Seasonal Changes in Plasma Membranes and Mitochondria Isolated From Jerusalem Artichoke Tubers. Possible Relationship to Cold Hardiness

Plasma membranes and mitochondria were isolated from Jerusalemartichoke tubers during cold acclimation from September to December.The protein and lipid contents of the membranes were analyzedwith reference to physiological properties of the tubers, especiallycold hardiness. As cold hardiness increased from autumn to winter,the content of phospholipids and sterols on a mg protein basisincreased by 20–30% in plasma membranes, but little changewas observed in mitochondria. Minor changes were observed inthe fatty acid composition of phospholipids either in plasmamembranes or mitochondria. Membrane fluidity, assessed by fluorescentpolarization of 1,6-diphenyl-1,3,5-hexatriene, was found tobe relatively constant in both membranes during the season.One dimensional SDS-polyacrylamide gel electrophoresis revealedseasonal changes in proteins and glycoproteins in plasma membranes,but not in mitochondrial membranes. Plasma membrane ATPase increasedin specific activity from September to December, which was morenoticeable at higher assay temperatures. However, irrespectiveof the season, the plasma membrane ATPase had an inflectionon the slope of the Arrhenius plot around 15C. These resultssuggest that plasma membranes, in contrast to mitochondria,undergo several molecular changes from autumn to winter, whichmay be related to cold acclimation of the tubers. ¹ Contribution No. 2668 from the Institute of Low TemperatureScience. ² Present address: Crop Development Centre, University of Saskatchewan,Saskatoon, Canada S7N 0W0.     

Reverse Changes in Plasma Membrane Properties upon Deacclimation of Mulberry Trees (Morus bombysis Koidz.)

To gain more insight into the relation between plasma membranechanges and cold hardiness in mulberry trees (Morus bombysisKoidz. cv Goroji), biochemical and biophysical changes in theplasma membrane were studied during cold deacclimation in spring.The majority of the changes in the plasma membranes that occurredduring the cold acclimation process in the fall/winter werereversed following deacclimation in the spring. Significantdecreases in phospholipid content, degree of unsaturation inphospholipid fatty acids, and membrane fluidity were observedin the plasma membranes during cold deacclimation. The sterolto phospholipid ratio increased with decreasing cold hardiness.Reverse changes were also detected in the majority of proteinand glycoprotein components. These reversible changes in theplasma membranes are considered to be involved in the mechanismof cold hardiness of plants. ¹Contribution No. 2766 from the Institute of Low TemperatureScience. (Received July 10, 1985; Accepted October 25, 1985)     

Protein and Lipid Compositions of Isolated Plasma Membranes from Orchard Grass (Dactylis glomerata L.) and Changes during Cold Acclimation

The chemical composition of plasma membrane fractions isolated from orchard grass seedlings (Dactylis glomerata L.) was analyzed and compared with endomembranes. The plasma membrane is characterized by an enrichment of sterols and a lower degree of unsaturation of phospholipids. Steryl glycosides were found to be one of the lipid components of the plasma membrane, but steryl esters and galactolipids were barely detectable. Diphosphatidyl glycerol was characteristically detected in the mitochondrial membrane, but not in the plasma membrane fraction. Plasma mambrane fraction was also characterized by its `lower fluidity' in comparison with the endomembranes. This may be due to the large amount of sterols and the lower degree of phospholipid unsaturation in plasma membranes.

Electrophoretic comparison of polypeptides was also made between different membranes. The distribution patterns of polypeptides revealed on one- and two-dimensional SDS-slab gels were characteristic for those membranes. The presence of glycopeptide complements was a useful criterion for distinguishing plasma membrane from other membranes. The plasma membrane and the ER + Golgi membranes were enriched in glycopeptides. However, a marked difference was revealed in the total number and the molecular weights of the peptides.

During cold acclimation of orchard grass seedlings, the degree of fatty acid unsaturation increased only slightly in plasma membrane, unlike in endomembranes. The change in sterols-to-phospholipids ratio in plasma membrane was also slight. On the other hand, the phospholipid-to-protein ratio increased significantly in the plasma membrane as cold hardiness increased. A significant change in the polypeptide complements of plasma membrane was also demonstrated during cold acclimation.

