPase acitivity of treated fruit was two times higher than that of control fruit. After storage at chilling temperature (2°C), the control fruit developed more chilling iniury, and had greater weight loss and a higher rate of K⁺ leakage than paclobulrazol treated fruit. Plasma membrane phospholipid content decreased and saturation of phospholipid fatty acids was higher than in control fruit. These two changes were largely absent in plasma membrane from treated fruit. At harvest antioxidant levels in the plasma membrane of paclobutrazol treated peppers were higher than in those of controls and changed little during storage, whereas levels in control fruit plasma membrane decreased 66%. ATPase activity increased and then decreased in control fruit held at low temperature, whereas in treated fruit activity was constant. The protective effect of paclobutrazol against chilling injury of pepper fruit may result from a combination of its effect on fruit morphology, and protection of the lipids against oxidative stress.     

Understanding the cellular mechanism of recovery from freeze–thaw injury in spinach: possible role of aquaporins,heat shock proteins,dehydrin and antioxidant system

Recovery from reversible freeze–thaw injury in plants is a critical component of ultimate frost survival. However, little is known about this aspect at the cellular level. To explore possible cellular mechanism(s) for post‐thaw recovery (REC), we used Spinacia oleracea L. cv. Bloomsdale leaves to first determine the reversible freeze–thaw injury point. Freeze (–4.5°C)–thaw‐injured tissues (32% injury vs <3% in unfrozen control) fully recovered during post‐thaw, as assessed by an ion leakage‐based method. Our data indicate that photosystem II efficiency (Fv/Fm) was compromised in injured tissues but recovered during post‐thaw. Similarly, the reactive oxygen species (O₂^?? and H₂O₂) accumulated in injured tissues but dissipated during recovery, paralleled by the repression and restoration, respectively, of activities of antioxidant enzymes, superoxide dismutase (SOD) (EC. 1.14.1.1), and catalase (CAT) (EC.1.11.1.6) and ascorbate peroxidase (APX) (EC.1.11.1.11). Restoration of CAT and APX activities during recovery was slower than SOD, concomitant with a slower depletion of H₂O₂ compared to O₂^??. A hypothesis was also tested that the REC is accompanied by changes in the expression of water channels [aquaporines (AQPs)] likely needed for re‐absorption of thawed extracellular water. Indeed, the expression of two spinach AQPs, SoPIP2;1 and SoδTIP, was downregulated in injured tissues and restored during recovery. Additionally, a notion that molecular chaperones [heat shock protein of 70 kDa (HSP70s)] and putative membrane stabilizers [dehydrins (DHNs)] are recruited during recovery to restore cellular homeostasis was also tested. We noted that, after an initial repression in injured tissues, the expression of three HSP70s (cytosolic, endoplasmic reticulum and mitochondrial) and a spinach DHN (CAP85) was significantly restored during the REC.     

Proteomic changes associated with freeze‐thaw injury and post‐thaw recovery in onion (Allium cepa L.) scales

The ability of plants to recover from freeze‐thaw injury is a critical component of freeze‐thaw stress tolerance. To investigate the molecular basis of freeze‐thaw recovery, here we compared the proteomes of onion scales from unfrozen control (UFC), freeze‐thaw injured (INJ), and post‐thaw recovered (REC) treatments. Injury‐related proteins (IRPs) and recovery‐related proteins (RRPs) were differentiated according to their accumulation patterns. Many IRPs decreased right after thaw without any significant re‐accumulation during post‐thaw recovery, while others were exclusively induced in INJ tissues. Most IRPs are antioxidants, stress proteins, molecular chaperones, those induced by physical injury or proteins involved in energy metabolism. Taken together, these observations suggest that while freeze‐thaw compromises the constitutive stress protection and energy supply in onion scales, it might also recruit ‘first‐responders’ (IRPs that were induced) to mitigate such injury. RRPs, on the other hand, are involved in the injury‐repair program during post‐thaw environment conducive for recovery. Some RRPs were restored in REC tissues after their first reduction right after thaw, while others exhibit higher abundance than their ‘constitutive’ levels. RRPs might facilitate new cellular homeostasis, potentially by re‐establishing ion homeostasis and proteostasis, cell‐wall remodelling, reactive oxygen species (ROS) scavenging, defence against possible post‐thaw infection, and regulating the energy budget to sustain these processes.     

