The significance of glutathione for photoprotection at contrasting temperatures in the alpine plant species Soldanella alpina and Ranunculus glacialis

The significance of total glutathione content was investigated in two alpine plant species with highly differing antioxidative scavenging capacity. Leaves of Soldanella alpina and Ranunculus glacialis incubated for 48 h in the presence of buthionine-sulfoximine had 50% lower glutathione contents when compared with leaves incubated in water. The low leaf glutathione content was not compensated for by activation of other components involved in antioxidative protection or electron consumption. However, leaves with normal but not with low glutathione content increased their ascorbate content during high light (HL) treatment (S. alpina) or catalase activity at low temperature (LT) (R. glacialis), suggesting that the mere decline of the leaf glutathione content does not act as a signal to ameliorate antioxidative protection by alternative mechanisms. CO(2)-saturated oxygen evolution was not affected in glutathione-depleted leaves at various temperatures, except at 35°C, thereby increasing the high temperature (HT) sensitivity of both alpine species. Leaves with low and normal glutathione content were similarly resistant to photoinhibition and photodamage during HL treatment at ambient temperature in the presence and absence of paraquat or at LT. However, HL- and HT-induced photoinhibition increased in leaves with low compared to leaves with normal glutathione content, mainly because the recovery after heat inactivation was retarded in glutathione-depleted leaves. Differences in the response of photosystem II (PSII) activity and CO(2)-saturated photosynthesis suggest that PSII is not the primary target during HL inactivation at HT. The results are discussed with respect to the role of antioxidative protection as a safety valve for temperature extremes to which plants are not acclimated.     

The effects of acclimation to sunlight on the xylem vulnerability to embolism in Fagus sylvatica L.

We assessed the effects of irradiance received during growth on the vulnerability of Fagus sylvatica L. xylem vessels to water-stress-induced embolism. The measurements were conducted on (1) potted saplings acclimated for 2 years under 100% and 12% incident global radiation and (2) branches collected from sun-exposed and shaded sides of adult trees. Both experiments yielded similar results. Light-acclimated shoots were less vulnerable to embolism. Xylem water potential levels producing 50% loss of hydraulic conductivity were lower in sun-exposed branches and seedlings than in shade-grown ones (–3·0 versus –2·3 MPa on average). The differences in vulnerability were not correlated with differences in xylem hydraulic conductivity nor vessel diameter. Resistance to cavitation was correlated with transpiration rates, midday xylem and leaf water potentials in adult trees. We concluded that vulnerability to cavitation in Fagus sylvatica may acclimate to contrasting ambient light conditions.     

Past,present and future of mountain species of the French Massif Central – the case of Soldanella alpina L. subsp. alpina (Primulaceae) and a review of other plant and animal studies

Aim Our goals were: (1) to investigate patterns of genetic variation in the French Massif Central (MC) of Soldanella alpina (Primulaceae), an alpine plant species that has only one known population in the region; (2) to analyse these patterns in order to deduce the Quaternary history of the population and to predict how current climatic warming may affect it; and (3) to review molecular analyses from the MC to evaluate the importance of the region for the conservation of genetic diversity. Location Europe, with a special focus on the French Massif Central and adjacent regions. Methods Amplified fragment length polymorphisms (AFLPs) were analysed for 192 individuals (nine populations) of S. alpina (subsp. alpina) representing the MC, Pyrenees and south‐western Alps. Population genetic diversity was assessed by various parameters (e.g. H_E, Shannon’s I). Neighbor‐Net and Bayesian approaches, and analysis of molecular variance (AMOVA) were used to infer population genetic relationships and structure. Results Individuals generally clustered according to populations within mountain regions. Hierarchical AMOVA indicated significant variation among mountain ranges (33.2% of the total variance), but there was also strong differentiation between populations (26.3%). The single population of S. alpina from the MC was identified as a distinct lineage of high genetic diversity. Our literature survey indicated that taxa with low and with high genetic diversity exist in the MC, and that genetic relationships to surrounding regions are diverse. Main conclusions The high genetic diversity and distinctiveness of S. alpina in the MC suggests the long‐term persistence of the single population in this region, which might have been favoured through elevational range shifts in response to past climatic change. This interpretation partly accords with other studies indicating that several plant and animal populations in the MC contain comparatively high genetic diversity, represent genetically independent lineages, and/or are likely descendants of populations that persisted in the MC throughout the Quaternary. These data underline the conservation importance of the MC as a key area for the long‐term persistence of species with often high levels of intraspecific genetic diversity.     

