Membrane lipid changes in root cells of rape (Brassica napus) as a function of water-deficit stress

Rape seedlings ( Brassica napus L. cv. Brink) were exposed to repeated water-deficit stress. The water-stress program started after 19 days of growth and consisted of three 24 h stress periods interspersed with 24 h rewatering periods. After the third stress period the seedlings were harvested and the membrane lipids of the roots were extracted, isolated and quantified. The stress caused an increased ratio of dry weight roots/shoot. Furthermore, the total amount of acyl lipids as well as phospholipids decreased drastically. However, the relative distribution of individual phospholipids was constant and independent of stress. Free and esterified sterols showed only a small decrease in response to water stress. As a consequence the ratio free sterols/phospholipids increased from 0.07 in the control root cells to 0.15 in the stressed cells. The lipid changes are discussed in relation to membrane activity.     

Sterols in Germinating Seeds and Developing seedlings of Longleaf Pine, Pinus palustris

Sterols in germinating embryos and young seedlings of longleaf pine (Pinus palustris Mill.) were identified and quantities determined for different periods after germination. Sterol analyses were performed by gas-liquid chromatography (GLC) and verified by combination of GLC-mass spectrometry. Campesterol and β-sitosterol were two major sterols which accounted for most of the sterol composition while stigmasterol was present in very small amounts. No cholesterol was revealed by GLC-mass spectrometry although there was a minor peak appearing on the sterol gas-liquid chromatograms with a retention time close to that of authentic cholesterol. By fractionation, three different forms of sterols were obtained: steryl esters, steryl glycosides, and free sterols. The sterols were mainly found in the esterified fraction, while steryl glycosides and free sterols only made up a small portion of the total sterol value. The total sterol content in general increased during seedling development, and this increase reflected mainly a change in steryl esters. The low levels of both free and glycosidic sterols remained nearly unchanged throughout the experimental germination period.     

STEROLIC BIOMARKERS IN MARINE PHYTOPLANKTON. II. FREE AND CONJUGATED STEROLS OF SEVEN SPECIES USED IN MARICULTURE

The sterols of seven unicellular algae widely used in mariculture and belonging to different classes (Prasinophyceae, Haptophyceae, Eustigmatophyceae, and Diatomophyceae) were examined for free and combined forms. Under standard culture conditions, all synthesized free sterols and combined forms (steryl esters, acyl steryl glycosides, and steryl glycosides). Free sterols were dominant in five of the species. In contrast, sterols were mostly esterified in the eustigmatophyte Nannochloropsis oculata (Droop) Hibberd and the diatom Thalassiosira pseudonana Hasle et Heimdal, whereas in another diatom, Chaetoceros calcitrans (Paulsen) Takano, glycosylated forms represented over 60% of total sterols. The pennate diatom Haslea ostrearia (Gaillon) Simonsen synthesized large amounts of steryl glycosides consisting mainly of an unusual sterol, 23,24-dimethylcholest-5-en-3β-ol, occurring in some dinoflagellates.     

Lipids of plasma membranes prepared from oat root cells : effects of induced water-deficit tolerance

Plasma membranes were isolated from oat (Avena sativa) roots by the phase-partitioning method. The membranes were exposed to repeated periods of moderate water-deficit stress, and a water-deficit tolerance was induced (acclimated plants). The plasma membranes of the controls (nonacclimated plants) were characterized by a high phospholipid content, 79% of total lipids, cerebrosides (9%) containing hydroxy fatty acids (>90% 24:1-OH) and free sterols, acylated sterylglucosides, sterylglucosides, and steryl esters, together amounting to 12%. Major phospholipids were phosphatidylcholine and phosphatidylethanolamine with lesser amounts of phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid. After the membranes were acclimated to dehydration, the lipid to protein ratio decreased from 1.3 to 0.7 micromoles per milligram. Furthermore, the cerebrosides decreased to 5% and free sterols increased from 9% (nonacclimated plants) to 14%. Because the total phospholipids did not change significantly, the free sterol to phospholipid ratio increased from 0.12 to 0.19. There was no change in the relative distribution of sterols after acclimation. The ratio of phosphatidylcholine to phosphatidylethanolamine changed from 1.1 in the nonacclimated plants to 0.69 in the acclimated plants. The results show that acclimation to dehydration implies substantial alterations in the lipid composition of the plasma membrane.     

