Enzymatic Conversion of 1-Hydroxy-2-naphthoate in Phenanthrene-grown Aeromonas sp. S45P1

Soybean seedlings were grown at 28°C in the dark or the light for 12 days, and four classes of sterol lipids, sterol esters (SE), free sterols (St), acylated steryl glycosides (ASG) and steryl glycosides (SG), were isolated from the cotyledons by solvent extractions, Florisil column chromatography, and thin-layer chromatography (TLC), successively. Each sterol lipid (SE, ASG and SG) obtained was hydrolyzed and then separately divided into sterol, fatty acid and/ or sugar fractions. The hydrolysates and St were analyzed mainly by gas-liquid chromatography (GLC).

Under the two conditions tested, the main sterol lipid class was St during germination, the minor one being SG. With the progress of germination, St and ASG decreased under both conditions tested, whereas SE and SG increased, especially SE in the light-grown seedlings. The changing patterns of sterol and sugar compositions of ASG resembled those of SG, but those of fatty acid composition differed between SE and ASG. In general, the changes in fatty acid compositions of SE and ASG were more marked in the light-grown seedlings than in the dark-grown ones.     

Biosynthesis and metabolism of steryl glycosides in Sinapis alba seedlings

With ¹⁴CO₂, d-glucose-[U-¹⁴C] and dl-mevalonate-[4R-4-³H₁] used as precursors, a study was made of the labelling dynamics of the steryl glucosides (SG) and steryl acylglucosides (ASG) in Sinapis alba seedlings. The radioactivity of the sterol and sugar moieties, as well as of the fatty acid moieties in the case of ASG, was analysed separately. The course of incorporation of ¹⁴C from ¹⁴ CO₂ and glucose-[U-¹⁴C] into the sugar part of SG and ASG indicated that about $\text{2}\text{3}$ of the whole pool of the newly synthesized sterol glycosides of both types underwent rapid deglucosylation. Likewise, fatty acids in the ASG pool were rapidly exchanged. The present results point to a high metabolic activity of the sterol glycoside derivatives in plant cells.     

Concentration gradients of free sterols,steryl esters and lipid phosphorus in the trunkwood of Scot's pine (Pinus sylvestris L.)

The amounts of free sterols, steryl esters and lipid phosphorus were determined in the sapwood and heartwood of mature, and in the outer and inner sapwood of young Pinus sylvestris trees. In the mature trees (up to 70 years old) the heartwood contains significantly higher amounts of free sterols than the sapwood. No radial gradient can be demonstrated in the amounts of steryl esters. Lipids extracted from the sapwood contain higher amounts of phosphorus than those from the heartwood. Stems of young Pinus sylvestris trees (up to 13 years old) show in the inner sapwood higher amounts of both free sterols and steryl esters than the peripheral younger wood zone. The inner sapwood of the young stems shows slightly higher amounts of lipid phosphorus than the outer sapwood. The results indicate that Pinus sylvestris accumulates both free sterols and steryl esters in the stems at a very early stage of the life cycle. Sterol accumulation in the innermost parts of the stems seems not to depend on heartwood formation.     

Concept of sequential analysis of free and conjugated phytosterols in different plant matrices

A unique concept and method for the determination of the total plant sterol content as sum of free sterols (FS), steryl esters (SE), steryl glycosides (SG) and acylated steryl glycosides (ASG) in different plant materials (pumpkin seeds, lecithins) and phytopharmaceuticals derived thereof, was developed. For this purpose, a multidimensional sample clean-up protocol based on efficient solid-phase extraction materials was elaborated and the SG were isolated employing a novel phenyl boronic acid modified silica gel material. Along this line also a set of steryl glucosides was synthesised and employed as internal standard and for calibration in the course of quantitative analysis. Final quantification of SG was carried out with reversed-phase HPLC in combination with evaporative light scattering detection (ELSD); the ASG were determined after conversion to SG by mild alkaline hydrolysis. In order to determine the total plant sterol profile the sum of FS and SE was additionally analysed from the unsaponifiable lipid fraction by GC-FID. The yields obtained from recovery tests for the determination of SG using soya lecithin as matrix to which 2, 20 and 40 mg/g of cholesterol-beta-D-glucoside was added were 99.10, 98.07 and 90.00%, and the RSDs were 4.11, 2.62 and 4.50%, respectively. Application related to the qualitative and quantitative analysis of total phytosterol profiles in different plant matrices and extracts demonstrate the validity of the method.     

