Developmental regulation of sterol biosynthesis inCucurbita maxima L.

Twenty-two sterols were identified by capillary gas chromatography and capillary gas chromatography/mass spectroscopy inCucurbita maxima grown under green-house conditions. Both whole plants and individual tissues (leaves, stems, roots, cotyledons, flowers) were analyzed at weekly intervals during the 12-week development of the plant. In whole plants, sterol accumulation parallels plant growth except for a period in the mid-life cycle where there is a reduction in the amount of sterol accumulated on a total sterol/plant and mg sterol/g dry wt basis. This reduction in the amount of sterol is coincident with the visual onset of flowering. During development, the percent contribution of each class of sterol (Δ^5_, Δ^7_, Δ^0_-sterols) remains relatively constant. However, the percent contribution of an individual sterol species varies depending on the tissue examined and the developmental period selected for analysis. While the young plant (<2 weeks) possesses elevated levels of sterols with the Δ²⁵⁽²⁷⁾-double bond, the trend was toward a reduction in the amounts of these sterols with development. Leaves and stems accumulate large quantities of 24ζ-ethyl-5α-cholesta-7,22-dien-3β-ol (7,22-stigmastadienol) and 24ζ-ethyl-5α-cholest-7-en-3β-ol (7-stigmastenol), while roots accumulate only 7,22-stigmastadienol as their principal sterol. Male flowers and roots were found to contain elevated levels of Δ^5_-sterols.     

Cholesterol synthesis in the vertebrate retina: Effects of U18666A on rat retinal structure, photoreceptor membrane assembly, and sterol metabolism and composition

Treatment of neonatal rats with U18666A, an inhibitor of desmosterol Δ²⁴-reductase, results in accumulation of desmosterol (Δ^5,24) and depletion of cholesterol (Δ⁵) in various bodily tissues and also causes cataracts. We evaluated the effects of U18666A on the sterol composition, de novo sterol synthesis, and histological structure of the retina. Neonatal Sprague-Dawley rats were injected subcutaneously with U18666A (15 mg/kg, in olive oil) every other day from birth through 3 wk of age; in parallel, control rats received olive oil alone. At 21 d, treated and control groups each were subdivided into two groups: one group of each was injected intravitreally with [³H]acetate; retinas were removed 20 h later and non-saponifiable lipids (NSL) were analyzed by radio-high-performance liquid chromatography. The other group was injected intravitreally with [³H]leucine; 4 d later, one eye of each animal was evaluated by light and electron microscopy and light microscopic autoradiography, while contralateral retinas and rod outer segment (ROS) membranes prepared thereform were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/fluorography. In the treated group, the Δ⁵/Δ^5,24 mole ratio of retinas was ca. 1.0, and >88% of the NSL radioactivity was in Δ^5,24; in contrast, control retinas had Δ⁵/Δ^5,24 >170, with >80% of the NSL radioactivity in Δ⁵. Retinal histology, ultrastructure, ROS renewal rates, and rhodopsin synthesis and intracellular trafficking were comparable in both treated and control animals. These results suggest that desmosterol can either substitute functionally for cholesterol in the retina or it can complement subthreshold levels of cholesterol by sterol synergism.     

Comparison of effects of U18666A and enantiomeric U18666A on sterol synthesis and induction of apoptosis

Treatment of animals or cells with the amphipathic tertiary amine U18666A {3β-[2-(diethylamino) ethoxy]androst-5-en-17-one} provides models for several human diseases (e.g., cataracts, Niemann-Pick disease, and epilepsy). Although U18666A can inhibit several enzymes in the cholesterol synthesis pathway, we hypothesized that induction of these varied conditions was due to physical effects of the amine rather than to inhibition of specific proteins. To test this possibility we compared the capacity of U18666A and its enantiomer, ent-U18666A, to inhibit net sterol synthesis and induce apoptosis in cultured bovine lens epithelial cells. Nonenantiospecific actions dependent on the physical properties of these mirror image molecules would be identical, but effects dependent upon enantiospecific interactions would be different for the enantiomers. At the same concentrations, both forms of the compound equally inhibited sterol synthesis and induced apoptosis. These observations supported a generalized mechanism of enzyme inhibition such as perturbation of the microenvironment of endoplasmic enzymes and alteration of membrane order, perhaps of the mitochondrial membrane, to explain induction of apoptosis.     