Plant sterols: Diversity,biosynthesis, and physiological functions

Sterols, which are isoprenoid derivatives, are structural components of biological membranes. Special attention is now being given not only to their structure and function, but also to their regulatory roles in plants. Plant sterols have diverse composition; they exist as free sterols, sterol esters with higher fatty acids, sterol glycosides, and acylsterol glycosides, which are absent in animal cells. This diversity of types of phytosterols determines a wide spectrum of functions they play in plant life. Sterols are precursors of a group of plant hormones, the brassinosteroids, which regulate plant growth and development. Furthermore, sterols participate in transmembrane signal transduction by forming lipid microdomains. The predominant sterols in plants are β-sitosterol, campesterol, and stigmasterol. These sterols differ in the presence of a methyl or an ethyl group in the side chain at the 24th carbon atom and are named methylsterols or ethylsterols, respectively. The balance between 24-methylsterols and 24-ethylsterols is specific for individual plant species. The present review focuses on the key stages of plant sterol biosynthesis that determine the ratios between the different types of sterols, and the crosstalk between the sterol and sphingolipid pathways. The main enzymes involved in plant sterol biosynthesis are 3-hydroxy-3methylglutaryl-CoA reductase, C24-sterol methyltransferase, and C22-sterol desaturase. These enzymes are responsible for maintaining the optimal balance between sterols. Regulation of the ratios between the different types of sterols and sterols/sphingolipids can be of crucial importance in the responses of plants to stresses.     

Changes in cuticular transpiration rate and cuticular lipids of oat (Avena sativa) seedlings induced by water stress

Two cultivars of oat ( Avena sativa L. cvs Pendek and Stormogul II) were exposed to short periods of water-deficit stress on five consecutive days. The plants responded to the stress by decreasing their cuticular transpiration rate. After two stress periods the cuticular transpiration rate was reduced by 30% for Pendek and by 47% for Stormogul II, and after another three stress periods by 30% and 20%, respectively. These reductions were correlated neither to changes in the total amount of what is generally called epicuticular lipids, nor to changes in any of the major individual constituents of the epicuticular lipids (alkanes, free and esterified fatty acids or free primary alcohols). After removal of the epicuticular lipids the long chain free primary alcohols of the leaves were extracted and determined. The amount of these presumably intracuticular alcohols increased after stress and changed to shorter chain length. From these results it is concluded that the intra- as well as the epicuticular lipids must be taken into consideration when discussing leaf surface lipids as protecting agents against water loss.     

24-methylenepollinastanol and 24-dehydropollinastanol—two sterols of pollen

The esterified and unesterified sterol fractions of bee-gathered mixed pollens were examined, and total sterol composition was determined. Two new sterols of pollens, 14α-methyl-9β,19-cyclo-5α-cholest-24-en-3β-ol (24-dehydropollinastanol) and 14α-methyl-5α-ergost-24(28)-en-3β-ol (24-methylenepollinastanol) were isolated and identified. Both sterols were found primarily in the esterified sterol fraction, and 24-methylenepollinastanol accounted for 43% of the sterols of this fraction. 24-Dehydropollinastanol and four other sterols which also contain a 9β,19-cyclopropane ring were found only in the esterified sterol fraction. 24-Methylenecholesterol was the major sterol of the unesterified sterol fraction.     

