Characterization of Soybean Plasma Membrane during Development: FREE STEROL COMPOSITION AND CONCANAVALIN A BINDING STUDIES

Plasma membrane preparations from soybean root and hypocotyl contained the following free sterols: cholesterol, campesterol, stigmasterol, and sitosterol. The cholesterol level was relatively low in root plasma membrane (less than 0.5%) but was 1.4 to 2.4% in hypocotyl membrane. The relative levels of the three other sterols fluctuated with cellular development and tissue source. Campesterol level decreased with the development of both root and hypocotyl membrane. With development, stigmasterol increased greatly in root membrane but remained constant in hypocotyl membrane, and sitosterol, the major free sterol component of all membrane preparations, decreased in root membrane but increased slightly in hypocotyl membrane.     

Distribution and movement of sterols with different side chain structures between the two leaflets of the membrane bilayer of mycoplasma cells

Mycoplasma gallisepticum was adapted to grow with delta 5-sterols modified in the aliphatic side chain, and stopped-flow kinetic measurements of filipin association were made to estimate the sterol distribution between the two leaflets of the membrane. Cholesterol derivatives with unsaturated side chains (desmosterol, cis- and trans-22-dehydrocholesterol, and cholesta-5,22E,24-trien-3 beta-ol) or an alkyl substituent (beta-sitosterol) were predominantly (86-94%) localized in the outer leaflet of the bilayer. However, cholesterol, 20-isocholesterol, and sterols with side chains of varying lengths (in the 20(R)-n-alkylpregn-5-en-3 beta-ol series where the alkyl group ranged from ethyl to undecyl) were distributed nearly symmetrically between the two halves of the bilayer. Kinetic measurements of beta-[14C]sitosterol and [14C]desmosterol exchange between M. gallisepticum cells and an excess of sonicated sterol/phosphatidylcholine vesicles confirmed the filipin-binding studies. More than 90% of these radiolabeled sterols underwent exchange at 37 degrees C with unlabeled sterols in vesicles over a period of 12-14 h in the presence of 2% (w/v) albumin. beta-[14C]Sitosterol exchange was characterized by biphasic exchange kinetics, indicative of two pools of sitosterol molecules in the cell membrane. Only a single kinetic pool was detected for [14C]desmosterol exchange. Stopped flow measurements of filipin binding to beta-sitosterol and stigmasterol also revealed an asymmetrical localization of these sterols in membranes of growing Mycoplasma. capricolum cells. When an early exponential culture of beta-sitosterol- or stigmasterol-adapted M. capricolum was transferred to a sterol-rich medium at 37 degrees C, approximately three-quarters of the beta-sitosterol or stigmasterol was localized in the outer leaflet after growth was continued for 6 h; in contrast, cholesterol was distributed symmetrically after about 1 h. The asymmetric localization of sterols with alkylated or unsaturated side chains suggests that growth-supporting sterols need not be translocated extensively into the inner leaflet of the bilayers of M. gallisepticum and M. capricolum.     

Sterol Accumulation and Composition in Developing Zea mays L. Kernels

Kernels were collected from three maize (Zea mays L.) inbreds from 10 days after pollination until kernel maturity. Sitosterol, campesterol, and stigmasterol were the major sterols at all stages of kernel development. Cholesterol was less than 1% of the dry weight. The three major sterols accumulated during kernel development, but at a rate slower than dry weight. The ratio of the sterols did not vary greatly among the inbreds. At maturity, the three inbreds, Wf9, Oh43, and Ky226, had sterol levels of 325, 228, and 173 micrograms per kernel, respectively. Sitosterol accounted for 75 to 85% of the sterol. The relative amount of stigmasterol decreased during the linear phase of development, while sitosterol increased in the free fraction and campesterol increased in the steryl ester fraction.     