Sterol composition and biosynthesis in sorghum: Importance to developmental regulation

Sterol composition and biosynthesis have been examined in seeds, germinating seeds and blades from fally matured leaves ofSorghum bicolor in various stages of development’from seedlings (seven-day plants) to flowering (66-day) plants. The profile of the dominant free sterols of seeds was similar to that of leaf blades; both contained cholesterol, 24α-methylcholesterol (campesterol), 24β-methylcholesterol (dihydrobrassicasterol), 24α-ethylcholesterol (sitosterol) and 24α-ethylcholesta-5,22-dienol (stigmasterol). Sufficient sterol intermediates were identified in the plant to indicate separate post-cycloartenol pathways to sterolic end products. The total free sterol content of the seed (μg/seed) increased somewhat during the 20 hr germination period. However, as the plant developed (seven to 48 days), there was a logarithmic increase in the leaf blade sterol content (μg/leaf blade) which plateaued at the onset of floral differentiation (ca. day 41). Over the next 18 days (48 to 66 days—period of inflorescense development), the sterol content rapidly decreased. In the early stages of plant development, the leaf blade pentacyclic triterpenoid (PT) content was negligible. With the onset of floral differentiation, PT content increased logarithmically, reaching a plateau level that surpassed the sterol content as flowering progressed. These results imply that a critical mass of sterol is associated with sorghum for floral induction. Sterol loss from the leaves of the flowering plants presumably was compensated for by the diversion of 2,3-oxidosqualene (SO) from sterol synthesis to PT production. Additional feeding and trapping experiments with [2-¹⁴C]mevalonic acid, [2-³H]cycloartenol, [24-³H]lanosterol [4-¹⁴C]sitosterol and [4-¹⁴C]cholesterol fed to germinating seeds and leaves from flowering plants demonstrated that sorghum possessed a cycloartenolbased pathway; germinating seeds synthesized 24-alkylsterols but not cholesterol, although cholesterol was identified in both dry and germinating seeds by gas chromatography-mass spectroscopy (GC-MS); and mature leaves synthesized cholesterol and 24α-alkylsterols but not 24β-methylcholesterol.     

Multiple inhibitory effects of garlic extracts on cholesterol biosynthesis in hepatocytes

Exposure of primary rat hepatocytes and human HepG2 cells to water-soluble garlic extracts resulted in the concentration-dependent inhibition of cholesterol biosynthesis at several different enzymatic steps. At low concentrations, sterol biosynthesis from [¹⁴C]acetate was decreased in rat hepatocytes by 23% with an IC₅₀ (half-maximal inhibition) value of 90μg/mL and in HepG2 cells by 28% with an IC₅₀ value of 35 μg/mL. This inhibition was exerted at the level of hydroxymethylglutaryl-COA reductase (MHG-CoA reductase) as indicated by direct enzymatic measurements and the absence of inhibition if [¹⁴C]mevalonate was used as a precursor. At high concentrations (above 0.5 mg/mL), inhibition of cholesterol biosynthesis was not only seen at an early step where it increased considerably with dose, but also at later steps resulting in the accumulation of the precursors lanosterol and 7-dehydrocholesterol. No desmosterol was formed which, however, was a major precursor accumulating in the presence of triparanol. Thus, the accumulation of sterol precursors seem to be of less therapeutic significance during consumption of garlic, because it requires concentrations one or two orders of magnitude above those affecting HMG-CoA reductase. Alliin, the main sulfur-containing compound of garlic, was without effect itself. If converted to allicin, it resulted in similar changes of the sterol pattern. This suggested that the latter compound might contribute to the inhibition at the late steps. In contrast, nicotinic acid and particularly adenosine caused moderate inhibition of HMG-CoA reductase activity and of cholesterol biosynthesis suggesting that these compounds participate, at least in part, in the early inhibition of sterol synthesis by garlic extracts. Dedication: This article is dedicated to Prof. Dr. D. Mecke on the occasion of his 60th birthday.     

Occurrence and levels of ecdysteroids in spinach

The dominant ecdysteroid in spinach,Spinacia oleracea L., is 20-hydroxyecdysone (2β,3β,14α, 20R,22R,25-hexahydroxy-5β-cholest-7-en-6-one) with the presence of a smaller amount of polypodine B (2β,3β,5β,14α,20R,22R,25-heptahydroxycholest-7-en-6-one). Ecdysteroids are present in the seed embryo at 14 μg ecdysteroid/seed, and ecdysteroid levels increase, in the plant during growth and development. During the onset of ecdysteroid production, the ratio of ecdysteroid to total sterol shifts from a value of one to about ten over the course of a week. Spinach may serve as a model to study ecdysteroid biosynthesis in plants. Based on a paper presented at the symposium on Plant and Fungal Sterols: Biosynthesis, Metabolism and Function, held at the AOCS Annual Meeting, Baltimore, MD, April 1990.     