Sterol conjugate interconversions during germination of white mustard (Sinapis alba)

Changes in the content of free sterols (FS), steryl esters (SE), steryl glucosides (SG) and acylated steryl glucosides (ASG) in germinating seeds of white mustard (Sinapis alba) were studied together with parrallel changes in specific activities of some enzymes involved in sterol conjugate transformation. It has been found that a distinct increase in the net SE content and a similar, but less pronounced, increase in SG content at the beginning of germination can be correlated with a distinctly earlier appearance of SE and SG synthesizing enzymes, i.e. triacylglycerol: sterol acyltransferase and UDPG: sterol glucosyltransferase in comparison with hydrolytic activities, i.e. SE hydrolase and SG hydrolase. Our results suggest that metabolism of SG and ASG takes place mainly in the cotyledons while SE metabolism takes place mainly in the roots.     

Variation in the amount of triacylglycerols and steryl esters in the outer sapwood of Pinus sylvestris L.

Variation in the amount of triacylglycerols and steryl esters was analysed enzymatically in the outer sapwood of Scots pine (Pinus sylvestris L.). Increment borings were taken at breast height from 40 stems of different diameter. Wood samples from outer sapwood (10 mm from the cambium) were extracted with acetone. Triacylglycerols and steryl esters were separated on TLC and the levels of glycerol and sterol were analysed enzymatically. The average amount of triacylglycerols and steryl esters was approximately 25 and 0.83 mg/g dry weight, respectively. However, variation between the stems studied was fairly large. The sapwood of young and small-diameter stems was not found to store larger amounts of either triacylglycerols or steryl esters than the sapwood of old stems. Neither was there any relationship between the amount of triacylglycerols and steryl esters in the outer sapwood. The possible role of these compounds in heartwood formation is discussed.     

Occurrence of steryl glycosides and acylated steryl glycosides in some marine algae

There is some controversy concerning the presence of steryl glycosides and acylated steryl glycosides in eucaryotic algae. These two classes of sterol compounds were investigated in species belonging to the three major groups of eucaryotic algae: green algae (Ulva gigantea, Cladophora rupestris), brown algae (Fucus vesiculosus, Ascophyllum nodosum), and red algae (Rhodymenia palmata, Porphyridium sp.). All these algae contain both steryl glycosides and acylated steryl glycosides. The sterol components of these compounds vary according to the alga but they are always the same as the free sterols of the alga in question. The most common sugar moiety is glucose. In the acylated steryl glycosides, the fatty acid is mainly palmitic acid. The percentage of these compounds (as a percentage of the total sterol content) is often low.     

Modulation of proton pumping across proteoliposome membranes reconstituted with tonoplast H(+)-ATPase from cultured rice (Oryza sativa L. var. Boro) cells by acyl steryl glucoside and steryl glucoside

