Enhancement of seed phytosterol levels by expression of an N-terminal truncated Hevea brasiliensis (rubber tree) 3-hydroxy-3-methylglutaryl-CoA reductase

Dietary intake of phytosterols (plant sterols) has been shown to be effective in reducing blood cholesterol levels, thereby reducing the risk of cardiovascular disease. Phytosterols are most commonly sourced from vegetable oils, where they are present as minor components. We report here the generation of transgenic tobacco seeds substantially enhanced in phytosterol content by the expression of a modified form of one of the key sterol biosynthetic enzymes, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). The constitutive expression of an N-terminal truncated Hevea brasiliensis HMGR (t-HMGR), lacking the membrane binding domain, enhanced seed HMGR activities by 11-fold, leading to increases in total seed sterol of 2.4-fold. Seed-specific expression of t-HMGR enhanced total seed sterol levels by 3.2-fold, to 1.36% dry weight or 3.25% of oil. 4-desmethylsterols were increased by 2.2-fold, whilst certain sterol biosynthetic intermediates, in particular cycloartenol and 24-ethylidene lophenol, also accumulated. The additional sterol in seed tissue was present in the form of fatty acid esters. Constitutive expression of t-HMGR increased leaf phytosterol sterol levels by 10-fold, representing 1.8% dry weight, and the sterol was sequestered, in acyl ester form, as cytoplasmic 'oil droplets'. These studies establish HMGR as a key enzyme controlling overall flux into the sterol biosynthesis pathway in seed tissue, but the accumulation of certain intermediates suggests additional slow steps in the pathway. The expression of an N-truncated HMGR activity has generated novel phytosterol-enriched raw materials that may provide the basis of new sourcing opportunities for this important class of cholesterol-lowering actives.     

Sterol C-24 methyltransferase type 1 controls the flux of carbon into sterol biosynthesis in tobacco seed

The first committed step in the conversion of cycloartenol into Delta(5) C24-alkyl sterols in plants is catalyzed by an S-adenosyl-methionine-dependent sterol-C24-methyltransferase type 1 (SMT1). We report the consequences of overexpressing SMT1 in tobacco (Nicotiana tabacum), under control of either the constitutive carnation etched ring virus promoter or the seed-specific Brassica napus acyl-carrier protein promoter, on sterol biosynthesis in seed tissue. Overexpression of SMT1 with either promoter increased the amount of total sterols in seed tissue by up to 44%. The sterol composition was also perturbed with levels of sitosterol increased by up to 50% and levels of isofucosterol and campesterol increased by up to 80%, whereas levels of cycloartenol and cholesterol were decreased by up to 53% and 34%, respectively. Concomitant with the enhanced SMT1 activity was an increase in endogenous 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, from which one can speculate that reduced levels of cycloartenol feed back to up-regulate 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and thereby control the carbon flux into sterol biosynthesis. This potential regulatory role of SMT1 in seed sterol biosynthesis is discussed.     

Metabolic engineering of soybean affords improved phytosterol seed traits

Different combinations of three rate‐limiting enzymes in phytosterol biosynthesis, the Arabidopsis thaliana hydroxyl methylglutaryl CoA1 (HMGR1) catalytic subunit linked to either constitutive or seed‐specific β‐conglycinin promoter, and the Glycine max sterol methyltransferase1 (SMT1) and sterol methyltransferase2‐2 (SMT2‐2) genes, under the control of seed‐specific Glycinin‐1 and Beta‐phaseolin promoters, respectively, were engineered in soybean plants. Mature seeds of transgenic plants displayed modest increases in total sterol content, which points towards a tight control of phytosterol biosynthesis. However, in contrast to wild‐type seeds that accumulated about 35% of the total sterol in the form of intermediates, in the engineered seeds driven by a seed‐specific promoter, metabolic flux was directed to Δ⁵‐24‐alkyl sterol formation (99% of total sterol). The engineered effect of end‐product sterol (sitosterol, campesterol, and stigmasterol) over‐production in soybean seeds resulted in an approximately 30% increase in overall sitosterol synthesis, a desirable trait for oilseeds and human health. In contradistinction, increased accumulation of cycloartenol and 24(28)‐methylencylartanol (55% of the total sterol) was detected in plants harbouring the constitutive t‐HMGR1 gene, consistent with the previous studies. Our results support the possibility that metabolic flux of the phytosterol family pathway is differentially regulated in leaves and seeds.     