Low temperature alters plasma membrane lipid composition and ATPase activity of pineapple fruit during blackheart development

Plasma membrane (PM) plays central role in triggering primary responses to chilling injury and sustaining cellular homeostasis. Characterising response of membrane lipids to low temperature can provide important information for identifying early causal factors contributing to chilling injury. To this end, PM lipid composition and ATPase activity were assessed in pineapple fruit (Ananas comosus) in relation to the effect of low temperature on the development of blackheart, a form of chilling injury. Chilling temperature at 10 °C induced blackheart development in concurrence with increase in electrolyte leakage. PM ATPase activity was decreased after 1 week at low temperature, followed by a further decrease after 2 weeks. The enzyme activity was not changed during 25 °C storage. Loss of total PM phospholipids was found during postharvest senescence, but more reduction was shown from storage at 10 °C. Phosphatidylcholine and phosphatidylethanolamine were the predominant PM phospholipid species. Low temperature increased the level of phosphatidic acid but decreased the level of phosphatidylinositol. Both phospholipid species were not changed during storage at 25 °C. Postharvest storage at both temperatures decreased the levels of C18:3 and C16:1, and increased level of C18:1. Low temperature decreased the level of C18:2 and increased the level of C14:0. Exogenous application of phosphatidic acid was found to inhibit the PM ATPase activity of pineapple fruit in vitro. Modification of membrane lipid composition and its effect on the functional property of plasma membrane at low temperature were discussed in correlation with their roles in blackheart development of pineapple fruit.     

Properties of Plasma Membrane Isolated from Chilling-Sensitive Etiolated Seedlings of Vigna radiata L

Plasma membrane was isolated in a uniform population and with a high purity from chilling-sensitive etiolated young seedlings of Vigna radiata (mung bean) utilizing an aqueous two polymer phase separation system and subsequent sucrose density gradient. The isolated plasma membrane was associated with vanadate-sensitive and KNO₃-insensitive ATPase. The ATPase has high specificities both for substrate and Mg²⁺ ion with optimum pH at 6.5. It was slightly stimulated by monovalent anions, especially Cl⁻. Proton ionophores such as gramicidin D and carbonyl cyanide p-trifluoromethoxyphenylhydrazone did not stimulate the enzyme activity. The ATPase is apparently latent and highly stimulated by the addition of detergents such as Triton X-100. A maximum stimulation was achieved by the addition of 0.02% Triton X-100. After treatment with proteinase K in an isotonic buffer solution, the enzyme activity was less affected, whereas the peptides were specifically digested. Based on these facts, the isolated plasma membrane vesicles appear to be tightly sealed and in a right-side-out orientation. The plasma membrane ATPase had two inflection points at higher (18.9°C) and lower (6.7°C) temperatures on the Arrhenius plots of the activity. The lower inflection temperature apparently coincided with that of the anisotropy parameter of embedded 1,6-diphenyl-1,3,5-hexatriene, indicating that the membrane bound ATPase activity was affected by a phase transition of membrane lipids and/or temperature-dependent conformational changes in the enzyme molecules per se. Considering the fact that the plant material used here is highly sensitive to chilling temperatures and injured severely by exposure to temperatures below 5°C for a relatively short period, the thermotropic properties of membrane molecules are considered to be involved in the mechanism of chilling injury.     