High Temperature Effects on Light Sensitivity in the Two High Mountain Plant Species Soldanella alpina (L.) and Rannunculus glacialis (L.)

Abstract: The susceptibility to high temperature‐induced photoinhibition was investigated in leaves of two high mountain plant species, S. alpina and R. glacialis. In both species, PSII was similarly photoinactivated at 38 °C in the light. However, recovery from damage was much faster in S. alpina and depended on protein synthesis. In contrast, recovery was independent from protein synthesis in R. glacialis. Heat‐induced photoinactivation in both species was accompanied by: (1) a decrease in relative photosynthetic electron transport rates, (2) an increase in non‐photochemical chlorophyll fluorescence quenching, (3) a strong accumulation of zeaxanthin, (4) a marked decrease in soluble carbon metabolites and (5) an increase in lipid metabolism products, which was more pronounced in R. glacialis than in S. alpina. These results indicate that carbon assimilation was inhibited and that membranes were affected. Lipid peroxidation and possible membrane disintegration might limit the repair of damaged PSII in R. glacialis, while S. alpina appears to be protected by carotenoids and antioxidants. A marked decrease in α‐tocopherol content and an increase in reduced ascorbate indicated lipid peroxide scavenging activity in S. alpina. When zeaxanthin synthesis was impaired by DTT, photoinhibition increased and α‐tocopherol accumulated in R. glacialis. The increased susceptibility of R. glacialis leaves to light‐induced photoinhibition after growth at moderate temperature (Streb et al., 2003a) and the inability to repair heat‐induced damage might limit the distribution of R. glacialis to lower altitudes in the Alps.     

Parallel evolution of flower reduction in two alpine Soldanella species (Primulaceae)

The European endemic Soldanella has traditionally been divided into two morphologically well‐defined sections. Section Tubiflores contains two species growing in high‐elevation habitats, whereas most of the 14 species of section Soldanella inhabit montane forests. Section Tubiflores has a reduced floral morphology compared with section Soldanella. A previous phylogenetic study based on internal transcribed spacer (ITS) and AFLP data has revealed that, although the genus Soldanella itself is monophyletic, both sections are paraphyletic. Soldanella alpina (section Soldanella) forms a clade with S. minima and S. pusilla (section Tubiflores), and the grouping of S. alpina with S. pusilla has been hypothesized to be the result of hybridization between S. pusilla and an unidentified species of section Soldanella. We re‐examined the phylogenetic relationships among the above species using additional sequence data (plastid DNA) and increasing the sample for ITS and AFLP data. Our new data confirmed that S. alpina is most closely related to S. pusilla. However, our data do not provide any evidence in support of the hypothesis that S. alpina is a hybrid species. In consequence, we consider it likely that the two species of section Tubiflores originated independently. The reduced floral morphology of these two alpine species is probably evidence for increased levels of self‐pollination, ensuring reproductive success in a high‐altitude habitat. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 175 , 409–422.     

Genetic diversity in an endangered alpine plant, Eryngium alpinum L. (Apiaceae), inferred from amplified fragment length polymorphism markers

Eryngium alpinum L. is an endangered species found across the European Alps. In order to obtain base-line data for the conservation of this species, we investigated levels of genetic diversity within and among 14 populations from the French Alps. We used the amplified fragment length polymorphism (AFLP) technique with three primer pairs and scored a total of 62 unambiguous, polymorphic markers in 327 individuals. Because AFLP markers are dominant, within-population genetic structure (e.g. FIS) could not be assessed. Analyses based either on the assumption of random-mating or on complete selfing lead to very similar results. Diversity levels within populations were relatively high (mean Nei's expected heterozygosity = 0.198; mean Shannon index = 0.283), and a positive correlation was detected between both genetic diversity measurements and population size (Spearman rank correlation: P = 0. 005 and P = 0.002, respectively). Moreover, FST values and exact tests of differentiation revealed high differentiation among populations (mean pairwise FST = 0.40), which appeared to be independent of geographical distance (nonsignificant Mantel test). Founder events during postglacial colonizations and/or bottlenecks are proposed to explain this high but random genetic differentiation. By contrast, we detected a pattern of isolation by distance within populations and valleys. Predominant local gene flow by pollen or seed is probably responsible for this pattern. Concerning the management of E. alpinum, the high genetic differentiation leads us to recommend the conservation of a maximum number of populations. This study demonstrates that AFLP markers enable a quick and reliable assessment of intraspecific genetic variability in conservation genetics.     

