The role of abscisic acid and low temperature in chickpea (Cicer arietinum) cold tolerance. II. Effects on plasma membrane structure and function

The frost hardiness of many plants such as chickpea can be increased by exposure to low non-freezing temperatures and/or the application of abscisic acid (ABA), a process known as frost acclimation. Experiments were conducted to study the response over a 14 d period of enriched plasma membrane fractions isolated from chickpea plants exposed to low temperature and sprayed with exogenous ABA. Measurement of the temperatures inducing 50% foliar cell death (LT50), and subsequent statistical analysis suggest that, like many plants, exposure to low temperatures (5/-2 degrees C; day/night) induces a significant level (P <0.05) of frost acclimation in chickpea when compared with control plants (20/7 degrees C; day/night). Spraying plants with exogenous ABA also increased frost tolerance (P <0.05), but was not as effective as low temperature-induced frost acclimation. Both pre-exposure to low temperatures and pre-treatment with ABA increased the levels of fatty acid desaturation in the plasma membrane (measured as the double bond index, DBI). Exposure of chickpea plants to low temperatures increased the DBI by 15% at day 4 and 19% at day 14 when compared with untreated control plants. Application of ABA alone did not increase the DBI by more than 6% at any time; the effects of both treatments applied together was more than additive, inducing a DBI increase of 27% at day 14 when compared with controls. There was a good correlation (P <0.05) between the DBI and LT50, suggesting that the presence of more unsaturated lipid in the plasma membrane may prevent cell lysis at low temperatures. Both pre-exposure to low, non-freezing temperatures and pre-treatment with ABA induced measurable changes in membrane fluidity, but these changes did not correlate with changes in LT50, suggesting that physical properties of the plasma membrane other than fluidity are involved in frost acclimation in chickpea.     

Alterations in Detergent-Resistant Plasma Membrane Microdomains in Arabidopsis thaliana During Cold Acclimation

Microdomains in the plasma membrane (PM) have been proposedto be involved in many important cellular events in plant cells.To understand the role of PM microdomains in plant cold acclimation,we isolated the microdomains as detergent-resistant plasma membranefractions (DRMs) from Arabidopsis seedlings and compared lipidand protein compositions before and after cold acclimation.The DRM was enriched in sterols and glucocerebrosides, and theproportion of free sterols in the DRM increased after cold acclimation.The protein-to-lipid ratio in the DRM was greater than thatin the total PM fraction. The protein amount recovered in DRMsdecreased gradually during cold acclimation. Cold acclimationfurther resulted in quantitative changes in DRM protein profiles.Subsequent mass spectrometry and Western blot analyses revealedthat P-type H⁺-ATPases, aquaporins and endocytosis-related proteinsincreased and, conversely, tubulins, actins and V-type H⁺-ATPasesubunits decreased in DRMs during cold acclimation. Functionalcategorization of cold-responsive proteins in DRMs suggeststhat plant PM microdomains function as platforms of membranetransport, membrane trafficking and cytoskeleton interaction.These comprehensive changes in microdomains may be associatedwith cold acclimation of Arabidopsis.     

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

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

Oryzalin-Induced Changes in Water Status and Cytoskeleton Proteins of Winter Wheat Seedlings upon Cold Acclimation and ABA Treatment

The effect of oryzalin (a specific inhibitor of tubulin polymerization in plant cells) on water retention by the leaves and roots of winter wheat (Triticum aestivum L.) seedlings was studied. The cultivars differing in their frost resistance were compared after their acclimation to low temperature (3°C for 3 or 7 days) and after treatment with ABA. In control untreated plants, oryzalin reduced the water-retaining capacity (WRC) of leaves and roots. Both hardening and ABA lowered the effect of the inhibitor on WRC in leaves, whereas their effects on water retention by roots were opposite, i.e., hardening weakened and ABA intensified the effect of oryzalin. Oryzalin-induced reduction of WRC decreased in the following sequence of cultivars: weakly frost resistant moderately frost resistant highly frost resistant. It was more pronounced in the leaves than in the roots, the latter being characterized by the lower WRC and lower frost resistance. After three-day-long hardening of plants, an additive effect of hypothermia and ABA on oryzalin-induced decrease in WRC of leaves and the lack of such effect in the roots were observed. The immunochemical analysis of the composition and content of cytoskeletal proteins with Western blotting showed that in the leaves the actin/tubulin ratio was higher than in the roots. The treatment of nonacclimated plants with ABA lowered the content of - and -tubulins and actin in roots but did not affect the level of actin in leaves. Hardening negated the effects of ABA on cytoskeletal proteins. Oryzalin produced the greatest inhibitory effect on WRC and an increase in frost resistance in ABA-treated plants in the experiments with leaves of the weakly frost resistant cultivar before and after hardening. Organ- and cultivar-specific and ABA-mediated dependence of WRC on cytoskeletal proteins and microtubules and microfilaments formed by them is supposed to result from their effect on the state of intracellular water and water permeability of the plasma membrane. In the course of cold acclimation of plants and upon their treatment with ABA, this dependence was more distinctly expressed in leaves than in roots, and especially in the plants of the weakly frost resistant cultivar.     