Selective production of sealed plasma membrane vesicles from red beet (Beta vulgaris L.) storage tissue

Modification of our previous procedure for the isolation of microsomal membrane vesicles from red beet (Beta vulgaris L.) storage tissue allowed the recovery of sealed membrane vesicles displaying proton transport activity sensitive to both nitrate and orthovanadate. In the absence of a high salt concentration in the homogenization medium, contributions of nitrate-sensitive (tonoplast) and vanadate-sensitive (plasma membrane) proton transport were roughly equal. The addition of 0.25 M KCl to the homogenization medium increased the relative amount of nitrate-inhibited proton transport activity while the addition of 0.25 M KI resulted in proton pumping vesicles displaying inhibition by vanadate but stimulation by nitrate. These effects appeared to result from selective sealing of either plasma membrane or tonoplast membrane vesicles during homogenization in the presence of the two salts. Following centrifugation on linear sucrose gradients it was shown that the nitrate-sensitive, proton-transporting vesicles banded at low density and comigrated with nitrate-sensitive ATPase activity while the vanadate-sensitive, proton-transporting vesicles banded at a much higher density and comigrated with vanadate-sensitive ATPase. The properties of the vanadate-sensitive proton pumping vesicles were further characterized in microsomal membrane fractions produced by homogenization in the presence of 0.25 M KI and centrifugation on discontinuous sucrose density gradients. Proton transport was substrate specific for ATP, displayed a sharp pH optimum at 6.5, and was insensitive to azide but inhibited by N'-N-dicyclohexylcarbodiimide, diethylstilbestrol, and fluoride. The Km of proton transport for Mg:ATP was 0.67 mM and the K0.5 for vanadate inhibition was at about 50 microM. These properties are identical to those displayed by the plasma membrane ATPase and confirm a plasma membrane origin for the vesicles.     

Purification and characterization of the proton translocating plasma membrane ATPase of red beet storage tissue

Plasma membranes were prepared from red beet (Beta vulgaris L.) storage tissue by partition in an aqueous two-phase system. A highly active proton-translocating ATPase was purified from these membranes by lysophosphatidylcholine extraction and glycerol density gradient centrifugation. The ATPase activity was inhibited by vanadate or dicyclohexyl carbodiimide, but was insensitive to azide, nitrate and molybdate at concentrations which inhibit the F1ATPase, the tonoplast ATPase, and acid phosphatase. Inhibition by vanadate was consistent with a non-competitive mechanism, with Ki = 10 microM. The Km for Mg-ATP was about 1 mM, magnesium ions were required, and the activity was stimulated by KCl and by lysophosphatidylcholine. The optimal pH was 6.5. The molecular mass by gel filtration in the presence of 2 g/liter octyl glucoside was 600 kDa, while dodecyl sulfate gel electrophoresis gave a polypeptide molecular mass of 100 kDa. After blotting onto nitrocellulose, the purified enzyme did not bind concanavalin A, although a concanavalin A-binding peptide of the plasma membrane runs to nearly the same position on the gel and showed some tendency to co-purify with the ATPase. Phospholipid vesicles into which the purified ATPase had been incorporated by the freeze-thaw technique showed vanadate-sensitive, ATP-dependent proton uptake. When the ATPase was reconstituted into lipid membranes at high protein to lipid ratios and incubated with ATP, two-dimensionally crystalline arrays of protein molecules were formed.     

Epidermal growth factor accelerates recovery of LLC-PK1 cells following oxidant injury