Is the Reaction Catalyzed by 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase a Rate-Limiting Step for Isoprenoid Biosynthesis in Plants?

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyzes the irreversible conversion of 3-hydroxy-3-methylglutaryl coenzyme A to mevalonate and is considered a key regulatory step controlling isoprenoid metabolism in mammals and fungi. The rate-limiting nature of this enzyme for isoprenoid biosynthesis in plants remains controversial. To investigate whether HMGR activity could be limiting in plants, we introduced a constitutively expressing hamster HMGR gene into tabacco (Nicotiana tabaccum L.) plants to obtain unregulated HMGR activity. The impact of the resulting enzyme activity on the biosynthesis and accumulation of particular isoprenoids was evaluated. Expression of the hamster HMGR gene led to a 3- to 6-fold increase in the total HMGR enzyme activity. Total sterol accumulation was consequently increased 3- to 10-fold, whereas end-product sterols such as sitosterol, campesterol, and stigmasterol were increased only 2-fold. The level of cycloartenol, a sterol biosynthetic intermediate, was increased more than 100-fold. Although the synthesis of total sterols appears to be limited normally by HMGR activity, these results indicate that the activity of one or more later enzyme(s) in the pathway must also be involved in determining the relative accumulation of end-product sterols. The levels of other isoprenoids such as carotenoids, phytol chain of chlorophyll, and sesquiterpene phytoalexins were relatively unaltered in the transgenic plants. It appears from these results that compartmentation, channeling, or other rate-determining enzymes operate to control the accumulation of these other isoprenoid end products.     

Overexpression of Brassica juncea wild-type and mutant HMG-CoA synthase 1 in Arabidopsis up-regulates genes in sterol biosynthesis and enhances sterol production and stress tolerance

Brassica juncea 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) is encoded by four isogenes (BjHMGS1-BjHMGS4). In vitro enzyme assays had indicated that the recombinant BjHMGS1 H188N mutant lacked substrate inhibition by acetoacetyl-CoA (AcAc-CoA) and showed 8-fold decreased enzyme activity. The S359A mutant demonstrated 10-fold higher activity, while the H188N/S359A double mutant displayed a 10-fold increased enzyme activity and lacked inhibition by AcAc-CoA. Here, wild-type and mutant BjHMGS1 were overexpressed in Arabidopsis to examine their effects in planta. The expression of selected genes in isoprenoid biosynthesis, isoprenoid content, seed germination and stress tolerance was analysed in HMGS overexpressors (OEs). Those mRNAs encoding enzymes 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), sterol methyltransferase 2 (SMT2), delta-24 sterol reductase (DWF1), C-22 sterol desaturase (CYP710A1) and brassinosteroid-6-oxidase 2 (BR6OX2) were up-regulated in HMGS-OEs. The total sterol content in leaves and seedlings of OE-wtBjHMGS1, OE-S359A and OE-H188N/S359A was significantly higher than OE-H188N. HMGS-OE seeds germinated earlier than wild-type and vector-transformed controls. HMGS-OEs further displayed reduced hydrogen peroxide (H(2) O(2) )-induced cell death and constitutive expression of salicylic acid (SA)-dependent pathogenesis-related genes (PR1, PR2 and PR5), resulting in an increased resistance to Botrytis cinerea, with OE-S359A showing the highest and OE-H188N the lowest tolerance. These results suggest that overexpression of HMGS up-regulates HMGR, SMT2, DWF1, CYP710A1 and BR6OX2, leading to enhanced sterol content and stress tolerance in Arabidopsis.     