Tonoplast H(+)-ATPase purified from cultured rice cells (Oryza sativa L. var. Boro) was reconstituted into asolectin liposomes containing steryl glucoside (SG) or acyl steryl glucoside (ASG), and the effects of SG and ASG on proton pumping, ATP-hydrolysis activity and proton permeability of the proteoliposome membranes were investigated. In the proteoliposomes containing 10 mol% SG, proton pumping and ATP-hydrolysis activity were increased to around 140% of those in SG-free proteoliposomes. In the proteoliposomes containing ASG, proton pumping and ATP-hydrolysis activity were decreased to one-tenth of those in ASG-free proteoliposomes at 15 mol% ASG; however, activity increased again slightly in the range between 20 and 40 mol% ASG. The change in proton pumping across the proteoliposome membrane is not due to a change of proteoliposome size nor to the location of the catalytic site of the tonoplast H(+)-ATPase in the proteoliposomes. SG and ASG also reduced the passive proton permeability of the proteoliposomes. These results show that SG and ASG modulate proton pumping across the tonoplast toward stimulation and depression, respectively, and they reduce the passive proton permeability of the tonoplast.     

Ripening, storage temperature, ethylene action, and oxidative stress alter apple peel phytosterol metabolism

The chilling conditions of apple cold storage can provoke an economically significant necrotic peel disorder called superficial scald (scald) in susceptible cultivars. Disorder development can be reduced by inhibiting ethylene action or oxidative stress as well as intermittent warming. It was previously demonstrated that scald is preceded by a metabolomic shift that results in altered levels of various classes of triterpenoids, including metabolites with mass spectral features similar to β-sitosterol. In this study, a key class of phytosterol metabolites was identified. Changes in peel tissue levels of conjugates of β-sitosterol and campesterol, including acylated steryl glycosides (ASG), steryl glycosides (SG) and steryl esters (SE), as well as free sterols (FS), were determined during the period of scald development. Responses to pre-storage treatment with the ethylene action inhibitor, 1-methylcyclopropene, or an antioxidant (diphenylamine), rapid temperature elevation, and cold acclimation using intermittent warming treatments were evaluated. Diphenylamine, 1-MCP, and intermittent warming all reduced or prevented scald development. ASG levels increased and SE levels decreased in untreated control fruit during storage. Removing fruit from cold storage to ambient temperature induced rapid shifts in ASG and SE fatty acyl moieties from unsaturated to saturated. FS and SG levels remained relatively stable during storage but SG levels increased following a temperature increase after storage. ASG, SE, and SG levels did not increase during 6 months cold storage in fruit subjected to intermittent warming treatment. Overall, the results show that apple peel phytosteryl conjugate metabolism is influenced by storage duration, oxidative stress, ethylene action/ripening, and storage temperature.     

Lipids and sterols of Pinus sylvestris L. sapwood and heartwood

Summary The lipid and sterol content and composition of three lipid fractions (free fatty acids/ sterols, triacylglycerols and sterol/triterpenoid esters) extracted from three stem discs of Pinus sylvestris were assessed to investigate metabolic changes related to heartwood formation. The wood was separated into (1) cambial zone, (2) outer sapwood, (3) inner sapwood, (4) transition zone, (5) outer heartwood and 6) inner heart-wood. The fractions were separated by thin-layer chromatography (TLC) and analysed by gas-liquid chromatography (GLC). The amount of fatty acids of sapwood triacylglycerols was about 1.5% (dry wt.) but a large reduction occurred in the transition zone. In contrast, noticeable amounts of free fatty acids were present only in the heart-wood. The most important fatty acids in the sapwood fractions were 16:0, 18:0, 18:1, 18:2 (the dominant fatty acid in all fractions), 18:3 and 20:3. Together 18:1 and 18:2 formed about 70% of the total triacylglycerol fatty acids. Of the sterol/ triterpenoid esters, 18:2 and 18:3 were predominant. The fatty acid composition of all fractions changed in the transition zone. The sterols found were sitosterol, stigmastanol, campesterol and campestanol. The amount of sterol esters increased towards the heartwood, and the amount of free sterols was lowest in the inner sapwood. Sitosterol was the dominant sterol in both free sterols and sterol esters.     