Low Concentrations of Salicylic Acid Stimulate Insect Elicitor Responses in <Emphasis Type="Italic">Zea mays</Emphasis> Seedlings

Salicylic acid (SA) generally is thought to suppress jasmonic acid (JA) related signaling events. However, when we treated the roots of corn seedlings overnight with low physiological concentrations of SA (50 μM), we found a priming effect of this pretreatment on typical insect elicitor (IE)-induced responses in the leaves of these plants. IE-induced JA was more than 2-fold up regulated in SA-pretreated plants. Consequently, IE-induced volatile organic compounds (VOC) release also was significantly increased. In contrast, when corn seedlings were treated with SA overnight and then mechanically damaged, we found no significant differences in JA accumulation. We also found that the application of even lower concentrations of SA (5 μM) had no significant effect on IE-induced responses, while higher concentrations (500 μM) inhibited IE-induced JA accumulation. Likewise, shorter exposure to SA did not affect subsequent JA accumulation induced by IE or mechanical wounding. These results provide evidence for the existence of non-compatible defense priming by signaling molecules that usually are involved in a conflictive defense signaling pathway and suggests common elements in the regulation of priming plant defense responses.     

AY-9944 inhibition of sterol biosynthesis inChlorella emersonii

WhenChlorella emersonii, a green alga, was cultured in the presence of 20 ppm AY-9944, a number of sterols accumulated which appear to be intermediates of sterol biosynthesis in this organism. The sterols isolated include 14α-methyl-ergost-8-en-3β-ol, 14α-methyl 24S-stigmast-8-en-3β-ol, 14α-methyl ergosta-8,24(28)-dien-3β-ol and 4α, 14α-dimethyl 24S-stigmast-8-en-3β-ol. Smaller quantities of several other sterols were found in addition to the normally occurring Δ⁷, chondrillasterol and Δ⁷. Control cultures were found to contain, in addition to the normally occurring sterols, smaller quantities of most of the sterols isolated from AY-9944 inhibited cultures. AY-9944 is a specific inhibitor of Δ⁷ in cholesterol biosynthesis in animals. However, sinceC. emersonii terminates sterol biosynthesis one step prior to the Δ⁷ step, AY-9944 apparently inhibits sterol biosynthesis prior to this step in this organism. The accumulation of 14α-methyl sterols in treated cultures suggests that AY-9944 is an effective inhibitor of the 14α-methyl removal inC. emersonii. Scientific Article No. A1865, Contribution No. 4775 of the Maryland Agricultural Experiment Station.     

Effects of triarimol,tridermorph and triparanol on sterol biosynthesis in carrot,tobacco and soybean suspension cultures

The effects of triarimol, tridemorph and triparanol on sterol biosynthesis in carrot, tobacco and soybean suspension cultures were studied. The 3 plant species normally contain campesterol, stigmasterol and sitosterol as major sterols. Triarimol inhibited demethylation at C 14 and the second alkylation of the side chain in all 3 species. The primary effects of tridemorph were the inhibition of the opening of the 9β,19-cyclopropane ring and the second alkylation of the side chain. Triparanol treatments resulted in the accumulation of 14α-methyl sterols, and the inhibition of second alkylation in the side chain in carrot and tobacco cultures. Cyclopropyl sterols also accumulated in carrot and tobacco cultures treated with triparanol. Triparanol did not alter the sterol composition of soybean cultures except for decreasing concentrations of campesterol and stigmasterol and increasing amounts of sitosterol. Scientific article number A-3713, contribution number 6689 of the Maryland Agricultural Experiment Station.     

Accumulation of ergosta-8,14-dien-3β-ol bySaccharomyces cerevisiae cultured with an azasterol antimycotic agent

15-Aza-24-methylene-D-homocholesta-8,14-dien-3β-ol, an antimycotic agent, at a concentration of 75 ng/ml inhibited ergosterol biosynthesis inSaccharomyces cerevisiae strain 3701 B resulting in the accumulation of an unusual sterol. Experimental data presented indicate that this sterol is ergosta-8,14-dien-3β-ol. The accumulation of the compound is supportive of current models of biosynthetic pathways for sterols in yeast and is consistent with inhibition by the azasterol of the Δ¹⁴ sterol reductase.     