     

Cold acclimation in Pinus sylvestris: Phospholipids in purified plasma membranes from needles of pine

Effects of cold acclimation on needles of Scots pine ( Pinus sylvestris L., Provenance Södra Ydre) were studied at the membrane level. Before and after a period of cold acclimation the plasma membranes were isolated from the needles by a aqueous polymer two-phase partition technique. Fatty acid composition of total lipids or of individual phospholipids from the plasma membrane showed that the plasma membrane fraction was different from the other microsomal fractions analyzed, especially the 18:2 levels of the individual phospholipids. Furthermore, the cold acclimation period did not result in a decreased saturation level in the plasma membranes. Different steps in cold acclimation reactions at the membrane level are discussed.     

Temperature and Aging Effects on Leaf Membranes of a Cold Hardy Perennial, Fragaria virginiana

The lipid composition of leaves of wild strawberry (Fragaria virginiana Duchesne) was analyzed throughout an annual growth cycle in the field. Cellular hardiness to temperature stress was assessed concomitantly by a solute leakage technique. Leaves were shown to be very sensitive to an applied temperature of −5°C during the summer months but insensitive to a 35°C treatment. This general pattern was also seen in young overwintering leaves but was reversed after a period of low-temperature hardening of these same leaves. Associated with cold hardening of the overwintering leaves was a twofold increase in the phospholipid content of the leaf membranes with a proportionately smaller increase in free sterols. The large increase in phospholipids presumably is due primarily to the proliferation of a sterol-poor membrane fraction, probably the endoplasmic reticulum. These quantitative changes in membrane material may be important in increasing freezing tolerance in the overwintering leaf cells by enhancing the overall capacity of the cell for plasma membrane and tonoplast extension through vesicle fusion using components from this endomembrane pool. Analysis of electron micrographs of hardened leaf cells showed an increase in vesiculated smooth endoplasmic reticulum and tonoplast membrane over nonhardened leaf cells, the latter resulting in an enhanced tonoplast surface area to vacuolar volume ratio. During this same period, no changes in the fatty acid or free sterol composition were detectable, suggesting that regulation of membrane fluidity via these components is not required for cold acclimation in this species. During aging and senescence of both the overwintering and the summer leaves, the cellular membranes remained functionally intact but became progressively more vulnerable to temperature stress. Free sterol content increased during this time. This feature may be related to the inability of the older leaves to withstand environmental stress. Increasing sensitivity of the cellular membranes to stress may, in turn, be causally related to the actual onset of senescence in these leaves, thus explaining why only the older leaves senesce when the plant is challenged by periodic environmental stress.     

A Contrast of the Plasma Membrane Lipid Composition of Oat and Rye Leaves in Relation to Freezing Tolerance

The lipid composition of the plasma membrane isolated from leaves of spring oat (Avena sativa L. cv Ogle) was vastly different from that of winter rye (Secale cereale L. cv Puma). The plasma membrane of spring oat contained large proportions of phospholipids (28.8 mol% of the total lipids), cerebrosides (27.2 mol%), and acylated sterylglucosides (27.3 mol%) with lesser proportions of free sterols (8.4 mol%) and sterylglucosides (5.6 mol%). In contrast, the plasma membrane of winter rye contained a greater proportion of phospholipids (36.6 mol%), and there was a lower proportion of cerebrosides (16.4 mol%); free sterols (38.1 mol%) were the predominant sterols, with lesser proportions of sterylglucosides (5.6 mol%) and acylated sterylglucosides (2.9 mol%). Although the relative proportions of individual phospholipids, primarily phosphatidylcholine and phosphatidylethanolamine, and the molecular species of these two phospholipids were similar in oat and rye, the relative proportions of di-unsaturated species of these two phospholipids were substantially lower in oat than in rye. The relative proportions of sterol species in oat were different from those in rye; the molecular species of cerebrosides were similar in oat and rye, with only slight differences in the proportions of the individual species. After 4 weeks of cold acclimation, the proportion of phospholipids increased significantly in both oat (from 28.8 to 36.8 mol%) and rye (from 36.6 to 43.3 mol%) as a result of increases in the proportions of phosphatidylcholine and phosphatidylethanolamine. For both oat and rye, the relative proportions of di-unsaturated species increased after cold acclimation, but the increase was greater in rye than in oat. In both oat and rye, this increase occurred largely during the first week of cold acclimation. During the 4 weeks of cold acclimation, there was a progressive decrease in the proportion of cerebrosides in the plasma membrane of rye (from 16.4 to 10.5 mol%), but there was only a small decrease in oat (from 27.2 to 24.2 mol%). In both oat and rye, there were only small changes in the proportions of free sterols and sterol derivatives during cold acclimation. Consequently, the proportions of both acylated sterylglucosides and cerebrosides remained substantially higher in oat than in rye after cold acclimation. The relationship between these differences in the plasma membrane lipid composition of oat and rye and their freezing tolerance is presented.     