Steryl glucoside biosynthesis in the alga Prototheca zopfii

A particulate enzyme fraction from the Chlorophyta Prototheca zopfii catalysed the transfer of glucose-[U-¹⁴C]from UDP-Glc-[U-¹⁴C] to endogenous sterol acceptors and the esterification of steryl glucosides with fatty acids from an endogenous acyl donor. Glucose was the only sugar present, and it appeared to have the β-configuration. In the acylated derivatives the glucose-acyl linkage appeared in the C-6 position of glucose, as indicated by periodate oxidation. UDP-Glc:sterol glucosyltransferase was solubilized with detergent and purified 34-fold. The solubilized enzyme showed no specificity for the sterol but a high affinity for the sugar nucleotide UDP-Glc. Time-course incorporation into steryl glucoside (SG) and the acylderivative (ASG) indicated that SG was the precursor of ASG and that phosphatidyl ethanolamine stimulated the formation of the latter compound, presumably acting as acyl donor. A high sterol glucosylating activity was found in the Golgirich fraction. All this evidence indicates that steryl glucosides and their acylated derivatives were synthesized by algae. The early assumption that these compounds were not present in algae must be revised.     

Wheat leaf lipids during heat stress: I. High day and night temperatures result in major lipid alterations

Understanding how wheat (Triticum aestivum L.) plants under high temperature (HT) regulate lipid composition is critical to developing climate‐resilient varieties. We measured 165 glycerolipids and sterol derivatives under optimum and high day and night temperatures in wheat leaves using electrospray ionization‐tandem mass spectrometry. Levels of polar lipid fatty acyl chain unsaturation were lower in both heat‐tolerant genotype Ventnor and susceptible genotype Karl 92 under HT, compared with optimum temperature. The lower unsaturation was predominantly because of lower levels of 18:3 acyl chains and higher levels of 18:1 and 16:0 acyl chains. Levels of 18:3‐containing triacylglycerols increased threefold/more under HT, consistent with their possible role in sequestering fatty acids during membrane lipid remodelling. Phospholipids containing odd‐numbered or oxidized acyl chains accumulated in leaves under HT. Sterol glycosides (SG) and 16:0‐acylated sterol glycosides (ASG) were higher under HT than optimum temperatures. Ventnor had lower amounts of phospholipids with oxidized acyl chains under HT and higher amounts of SG and 16:0‐ASG than Karl 92. Taken together, the data demonstrate that wheat leaf lipid composition is altered by HT, in which some lipids are particularly responsive to HT, and that two wheat genotypes, chosen for their differing physiological responses to HT, differ in lipid profile under HT.     

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.     

EDU and ozone protection: Foliar glycerolipids and steryl lipids in snapbean exposed to O3

Effects of the antiozonant EDU, N-[2-(2-oxo-1-imidazolidinyl) ethyl]-N'-phenylurea, on the content and composition of foliar lipids in snapbean ( Phaseolus vulgaris L. cv. Bush Blue Lake 290) before and after a single, acute ozone (O₃) exposure were assessed. Pretreatment with EDU conferred protection against O₃-induced necrosis and losses of glycerolipids and chlorophyll. Systemic treatment of snapbean plants with EDU did not significantly alter membrane lipids in the first trifoliate leaf. Leaves of untreated controls had lost ca 50% of both galacto- (GL) and phospholipids (PL) by the end of a 3 h exposure to 0.4 μl l⁻¹ O₃. A decline in the ratio of mono- to di-galactosyldiacylglycerol (MGDG/DGDG) was associated with the loss of GL, and a decline in the ratio of linoleic to linolenic acid (18:2/18:3) was associated with the loss of PL in untreated controls. EDU-treated plants showed no significant loss of foliar GL and PL. The MGDG/DGDG ratio declined only slightly, and the 18:2/18:3 ratio in PL increased during O₃ exposure of EDU-treated seedlings. The level of total membrane sterols, including free sterols (FS), acylated steryl glycosides (ASG) and steryl glycosides (SG), did not change during O₃ exposure of either treated or untreated plants. However, in the controls the proportions of ASG and SG increased at the expense of FS, and the ratio of stigmasterol/sitolsterol increased in FS and SG. In EDU-treated plants, a relatively small increase in SG was offset by a decrease in FS, and there was no change in the stigmasterol/sitosterol ratio in ASG, SG or FS. The results indicate that EDU may confer tolerance to O₃ through induction of enzyme systems involved in the elimination of activated oxygen species and free radicals.     