Co-ordinate regulation of sterol biosynthesis enzyme activity during accumulation of sterols in developing rape and tobacco seed

The activities of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, sterol methyl transferase 1 and sterol acyltransferase, key enzymes involved in phytosterol biosynthesis were shown to be co-ordinately regulated during oilseed rape ( Brassica napus L.) and tobacco ( Nicotiana tabacum L.) seed development. In both plants, enzyme activities were low during the initial stages of seed development, increasing towards mid-maturation where they remained stable for a time, before declining rapidly as the oilseeds reached maturity. During seed development, the level of total sterols increased 12-fold in tobacco and 9-fold in rape, primarily due to an increase in steryl ester production. In both seed tissues, stages of maximum enzyme activity coincided with periods of high rates of sterol production, indicating developmental regulation of the enzymes to be responsible for the increases in the sterol content observed during seed development. Consistent with previous studies the data presented suggest that sterol biosynthesis is regulated by two key steps, although there may be others. The first is the regulation of carbon flux into the isoprenoid pathway to cycloartenol. The second is the flux from cycloartenol to Delta(5)-end-product sterols. The implications of the results in terms of enhancing seed sterol levels by genetic modification are also discussed.     

Biochemical and physiological studies of Arabidopsis thaliana transgenic lines with repressed expression of the mitochondrial pyruvate dehydrogenase kinase

Pyruvate dehydrogenase kinase (PDHK), a negative regulator of the mitochondrial pyruvate dehydrogenase complex (mtPDC), plays a pivotal role in controlling mtPDC activity, and hence, the TCA cycle and cell respiration. Previously, the cloning of a PDHK cDNA from Arabidopsis thaliana and the effects of constitutively down-regulating its expression on plant growth and development has been reported. The first detailed analyses of the biochemical and physiological effects of partial silencing of the mtPDHK in A. thaliana using antisense constructs driven by both constitutive and seed-specific promoters are reported here. The studies revealed an increased level of respiration in leaves of the constitutive antisense PDHK transgenics; an increase in respiration was also found in developing seeds of the seed-specific antisense transgenics. Both constitutive and seed-specific partial silencing of the mtPDHK resulted in increased seed oil content and seed weight at maturity. Feeding 3-(14)C pyruvate to bolted stems containing siliques (constitutive transgenics), or to isolated siliques or immature seeds (seed-specific transgenics) confirmed a higher rate of incorporation of radiolabel into all seed lipid species, particularly triacylglycerols. Neither constitutive nor seed-specific partial silencing of PDHK negatively affected overall silique and seed development. Instead, oil and seed yield, and overall plant productivity were improved. These findings suggest that a partial reduction of the repression of the mtPDC by antisense PDHK expression can alter carbon flux and, in particular, the contribution of carbon moieties from pyruvate to fatty acid biosynthesis and storage lipid accumulation in developing seeds, implicating a role for mtPDC in fatty acid biosynthesis in seeds.     

Regulation of partitioned sterol biosynthesis in Saccharomyces cerevisiae.

Using yeast strains with null mutations in structural genes which encode delta-aminolevulinic acid synthetase (HEM1), isozymes of 3-hydroxy-3-methylglutaryl coenzyme A (HMG1 and HMG2), squalene epoxidase (ERG1), and fatty acid delta 9-desaturase (OLE1), we were able to determine the effect of hemes, sterols, and unsaturated fatty acids on both sterol production and the specific activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) in Saccharomyces cerevisiae. We found that the HMGR isozymes direct essentially equal amounts of carbon to the biosynthesis of sterols under heme-competent conditions, despite a huge disparity (57-fold) in the specific activities of the reductases. Our results demonstrate that palmitoleic acid (16:1) acts as a rate-limiting positive regulator and that ergosterol acts as a potent inhibitor of sterol production in strains which possess only the HMGR1 isozyme (HMG1 hmg2). In strains which contain only the HMGR2 isozyme (hmg1 HMG2), sterol production was inhibited by oleic acid (18:1) and to a lesser degree by ergosterol. The specific activities of the two reductases (HMGR1 and HMGR2) were found to be differentially regulated by hemes but not by ergosterol, palmitoleic acid, or oleic acid. The disparate effects of unsaturated fatty acids and sterols on these strains lead us to consider the possibility of separate, compartmentalized isoprenoid pathways in S. cerevisiae.     