Dynamic changes in plant secondary metabolites during UV acclimation in Arabidopsis thaliana

Plants respond to environmental stress by synthesizing a range of secondary metabolites for defense purposes. Here we report on the effect of chronic ultraviolet (UV) radiation on the accumulation of plant secondary metabolites in Arabidopsis thaliana leaves. In the natural environment, UV is a highly dynamic environmental parameter and therefore we hypothesized that plants are continuously readjusting levels of secondary metabolites. Our data show distinct kinetic profiles for accumulation of tocopherols, polyamines and flavonoids upon UV acclimation. The lipid‐soluble antioxidant α‐tocopherol accumulated fast and remained elevated. Polyamines accumulated fast and transiently. This fast response implies a role for α‐tocopherol and polyamines in short‐term UV response. In contrast, an additional sustained accumulation of flavonols took place. The distinct accumulation patterns of these secondary metabolites confirm that the UV acclimation process is a dynamic process, and indicates that commonly used single time‐point analyses do not reveal the full extent of UV acclimation. We demonstrate that UV stimulates the accumulation of specific flavonol glycosides, i.e. kaempferol and (to a lesser extent) quercetin di‐ and triglycosides, all specifically rhamnosylated at position seven. All metabolites were identified by Ultra Performance Liquid Chromatography (UPLC)‐coupled tandem mass spectrometry. Some of these flavonol glycosides reached steady‐state levels in 3–4 days, while concentrations of others are still increasing after 12 days of UV exposure. A biochemical pathway for these glycosides is postulated involving 7‐O‐rhamnosylation for the synthesis of all eight metabolites identified. We postulate that this 7‐O‐rhamnosylation has an important function in UV acclimation.     

Cold- and light-induced changes of metabolite and antioxidant levels in two high mountain plant species Soldanella alpina and Ranunculus glacialis and a lowland species Pisum sativum

Leaves of the two cold-acclimated alpine plant species Ranunculus glacialis and Soldanella alpina and, for comparison, of the non-acclimated lowland species Pisum sativum were illuminated with high light intensity at low temperature. The light- and cold-induced changes of antioxidants and of the major carbon and phosphate metabolites were analysed to examine which metabolic pathways might be limiting in non-acclimated pea leaves and whether alpine plants are able to circumvent such limitation. During illumination at low temperature pea leaves accumulated high quantities of sucrose, glucose-6-phosphate, fructose-6-phosphate, mannose-6-phosphate and phosphoglycerate (PGA) whereas ATP/ADP-ratios decreased. Although the PGA content also increased in leaves of R. glacialis the other metabolites did not accumulate and ATP/ADP-ratios remained fairly constant in either alpine species. These data indicate a inorganic phosphate (Pi)-limitation in the chloroplasts of pea leaves but not in the alpine species. However, the total phosphate pool and the percentage of free Pi were highest in pea and did not change during illumination in cold. In contrast, free Pi contents declined markedly in R. glacialis leaves, suggesting that Pi is available for metabolism in this species. In S. alpina leaves contents of ascorbate and glutathione doubled in light and cold, while the contents of sugars did not increase. Obviously, S. alpina leaves can use assimilated carbon for ascorbate synthesis, rather than for the synthesis of sugars. A high capacity for ascorbate synthesis might prevent the accumulation of mannose-6-phosphate and Pi-limitation.     

