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.     

Expression of freezing tolerance in the interspecific F1 and somatic hybrids of potatoes

 The expression of freezing tolerance was examined in interspecific F₁ and somatic hybrids of potatoes using 20 species and 34 different combinations between hardy and sensitive species. In the field, the frost tolerance of hybrids resembled either that of the hardy parent, the sensitive parent, or the parental mean, depending on the species combination and the genomic ratio (ratio of the number of sets of chromosomes contributed from each parent). Similar phenomena were observed when the non-acclimated freezing tolerance (NA) and the acclimation capacity (ACC) (two independent genetic components of freezing tolerance) were evaluated separately under controlled environments. In general, the expression level of freezing tolerance was higher in hybrids with more genomes contributed from the hardy parent than from the sensitive parent. In addition, the effectiveness or combining ability of genes conferring freezing tolerance from the hardy species also showed some influence on the expression of freezing tolerance. All three parameters, namely NA, ACC and acclimated freezing tolerance (AA) (NA plus ACC), were significantly correlated to the frost tolerance exhibited in the field. This indicates that the controlled freezing test used in this study could provide a good estimate of field performance. The implications of these results in breeding for freezing tolerance in potatoes are discussed. Received: 21 July 1998 / Accepted: 29 September 1998     

Freezing tolerance and tuber production in selfed and backcross progenies derived from somatic hybrids between Solanum tuberosum L. and S. commersonii Dun.

 Selfed and backcross progenies developed from tetraploid somatic hybrids between Solanum tuberosum (tbr) and S. commersonii (cmm) were characterized for nonacclimated freezing tolerance (NA) and acclimation capacity (ACC) (two independent genetic components of freezing tolerance) under controlled environments. The segregation covered 28% and 71% of the parental range for NA and ACC, respectively, with the distribution skewed toward the tbr parent. Therefore, ACC appeared to be relatively easier to recover in the segregating generation. Some first backcross progeny had greater freezing tolerance than the cultivated parent primarily through the increase in ACC. When grown in the field, the improved freezing tolerance observed in the selfed progeny under controlled conditions was confirmed. Among NA, ACC, and freezing tolerance after acclimation (AA, which is the cumulative performance of NA and ACC), AA exhibited the highest correlation coefficient with field frost tolerance. In addition to freezing tolerance, vine maturity and tuber traits including tuber yield, tuber number per plant, mean tuber weight, and specific gravity were also segregating. No significant correlation between undesirable tuber traits and freezing tolerance was detected. Vine maturity and freezing tolerance were significantly correlated, so more careful selection for earliness was necessary in incorporating freezing tolerance. Yield comparable or superior to the backcross parent Wis AG 231 and an early Canadian cultivar, ‘Sable’, was found in many backcross progeny and some selfed progeny. The observed high yield can be attributed to the increase in mean tuber weight as well as tuber number. Moreover, a high portion of progeny had a specific gravity higher than 1.085, and some greater than 1.1. The implications derived from this study in breeding for freezing tolerance and further use of these materials are discussed. Received: 22 August 1998 / Accepted: 4 January 1999     

Isolation and characterization of a glycolipid from Dictyostelium discoideum

A glycolipid was isolated from a lipid extract of the cellular slime mold Dictyostelium discoideum and characterized. From the results of analyses by thin-layer chromatography and infrared spectrometry, it was identified as a steryl glycoside. The steryl glycoside was further analyzed by gas-liquid chromatography/mass spectrometry as a trimethylsilyl ether derivative, and its quantitative and qualitative changes during the development of D. discoideum were examined. Δ²²-Stigmastenyl-d-glucoside was the major constituent of the steryl glycoside and comprised more than 90% of the total steryl glycoside fraction in cells at all stages of development. The content of the steryl glycoside was higher in vegetative-stage cells, late aggregation-stage cells, and 1-day sorocarps than in cells of other stages. The glycolipid fraction was often contaminated by a lipid which was also isolated and identified as a ceramide containing 2-hydroxy fatty acids and 4D-hydroxysphinganine.     