Sterol methyltransferase 1 controls the level of cholesterol in plants

The side chain in plant sterols can have either a methyl or ethyl addition at carbon 24 that is absent in cholesterol. The ethyl addition is the product of two sequential methyl additions. Arabidopsis contains three genes-sterol methyltransferase 1 (SMT1), SMT2, and SMT3-homologous to yeast ERG6, which is known to encode an S-adenosylmethionine-dependent C-24 SMT that catalyzes a single methyl addition. The SMT1 polypeptide is the most similar of these Arabidopsis homologs to yeast Erg6p. Moreover, expression of Arabidopsis SMT1 in erg6 restores SMT activity to the yeast mutant. The smt1 plants have pleiotropic defects: poor growth and fertility, sensitivity of the root to calcium, and a loss of proper embryo morphogenesis. smt1 has an altered sterol content: it accumulates cholesterol and has less C-24 alkylated sterols content. Escherichia coli extracts, obtained from a strain expressing the Arabidopsis SMT1 protein, can perform both the methyl and ethyl additions to appropriate sterol substrates, although with different kinetics. The fact that smt1 null mutants still produce alkylated sterols and that SMT1 can catalyze both alkylation steps shows that there is considerable overlap in the substrate specificity of enzymes in sterol biosynthesis. The availability of the SMT1 gene and mutant should permit the manipulation of phytosterol composition, which will help elucidate the role of sterols in animal nutrition.     

Loss of function of 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 (HMG1) in Arabidopsis leads to dwarfing, early senescence and male sterility, and reduced sterol levels

3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the first committed step in the cytosolic isoprenoid biosynthesis pathway in higher plants. To understand the contribution of HMGR to plant development, we isolated T-DNA insertion mutants for HMG1 and HMG2. The hmg1 and hmg2 mutants were both more sensitive than the wild type (WT) to lovastatin, an inhibitor of HMGR. The hmg2 mutant showed no visible phenotype under normal growth conditions. In contrast, the hmg1 mutant exhibited dwarfing, early senescence, and sterility. Expression of senescence-associated genes 12 (SAG12), a marker gene for senescence, was induced in the hmg1 mutant at an earlier stage than in the WT. Levels of trans-cytokinins--hormones known to inhibit senescence--were not lower in hmg1. The mutant did not have the typical appearance of brassinosteroid (BR)-deficient mutants, except for a dwarf phenotype, because of the suppression of cell elongation. The expression of several genes involved in cell elongation was suppressed in hmg1. WT plants treated exogenously with inhibitors of sterol biosynthesis had similar gene expression and sterility characteristics as the hmg1 mutants. Pleiotropic phenotypes were rescued by feeding with squalene, the precursor of sterols and triterpenoids. The sterol levels in hmg1 mutants were lower than in the WT. These findings suggest that HMG1 plays a critical role in triterpene biosynthesis, and that sterols and/or triterpenoids contribute to cell elongation, senescence, and fertility.     

Up-regulation of an N-terminal truncated 3-hydroxy-3-methylglutaryl CoA reductase enhances production of essential oils and sterols in transgenic Lavandula latifolia