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.     

Nucleic Acid and protein changes in relation to cold acclimation and freezing injury of korean boxwood leaves

Quantitative and qualitative differences in nucleic acids of Korean boxwood (Buxus microphylla var. Koreana) leaves were determined by methylated albumin kieselguhr chromatography at different levels of cold hardiness. During cold acclimation there was an increase in RNA, mainly ribosomal RNA, with little or no change in DNA. The increase in ribosomal RNA was closely paralleled by an increase in water soluble and membrane bound proteins. As cold hardiness increased, ribonuclease activity declined.     

Mass spectrometric approach for identifying putative plasma membrane proteins of Arabidopsis leaves associated with cold acclimation

Although enhancement of freezing tolerance in plants during cold acclimation is closely associated with an increase in the cryostability of plasma membrane, the molecular mechanism for the increased cryostability of plasma membrane is still to be elucidated. In Arabidopsis, enhanced freezing tolerance was detectable after cold acclimation at 2 degrees C for as short as 1 day, and maximum freezing tolerance was attained after 1 week. To identify the plasma membrane proteins that change in quantity in response to cold acclimation, a highly purified plasma membrane fraction was isolated from leaves before and during cold acclimation, and the proteins in the fraction were separated with gel electrophoresis. We found that there were substantial changes in the protein profiles after as short as 1 day of cold acclimation. Subsequently, using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS), we identified 38 proteins that changed in quantity during cold acclimation. The proteins that changed in quantity during the first day of cold acclimation include those that are associated with membrane repair by membrane fusion, protection of the membrane against osmotic stress, enhancement of CO2 fixation, and proteolysis.     

抗寒锻炼中不同抗寒性小麦细胞膜糖蛋白的细胞化学研究

本研究根据植物细胞的特点,修改了在动物和人体细胞方面立的酶标Con A的电镜细胞化学方法,成功地展现了2个不同抗寒性冬小麦品种幼苗在抗寒锻炼和脱锻炼过程中细胞膜系统上糖蛋白的分布动态,显示与Con A连接的标志酶-辣根过氧化物酶活性的反应产物呈颗粒状分散分布在质膜、内质网、核膜及液泡膜的一些部位上,揭示糖蛋白在冬小麦细胞膜系统上的分布似有其特定的位点。经抗寒锻炼后,强抗寒性品种燕大1817细胞内的糖蛋白在内质网和核膜上的分布量明显地增加;同时,几乎所有的胞间连丝通道中都有糖蛋白的分布。脱锻炼后,内质网和核膜上的糖蛋白分布量又减少,胞间连丝通道中的糖蛋白也消失,基本上回复到抗寒锻炼前的分布状态。抗寒性弱的冬小麦品种郑州39-1幼苗在同样的抗寒锻炼和脱锻炼过程中不产生这些明显的变化。这些结果说明,抗寒锻炼中内质网和核膜上糖蛋白分布量的增加,以及糖蛋白输入胞间连丝的动态变化是与植物抗寒力的提高和保持稳定密切相关的。     

Changes in parameters of the plasmalemma ATPase during cold acclimation of apple (Mains domestica) tree bark tissues

A plasmalemma fraction was isolated from homogenized apple tree (Mains domestica Borkh 'Golden Delicious') hark tissues using aqueous phase partitioning and ultra-centrifugation. Results of marker enzyme assays indicated that a membrane preparation highly enriched in plasma membranes was obtained. ATPase activity in this preparation possessed a high specificity for ATP as substrate, was inhibited by vanadate, diethylstilbcsterol and dicyclohexylcarbocHimide, and was insensitive to inhibitors of mitochondria! and tonoplasl ATPases. Specific activity of the plasma-lemma ATPase increased during cold acclimation prior to the attainment of vegetative maturity. Kinetic parameters (Km, Vln) determined from assays performed at different temperatures (10, 30°C) indicated a differential effect of cold acclimation on enzyme activity. Vm increased during cold acclimation, whereas Km increased when determined at 30°C but declined at 10°C. Acclimation treatments during April and May resulted in alteration of ATPase kinetics in the absence of any increase in bark frost hardiness. Changes in ATPase kinetics may be related more to enhanced low temperature metabolism than to frost hardiness per se.     