Permeability behaviour of lipid vesicles prepared from plant plasma membranes--impact of compositional changes

Exposure of oat seedlings to repeated moderate water deficit stress causes a drought acclimation of the seedlings. This acclimation is associated with changes in the lipid composition of the plasma membrane of root cells. Here, plasma membranes from root cells of acclimated and control plants were isolated using the two-phase partitioning method. Membrane vesicles were prepared of total lipids extracted from the plasma membranes. In a series of tests the vesicle permeability for glucose and for protons were analysed and compared with the permeability of model vesicles. Further, the importance of critical components for the permeability properties was analysed by modifying the lipid composition of the vesicles from acclimated and from control plants. The purpose was to add specific lipids to vesicles from acclimated plants to mimic the composition of the vesicles from control plants and vice versa. The plasma membrane lipid vesicles from acclimated plants had a significantly increased permeability for glucose and decreased permeability for protons as compared to control vesicles. The results point to the importance of the ratio phosphatidylcholine (PC)/phosphatidylethanolamine (PE), the levels of cerebrosides and free sterols and the possible interaction of these components for the plasma membrane as a permeability barrier.     

植物抗寒及其基因表达研究进展

植物经过逐渐降低的温度从而提高抗寒能力 ,这个过程被人们称为低温驯化。植物低温驯化过程是一个复杂的生理、生化和能量代谢变化过程 ,这些变化主要包括膜系统的稳定性、可溶性蛋白的积累和小分子渗透物质 ,比如脯氨酸、糖等 ,这些变化中的一些是植物抗寒必需的 ,而另外一些变化不是必需的。主要对冷害和低温生理生化变化、低温诱导表达基因的功能和作用、低温驯化的调节机制及其信号转导方面进行了综述。通过差别筛选 c DNA文库的方法已经鉴定了许多低温诱导表达、进而提高植物抗寒能力的基因 ,其中有脱水素、COR基因和 CBF1转录因子等。低温信号的感受、转导和调节表达是低温驯化的关键环节 ,低温信号的转导过程与干旱胁迫之间具有一定的交叉 ,这为利用 ABA等来提高植物抗寒能力成为可能 ,相信不久的将来人们可以通过提高植物抗寒能力从而增加经济产量成为现实。     

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

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

Effects of sterol-binding agent nystatin on wheat roots: the changes in membrane permeability, sterols and glycoceramides

Plant sterols are important multifunctional lipids, which are involved in determining membrane properties. Biophysical characteristics of model lipid and isolated animal membranes with altered sterol component have been intensively studied. In plants however, the precise mechanisms of involvement of sterols in membrane functioning remain unclear. In present work the possible interactions between sterols and other membrane lipids in plant cells were studied. A useful experimental approach for elucidating the roles of sterols in membrane activity is to use agents that specifically bind with endogenous sterols, for example the antibiotic nystatin. Membrane characteristics and the composition of membrane lipids in the roots of wheat (Triticum aestivum L.) seedlings treated with nystatin were analyzed. The application of nystatin greatly increased the permeability of the plasma membrane for ions and SH-containing molecules and decreased the total sterol level mainly as a consequence of a reduction in the amount of β-sitosterol and campesterol. Dynamic light-scattering was used to confirm the in vitro formation of stable complexes between nystatin and β-sitosterol or cholesterol. Sterol depletion was accompanied by a significant rise in total glycoceramide (GlCer) content after 2h treatment with nystatin. Analysis of the GlCer composition using mass spectrometry with electrospray ionization demonstrated that nystatin induced changes in the ratio of molecular species of GlCer. Our results suggest that changes in the sphingolipid composition can contribute to the changes in plasma membrane functioning induced by sterol depletion.     