Spike lavender ( Lavandula latifolia ) essential oil is widely used in the perfume, cosmetic, flavouring and pharmaceutical industries. Thus, modifications of yield and composition of this essential oil by genetic engineering should have important scientific and commercial applications. We generated transgenic spike lavender plants expressing the Arabidopsis thaliana HMG1 cDNA, encoding the catalytic domain of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR1S), a key enzyme of the mevalonic acid (MVA) pathway. Transgenic T₀ plants accumulated significantly more essential oil constituents as compared to controls (up to 2.1- and 1.8-fold in leaves and flowers, respectively). Enhanced expression of HMGR1S also increased the amount of the end-product sterols, β-sitosterol and stigmasterol (average differences of 1.8- and 1.9-fold, respectively), but did not affect the accumulation of carotenoids or chlorophylls. We also analysed T₁ plants derived from self-pollinated seeds of T₀ lines that flowered after growing for 2 years in the greenhouse. The increased levels of essential oil and sterols observed in the transgenic T₀ plants were maintained in the progeny that inherited the HMG1 transgene. Our results demonstrate that genetic manipulation of the MVA pathway increases essential oil yield in spike lavender, suggesting a contribution for this cytosolic pathway to monoterpene and sesquiterpene biosynthesis in leaves and flowers of the species.     

Expression of the Hevea brasiliensis (H.B.K.) Mull. Arg. 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase 1 in Tobacco Results in Sterol Overproduction

A genomic fragment encoding one (HMGR1) of the three 3-hydroxy-3-methylglutaryl coenzyme A reductases (HMGRs) from Hevea brasiliensis (H.B.K.) Mull. Arg. (M.-L. Chye, C.-T. Tan, N.-H. Chua [1992] Plant Mol Biol 19: 473-484) was introduced into Nicotiana tabacum L. cv xanthi via Agrobacterium transformation to study the influence of the hmg1 gene product on plant isoprenoid biosynthesis. Transgenic plants were morphologically indistinguishable from control wild-type plants and displayed the same developmental pattern. Transgenic lines showed an increase in the level of total sterols up to 6-fold, probably because of an increased expression level of hmg1 mRNA and a corresponding increased enzymatic activity for HMGR, when compared with the level of total sterols from control lines not expressing the hmg1 transgene. In addition to the pathway end products, campesterol, sitosterol, and stigmasterol, some biosynthetic intermediates such as cycloartenol also accumulated in transgenic tissues. Most of the overproduced sterols were detected as steryl-esters and were likely to be stored in cytoplasmic lipid bodies. These data strongly support the conclusion that plant HMGR is a key limiting enzyme in phytosterol biosynthesis.     

The ubiquitin-proteasome pathway mediates the regulated degradation of mammalian 3-hydroxy-3-methylglutaryl-coenzyme A reductase

3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), the key regulatory enzyme in the mevalonate (MVA) pathway, is rapidly degraded in mammalian cells supplemented with sterols or MVA. This accelerated turnover was blocked by N-acetyl-leucyl-leucyl-norleucinal (ALLN), MG-132, and lactacystin, and to a lesser extent by N-acetyl-leucyl-leucyl-methional (ALLM), indicating the involvement of the 26 S proteasome. Proteasome inhibition led to enhanced accumulation of high molecular weight polyubiquitin conjugates of HMGR and of HMGal, a chimera between the membrane domain of HMGR and beta-galactosidase. Importantly, increased amounts of polyubiquitinated HMGR and HMGal were observed upon treating cells with sterols or MVA. Cycloheximide inhibited the sterol-stimulated degradation of HMGR concomitantly with a marked reduction in polyubiquitination of the enzyme. Inhibition of squalene synthase with zaragozic acid blocked the MVA- but not sterol-stimulated ubiquitination and degradation of HMGR. Thus, similar to yeast, the ubiquitin-proteasome pathway is involved in the metabolically regulated turnover of mammalian HMGR. Yet, the data indicate divergence between yeast and mammals and suggest distinct roles for sterol and nonsterol metabolic signals in the regulated ubiquitination and degradation of mammalian HMGR.     