Investigation of the biogenetic reaction sequence of cholesterol in rat tissues,through inhibition with AY-9944

An inhibitor of Δ⁷-reductase, AY-9944 (trans-1,4-bis(2-dichlorobenzylaminomethyl cyclohexane dihydrochloride), was used to investigate the last steps of cholesterol formation in brain and liver of adult and newborn rats. The accumulation of different sterols in the two tissues of the same animals was observed. Δ^5,7-Cholestadien-3β-ol, Δ^7,24-cholestadien-3β-ol and Δ^5,7,24-cholestatrien-3β-ol, which are not present in detectable amounts in control brains, were identified in brains of growing rats treated with AY-9944. An accumulation of Δ^5,7-cholestadien-3β-ol only was found in adult rat tissues. These differences in sterol accumulation are discussed in relation with the possible in vivo pathways of cholesterol biosynthesis.     

The effect of AY-9944 on yeast sterol and sterol ester metabolism

The effects of the hypocholesterolemic drug AY-9944 (trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane dihydrochloride) at two concentrations (10⁻⁴M and 5×10⁻⁴M), on the synthesis of sterols and sterol esters bySaccharomyces cerevisiae were investigated. Although growth was not markedly affected by the drug, there was a decrease in the free sterol to sterol ester ratio with increased drug concentration. A concomitant increase in the saturated fatty acids esterified to sterol relative to the unsaturated fatty acids was also noted in response to increased drug concentration. Ergosterol accounted for 94.7% of the free sterol in the control culture and for 87.8% of the 5×10⁻⁴M drug-treated culture, respectively. However, in the sterol ester fraction, the ergosterol content decreased from a value of 45.1% in the control culture to 2.4% in the 5×10⁻⁴M AY-9944 treated culture. The sterol ester fraction simultaneously showed increased levels of the Δ⁸ sterol, fecosterol, in response to increased drug concentration from a 7.4% control value to 57.4% in the 5×10⁻⁴M drug-treated culture. The accumulation of the Δ⁸ sterol suggests that the site of action of the drug is probably at the Δ⁸ to Δ⁷ isomerase step in the biosynthesis of ergosterol. The fact that ergosterol is retained as the major free sterol suggests a biological advantage to the retention of this particular sterol. In addition, the near normal growth in the presence of the drug, in spite of the occurrence of an altered sterol ester profile, indicates that the composition of the sterol ester fraction is not as critical as the free sterol fraction. These results form a portion of a dissertation submitted to the Graduate School, University of Maryland, College Park, MD, by Ravi Pereira, in partial fulfillment of the requirements for the Ph.D. degree in Biochemistry.     

Inhibition of sterol biosynthesis in chlorella ellipsoidea by AY-99441

WhenChlorella ellipsoidea was grown in the presence of 4 ppm AY-9944, complete inhibition of Δ⁵-sterol biosynthesis was achieved. However total sterol production remained unaltered. As a result a number of sterols accumulated that appear to be intermediates in sterol biosynthesis. These sterols were described and identified as (24S)-5α-ergost-8(9)-en3β-ol, (24S)-5α-stigmast-8(9)-en-3β-ol, 4α-methyl-(24S)-5α-ergosta-8, 14-dien-3β-ol, 4α-methyl-(24S)-5α-stigmasta-8, 14-dien-3β-ol, 4α-methyl-(24S)-5α-ergost-8(9)-en-3β-ol and (24S)-4α-methyl-5α-stigmast-8(9)-en-3β-ol. The occurrence of these sterols inChlorella ellipsoidea is the first time they have been noted in biological material. The accumulation of these sterols in treated cultures indicates that AY-9944 is an extremely effective inhibitor of the Δ⁸→Δ⁷ isomerase and the Δ¹⁴ reductase of these plants. The occurrence of small amounts of other sterols in treated cultures has led to a proposed pathway for thebiosynthesis of sterols inChlorella ellipsoidea. Scientific Article No. A1775, Contribution No. 4565 of the Maryland Agricultural Experiment Station.     

Lack of specificity in accumulation of sterols byPhytophthora cactorum

The kinetics of sterol uptake inPhytophthora cactorum were examined. A V_max of 4.81×10⁻⁵ μmol/min/mg dry wt and an average apparent Km of 18.0 μM were determined for both cholesterol and sitosterol accumulation. Selectivity for specific sterols was not apparent in the accumulation, esterification, or distribution of sterols byPhytophthora cactorum.     