The Relationship of Carbohydrates to Cold Acclimation of Hedera helix L. cv. Thorndale

While both the total sugar content and cold hardiness greatly increased during artificial cold acclimation, no direct parallelism was demonstrated. In fact, plants hardened in the dark exhibited an increase in hardiness during the period when the total sugar content declined, furthermore, while there was evidence for an accumulation of sugars (especially sucrose) incorporation of ¹⁴C indicated that all of the isolated fractions were in a dynamic state. Dehardening, paralleled by a large increase in the starch content, was more rapid in the light than in the dark. Furthermore, in stems the sugar content increased after 3 days of dehardening but declined after 7 days. This increase may represent the release of sugars from a previously unextractable form such as a glycoprotein complex. Starvation experiments indicate that photosynthates produced during the cold acclimation period are preferentially used during cold acclimation rather than reserve carbohydrates. This was also indicated by the smaller amount of starch hydrolysis in plants hardened in the light. Thus, while there appears to be a role for carbohydrates in the cold acclimation process, the lack of parallelism between sugar content and hardiness may be interpreted as indicating 1) cold acclimation is not merely an accumulation of sugars or an osmotic effect per se, and 2) under normal conditions, the level of carbohydrates is not limiting the rate or degree of cold acclimation.     

Relationship of Electrical Conductance at Two Frequencies to Cold Injury and Acclimation in Cornus stolonifera Michx

The ratio of electrical conductance measured at two frequencies can be used to predict the cold hardiness of stem sections of Cornus stolonifera Michx. during the first stage of cold acclimation. Electrical conductance at 50 hertz divided by electrical conductance at 100 kilohertz gave a better estimate of hardiness than measurements at either frequency alone. The observed increase in the electrical conductance ratio as hardiness increased is consistent with an increase in membrane permeability. After plants were exposed to nonlethal frost, hardiness increased rapidly, and the relation between the conductance ratio and hardiness changed. This change indicates that ice crystallization induces a significant physiological alteration in the plants. Contrary to expectations, stem sections exposed to lethal temperatures could not consistently be separated from sections exposed to nonlethal temperatures by electrical conductance ratio measurements made immediately after thawing.     

Cold Acclimation of Hedera helix: Evidence for a Two Phase Process

The light-enhanced production and accumulation of sugars is only one step in the process of cold acclimation in Hedera helix L. var. Thorndale (English ivy). Applications of 2,4-dinitrophenol to plants with different portions exposed to light and dark indicated that the mere presence or accumulation of the light-generated promoters did not invoke an increase in hardiness. Kinetics of cold acclimation during alternating periods of light and dark also indicate that the light stimulation of cold acclimation is only a partial component of the total process. Incubation on 50 mm solutions of sucrose can replace the light requirement. A second phase which can proceed in the dark is thought to result in the production of proteins which, due to an altered composition or configuration, have a greater capacity to bind sugars. This is evidenced by the fact that protein from cold acclimated tissue exhibited a higher sugar-binding capacity than protein from nonacclimated tissue. Furthermore, the two phases can proceed independently of each other, but only upon complementation of the products of the two phases is an increase in cold hardiness manifested.     

The Role of Light in Cold Acclimation of Hedera helix L. var. Thorndale

The role of light in cold acclimation of Hedera helix L. var. Thorndale appears to differ from that reported for winter annuals. Although light greatly enhances the degree of hardiness attained, cold acclimation is not obligatorily linked to a light requirement. Photoperiods, varying from 8 to 24 hours, received during the cold acclimation period were equally effective in promoting maximum hardiness. Relatively low light intensities and short photoperiods stimulated maximum hardiness, and proportional increases in hardiness in response to increased photoperiods were demonstrated only in stems of prestarved plants. Exclusion of CO₂ and high concentrations of photosynthetic inhibitors decreased hardiness, but in no instance was hardiness reduced to the level of the dark control. The data are only compatible with a photosynthetic role of light if it is assumed that only a small portion of the total photosynthates are required to elicit maximum hardiness. Alternatively, the light stimulation which was elicited by low light intensities, short photoperiods, in the absence of CO₂, and in the presence of photosynthetic inhibitors, may be a light signal similar to a phytochrome response.     