Gamma-ray-induced changes in hypodermal mesocarp tissue plasma membrane of pre- and post-storage muskmelon

Gamma irradiation (1.0 kGy) of intact, newly harvested, mature muskmelon fruit (Cucumis melo L. var. reticulatus Naud.) appears to have an immediate deleterious effect, but also a long-term beneficial effect, on the integrity and function of the plasma membrane (PM) of hypodermal mesocarp tissue. The initial consequences of gamma irradiation included an increase in the free sterol:phospholipid ratio, resulting at least in part from deglycosylation of steryl glycosides, a decrease in the spinasterol:7-stigmastenol ratio in each of the PM steryl lipids (free sterols, steryl glycosides, and acylated steryl glycosides), and a decrease in H⁺-ATPase activity. Irradiation did not increase protein loss, suggesting that the decrease in H⁺-ATPase activity resulted from either direct inactivation of the enzyme or altered PM ordering caused by the steryl lipid modifications. The long-term beneficial effects of irradiation, observed following 10 days of commercial storage, included greater retention of total PM protein, a diminished decline in total PM phospholipids (PL) and in the PL:protein ratio, and maintenance of greater overall H⁺-ATPase activity (activity was the same as in controls on a per mg protein basis, but there was > 30% more protein in the PM of stored irradiated fruit). These results indicate that 1 kGy gamma irradiation administered prior to storage slowes the progression of two key parameters of senescence, PM protein loss and PL catabolism.     

Effects of Cadmium on Polar Lipid Composition and Unsaturation in Maize （Zea mays） in Hydroponic Culture

This study aimed to evaluate the effect of Cd exposure （100 μmol/L） on polar lipid composition, and to examine the level of fatty acid unsaturation in maize （Zea mays L.）. In roots, the level of 16：0 and monounsaturated fatty acids （16：1 ＋ 18：1） decreased in phosphatidylcholine （PC） and phosphatidylethanolamine （PE）. In contrast, the proportion of unsaturated 18-C fatty acid species showed an opposite response to Cd. The content, on the other hand, of PC, PE, digalactosyldiacylglycerol （DGDG）, and steryl lipids increased in roots （2.9-, 1.6-, 5.3-, and 1.7-fold increase, respectively）. These results suggest that a more unsaturated fatty acid composition than found in control plants with a concomitant increase in polar lipids may favor seedling growth during Cd exposure. However, the observed increase in the steryl lipid （SL） ： phospholipid （PL） ratio （twofold）, the decrease in monogalactosyldiacylglycerol （MGDG） ： DGDG ratio, as well as the induction of lipid peroxidation in roots may represent symptoms of membrane injury. In shoots, the unsaturation level was markedly decreased in PC and phosphatidylglycerol （PG） after Cd exposure, but showed a significant increase in sulfoquinovosyldiacylglycerol （SQDG）, MGDG and DGDG. The content of PG and MGDG was decreased by about 65%, while PC accumulated to higher levels （4.4-fold increase）. Taken together, these changes in the polar lipid unsaturation and composition are likely to be due to alterations in the glycerolipid pathway. These results also support the idea that the increase in overall unsaturation plays some role in enabling the plant to withstand the metal exposure.     

Aluminum mediates compositional alterations of polar lipid classes in maize seedlings

Changes in lipid composition were investigated on maize roots and shoots under aluminum stress. After 4d exposure to 100 microM Al, root growth was inhibited while shoot growth was not affected. In roots, the decrease of the DBI (double bond index) of total fatty acids may signal a decrease in membrane fluidity. The total lipids (TL) decreased by 49%, but phospholipids (PL), phosphatidylcholine (PC) and phosphatidylinositol (PI) increased to approximately 3-fold. The MGDG increased to 2-fold but no significant change was found in the DGDG. The steryl lipids (SL) increased by 69%. The SL/PL ratio decreased from 2.64 to 1.52 and the MGDG/DGDG ratio increased from 0.45 to 1.06 in roots of Al-stressed plants. Al leads to oxidative stress in roots of treated plants as indicated by the increase of malondialdehyde (MDA) concentrations. In shoots, changes in fatty acid composition were associated with an increase of the DBI in all lipid classes except that of the DGDG decreased. The PG was the lipid class which shows the large variation of fatty acid composition. No significant changes were found either for TL, PL, SL or MDA concentrations in shoots of Al-treated plants. While PE levels did not show significant change, PI and PG increased and PC decreased. However, the Al caused 87% decrease in the GL levels. The MGDG and DGDG decreased to 19- and 8-fold, respectively. The deleterious effects of Al on polar lipids could be caused by a direct intervention of Al on plasma membrane and/or alteration of cell metabolism.     