Soluble carbohydrates of Pinus sylvestris L. sapwood and heartwood

Summary The amounts of glucose, fructose, sucrose, arabinose/galactose, raffinose/stachyose and starch were investigated in the outer sapwood, innermost sapwood, transition zone and heartwood of four stems of Pinus sylvestris L. The samples were taken in October and the determination of the compounds was done enzymatically. It was not possible to distinguish arabinose from galactose and raffinose from stachyose. The amounts of glucose, fructose and sucrose were greatest in the outer sapwood and decreased gradually towards the innermost sapwood and the heartwood. In the outermost heartwood glucose, fructose and sucrose were only present in trace amounts. Raffinose/stachyose showed highest concentrations in the outer sapwood and decreased towards the heartwood. In contrast, the concentrations of arabinose/galactose increased towards the heartwood and the greatest amount was found in the inner heartwood. When identified by thin-layer chromatography (TLC), arabinose was found to be present in greater amounts than galactose. The amount of starch decreased markedly towards heartwood. However, the amounts of sugars in all the studied stems was very variable. The changes in the amounts of carbohydrates in the different zones of the stems and the possible relationships of these phenomena with heartwood formation are discussed.     

Sterol glucosylation by the plasma membrane from cotyledons of Phaseolus aureus

Membrane fractions were isolated from dark grown cotyledons of Phaseolus auneus by differential and sucrose density gradient centrifugation. Endoplasmic reticulum-, Golgi apparatus- and plasma membrane-rich fractions were identified by their respective enzymic activities and tested for their ability to transfer glucose from UDP-glucose to endogenous sterols to form steryl glucosides. The glucosyltransferase activity was shown to be located mainly at the plasma membrane.ABBREVIATIONS SG steryl glucoside - ASG acylated steryl glucoside - UDP-glc Uridine diphosphoglucose     

Steryl glucoside and acyl steryl glucoside formation in the amyloplast membrane during the development of potato tuber

Steryl glucoside (SG) and acylated steryl glucoside (ASG) synthesis was investigated in amyloplast membranes from young, intermediate and mature potato tubers. The synthesis ratio SG/ASG was lowest in young tubers (3:2) and highest in mature tubers (6:1).About a 3-fold stimulation of [¹⁴C] glucose incorporation into a mixture of SG was observed in amyloplast membranes from mature tubers in the presence of β-sitosterol, while radioactivity incorporation in young tubers was unaffected, thus indicating that different availabilities of endogenous acceptors occur in the membranes.The enzymes involved in sterol modification exhibit a different behavior towards Triton X-100, depending on the developmental stage of the tubers. Low concentrations of the detergent (0.045%) are required to stimulate the enzymes present in young tuber membranes (2-fold). On the other,hand, 0.15% of Triton increased the enzymatic activity in mature tubers 5-fold. These results, together with those obtained after studies of pH dependence, could be related to the lipidic structure of the vesicles formed at different developmental stages of the tubers.It is concluded that the major changes in the enzymatic activities occur as a consequence of the sterol acceptors and acyl donor content during potato tuber growth.     

