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.     

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.     

Steryl glycosides and acylated steryl glycosides ofPinus sylvestris L. sapwood and heartwood

Summary The amounts of steryl glycosides (SG) and acylated steryl glycosides (ASG) were investigated in the sapwood, transition zone, inner heartwood and outer heartwood ofPinus sylvestris L. Only traces of both sterol derivates were present and their amounts decreased slightly towards the heartwood. The amount of SG decreased nearly to zero in the inner heartwood but the amount of ASG in the inner heartwood increased slightly. The suitability of enzymatic methods in SG and ASG hydrolysis, and sterol and glucose quantitative determinations, is discussed.     

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.     

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.     

Changes in Composition of Non-polar Lipids of the Axis and Root of Germinating Soybeans

Soybean seedlings were grown at 28°C under dark or light conditions for 12 days. Non-polar lipids (NPL) were separated by silicic acid column chromatography from total lipids in epicotyl containing young leaves, hypocotyl and root. The glyceride (TG, DG, and MG), free fatty acid (FFA) and sterol lipid (SE) components in NPL were analyzed mainly by thin-layer and gas-liquid chromatographies (TLC and GLC).

During germination, the amounts of polar lipids (PL) markedly increased in the tissues of soybean seedlings, especially in light-grown seedlings, whereas these of NPL increased slightly or maintained constant values. The features of the compositions and changing patterns of NPL in the tissues were more clarified in light-grown seedlings than in dark-grown ones. The pattern of change in fatty acid composition was similar in TG and 1,2-DG, which showed higher proportions of linoleic and linolenic acids, whereas FFA, 1,3-DG or MG had high proportions of saturated fatty acids. These results indicate that the compositions and changing patterns of NPL and their fatty acids in the tissues depend on the differences under two germinating conditions tested.     

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.     

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.     

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.     

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     

An enhanced plant lipidomics method based on multiplexed liquid chromatography–mass spectrometry reveals additional insights into cold‐ and drought‐induced membrane remodeling

Within the lipidome of plants a few bulk molecular species hamper the detection of the rest, which are present at relatively low levels. In addition, low‐abundance species are often masked by numerous isobaric interferences, such as those caused by isoelemental species and isotopologues. This scenario not only means that minor species are underrepresented, but also leads to potential misidentifications and limits the structural information gathered by lipidomics approaches. In order to overcome these limitations we have developed a multiplexed liquid chromatography–mass spectrometry lipidomics platform able to achieve an enhanced coverage of plant lipidomes. The platform is based on a single extraction step followed by a series of ultra‐performance liquid chromatography separations. Post‐column flow is then directed to both a triple quadrupole analyzer for targeted profiling and a time‐of‐flight analyzer for accurate mass analysis. As a proof of concept, plants were subjected to cold or drought, which are known to trigger widespread remodeling events in plant cell membranes. Analysis of the leaf lipidome yielded 393 molecular species within 23 different lipid classes. This enhanced coverage allowed us to identify lipid molecular species and even classes that are altered upon stress, allowing hypotheses on role of glycosylinositolphosphoceramides (GIPC), steryl glycosides (SG) and acylated steryl glycosides (ASG) in drought stress to be addressed and confirming the findings from numerous previous studies with a single, wide‐ranging lipidomics approach. This extended our knowledge on membrane remodeling during the drought response, integrating sphingolipids and sterol lipids into the current glycerolipid‐based model.     

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.     

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.     

Modulation of activities of steryl glucoside hydrolase and UDPG: Sterol glucosyltransferase from Sinapis alba by detergents and lipids

Interactions of detergents and lipid compounds on the activity of delipidated preparations of UDPG: sterol glucosyltransferase and steryl β-d-glucoside hydrolase (SG hydrolase) isolated from white mustard seedlings were studied. It has been found that various lipids exert diverse effects on the activity of SG hydrolase. This activity was distinctly stimulated by several neutral, relatively unpolar compounds such as phytol, tripalmitoylglycerol, methyl stearate or cholesteryl acetate and, to a lesser extent, by free fatty acids. On the other hand a number of phospho- and glycolipids were inhibitory. A particularly strong inhibition was observed with charged, zwitterionic phospholipids such as PC, PE or their 2-lyso derivatives. These results point to the possibility of in vivo regulation of the membrane-bound SG hydrolase by its lipid microenvironment. In contrast to SG hydrolase no evidence was found for a clear-cut effect of lipids on the activity of UDPG: sterol glucosyltransferase even after a pretreatment of the enzyme preparation with phospholipase C.     