Reversibility of cold- and light-stress tolerance and accompanying changes of metabolite and antioxidant levels in the two high mountain plant species Soldanella alpina and Ranunculus glacialis

Two high mountain plants Soldanella alpina (L.) and Ranunculus glacialis (L.) were transferred from their natural environment to two different growth conditions (22 degrees C and 6 degrees C) at low elevation in order to investigate the possibility of de-acclimation to light and cold and the importance of antioxidants and metabolite levels. The results were compared with the lowland crop plant Pisum sativum (L.) as a control. Leaves of R. glacialis grown for 3 weeks at 22 degrees C were more sensitive to light-stress (defined as damage to photosynthesis, reduction of catalase activity (EC 1.11.1.6) and bleaching of chlorophyll) than leaves collected in high mountains or grown at 6 degrees C. Light-stress tolerance of S. alpina leaves was not markedly changed. Therefore, acclimation is reversible in R. glacialis leaves, but constitutive or long-lasting in S. alpina leaves. The different growth conditions induced significant changes in non-photochemical fluorescence quenching (qN) and the contents of antioxidants and xanthophyll cycle pigments. These changes did not correlate with light-stress tolerance, questioning their role for light- and cold-acclimation of both alpine species. However, ascorbate contents remained very high in leaves of S. alpina under all growth conditions (12-19% of total soluble carbon). In cold-acclimated leaves of R. glacialis, malate represented one of the most abundant compounds of total soluble carbon (22%). Malate contents declined significantly in de-acclimated leaves, suggesting a possible involvement of malate, or malate metabolism, in light-stress tolerance. Leaves of the lowland plant P. sativum were more sensitive to light-stress than the alpine species, and contained only low amounts of malate and ascorbate.     

Soldanella rugosa is synonym to real S. marmarossiensis

Soldanella rugosa Zhang & Kadereit is a synonym to the real S. marmarossiensis Klášt. Correct name for the taxon called S. marmarossiensis by Zhang and Kadereit as defined by them is S. haretii G. Grint. but it comprises at least three different taxa.     

Hierarchical responses of plant stoichiometry to phosphorus addition in an alpine meadow community

为了探明氮(N)限制的植物群落中物种水平和功能群水平的碳(C)、N、磷(P)含量以及C:N:P对P添加的响应是否一致,明确P添加对群落物种构成改变的内在机制。以青藏高原高寒草甸为研究对象, 通过P添加试验, 研究了功能群水平和物种水平生态化学计量比对P添加的响应, 以及P添加对物种水平的优势度和功能群水平生物量的影响。结果表明: 在青藏高原高寒草甸连续5年添加P显著改变了植物的C、N、P含量以及C:N:P, 且在物种水平和功能群水平(不含典型物种)的响应规律基本一致。在禾本科、莎草科和杂类草功能群(不含典型物种)和相应物种水平上P添加对C含量影响不显著。P添加显著增加了禾本科、莎草科、豆科和杂类草4个功能群(不含典型物种)和相应物种水平的植物P含量, 降低了C:P和N:P。禾本科和莎草科的N含量和C:N对P添加在物种水平和功能群水平上(不含典型物种)的响应规律一致, 表现为N含量显著降低, C:N显著增加; P添加使豆科物种水平上N含量显著增加而C:N显著降低, 但在功能群水平上(不含典型物种)无显著作用; 杂类草的N含量和C:N对P添加在物种水平和功能群水平上(不含典型物种)的响应规律均不一致。在N限制的生境中添加P, 禾本科物种在群落中逐渐占据优势跟其增高的N、P利用效率相关, 而杂类草由于逐渐降低的N和P利用效率使其生物量在群落中所占的比重逐渐下降。     