Sterol composition and growth of transgenic tobacco plants expressing type-1 and type-2 sterol methyltransferases

Transgenic tobacco (Nicotiana tabacum L.) plants with altered sterol composition were generated by transformation with plant cDNAs encoding type-1 and type-2 sterol methyltransferases (SMTs; EC 2.1.1.41). For both SMT1 and SMT2 transformants, the transformation was associated with a reduction in the level of cholesterol, a non-alkylated sterol. In SMT1 transformants a corresponding increase of alkylated sterols, mainly 24-methyl cholesterol, was observed. On the other hand, in SMT2 transformants the level of 24-methyl cholesterol was reduced, whereas the level of sitosterol was raised. No appreciable alteration of total sterol content was observed for either genotype. The general phenotype of transformants was similar to that of controls, although SMT2 transformants displayed a reduced height at anthesis. The results show that plant sterol composition can be altered by transformation with an SMT1 cDNA without adverse effects on growth and development, and provide evidence, in planta, that SMT1 acts at the initial step in sterol alkylation. Received: 27 June 2000 / Accepted: 22 July 2000     

Effects of a novel lanosterol 14 alpha-demethylase inhibitor on the regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in Hep G2 cells.

A 32-carboxylic acid derivative of lanosterol (SKF 104976) was found to be a potent inhibitor of lanosterol 14 alpha-demethylase (14 alpha DM). 14 alpha DM activity in a Hep G2 cell extract was inhibited 50% by 2 nM SKF 104976. Exposure of intact cells to similar concentrations of the compound resulted in the inhibition of incorporation of [14C]acetate into cholesterol with concomitant accumulation of lanosterol as well as a 40-70% decrease in 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) activity. SKF 104976 did not effect low density lipoprotein uptake and degradation in Hep G2 cells, suggesting that HMGR and low density lipoprotein receptor activity were not coordinately regulated under these conditions. Reduction of the flux of carbon units in the sterol synthetic pathway by as much as 80% did not alter the suppressing effect of SKF 104976 on HMGR activity. However, under conditions where sterol synthesis was almost completely blocked by lovastatin, HMGR activity was not suppressed by SKF 104976. Mevalonate, at concentrations that did not decrease HMGR activity, was able to restore the inhibiting effect of SKF 104976 on HMGR activity. The rapid inhibition (2-3 h) of HMGR activity by SKF 104976 to 30-60% of the level in controls was not dependent on the initial amount of HMGR enzyme present. These findings suggest that upon inhibition of 14 alpha DM by SKF 104976, a mevalonate-derived precursor regulates HMGR activity, even when the sterol synthetic rate is considerably reduced and when HMGR protein levels are very high. In Hep G2 cells, formation of oxylanostenols from [3H]mevalonate reached a maximum between 1 and 10 nM SKF 104976 and was negligible at higher concentrations. This result suggests that oxylanostenols are not the key mediators of the modulation of HMGR in Hep G2 cells upon 14 alpha DM inhibition.     

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.     

Steroid profiles of transgenic tobacco expressing an Actinomyces 3-hydroxysteroid oxidase gene

Previously, we have shown that the expression of a 3-hydroxysteroid-oxidase gene in transgenic tobacco initiated a series of biochemical events leading to the conversion of sterol to stanol. As a result, the plants maintained a diminished sterol pool and a modified relative sterol ratio but demonstrated no observable morphological abnormalities. The maintenance of normal higher plant physiology in the absence of particular sterols or in the presence of modified sterol ratios is controversial. In this report, we present additional biochemical and physiological characteristics of transgenic tobacco expressing an Actinomyces 3-hydroxysteroid-oxidase gene. The total steroid accumulated in the transgenic plants is 6-fold higher than in control plants and consists of sterol, 3-ketosteroid and stanol. The relative abundance of sterols within whole plant and individual organs is grossly altered as ethylated side chain sterols account for 99% of the total sterol pool in the transgenic tobacco. Stigmasterol is readily apparent in all tissues and cholesterol is found at measurable levels in specific organs, while campesterol and sitosterol are detected at trace levels in the transgenic plants. Stanols and 3-ketosteroids accumulate in all tissues and represent 77% of the measurable steroid pool in the transgenic plants. The sum of sterol, the respective 3-ketosteroid plus stanol provide a relative abundance of steroid, which is similar to the abundance of sterol accumulated in control tissue. In vitro photosynthetic electron transport measurements demonstrate altered activity of chloroplasts under a variety of reaction conditions, indicating a link between the modified steroid pool and a modulation of chloroplast membrane function.