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.     

Differential degradation of extraplastidic and plastidic lipids during freezing and post-freezing recovery in Arabidopsis thaliana

Changes in membrane lipid composition play important roles in plant adaptation to and survival after freezing. Plant response to cold and freezing involves three distinct phases: cold acclimation, freezing, and post-freezing recovery. Considerable progress has been made toward understanding lipid changes during cold acclimation and freezing, but little is known about lipid alteration during post-freezing recovery. We previously showed that phospholipase D (PLD) is involved in lipid hydrolysis and Arabidopsis thaliana freezing tolerance. This study was undertaken to determine how lipid species change during post-freezing recovery and to determine the effect of two PLDs, PLDalpha1 and PLDdelta, on lipid changes during post-freezing recovery. During post-freezing recovery, hydrolysis of plastidic lipids, monogalactosyldiacylglycerol and plastidic phosphatidylglycerol, is the most prominent change. In contrast, during freezing, hydrolysis of extraplastidic phospholipids, phosphatidylcholine and phosphatidylethanolamine, occurs. Suppression of PLDalpha1 decreased phospholipid hydrolysis and phosphatidic acid production in both the freezing and post-freezing phases, whereas ablation of PLDdelta increased lipid hydrolysis and phosphatidic acid production during post-freezing recovery. Thus, distinctly different changes in lipid hydrolysis occur in freezing and post-freezing recovery. The presence of PLDalpha1 correlates with phospholipid hydrolysis in both freezing and post-freezing phases, whereas the presence of PLDdelta correlates with reduced lipid hydrolysis during post-freezing recovery. These data suggest a negative role for PLDalpha1 and a positive role for PLDdelta in freezing tolerance.     

Protein, leucine aminopeptidase, esterase, acid phosphatase and photosynthetic responses of oleander (Nerium oleander L.) during cold acclimation and freezing treatments

Six-month-old oleander (Nerium oleander L.) pot plants, derived from vegetative propagation by cuttings, were tested for their ability to cold hardening. Damage of the non-acclimated (NA) plants was visible when treated by low freezing temperatures (below -2 degrees C). The responses of total proteins, leucine aminopeptidase (LAP), esterase (EST) and acid phosphatase (ACP) isoforms of NA and cold-acclimated (CA; 4 degrees C for 14 days) plants were compared using polyacrylamide gel electrophoresis. These molecular markers were also compared in NA and CA plants which received for 2h temperatures of 0, -2, -4, -6 and -8 degrees C. A new 38-kDa polypeptide appeared from day 7 to 14 during the acclimation treatment in the bark extracts and on day 14 in the leaf extracts. The above-mentioned polypeptide band (38 kDa) strongly appeared in all freezing treatments (0, -2, -4, -6 and -8 degrees C) in both bark and leaf extracts of the CA plants. Alterations in the number and the intensity of LAP and EST isoforms as well as in the intensity of ACP isoforms were observed in both bark and leaf of the CA oleander plants. A newly expressed EST isoform is proposed as biochemical marker for the cold acclimation treatment. CO2 assimilation rates (A) as well as transpiration rates (E) in NA plants were positive in 0 degrees C and negative in all temperatures below zero in the freezing treatments. In contrast, CO2 assimilation rates (A) and transpiration rates (E) were positive in CA plants in all temperatures of freezing treatment. A significant decrease (P<0.05) in chlorophyll (Chl) a, Chl a+b concentration and Chl a/b ratio were noticed in oleander plants during the acclimation treatment (from day 0 to 14), while Chl b concentration was unchanged at the respective time. On the other hand, no significant (P<0.05) differences were observed in the freezing treatments.     