Soybean phosphatidylcholine vesicles containing plant sterols: a fluorescence anisotropy study

The typical plant sterols (sitosterol, stigmasterol and campesterol) were compared with respect to their ability to regulate membrane fluidity of soybean phosphatidylcholine (PC) vesicles. Fluidity changes were monitored by the steady-state fluorescence anisotropy with 1,6-diphenyl-1,3,5-hexatriene as a probe and assigned to a measure of the acyl chain orientational order. Sitosterol and campesterol appear to be the most suitable sterols in ordering the acyl chains of soybean lecithin bilayers, even more efficient than cholesterol, the standard of reference for sterol effects on membranes, suggesting that they play a significant role in the regulation of plant membrane properties. Stigmasterol is shown to be much less active. Cycloartenol, a biosynthetic precursor of plant sterols, increases the acyl chain order with the same efficiency as cholesterol. We also investigated the effects of two unusual sterols, 24-methylpollinastanol and 14 alpha,24-dimethylcholest-8-en-3 beta-ol, which were shown to accumulate in plants treated with fungicides belonging to two important classes, N-substituted morpholines and triazoles, respectively. These two sterols exhibit a behavior very similar to that of stigmasterol. The results are discussed in terms of sterol effects on the molecular packing of soybean PC bilayers.     

Sterol utilization in honey bees fed a synthetic diet: Analysis of prepupal sterols

The sterols of prepupal honey bees, Apis mellifera L., from brood reared by workers fed chemically-defined synthetic diets containing cholesterol, campesterol, sitosterol, stigmasterol, 24-methylenecholesterol, or no sterol over a 12-week period were isolated, identified, and quantified. The major sterol present in each prepupal sample was 24-methylenecholesterol, but significant levels of sitosterol and isofucosterol were also present in every case, as was a very small percentage of desmosterol (usually < 1%). This is the first report of isofucosterol being identified in the sterols of the honey bee. A considerably larger percentage of each dietary sterol was found in prepupae reared by workers fed that particular sterol in the diet. This was most dramatic in the case of the cholesterol diet in which case cholesterol content increased to as much as 17.2% of the prepupal sterols, whereas cholesterol had not exceeded 2.2% in samples from other diet regimens. However, stigmasterol comprised no more than 6.3% of the total sterols in any sample from prepupae fed the stigmasterol diet. The preponderance of 24-methylenecholesterol in all prepupae, regardless of the dietary sterol provided to the workers, as well as the lesser quantities of sitosterol and isofucosterol present in all samples, suggest a unique system of utilization and metabolism of these dietary sterols by the worker bees. Apparently they make available to the brood varying amounts of unchanged dietary sterol plus considerable and fairly constant portions of 24-methylenecholesterol, sitosterol, and isofucosterol drawn from their own sterol pools.     

Soybean sterol composition and utilization by Phytophthora sojae

The sterol fraction of Glycine max (soybean) was found to contain a mixture of 13 major sterols which differed dramatically in composition between seeds and shoots. Typical C4-desmethyl Delta(5)-sterols, including sitosterol, predominate the sterol mixture of shoots, whereas C4-methyl sterol intermediates, cycloartenol and 24(28)-methylene cycloartanol, accumulate in seeds. The significance of modified sterol profile of shoot compared to seed was relevant to the physiology of Phytophthora sojae, a phytopathogen of soybean shown to be auxotrophic for sterol. Sterols native to the host plant containing a C4-methyl group, such as cycloartenol, were not utilized by the fungus. Alternatively, all Delta(5)-sterols added to the culture media of P. sojae supported normal growth and promoted viable oospore production. The results demonstrate the importance of sterols in plant-fungal interactions and offer the possibility of bioengineering the phytosterol pathway for resistance to phytopathogens which scavenge specific sterols of the host plant to complete the life cycle.     

Codisterol and other Δ5-sterols in the seeds of Cucurbita maxima

A substantial amount (ca 18%) of the sterol found in the seeds of Cucurbita maxima had a Δ-bond and consisted of seven components. They were identified as 25(27)-dehydroporiferasterol, clerosterol, isofucosterol, stigmasterol, sitosterol, campesterol and codisterol. The C-24 configuration of each of the sterols was unequivocally established by a ¹H NMR spectral comparison with authentic standards. This is the first time codisterol has been found in a higher plant and also the first time the structures and configurations of the Δ⁵-sterols from a Cucurbitaceae species have been clearly characterized.     

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.     