Sterols and sterylglycosides of oats (Avena sativa). Distribution in the leaf tissue and medium-induced glycosylation of sterols during protoplast isolation

Sterols, sterylglycosides (SG), acylated sterylglycosides (ASG) and steroidal saponins of primary leaves of oat ( Avena sativa L. cv. Flämingskrone) were analyzed by thin-layer chromatography, gas-liquid chromatography and high-performance liquid chromatography. Intact leaves, epidermis preparations, epidermis-stripped leaves, isolated protoplasts and chloroplasts were compared. The mesophyll contained 79% of the total leaf sterols, 80% of the SG and 78% of the ASG, but only 33–67% of the saponins. Free sterols, SG and ASG were mainly localized within the mesophyll, whereas steroidal saponins were localized in the epidermis to a significantly higher extent. The sterol parts consisted mainly of sitosterol, stigmasterol. cholesterol. Δ⁵-avenasterol, Δ⁷-avenasterol, campesterol and Δ⁷-cholestenol, and were quantitatively different in different sterol groups. A higher percentage of sitosterol at the expense of stigmasterol was typical for SG and ASG as compared to free sterols. Only minor differences in the sterol composition were found in a given sterol group when isolated from different tissues. Isolated protoplasts contained only 5–9% of the sterols present in mesophyll cells, indicating that the major part of the free sterols was lost during isolation. Exposure of radioactively labelled leaf segments to either buffer or digestion medium induced rapid transformation of sterols to SG and ASG as shown by the shift of radioactivity from free sterols to the glyeosides. This suggests that two sterol pools exist in the cell: one in the plasmalemma, which is accessible to medium-induced transformation, and a second non-accessible pool in the interior membranes (e.g. chloroplasts) of the cell.     

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.     

Acylated Steryl Glycoside Synthesis in Seedlings of Nicotiana tabacum L

In tobacco seedlings (Nicotiana tabacum L.), glucose from supplied uridine diphosphate-[U-¹⁴C]glucose was first incorporated into steryl glycosides and later into acylated steryl glycosides. However, when [¹⁴C]cholesterol was used as substrate, the acylated steryl glycosides became labeled earlier than the steryl glycosides. With [¹⁴C]cholesteryl glucoside as substrate, most of the radioactive label was recovered as free sterol, and the acylated steryl glycosides were not readily labeled; however, palmitoyl [¹⁴C]cholesteryl glucoside was rapidly converted to steryl glycoside. In feeding experiments with free sterol, an unknown, highly radioactive steroid component was isolated. Incorporation of radioactivity into the unknown occurred before the acylated steryl glycosides were labeled.     

Lipids in plant tissue cultures IV. The characteristic patterns of lipid classes in callus cultures and suspension cultures

Lipids from callus cultures and suspension cultures of higher plants constitute 5 to 8% of the dry tissue's weight.The predominant lipid classes are the sterols, steryl esters, steryl glycosides and esterified steryl glycosides. Considerable amounts of a variety of sterylglycolipids, whose structures are not completely elucidated, are also present. Triglycerides and phospholipids occur in small proportions, whereas monogalactosyl diglycerides, digalactosyl diglycerides and sulfoquinovosyl diglycerides are present only in traces, if at all.β-Sitosterol is the predominant constituent sterol, stigmasterol and campesterol as well as a variety of as yet unidentified sterols occur in smaller proportions. The major constituent fatty acids are palmitic, oleic, linoleic and linolenic acids. Saturated very long-chain fatty acids are found in smaller proportions. Unusual fatty acids, such as epoxy acids, which occur in the seed lipids of certain plants, are not found in tissue cultures derived from these plants. Clucose and traces of galactose are the only sugars obtained by acid hydrolysis of the glycolipids occurring in plant tissue cultures.     

Xylanase treatment of plant cells induces glycosylation and fatty acylation of phytosterols

Treatment of tobacco suspension cells ( Nicotiana tabacum cv. KY 14) with a purified β -1,4-endoxylanase from Trichoderma viride [1 μg enzyme (ml cells)⁻¹] caused a 13-fold increase in the levels of acylated sterol glycosides and elicited the synthesis of phytoalexins. A commercial preparation of xylanase from Trichoderma viride caused an identical shift in sterols. In contrast, a commerical xylanase from Aureobasidium pullaulans had no effect on the levels of acylated sterol glycosides, but did elevate the levels of sterol esters. Treatment of the cells with Cu²⁺ or Ag⁺ also evoked a severalfold increase in the levels of acylated sterol glycosides. Analysis of the various sterol lipid classes revealed that the large xylanase-induced increase in acylated sterol glycosides occurred at the expense of sterol esters, free sterols and sterol glycosides. Further analyses revealed that the most abundant phytosterol in each of the four classes of sterol lipids was β -sitosterol. Linoleic acid was the most abundant fatty acid in the sterol esters, and palmitic and linoleic acids were the most abundant fatty acids in the acylated sterol glycosides. Glucose was the only sugar moiety in the sterol glycoside and acvlated sterol glycosides. Glucose was the only sugar moiety in the sterol glycoside and acylated sterol glycoside fractions. The results of the present study demonstrate that xylanase from Trichoderma viride induces a dramatic shift in the level of acylated sterol glycosides, indicating that endoxylanase was probably the active component in the cellulase enzyme preparations used in our previous study.