Klebsormidium flaccidum, a charophycean green alga, exhibits cold acclimation that is closely associated with compatible solute accumulation and ultrastructural changes

To elucidate the fundamental mechanisms and subsequent evolutionary aspects of plant cold acclimation, we examined the effect of cold acclimation on freezing tolerance in Klebsormidium flaccidum, a green alga belonging to Charophyceae, a sister group of land plants. Freezing tolerance of K. flaccidum was significantly enhanced by cold treatment: survival increased from 15% at -10 degrees C when grown at 18 degrees C to 55 and 85% after exposure at 2 degrees C for 2 and 7 d, respectively. Accompanying the development of freezing tolerance, soluble sugars (glucose and sucrose), a putative glycoside and amino acids, including gamma-aminobutyric acid (GABA), accumulated to high levels in the alga, suggesting that these solutes play a crucial role in the cold acclimation of K. flaccidum. Interestingly, the application of abscisic acid (ABA) did not change the freezing tolerance of the alga. We also observed changes in cell structure, including increased numbers and sizes of starch grains in chloroplasts, chloroplast enlargement, vacuole size reduction and cytoplasmic volume increase. These results suggest that K. flaccidum responds well to cold treatment and develops freezing tolerance in a process comparable to that of land plants.     

Lipid profiling and tolerance to low-temperature stress in Thellungiella salsuginea in comparison with Arabidopsis thaliana

Changes in membrane lipid composition is a fundamental strategy for plants to resist low-temperature stress. We compared members of 11 membrane glycerolipid classes in Thellungiella salsuginea and its close relative Arabidopsis thaliana at normal growth temperature, and during cold acclimation (CA), freezing (FR), and post-freezing recovery (PFR). The results showed several properties of T. salsuginea distinct from that in A. thaliana, which included: 1) low relative content of phosphatidic acid (PA) and a rapid increase and decrease of PA during FR and PFR respectively; 2) insensitivity of lyso-phospholipids to freezing; and 3) high ratio of phosphatidylcholine to phosphatidylethanolamine. All these properties were in favour of maintaining membrane integrity and stability and therefore enable T. salsuginea to be more tolerant to freezing than A. thaliana.     

Free radical and freezing injury to cell membranes of winter wheat

The symptoms of injury in microsomal membranes isolated from crowns of seedlings of Triticum aestivum , L. cultivar Fredrick after a lethal freeze-thaw stress included an increased lipid phase transition temperature, loss of lipid phosphate (lipid-P), and increased free fatty acid levels. However, minimal changes in fatty acid saturation were observed, suggesting minimal amounts of lipid peroxidation. All of these injury symptoms, including the lack of lipid peroxidation, were simulated in vitro by treatment of isolated membranes with oxygen free radicals, generated from either xanthine oxidase (EC 1.1.3.22) or paraquat (l,r-dimethyl-4,4'-bipyridinium dichloride). Further evidence indicating a relationship between free radicals and freezing injury comes from the observation that both protoplasts and microsomal membranes isolated from wheat seedlings, that had been acclimated to induce freezing tolerance, also had increased tolerance of oxygen free radicals, and contained higher lipid-soluble antioxidant levels, than those from non-acclimated seedlings. Lipid-soluble antioxidants accumulated in the crown tissue of the wheat seedling during the acclimation period. Freezing stress accelerated the formation of oxygen free radicals. Membranes isolated from crowns after a freeze–thaw stress tended to produce higher levels of superoxide as shown by the reduction of Tiron (1,2-dihydroxy-l,3-benzenedisulfonic acid). In protoplasts, increased superoxide production coincided with lethal freezing injury. These results are discussed in terms of the possible involvement of oxygen free radicals in mediating aspects of freezing injury to cell membranes.     

Computer aided morphometric analysis of oral leukoplakia and oral squamous cell carcinoma

We compared the changes in the cells in the basal layer of normal mucosa, oral leukoplakia with dysplasia and different grades of oral squamous cell carcinoma (OSCC) using computer aided image analysis of tissue sections. We investigated three morphometric parameters: nuclear area (NA), cell area (CA) and their ratio (NA:CA). NA and NA:CA ratio showed a statistically significant increase from dysplasia to increasing grades of OSCC. Nuclear size was useful for differentiating normal tissue, potentially malignant leukoplakia and OSCC.     