Summary In renal tubular epithelial cells, oxidant injury results in several metabolic alterations including ATP depletion, decreased Na⁺K⁺ ATPase activity, and altered intracellular sodium and potassium content. To investigate the recovery of LLC-PK1 cells following oxidant injury and to determine if recovery can be accelerated, we induced oxidant stress in LLC-PK1 cells with 500 μM hydrogen peroxide for 60 min. Identical cohorts of oxidant-stressed cells were incubated in recovery medium without epidermal growth factor (EGF) or recovery medium containing 25 ng EGF per ml. ATP levels, Na⁺K⁺ ATPase activity in whole cells, Na⁺K⁺ ATPase activity in disrupted cells, and intracellular sodium and potassium ion content were determined at 0, 5, 24, 48, and 72 h following oxidant injury in each cohort of cells. In oxidant-stressed cells recovering in medium without EGF, ATP levels, Na⁺K⁺ ATPase activity, and intracellular ion content improved but continued to remain substantially lower than control values at all time points following oxidant stress. In cells recovering in medium with EGF, ATP levels, Na⁺K⁺ ATPase activity, and the intracellular potassium-to-sodium ratio were significantly higher at nearly all time points than values in cells recovering in medium alone. In cells recovering with added EGF, Na⁺K⁺ ATPase activity had improved to control levels, whereas ATP levels and intracellular ion content approached control values by 72 h following oxidant stress. We conclude that oxidant-mediated ATP depletion, altered Na⁺K⁺ ATPase activity, and intracellular ion content remain depressed for several d following oxidant stress and that EGF accelerated recovery of LLC-PK1 cells from oxidant injury.     

Cytological and physiological changes in orthodox maize embryos during cryopreservation

Cytological and physiological changes during cryopreservation were studied in maize embryos at 35 days after pollination (DAP). Both dehydration and freezing caused cytological damage, such as plasmolysis, swelled mitochondria, increased heterochromatin, and nuclear shrinkage. Dehydration alone slightly impaired plasma membrane integrity while a drastic increase in electrolyte leakage was observed after freezing of embryos with moisture content above 23%. Damage to cellular ultrastructure and plasmalemma integrity was negatively related to moisture content in unfrozen embryos and positively related in frozen embryos. The pattern of changes in activity of antioxidant enzymes differed from one another during dehydration and/or freezing–thawing treatment. Dehydration increased activity of ascorbate peroxidase (APX) and glutathione reductase (GR) but decreased activity of superoxide dismutase (SOD) and dehydroascorbate reductase (DHAR). Freezing further decreased GR and SOD activity and resulted in extremely low DHAR activity. Embryos at intermediate moisture contents had low catalase (CAT) activity before freezing but highest CAT activity after freeze–thaw. Both dehydration and freezing promoted membrane lipid peroxidation which resulted in an approximately threefold increase at most in the malondialdehyde content in postthaw embryos. Changes in viability of postthaw embryos can be closely related to damage in cellular ultrastructure and plasmalemma integrity but directly related neither to antioxidants nor lipid peroxidation levels.     

Metabolic changes in rat skin during preservation and storage in glycerol buffer at -196 degrees C.

The penetrating cryoprotective glycerol dissolved in different concentrations in buffer medium effectively protects skin tissue against freeze-thaw injury when progressively cooled to ?196 °C followed by a fast warming-up rate. Upon incubation of the tissue after storage, the incorporation of [2-¹⁴C]glycine into the proteins, of [6-³H]thymidine into DNA, and α-[1-¹⁴C]aminoisobutyric acid transport through the cell membrane are reduced compared to freshly incubated skin. An essential loss in metabolic activity occurs during exposure of the skin to the preserving buffer. Although the length of storage does not seem to affect the final viability, the actual freezing and thawing procedures are particularly damaging to tissue already injured by previous exposure to buffer containing cryoprotective agents.     

Destabilization of the plasma membrane of isolated plant protoplasts during a freeze-thaw cycle: the influence of cold acclimation

In conclusion, isolated protoplasts are an excellent arena in which destabilization of the plasma membrane can be directly observed during a freeze-thaw cycle by cryomicroscopy. Destabilization is manifested in various ways--intracellular ice formation, loss of osmotic responsiveness, or expansion-induced lysis. The incidence of any particular form of injury will depend on the freeze-thaw protocol and hardiness of the tissue from which the protoplasts were isolated. In all cases, however, cold acclimation directly increases the stability of the plasma membrane to the multiple stresses that arise during a freeze-thaw cycle. Such observations provide for functional differences in the plasma membrane that may now be used to consider the significance of any compositional changes in the membrane that might be determined.     