高寒草甸植物化学计量比对磷添加的分层响应

为了探明氮(N)限制的植物群落中物种水平和功能群水平的碳(C)、N、磷(P)含量以及C:N:P对P添加的响应是否一致,明确P添加对群落物种构成改变的内在机制。以青藏高原高寒草甸为研究对象, 通过P添加试验, 研究了功能群水平和物种水平生态化学计量比对P添加的响应, 以及P添加对物种水平的优势度和功能群水平生物量的影响。结果表明: 在青藏高原高寒草甸连续5年添加P显著改变了植物的C、N、P含量以及C:N:P, 且在物种水平和功能群水平(不含典型物种)的响应规律基本一致。在禾本科、莎草科和杂类草功能群(不含典型物种)和相应物种水平上P添加对C含量影响不显著。P添加显著增加了禾本科、莎草科、豆科和杂类草4个功能群(不含典型物种)和相应物种水平的植物P含量, 降低了C:P和N:P。禾本科和莎草科的N含量和C:N对P添加在物种水平和功能群水平上(不含典型物种)的响应规律一致, 表现为N含量显著降低, C:N显著增加; P添加使豆科物种水平上N含量显著增加而C:N显著降低, 但在功能群水平上(不含典型物种)无显著作用; 杂类草的N含量和C:N对P添加在物种水平和功能群水平上(不含典型物种)的响应规律均不一致。在N限制的生境中添加P, 禾本科物种在群落中逐渐占据优势跟其增高的N、P利用效率相关, 而杂类草由于逐渐降低的N和P利用效率使其生物量在群落中所占的比重逐渐下降。     

Changes in reproductive investment with altitude in an alpine plant

Aims In perennial species, the allocation of resources to reproduction results in a reduction of allocation to vegetative growth and, therefore, impacts future reproductive success. As a consequence, variation in this trade-off is among the most important driving forces in the life-history evolution of perennial plants and can lead to locally adapted genotypes. In addition to genetic variation, phenotypic plasticity might also contribute to local adaptation of plants to local conditions by mediating changes in reproductive allocation. Knowledge on the importance of genetic and environmental effects on the trade-off between reproduction and vegetative growth is therefore essential to understand how plants may respond to environmental changes.Methods We conducted a transplant experiment along an altitudinal gradient from 425 to 1?921 m in the front range of the Western Alps of Switzerland to assess the influence of both altitudinal origin of populations and altitude of growing site on growth, reproductive investment and local adaptation in Poa alpina .Important findings In our study, the investment in reproduction increased with plant size. Plant growth and the relative importance of reproductive investment decreased in populations originating from higher altitudes compared to populations originating from lower altitudes. The changes in reproductive investment were mainly explained by differences in plant size. In contrast to genetic effects, phenotypic plasticity of all traits measured was low and not related to altitude. As a result, the population from the lowest altitude of origin performed best at all sites. Our results indicate that in P. alpina genetic differences in growth and reproductive investment are related to local conditions affecting growth, i.e. interspecific competition and soil moisture content.     

Low temperature acclimation of guard cell chloroplasts by the arctic plant Saxifraga cernua L.

Abstract. The potential for thermal acclimation of photosynthetic electron transport by guard cell chloropiasts (GC ch) was assessed in epidermal peels taken from the abaxial side of Saxifraga cernua leaves grown at 20°C and 10°C. Chlorophyll a fluorescence induction kinetics measured in pairs of guard cells in individual stomata from tissue grown at 10 °C demonstrated a rise in the fluorescence to a maximum and a larger amplitude in variable fluorescence when measured at temperatures below 18°C than was seen in GC ch from tissue grown at 20°C. The rates of fluorescence quenching in 10°C-grown tissue were also faster than in 20°C-grown tissue when measured at temperatures below 18°C. State 1-State 2 transitions by GC ch were measured at selected temperatures between 5 and 25 °C as changes in the magnitude of the fluorescence emission maxima at 685, 695 and 730nm (F685, F695 and F730) measured at 77K. At measuring temperatures of 5 and 10°C, GC ch in tissue grown at 10 °C showed a greater transition to State 2 (a larger F730/F695 ratio) than did GC ch in tissue grown at 20 °C. At measuring temperatures of 20 and 25 °C, there was no difference in either the kinetics or the magnitude of the State 1 to State 2 transition in the two tissues. The ultrastructure of GC ch from tissues grown at 10 and 20 °C was also examined using transmission electron microscopy. Less than half (48%) of the grana from the higher temperature grown tissue had more than nine thylakoids/grana. Grana in GC ch which had developed at 10 °C showed a dramatic reduction in stacking, such that 85% of the grana contained no more than two thylakoids. The reduction in grana stacking was also accompanied by a decrease in the degree of appression of thylakoid membranes. The results demonstrate a capacity for thermal acclimation of GC ch function to low temperatures. This acclimation is associated with alterations in the chloroplast ultrastructure.     

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.