Sterols in hardening winter wheat

The main sterols in winter wheat crowns and roots were sitosterol and campesterol, with significant amounts of stigmasterol and traces of cholesterol. The main groups of sterol-containing lipids were free sterols, steryl glucosides, steryl esters and esterified steryl glucosides. Sterol analysis within each group showed little difference between them. Steryl esters were relatively rich in cholesterol and poor in stigmasterol. Free sterols were rich in stigmasterol. Low temperature caused an increase in sterol content but had little effect on sterol composition and sterol to lipid P ratio. There was some increase in steryl esters and some decrease in free sterols. Cholesterol and stigmasterol decreased in the steryl ester and free sterol fractions, respectively. There was little evidence for involvement of sterols in winter wheat frost hardening.     

Characterization of Arabidopsis sterol glycosyltransferase TTG15/UGT80B1 role during freeze and heat stress

Sterol glycosyltransferases regulate the properties of sterols by catalyzing the transfer of carbohydrate molecules to the sterol moiety for the synthesis of steryl glycosides and acyl steryl glycosides. We have analyzed the functional role of TTG15/UGT80B1 gene of Arabidopsis thaliana in freeze/thaw and heat shock stress using T-DNA insertional sgt knockout mutants. Quantitative study of spatial as well as temporal gene expression showed tissue-specific and dynamic expression patterns throughout the growth stages. Comparative responses of Col-0, TTG15/UGT80B1 knockout mutant and p35S:TTG15/UGT80B1 restored lines were analyzed under heat and freeze stress conditions. Heat tolerance was determined by survival of plants at 42°C for 3 h, MDA analysis and chlorophyll fluorescence image (CFI) analysis. Freezing tolerance was determined by survival of the plants at -1°C temperature in non-acclimatized (NA) and cold acclimatized (CA) conditions and also by CFI analysis, which revealed that, p35S:TTG15/UGT80B1 restored plants were more adapted to freeze stress than TTG15/UGT80B1 knockout mutant under CA condition. HPLC analysis of the plants showed reduced sterol glycoside in mutant seedlings as compared to other genotypes. Following CA condition, both β-sitosterol and sitosterol glycoside quantity was more in Col-0 and p35S:TTG15/UGT80B1 restored lines, whereas it was significantly less in TTG15/UGT80B1 knockout mutants. From these results, it may be concluded that due to low content of free sterols and sterol glycosides, the physiology of mutant plants was more affected during both, the chilling and heat stress.     

Dimorphism-associated variations in the lipid composition of Candida albicans

Yeast and mycelial forms of Candida albicans ATCC 10231, growing together in 12 h and in 96 h cultures, were separated and their lipids were extracted and characterized. The total lipid content of the yeast forms was always lower than that of the mycelial forms. In 12 h cultures the lipids from the two morphological forms consisted mainly of polar compounds, viz, phospholipids and glycolipids. In 96 h cultures both the yeast and mycelial forms accumulated substantial amounts of apolar compounds, mainly steryl esters and triacylglycerols. The mycelial forms were more active than the yeast forms in this respect. Major differences in the lipid composition between the two morphological forms involved the contents of sterols and complex lipids that contain sterols. As a rule, the yeast lipids contained much larger proportions of free sterols than the mycelial lipids. However, the mycelial lipids contained several times more sterols than the yeast forms but bound as steryl glycosides, esterified steryl glycosides and steryl esters. Steryl glycosides and esterified steryl glycosides occurred in yeast lipids only in traces, if at all. The major steryl glycoside in the mycelial forms was unequivocally identified as cholesteryl mannoside. At both phases of growth the apolar and polar lipid fractions from the mycelial forms contained higher levels of polyunsaturated fatty acids (18:2 and 18:3) but lower levels of oleic acid (18:1) than the corresponding fractions from the yeast forms. The lipid content and composition of 12 h and 96 h yeast and mycelial forms of C. albicans KCCC 14172, a clinical isolate, were almost identical with those of C. albicans ATCC 10231.