Fatty acid metabolism inTaphrina deformans treated with sterol biosynthesis inhibitors

Changes in the unsaturated fatty acid content of the fungusTaphrina deformans as a function of growth, temperature, and sterol content were investigated. It was found that the highest growth rate was accompanied by a relatively high degree of fatty acid unsaturation (18∶1<18∶2+18∶3) and low sterol (brassicasterol) content. Also, a substantial shift in the degree of unsaturation from mainly 18∶2+18∶3 to 18∶1 occurred in the later stages (mid-linear) of culture development. Cells readily adapted from 18°C to 13°C, and exhibited a higher growth rate at the lower temperature after a period of acclimation. Growth was readily inhibited by the sterol biosynthesis inhibitors propiconazole and naftifine, which blocked brassicasterol biosynthesis at C-14 demethylation and squalene epoxidation, respectively. Growth was also inhibited by tunicamycin which did so without affecting sterol content. The shift in degree of fatty acid unsaturation did not occur in cells from cultures at reduced temperature or treated with any of the inhibitors. Since tunicamycin did not affect the sterol content, delay in the shift in the degree of fatty acid unsaturation was attributed to factors other than a reduction in sterol content. Based on a paper presented at the Symposium on Plant and Fungal Sterols: Biosynthesis, Metabolism and Function, held at the AOCS Annual Meeting, Baltimore, MD, April 1990.     

Sterols and fatty acids ofEpifagus virginiana,a nonphotosynthetic angiosperm

The sterol and fatty acid compositions ofEpifagus virginiana, which is a nonphotosynthetic flowering plant, have been examined by combined gas liquid chromatography-mass spectrometry (GLC-MS) and nuclear magnetic resonance (NMR). The organism exhibited a typical higher plant Δ⁵-sterol profile with the C-24 alkyl group oriented “α” for the major sterol Trace amounts of 24-methylenecycloartanol and cycloeucalenol established connection with the first cyclic intermediate (cycloartenol) in photosynthetic plant sterol biosynthesis. In a separate in vitro experiment, excised root and stem tissue were incubated with two labeled triterpenoid precursors, [2-³H] lanosterol and [2-³H]cycloartenol. Both the radioactive compounds were converted to labeled [³H]sitosterol indicating the presence of an active biosynthetic pathway inEpifagus. Characteristic photosynthetic higher plant fatty acids were also present, and the polyenic acid was identified as α-linolenic acid. Scientific Article No. A2451. Contribution No. 5480 of the Maryland Agricultural Experiment Station.     

Distribution of sterols in the fungi I. Fungal spores

The freely extractable sterols of spores ofLinderina pennispora, Spicaria elegans, Penicillium claviforme, Aspergillus niger, Ustilago nuda, U. maydis, Puccinia graminis, andP. striiformis were examined using mass spectrometric techniques. Each species contained at least 3–5 detectable sterol components in the 4-desmethyl sterol fraction, and, when present, ergosterol was generally the most abundant sterol produced by an individual species. Smaller relative concentrations of fungisterol (ergost-Δ⁷-enol) di- and tetraunsaturated C₂₈ sterols also were found. In some species, fungisterol was the most abundant sterol. In uredospores of rust fungi, stigmast-Δ⁷-enol (C₂₉) was predominant and was accompanied by lower relative concentrations of a diunsaturated C₂₉ sterol and fungisterol. Cholesterol was found only in the teliospores of the corn smut fungus (U. maydis). Application of glass capillary columns to the separation of yeast sterols by gas liquid chromatography is illustrated. One of eight papers presented in the symposium “Phytosterols,” AOCS Spring Meeting, New Orleans, April 1973.     