Review and future prospects on the mechanisms related to cold stress resistance and tolerance in medicinal plants

Medicinal plants play important role in industrial production of medicines. Moreover, they consume without complicated processes around the world. They are considered as healthy cure without any harmful side effects at least among ordinary people. Cold stress is one the harmful abiotic stresses and constrains medicinal plants yielding geographically. Cold acclimation is a process that induces cold stress resistance in temperate plants. Various structural and morphological alterations are involved in this process. Also, enzymatic and non-enzymatic agents play role in cold acclimation. Cell membrane modification and compatible solutes accumulation and so many other changes occur through cold acclimation. Growing under different stressful conditions, medicinal plants synthesize different components such as metabolites. Moreover, ROS can be generated in plant cells under stressful conditions. The accumulation of bioactive components, biosynthesis of phytohormones, ion hemostasis, osmolyte (compatible solutes) accumulation and changes in nutrient uptake, root system modification and systemic resistance are some of new investigations that are considered in this review.     

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.     

Light-dependent cold-induced fatty acid unsaturation, changes in membrane fluidity, and alterations in gene expression in Synechocystis

Cold stress causes unsaturation of the membrane lipids. This leads to adjustment of the membrane fluidity, which is necessary for cold acclimation of cells. Here we demonstrate that the cold-induced accumulation of PUFAs in the cyanobacterium Synechocystis is light-dependent. The desA(-)/desD(-) mutant, that lacks the genes for Δ12 and Δ6 desaturases, is still able to adjust the fluidity of its membranes in spite of its inability to synthesize PUFAs and modulate the fatty acid composition of the membrane lipids under cold stress. The expression of cold-induced genes, which are controlled by the cold sensor histidine kinase Hik33, depends on the fluidity of cell membranes and it is regulated by light, though it does not require the activity of the photosynthetic apparatus. The expression of cold-induced genes, which are not controlled by Hik33, does not depend on the membrane fluidity or light. Thus, membrane fluidity determines the temperature dependence of the expression of cold-induced genes that are under control of the Hik33, which might be the sensor of changes in the membrane fluidity. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Plasma Membrane Lipid Alterations Associated with Cold Acclimation of Winter Rye Seedlings (Secale cereale L. cv Puma)

Highly enriched plasma membrane fractions were isolated from leaves of nonacclimated (NA) and acclimated (ACC) rye (Secale cereale L. cv Puma) seedlings. Collectively, free sterols, steryl glucosides, and acylated steryl glucosides constituted >50 mole% of the total lipid in both NA and ACC plasma membrane fractions. Glucocerebrosides containing hydroxy fatty acids constituted the major glycolipid class of the plasma membrane, accounting for 16 mole% of the total lipid. Phospholipids, primarily phosphatidylcholine and phosphatidylethanolamine with lesser amounts of phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and phosphatidylinositol, comprised only 32 mole% of the total lipid in NA samples. Following cold acclimation, free sterols increased from 33 to 44 mole%, while steryl glucosides and acylated steryl glucosides decreased from 15 to 6 mole% and 4 to 1 mole%, respectively. Sterol analyses of these lipid classes demonstrated that free β-sitosterol increased from 21 to 32 mole% (accounting for the increase in free sterols as a class) at the expense of sterol derivatives containing β-sitosterol. Glucocerebrosides decreased from 16 to 7 mole% of the total lipid following cold acclimation. In addition, the relative proportions of associated hydroxy fatty acids, including 22:0 (h), 24:0 (h), 22:1 (h), and 24:1 (h), were altered. The phospholipid content of the plasma membrane fraction increased to 42 mole% of the total lipid following cold acclimation. Although the relative proportions of the individual phospholipids did not change appreciably after cold acclimation, there were substantial differences in the molecular species. Di-unsaturated molecular species (18:2/18:2, 18:2/18:3, 18:3/18:3) of phosphatidylcholine and phosphatidylethanolamine increased following acclimation. These results demonstrate that cold acclimation results in substantial changes in the lipid composition of the plasma membrane.     