Isolation of plasma membranes from mung bean hypocotyl sections by two-phase partitioning: suitability of the technique for ageing tissues

This paper discusses the application of a particular two-phase partitioning system to the isolation of plasma membranes from heterogeneous starting material, differing in physiological age. Plasma membranes were isolated from hypocotyl segments of mung beans ( Vigna radiata L. Wilczek) on four successive days in order to examine the variation caused by ageing of the seedling. Additionally, the segments were cut at different positions of the hypocotyl to measure variation caused by position-related ageing. To assess purity and degree of contamination of the plasma membrane-enriched preparations, a series of membrane enzyme markers were screened for all isolated fractions. Glucan synthetase II activities were enriched in the plasma membrane fractions, but enrichment and recovery became less pronounced with increasing age. Plasma membrane ATPase activity affected by VO₄^3-, Ca²⁺ and K⁺ was similar in all segments throughout the time-course of the experiment (4 days). However, control ATPase activity varied with segment origin: the physiologically oldest segments showed only 75% activity compared to the youngest ones. K_m and V_max values indicated a smaller proportion of active enzyme but higher substrate affinity as the age of the segments increased. Contamination by intracellular membranes was minimal and unrelated to tissue age.     

Protoplasmic Swelling as a Symptom of Freezing Injury in Onion Bulb Cells : Its Simulation in Extracellular KCl and Prevention by Calcium

Freezing injury, in onion bulb tissue, is known to cause enhanced K⁺ efflux accompanied by a small but significant loss of Ca²⁺ following incipient freezing injury and swelling of protoplasm during the postthaw secondary injury. The protoplasmic swelling of the cell is thought to be caused by the passive influx of extracellular K⁺ into the cell followed by water uptake. Using outer epidermal layer of unfrozen onion bulb scales (Allium cepa L. cv Big Red), we were able to stimulate the irreversible freezing injury symptoms, by bathing epidermal cells in 50 millimolar KCl. These symptoms were prevented by adding 20 millimolar CaCl₂ to the extracellular KCl solution. Our results provide evidence that loss of cellular Ca²⁺ plays an important role in the initiation and the progression of freezing injury.     

Insufficiency of Copper Ion Homeostasis Causes Freeze-Thaw Injury of Yeast Cells as Revealed by Indirect Gene Expression Analysis