The steric requirements for sterol inhibition of tetrahymanol biosynthesis

Many naturally occurring sterols are accumulated and metabolized byTetrahymena pyriformis. In most cases, the sterols are desaturated to give^Δ5,7,22-derivatives. Compounds with an ethyl, but not with a methyl, substituent at C-24 are dealkylated. Exposure of the ciliates to the appropriate sterol sharply curtails the synthesis of the native pentacyclic triterpenoid alcohols, tetrahymanol and diplopterol. An analysis with modified sterols has revealed several additional features that are required for desaturation at C-7,8 and C-22,23 and for inhibition of tetrahymanol biosynthesis. The presence of atrans-17(20)-double bond, which eliminates free rotation at C-20 and fixes C-22 to the right of the nucleus, does not interfere with desaturation, while thecis- or left-handed isomer is not metabolized. Thecis-Δ¹⁷⁽²⁰⁾-isomer is, however, an effective inhibitor of tetrahymanol biosynthesis, although less so that thetrans-counterpart. When a methyl or hydroxyl group at C-20 protrudes to the front of the molecule in the right-handed conformation, metabolism is reduced or abolished. Shortening (by one C-atom) or lengthening of the sterol side chain has little effect on the ability of the compounds to inhibit tetrahymanol biosynthesis or to support growth, as long as the overall length of the side chain does not exceed seven carbons from C-20. The presence of a 7α-, 7β-, 20α-, 20β-, or a 25-hydroxy group in the cholesterol molecule sharply inhibits desaturation and curtails the effectiveness of the compound as an inhibitor of tetrahymanol biosynthesis. The 7- or 22-keto derivatives seem to act in a fashion similar to the hydroxy derivatives, but these compounds show greater inhibition of growth. 20-Methylcholesterol, however, is a potent inhibitor of synthesis, which suggests that the polarity of the substituent of C-20 is more important than bulk. Many sterols can effectively replace tetrahymanol in the membranes of these ciliates. However, several of the compounds, which inhibit synthesis, appear to be physiologically inappropriate, and poor growth results. An example of the latter class is 20-methylcholesterol. Finally, a class of sterols, represented by 20α-hydroxycholesterol and 7-ketocholesterol, does not severly inhibit tetrahymanol synthesis but leads to growth inhibition and surface abnormalities. These sterols apparently lead to a disordered membrane, even in the presence of tetrahymanol.     

Phytosterol accumulation in canola, sunflower, and soybean oils: Effects of genetics, planting location, and temperature

To assess the potential of traditional selection breeding to develop varieties with increased phytosterol content, we determined concentrations of those sterols in canola, sunflower, and soybean seed oils produced from breeding lines of diverse genetic backgrounds. Seed oils were extracted and saponified, and the nonsaponifiable fractions were subjected to silylation. The major phytosterols brassicasterol, campesterol, stigmasterol and β-sitosterol, were quantified by capillary gas chromatography with flame-ionization detection. Canola contained approximately twice the amount of total phytosterols (4590–8070 μg g⁻¹) as sunflower (2100–4540 μg g⁻¹) or soybean (2340–4660 μg g⁻¹) oils. Phytosterol composition varied among crops as expected, as well as within a crop. Both genetic background and planting location significantly affected total phytosterol concentrations. Soybean plants were maintained from flower initiation to seed maturity under three temperature regimes in growth chambers to determine the effect of temperature during this period on seed oil phytosterol levels. A 2.5-fold variability in total phytosterol content was measured in these oils (3210–7920 μg g⁻¹). Total phytosterol levels increased with higher temperatures. Composition also changed, with greater percent campesterol and lower percent stigmasterol and β-sitosterol at higher temperatures. In these soybean oils, total phytosterol accumulation was correlated inversely with total tocopherol levels. Owing to the relatively limited variability in phytosterol levels in seed oils produced under field conditions, it is unlikely that a traditional breeding approach would lead to a dramatic increase in phytosterol content or modified phytosterol composition.     

Design of high energy intermediate analogues to study sterol biosynthesis in higher plants

Several enzymes of plant sterol biosynthesis involve during their catalysis postulated or demonstrated carbocationic high energy intermediates (HEI). The aim of this study was to interfere with plant sterol biosynthesis by means of rationally designed species able to mimic these carbocationic HEI. It has been demonstrated previously that the design of transition state (TS) or HEI analogues could lead to powerful and specific inhibitors of enzymes. We applied this approach to the following target enzymes: 2,3-epoxy-2,3-dihydroqualene cyclase, AdoMet-cycloartenol-C-24-methyltransferase (AdoMet CMT), cycloeucalenol-obtusifoliol isomerase (COI) and Δ⁸-Δ⁷-sterol isomerase. Very potent inhibitors have been obtained in the four cases. As an example, analogues of cycloartenol substituted at C-25 by a charged heteroatom (N, As, S) have been synthesized and shown to be able to mimic the C-25 carbocationic HEI involved in the reaction catalyzed by the AdoMet CMT. These compounds were shown to be very potent and specific inhibitors of this enzyme both in vitro (Ki=2.10⁻⁸ M, Ki/Km=10⁻³) and in vivo. The potent inhibitors described are powerful tools to control in vivo the sterol profile of plant cells and therefore to study the structural and functional roles of sterols in cell membranes. Moreover, these compounds constitute leader molecules of a new class of rationally designed inhibitors which could be of value in plant protection.