Changes in the levels and composition of sterols in different tissues of Lolium temulentum plants during floral development

Free, esterified and glycosylated sterols were analysed separately from the shoot apices, leaves, leaf sheaths and stems of Lolium temulentum L. (strain Ceres) plants during floral development. Short-day grown plants (50 days old) were induced to flower by exposure to a single long day. The four major sterols found by GC-MS analysis were sitosterol, cholesterol, campesterol and stigmasterol. The sterol levels in the shoot apex were much higher than those in the leaf, leaf sheath and stem. A much greater proportion of cholesterol was found in the shoot apex than in other tissues and this may reflect a specific association of cholesterol with meristematic and/or reproductive tissues.
During the inductive treatment, the sterol levels decreased in all four tissues. The major effect during early differentiation was the occurrence of transient increases in the free and esterified sterol levels in the leaf and the stem tissues. The steryl ester content peaked 24 h before the appearance of double ridges, followed by a peak in free sterol content at the double ridge stage. Similar changes could not be detected in the shoot apices. This is the first report of the sterol composition of developing shoot apices, and the results emphasize the dynamic nature of sterol metabolism during reproductive growth of L. temulentum.     

Sterol Composition of the Corn Cyst Nematode,Heterodera zeae,and Corn Roots

Sterols from free sterol and steryl ester fractions from Heterodera zeae and from total lipids of Zea mays roots were analyzed by gas-liquid chromatography (GLC) and by GLC-mass spectrometry. The major free sterols of H. zeae were 24-ethylcholesterol (54.4% of total free sterol), 24-ethylcholesta-5,22-dien-3β-ol (13.3%), 24-methylcholesterol (12.5%), and cholesterol (7.2%). The same four sterols comprised 34.6%, 7.2%, 30.3%, and 18.6%, respectively, of the esterified sterols of H. zeae. Corn root sterols included 46.6% 24-ethylcholesta-5,22-dien-3β-ol, 16.7% methylcholesterol, 16.4% cycloartenol, 12.7% 24-ethylcholesterol, and 0.5% cholesterol. The sterol 24-composition of H. zeae differed greatly from that of the only other cyst nematode previously investigated, Globodera solanacearum.     

Sterol Changes during Germination of Nicotiana tabacum Seeds

The identity, composition, and concentration of the total, free, esterified, and glycosidic sterol fractions were determined during germination of tobacco seeds. The total, free, and esterified sterols increased, with stigmasterol and campesterol accounting for most of the increase. Steryl glycosides decreased during germination, and stigmasteryl and sitosteryl glycosides showed the largest decrease. During germination, sitosterol was the major sterol in all fractions but stigmasterol and campesterol showed the greatest changes. The fatty acid composition of the steryl esters and acylated steryl glycosides most closely resembled the di- and triglycerides.     

The biosynthesis of the free sterols and sterol esters of Neurospora crassa.

The composition of the free and esterified sterols in Neurospora crassa was examined as a function of incubation time in starvation medium containing [2-14C]mevalonic acid. The 14C incorporation was monitored in nuclear methylated and 4,14-desmethyl sterol fractions. After 7 h incubation, sterol esterification had increased from an initial 5% in the log phase culture to 48% of the total sterol pool, with a concomitant decrease in free sterols. The relationship of the free and esterified sterol components in ergosterol biosynthesis is discussed.     

Metabolic interconversion of free sterols and steryl esters in Saccharomyces cerevisiae.

The interconversion of free and esterified sterols was followed radioisotopically with [U-14C]acetate and [methyl-14C]methionine. In pulse-chase experiments, radioactivity first appeared mainly in unesterified sterols in exponential-phase cells. Within one generation time, the label equilibrated between the free and esterified sterol pools and subsequently accumulated in steryl esters in stationary-phase cells. When the sterol pools were prelabeled by growing cells aerobically to the stationary phase and the cells were diluted into unlabeled medium, the prelabeled steryl esters returned to the free sterol form under several conditions. (i) During aerobic growth, the prelabeled sterols decreased from 80% to 45% esters in the early exponential phase and then returned to 80% esters as the culture reached the stationary phase. (ii) Under anaerobic conditions, the percentage of prelabeled steryl esters declined continuously. When growth stopped, only 15% of the sterols remained esterified. (iii) In the presence of an inhibitor of sterol biosynthesis, which causes accumulation of a precursor to ergosterol, prelabeled sterols decreased to 40% steryl esters while the precursor was found preferentially in the esterified form. These results indicate that the bulk of the free sterol and steryl ester pools are freely interconvertible, with the steryl esters serving as a supply of free sterols. Furthermore, there is an active cellular control over what types of sterol are found in the free and esterified sterol pools.     