A dynamic role for sterols in embryogenesis of Pisum sativum

Molecular roles of sterols in plant development remain to be elucidated. To investigate sterol composition during embryogenesis, the occurrence of 25 steroid compounds in stages of developing seeds and pods of Pisum sativum was examined by GC-MS analysis. Immature seeds containing very young embryos exhibited the greatest concentrations of sterols. Regression models indicated that the natural log of seed or pod fr. wt was a consistent predictor of declining sterol content during embryonic development. Although total sterol levels were reduced in mature embryos, the composition of major sterols sitosterol and campesterol remained relatively constant in all 12 seed stages examined. In mature seeds, a significant decrease in isofucosterol was observed, as well as minor changes such as increases in cycloartenol branch sterols and campesterol derivatives. In comparison to seeds and pods, striking differences in composition were observed in sterol profiles of stems, shoots, leaves, flowers and flower buds, as well as cotyledons versus radicles. The highest levels of isofucosterol, a precursor to sitosterol, occurred in young seeds and flower buds, tissues that contain rapidly dividing cells and cells undergoing differentiation. Conversely, the highest levels of stigmasterol, a derivative of sitosterol, were found in fully-differentiated leaves while all seed stages exhibited low levels of stigmasterol. The observed differences in sterol content were correlated to mRNA expression data for sterol biosynthesis genes from Arabidopsis. These findings implicate the coordinated expression of sterol biosynthesis enzymes in gene regulatory networks underlying the embryonic development of flowering plants.     

Expression of a Streptomyces 3-hydroxysteroid oxidase gene in oilseeds for converting phytosterols to phytostanols

Plant sterols and their hydrogenated forms, stanols, have attracted much attention because of their benefits to human health in reducing serum and LDL cholesterol levels, with vegetable oil processing being their major source in several food products currently sold. The predominant forms of plant sterol end products are sitosterol, stigmasterol, campesterol and brassicasterol (in brassica). In this study, 3-hydroxysteroid oxidase from Streptomyces hygroscopicus was utilized to engineer oilseeds from rapeseed (Brassica napus) and soybean (Glycine max), respectively, to modify the relative amounts of specific sterols to stanols. Each of the major phytosterols had its C-5 double bond selectively reduced to the corresponding phytostanol without affecting other functionalities, such as the C-22 double bond of stigmasterol in soybean seed and of brassicasterol in rapeseed. Additionally, several novel phytostanols were obtained that are not produced by chemical hydrogenation of phytosterols normally present in plants.     

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.     

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.     

Influence of Temperature on Sterol Biosynthesis in Triticum aestivum

Sitosterol, campesterol, stigmasterol, and cholesterol were isolated from green wheat (Triticium aestivum var. Monon) seedlings. Sitosterol was the predominant sterol extracted from the shoot, root, and crown tissue. Cholesterol accounted for less that 1% of sterol in shoot tissue with only trace amounts in the root. A temperature change from 10 to 1 C resulted in a general decrease in sitosterol, stigmasterol, and campesterol in the shoot tissue. The cholesterol level was not altered significantly by the temperature change. The sterols in the root responded in a manner very different from those in the shoots. With the reduction in temperature, sterols first decreased and then recovered over a period of 7 to 14 days to levels that were equal to or exceeded the original levels. From these experiments, it would appear that root tissue can acclimate to the lower temperatures and continue sterol synthesis at the normal rate. The level and response of sterols in the crown tissue were intermediate between the root and shoot tissue. At 10 C the crown response was similar to that of root tissue, whereas, at 1 C the response more closely resembled that of the shoot.     

Changes of fatty acids and sterols in apple buds during bud break induced by a plant bioregulator, thidiazuron

The effects of N-phenyl-N'-l,2,3,-thidiazol-5-ylurea (thidiazuron; Dropp; SN 49537) on fatty acids of membrane lipids and sterol content in apple ( Malus domestica Borkh cv. Golden Delicious) buds associated with bud break and bud development were determined. The predominant fatty acids in the membrane lipids of apple buds were palmitic acid (C16:0), linoleic acid (C18:2) and linolenic acid (C18:3). β -Sitosterol and sitosteryl ester were the predominant sterols. An accumulation of unsaturated polar membrane fatty acids started after thidiazuron treatment. A decrease in the percentage of the sitosterol was accompanied by an increase in campesterol and stigmasterol at the beginning of rapid growth. An increase in the ratio of campesterol and stigmasterol to sitosterol and a decrease in the ratio of free sterols to membrane lipids upon breaking of dormancy also occurred in apple buds induced by thidiazuron.     