Cold acclimation can induce microtubular cold stability in a manner distinct from abscisic acid

The response of cortical microtubules to low temperature was investigated for the Chinese winter wheat (Triticum aestivum L.) cultivar Jing Nong 934. Microtubules in the cortex of the root elongation zone disassembled rapidly in response to a cold shock of -7 degrees C and reassembled upon rewarming to 25 degrees C. The microtubules acquired resistance against this cold shock in response to cold acclimation in chilling, but non-freezing, temperature or after a treatment with abscisic acid (ABA). Cold acclimation and ABA differed with respect to the appearance of microtubules: fine, transverse strands were observed after cold acclimation, whereas ABA produced steeply oblique microtubule bundles. The findings are discussed in terms of an ABA-independent pathway for acquired cold stability of microtubules.     

ABA and gibberellin-like substances during prehardening,cold acclimation,de- and reacclimation of oilseed rape

Previously published results showed that high relative reduction state of PSII (PSII excitation pressure) during both early seedling growth (prehardening) as well as cold deacclimation caused significant changes in growth pattern. The differences in elongation growth rate were related to the cold acclimation of photosynthetic apparatus and to frost resistance. To study changes in the hormonal balance connected with alterations in elongation growth rate observed during prehardening and deacclimation under different PSII excitation pressure (modulated by day-temperatures), endogenous concentration of ABA, GA₃ and GA-like substances (GAs) were analysed. Analyses were also performed during cold acclimation and reacclimation of plants characterized by different elongation growth rate triggered by prehardening or deacclimation under different day-temperatures. Growth under high PSII excitation pressure (prehardening) resulted in a significant increase in ABA and a considerable decrease in GAs contents. On the other hand, different ABA content played almost no role in controlling growth rate during cold deacclimation and subsequent reacclimation, when the induction of elongation growth was connected with the changes in concentration of GAs including GA₃. The possible role of ABA and GAs in controlling prehardening, cold acclimation and deacclimation is discussed.     

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。     

Modifications of abscisic acid level in winter oilseed rape leaves during acclimation of plants to freezing temperatures

An almost twofold increase in abscisic acid (ABA) content was observed in the leaves of winter oilseed rape plants (Brassica napus L., var. oleifera L., cv. Jantar) grown in the cold (>0°C). This ABA increase took place during the first three days of cold treatment. After 6 days of plant growth in the cold, the level of ABA started to decline or remained constant, depending on the calculation basis: dry weight or disc area units, respectively. The exposure of cold-acclimated plants to night frost (–5°C for 18 h) induced a further increase (65%) in the ABA level, which begun during the first few hours after thawing. The comparison of time courses of frost resistance increments and ABA content changes showed that modifications of ABA level in the cold-treated leaves preceded those of frost resistance, whereas in the frost-pretreated tissues the ABA increase occurred later than that of frost tolerance. Possible interrelations between ABA content, frost tolerance and tissue water potential modifications in the low temperature-affected tissues are discussed.     

Sterols as dietary markers for Drosophila melanogaster

During cold acclimation fruit flies switch their feeding from yeast to plant food, however there are no robust molecular markers to monitor this in the wild. Drosophila melanogaster is a sterol auxotroph and relies on dietary sterols to produce lipid membranes, lipoproteins and molting hormones. We employed shotgun lipidomics to quantify eight major food sterols in total lipid extracts of heads and genital tracts of adult male and female flies. We found that their sterol composition is dynamic and reflective of fly diet in an organ-specific manner. Season-dependent changes observed in the organs of wild-living flies suggested that the molar ratio between yeast (ergosterol, zymosterol) and plant (sitosterol, stigmasterol) sterols is a quantifiable, generic and unequivocal marker of their feeding behavior suitable for ecological and environmental population-based studies. The enrichment of phytosterols over yeast sterols in wild-living flies at low temperatures is consistent with switching from yeast to plant diet and corroborates the concomitantly increased unsaturation of their membrane lipids.     

Maintenance of Membrane Fluidity during Development of Freezing Tolerance of Winter Wheat Seedlings

Fluidity of membrane lipids of shoot and root tissue and of chloroplasts from young wheat seedlings of contrasting freezing tolerance was investigated by measuring the motion and order parameters after spin labeling. A striking similarity was observed in membrane lipid fluidity of the five cultivars grown at 22 C. After cold hardening by growth at 2 C, a small change in membrane lipid fluidity was observed, but this was not correlated with the development of freezing tolerance, and there was no alteration in the transition temperature of membrane lipids. The results show that neither changes in membrane lipid fluidity nor transition temperature are a necessary feature of cold acclimation in wheat.     

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。