Structural and functional responses of wheat mitochondrial membranes to growth at low temperatures

The responses in membrane lipid composition, structure, and function of four cultivars of wheat (Triticum aestivum L.) to growth at low temperature have been investigated. Marked growth temperature-dependent alterations in the fatty acid composition and unsaturation of the mitochondrial phospholipids correlate with changes in respiratory activity in all the varieties. Parameters such as the respiratory control ratio and the phosphorylative efficiency decrease in cold-adapted seedlings. Three temperature-dependent structural transitions were identified in the mitochondria by the spin-labeling method. The structural transitions occur at lower temperatures in the cold-grown material. The shift in one transition appears to be quantitatively greater in the winter hardy varieties. Cold-induced changes in all of the other measured parameters were indistinguishable in hardy and nonhardy varieties. The results indicate major involvement of the phospholipid matrix in cold acclimation. A link between cold acclimation and winter survival may exist involving the structural and functional modifications in membrane structure which occur during acclimation.     

Potato cold hardiness development and abscisic acid. II. De novo synthesis of proteins is required for the increase in free abscisic acid during potato (Solanum commersonii) cold acclimation

During cold acclimation of potato plantlets ( Solanum commersonii Dun, PI 458317), there are two transitory increases in free ABA content corresponding to a three-fold increase on the 2nd day and a five-fold increase on the 6th day (Ryu and Li 1993). During this period, plantlets increased in cold hardiness from −5°C (killing temperature, control grown at 22/18°C, day/night) to −10°C by the 7th day of exposure to 4/2°C (day/night). This increase in free ABA was not found when cycloheximide (CHI), an inhibitor of cytoplasmic protein synthesis, was added to the culture medium 6 h before exposure to low temperatures. Plantlets treated with CHI did not acclimate to cold, maintaining a hardiness level (−5°C) similar to that of the 22/18°C-grown plantlets. When the CHI-treated plantlets were exposed to low temperatures for 3 days, transferred to CHI-free culture medium and grown at low temperatures, the plantlets showed a transitory increase in free ABA 2 days later. This increase was followed by the development of cold hardiness (−8°C). Application of CHI to the culture medium after 3 days of cold acclimation, when the first ABA peak and a partial development of cold hardiness (−8°C) had occurred, blocked the second transitory increase in free ABA and resulted in no further development of cold hardiness. These results suggest that de novo synthesis of proteins is required for these transitory increases in free ABA during cold acclimation of potato plantlets.     

CBF 3 基因过量表达的拟南芥细胞质膜组分的变化

通过提取过量表达CBF3基因和对照的拟南芥[Arabidopsis thaliana L．Heyn．(Columbia)]茎叶的质膜．分离并分析其脂类成分和蛋白质含量,从中探讨CBF3对膜脂成分的影响及与抗冷适应的关系。研究结果表明,过量表达CBF3植株的质膜膜脂总量和膜蛋白总量分别是对照的227％和190％,磷脂为105％,与冷适应诱导的效果相似。因此CBF3表达的变化可能对冷适应过程中质膜组成的改变起重要作用。     

Plasma Membrane Alterations in Callus Tissues of Tuber-bearing Solanum Species during Cold Acclimation

Plasma membrane alterations in two tuber-bearing potato species during a 20-day cold acclimation period were investigated. Leaf-callus tissues of the frost-resistant Solanum acaule Hawkes `Oka 3878' and the frost-susceptible, commonly grown Solanum tuberosum `Red Pontiac,' were used. The former is a species that can be hardened after subjecting to the low temperature, and the latter does not harden. Samples for the electron microscopy were prepared from callus cultures after hardening at 2 C in the dark for 0, 5, 10, 15, and 20 days. After 20 days acclimation, S. acaule increased in frost hardiness from −6 to − 9 C (killing temperature), whereas frost hardiness of S. tuberosum remained unchanged (killed at −3 C). Actually, after 15 days acclimation, a −9 C frost hardiness level in S. acaule callus cultures had been achieved.     

CBF3基因过量表达的拟南芥细胞质膜组分的变化

通过提取过量表达CBF3基因和对照的拟南芥[Arabidopsis thaliana L.Heyn.(Columbia)]茎叶的质膜,分离并分析其脂类成分和蛋白质含量,从中探讨CBF3对膜脂成分的影响及与抗冷适应的关系。研究结果表明,过量表达CBF3植株的质膜膜脂总量和膜蛋白总量分别是对照的227%和190%,磷脂为105%,与冷适应诱导的效果相似。因此CBF3表达的变化可能对冷适应过程中质膜组成的改变起重要作用。