Sterols of soybeans differing in insect resistance and maturity group

Eight soybean varieties and lines representing insect-susceptible and -resistant genotypes, and differing in plant maturity group, showed no difference in leaf dry wt. Over the growing season the total and bound sterols increased while the free sterols decreased. Sitosterol, stigmasterol and campesterol were the major sterols. Over the season sitosterol increased while stigmasterol decreased. No difference due to insect resistance could be established but early maturing plants showed a larger change in sterols.     

Metabolism of Δ0-, Δ5-, and Δ7-Sterols by Larvae of Heliothis zea

Heliothis zea was reared on artificial diets containing Δ⁵-sterols (cholesterol, campesterol, or sitosterol), Δ⁷-sterols (lathosterol, epifungisterol, or spinasterol), or Δ⁰-sterols (cholestanol, epicoprostanol, campestanol, or sitostanol) in order to determine how different dietary sterols affect the type of sterols present in the tissues of the late-sixth-instar larva. Although all of the dietary sterols (except epicoprostanol) supported the growth of the larvae, not all of the sterols were metabolized to the same end products. In each case, at least 80% of the sterols in the tissues of the larvae retained the same nucleus as that of the dietary sterol, indicating that H. zea carries out very little metabolism of ring B of Δ⁵-, Δ⁷-, and Δ⁰-sterols. The larvae dealkylated the Δ⁵-, Δ⁷-, and Δ⁰-alkylsterols to 24-desalkylsterols, but a greater percentage of the Δ⁵-alkylsterols were metabolized in this manner. The sterols present as free sterols in the larva were also present as esterifed sterols which accounted for 2–4% of the total sterols. Therefore, the sterol composition of the tissues of H. zea can be altered by varying the dietary sterols.     

Complex formation between solanaceous steroidal glycoalkaloids and free sterols in vitro

Both the steroidal glycoalkaloid mixture from potato (α-solanine and α-chaconine) and pure α-tomatine are able to complex with the sterols cholesterol, sitosterol, stigmasterol, campesterol and ergosterol in vitro. The sterol-complexing ability of tomatine was greater than that of the potato alkaloids and more akin to that of the steroidal saponin, digitonin. With all three compounds, cholesterol was the least-readily bound sterol while binding to other sterols was of a similar order. Complex formation with tomatine was not markedly influenced by temperature, and with the aglycone tomatidine did not appear to occur at all.     

Sterol composition of Aureoumbra lagunensis, the Texas brown tide alga

Aureoumbra lagunensis, the alga responsible for the "Texas brown tide", contains (E)-24-propylidenecholesterol (35.7% of total sterols) as its dominant sterol, in common with other members of the Pelagophyceae. Other major sterols are stigmasterol (22.2%), sitosterol (19.2%), cholesterol (14.1%), and (24R)-24-propylcholesterol (5.2%). Trace amounts of 24-methylenecholesterol, crinosterol, clerosterol, campesterol, dihydrobrassicasterol, and 24-isopropylcholesterol were also detected.     

Sterol utilization in honey bees fed a synthetic diet: Effects on brood rearing

The dietary sterols, cholesterol, campesterol, sitosterol, stigmasterol and 24-methylenecholesterol, were tested for their ability to support brood rearing in the honey bee, Apis mellifera L., by adding them singly to a chemically-defined worker bee diet. Diet supplemented with 24-methylenecholesterol supported the greatest survival of worker bees, but diet supplemented with either 24-methylenecholesterol or cholesterol supported the production of nearly equivalent amounts of sealed brood and more than any of the other three sterols tested. Diets containing stigmasterol, sitosterol, campesterol, or no supplement produced less sealed brood, in decreasing order.