Total, free and conjugated sterolic forms in three microalgae used in mariculture

Total, free and conjugated forms (steryl esters, steryl glycosides and acyl steryl glycosides) of sterols from three microalgae that are extensively used in mariculture (Tetraselmis chuii, Nannochloropsis salina and Skeletonema costatum) were examined. The results revealed that cholesterol is the only common fraction detected in all investigated species and distributed in free and all conjugated forms. However, the total sterol content of T. chuii was about 325 microg/g dry wt, most of it was concentrated amongst 24-methylcholesta-5,24-diene-3beta-ol and 24-methylcholest-5-en-3beta-ol. On the other hand, the majority of the fractions were distributed in the free form. The total sterol content of N. salina was about 180 microg/g dry wt, cholesterol was the major fraction that was detected. Nevertheless, the dominant distribution forms were esterified. While in S. costatum, the total sterol content was 76 microg/g dry wt, approximately most fractions are quantitatively alike and dominated in the free form. Furthermore, our study shows clearly that most sterols are not distributed regularly within each form, a result that encouraged us to suggest a distribution of specific sterol fraction as a free or conjugated can be used as a serving tool in chemotaxonomic studies.     

Sterol distribution in intracellular organelles isolated from tobacco leaves

All membrane-containing fractions isolated from tobacco leaves contained free sterols, sterol glycosides, and sterol esters. The three sterol forms increased, on a dry weight basis, with a decrease in particle size. The supernatant fraction contained only trace amounts of sterol. The major sterols in all cellular fractions, in the order of decreasing amounts, were: stigmasterol, β-sitosterol, campesterol, and cholesterol. The 500g pellet contained the largest percentage of free sterol, while the 46,000g pellet contained the largest percentage of esterified sterol. The individual sterol composition of the free sterol and sterol glycoside fraction was very similar; however, the composition of the sterol ester fraction varied widely among intracellular fraction. The intracellular distribution pattern of cholesterol-¹⁴C added to the isolation medium provided evidence that the intracellular sterol distribution pattern is not an artifact. These results support the suggestion that sterols in plant cells may have a physiological function associated with membranes.     

The sterols of growth and stationary phases of Aspergillus nidulans cultures

The accumulation of 4-desmethyl and 4,4-dimethyl sterols, as well as the triterpenoid beta-amyrin, was analysed during both exponential and stationary phases of Aspergillus nidulans growth. Throughout growth, the amount of 4-desmethyl sterol was proportional to the cellular dry weight, while the dimethyl sterols and beta-amyrin stopped accumulating after day 2. The sterols were found primarily as the free alcohol and not as fatty acid esters, the glycosides, or acyl glycosides. The amount of beta-amyrin in stationary phase cultures was affected by the concentrations of Mg2+ and Cu2+.     

Sterol Composition and Ecdysteroid Content of Eggs of the Root-knot Nematodes Meloidogyne incognita and M. arenaria

Free and esterified sterols of eggs of the root-knot nematodes Meloidogyne incognita races 2 and 3 and M. arenaria race 1 were isolated and identified by gas-liquid chromatography-mass spectrometry. The major sterols of eggs of each race were 24-ethylcholesterol (33.4-38.8% of total sterol), 24-ethylcholestanol (18.3-25.3%), 24-methylcholesterol (8.6-11.7%), 24-methylcholestanol (7.7-12.5%), and cholesterol (4.6-11.6%). Consequently, the major metabolic transformation performed by Meloidogyne females or eggs upon host sterols appeared to be saturation of the sterol nucleus. The free and esterified sterols of the same race did not differ appreciably, except for a slight enrichment of the steryl esters in cholesterol. Although the sterol composition of Meloidogyne eggs differed from that of other life stages of other genera of plant-parasitic nematodes, the three Meloidogyne races could not be distinguished from each other by their egg sterols. Ecdysteroids, compounds with hormonal function in insects, were not detected by radioimmunoassay in the Meloidogyne eggs either as free ecdysteroids or as polar conjugates.     