Lipids and lipolytic enzymes in the trunkwood ofRobinia pseudoacacia L. during heartwood formation

The radial distribution of membrane and storage lipids was determined in the trunkwood ofRobinia pseudoacacia L. The trees were felled in November at the time of heartwood formation and fluctuations in the amount and composition of phospholipids, free sterols, steryl esters, diand triacylglycerols, and free fatty acids were investigated across the sapwood-heartwood boundary. The individual compounds were identified and quantified by thin layer chromatography, enzymatic and colorimetric assays, and by capillary gas chromatography. Phospholipids show a significant decrease towards the boundary area, and in the heartwood only trace amounts can be detected. The same pattern is observed for free sterols in the sapwood; in the heartwood, however, they reach maximum values with increasing depth of the trunk. Steryl esters exhibit a complementary behaviour by accumulating at the periphery of the heartwood. No concentration changes are found in the total amounts of diacylglycerols and free fatty acids. In contrast, the triacylglycerol concentration declines steadily across the trunk. With regard to qualitative composition, free fatty acids and the fatty acid moieties of the esterified constituents vary in their chain length from 14 to 24 carbon atoms and have up to three double bonds. A radial gradient in the ratio saturated/unsaturated fatty acids can be observed: except for the phospholipid fraction the relative amounts of unsaturated fatty acids increase in centripetal direction. Seven phospholipids were identified: phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, diphosphatidylglycerol, and phosphatidic acid, which constitutes the major proportion. In the sterol group, sitosterol is the most abundant component. The composition of the esterified sterols remains constant across the trunk cross-section, whereas the relative frequencies of individual free sterols change markedly.     

Effects of tetcyclacis on growth and on sterol and sapogenin content in fenugreek

Tetcyclacis, a norbornanodiazetine plant growth retardant, used at 10 mg · L^–1 (36 m), caused greater growth inhibition in the shoots of fenugreek (Trigonella foenum-graecum L.) seedlings (60%) than in the roots (30%), compared with control. This greater retardation was reversed by a supplement of gibberellin (200 mg · L^–1). The total sterol composition of control and treated seedlings was analyzed and quantified. In the roots especially, treatment of seedlings with tetcyclacis resulted in a modification of the sterol profile, leading to an accumulation of 14-methyl sterols, presumably as a consequence of the inhibition of cytochrome P-450-dependent obtusifoliol 14-demethylase. In addition, tetcyclacis caused a significant increase in the cholesterol content of the roots: 38.1% of total sterols against 3.7% in the control roots. However, tetcyclacis was shown to be an ineffective inhibitor of the S-adenosyl-l-methionine (Adomet): cycloartenol-C24-methyltransferase (EC 2.1.1.41) in fenugreek microsomes indicating that cholesterol accumulation does not result from the inhibition of the sterol side chain-alkylating enzyme. Moreover, this accumulation was shown to be concomitant with a significant decrease of the sapogenin content in the treated roots. This last result is discussed with respect to the current proposed pathway by which cholesterol is metabolized to saponins.Abbreviations GA gibberellin - PGR plant growth regulator - GA₃ gibberellin acid - FW fresh weight(s) - DW dry weight(s) - Adomet-CMT S-adenosylmethionine-cycloartenol-C24-methyltransferase - GC gas chromatography - TLC thin layer chromatography - MS mass spectroscopy - FS free sterol(s) - SE steryl ester(s) - SG steryl glycoside(s) - ASG acylated steryl glycosides - SAM S-adenosylmethionine     

Sterols in relation to ageing of seeds of Helianthus annuus and Cicer arietinum

Under accelerated ageing at high relative humidity and high temperature for 4 days germination and membrane permeability remained unaffected both in sunflower and chick pea seeds. However, the steryl glycoside concentration in the pooled leachate increased progressively with ageing. Total sterols, as well as steryl glycosides and free sterols of the seeds, increased with a concomitant decline in steryl esters under accelerated ageing. Pretreatment with the sterol biosynthesis inhibitor SK & F 7997A₃ prevented the increase of total sterols under accelerated ageing conditions but there were increases in the amounts of steryl glycosides and free sterols and a decrease in steryl ester after such treatment, therefore, indicating interconversions of the various sterol types. Accelerated ageing also caused increases in free amino acids and soluble carbohydrate. Low relative humidity-high temperature and high relative humidity-low temperature failed to produce such effects.