Saccharomyces cerevisiae is exposed to freeze-thaw stress in commercial processes, including frozen dough baking. Cell viability and fermentation activity after a freeze-thaw cycle were dramatically decreased due to freeze-thaw injury. Because this type of injury involves complex phenomena, the injury mechanisms are not fully understood. We examined freeze-thaw injury by indirect gene expression analysis during postthaw incubation after freeze-thaw treatment using DNA microarray profiling. The results showed that genes involved in the homeostasis of metal ions were frequently contained in genes that were upregulated, depending on the freezing period. We assessed the phenotype of deletion mutants of the metal ion homeostasis genes that exhibited freezing period-dependent upregulation and found that the strains with deletion of the MAC1 and CTR1 genes involved in copper ion homeostasis exhibited freeze-thaw sensitivity, suggesting that copper ion homeostasis is required for freeze-thaw tolerance. We found that supplementation with copper ions during postthaw incubation increased intracellular superoxide dismutase activity and intracellular levels of reactive oxygen species were decreased. Moreover, cell viability was increased by supplementation with copper ions. These results suggest that insufficiency of copper ion homeostasis may be one of the causes of freeze-thaw injury.Yeast (Saccharomyces cerevisiae) cells are exposed to various environmental stresses such as freeze-thaw, high-temperature, osmotic, and air-drying stresses during commercial processes. Freeze-thaw stress is important in the bread-making process, because frozen dough baking has become a major technology (3). Frozen dough baking improves labor conditions for bakers and enables them to provide fresh baked goods for consumers (3). Because frozen dough baking involves freeze-thaw treatment, it exposes yeast cells to freeze-thaw stress, which leads to a significant decrease in the fermentation ability and viability of yeast cells (called “freeze-thaw injury”) (8). The freeze-thaw injury of yeast cells depends on many factors, including freezing periods, freezing temperature, and the physiology of yeast cells (2, 5, 16, 17). Clarification of the changes in the cell physiology of yeast cells caused by freeze-thaw stress is important, because bakers are eager to extend the shelf life of frozen dough. In this study, we attempted to determine the changes in yeast cell physiology due to freeze-thaw injury by indirect gene expression analysis, which is described below.Freezing subjects yeast cells to low temperature, ice crystal formation in the cells, and dehydration from the cells. This causes both physical damage to cellular components, such as the cell wall, membrane, and proteins, and formation of reactive oxygen species (ROS) (13, 15). Superoxide anions and free radicals are generated in yeast cells during the freeze-thaw process (18), and ROS generation during the thawing process is increased, depending on the freezing period (5, 18). Oxidative stress generated by freeze-thaw treatment enhances the damage to cellular components (9, 25). Because modulation of intracellular levels of ROS after freeze-thaw treatment is required to protect against toxicity, ROS scavenging systems such as glutathione, catalase, and superoxide dismutase (SOD) are believed to be important for freeze-thaw tolerance of yeast cells (1, 2, 17). In particular, copper/zinc SOD (Cu/Zn SOD), which plays a role in oxygen radical detoxification, is necessary to confer full tolerance to freeze-thaw injury (18). Heavy metal ions, such as iron ions and copper ions, are important transition metals for the detoxification of oxygen radicals in yeast cells (6).Although there have been several studies on the mechanisms of freeze-thaw injury (5, 17, 18, 24), the mechanisms are complex and have not yet been fully elucidated. We therefore examined freeze-thaw injury by indirect gene expression analysis, which was conducted during postthaw incubation after freeze-thaw treatment using DNA microarray profiling. Indirect gene expression analysis may be advantageous for such an examination, because changes of gene expression may reflect the physiology of the freezing-state cell. We hypothesized that the genes involved in freeze-thaw injury may upregulate during postthaw incubation. To elucidate the physiological changes during freeze-thaw injury, we carried out indirect gene expression analysis using yeast cells after they had been frozen for different periods. The upregulated genes in the indirect gene expression analysis were extracted by clustering methods. We found that the genes involved in metal ion homeostasis were specifically upregulated. The importance of the genes extracted by the clustering was confirmed by phenotypic analysis using the deletion strains of the extracted genes. We also showed, by physiological analysis, that insufficiency of copper ion homeostasis causes freeze-thaw injury.     

Change in plasma membrane ATPase activity during dormancy release of vegetative peach-tree buds

Plant dormancy and dormancy breaking depend, at least partially, on peculiar short distance relationships between buds and tissues underlying buds (bud stands). In peach-tree, it was previously observed that dormancy was related to a high nutrient absorption capacity in tissues underlying buds. This situation could be linked to higher plasma membrane ATPase activity (EC 3.6.1.3), inducing a higher nutrient absorption, in bud stands. This work consists of characterization of the plasma membrane ATPase activity in vegetative buds and bud stands during the rest period and dormancy release. During the dormant period (October and November), plasma membrane ATPase activity was found to be higher in bud stands than in buds. This was correlated with a lower amount of plasma membrane ATPase in buds compared to bud stands during this period. Moreover, plasma membrane ATPase activation by trypsin treatment was not the same in both tissues and different levels of ATPase activation could be noted within the same tissue during the different stages of dormancy release. According to these results, it can be postulated that dormancy release in peach-tree, is related to modifications of plasma membrane ATPase properties in buds and bud stands during winter time.