Annual Variation in the Sterol Content of Digitalis purpurea L. Seedlings

Seedings from a single lot of Digitalis purpurea L. seeds were germinated in batches over a period of 13 months. A total lipid extract was made which was resolved into esterified and unconjugated plus glycosylated sterol fractions. The amounts of sterol in each fraction and in the total were compared for seedlings germinated at different times of the year. The amount of esterified sterols reached a maximum value from March until June, and a low value from July until January. In January, a sharp increase began which lasted until March. Amounts of unconjugated and glycosylated sterols were elevated from March until June, low from July until October, and on the rise from November until March. These data correlate with an annual cycle in seed germination. The phase of maximum sterol content of seedlings is followed by a period of null germination.     

Variations of hepatic cholesterol precursors during altered flows of endogenous and exogenous squalene in the rat

Hepatic and serum levels of cholesterol precursors were analyzed in rats under basal (control) conditions and when cholesterol synthesis was activated by feeding 1% squalene or 5% cholestyramine. Exogenous squalene stimulated the activity of acyl-coenzyme A:cholesterol acyltransferase (ACAT) but strongly inhibited the activity of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase; cholestyramine did not affect ACAT but increased HMG-CoA reductase several-fold, indicating enhanced production of endogenous squalene. Activation of cholesterol synthesis by the two methods markedly increased the hepatic and serum contents of cholesterol precursor sterols. However, the sterol profiles were clearly different. Thus, exogenous squalene raised most significantly (up to 109-fold) free and esterified methyl sterols, and less so (up to 2-fold) demethylated C27 sterols (desmosterol and cholestenols) and also esterified cholesterol. Activation of endogenous squalene production by cholestyramine was associated with a depletion of esterified cholesterol and by a marked, up to 8-fold, increase of the free demethylated sterol precursor levels, whereas the increase of methyl sterols, up to 5-fold, was less conspicuous than during the squalene feeding. The changes were mostly insignificant for esterified sterols. The altered serum sterol profiles were quite similar to those in liver. Serum cholestenols and especially their portion of total serum precursor sterols were closely correlated with the hepatic activity of HMG-CoA reductase.     

Reduction of cholesterol and glycoalkaloid levels in transgenic potato plants by overexpression of a type 1 sterol methyltransferase cDNA

Transgenic potato (Solanum tuberosum cv Désirée) plants overexpressing a soybean (Glycine max) type 1 sterol methyltransferase (GmSMT1) cDNA were generated and used to study sterol biosynthesis in relation to the production of toxic glycoalkaloids. Transgenic plants displayed an increased total sterol level in both leaves and tubers, mainly due to increased levels of the 24-ethyl sterols isofucosterol and sitosterol. The higher total sterol level was due to increases in both free and esterified sterols. However, the level of free cholesterol, a nonalkylated sterol, was decreased. Associated with this was a decreased glycoalkaloid level in leaves and tubers, down to 41% and 63% of wild-type levels, respectively. The results show that glycoalkaloid biosynthesis can be down-regulated in transgenic potato plants by reducing the content of free nonalkylated sterols, and they support the view of cholesterol as a precursor in glycoalkaloid biosynthesis.     

Die quantitative verteilung der sterine und sterinderivate in organen von Solanum dulcamara

The following sterols were found in the roots, stems, leaves, unripe and ripe fruits of Solanum dulcamara: cholesterol, sitosterol, stigmasterol, campesterol, brassicasterol, isofucosterol and 24-methylenecholesterol. The most abundant components are cholesterol, sitosterol and stigmasterol (77–84%). In all parts of the plant the sterols are present in the free form and as esters, glycosides and acylated glycosides. The total sterol content and the content of combined forms were determined photometrically. In the leaves 58% of the sterols were found in the form of glycoside (26%), acylated glycoside (29%) and ester (2%). In the roots only 25% of the sterol were found in combined form. In the other organs the ratio of free and combined sterols was intermediate. In all cases, the ester fraction was the least.