Sterol C-24 Methyltransferase Type 1 Controls the Flux of Carbon into Sterol Biosynthesis in Tobacco Seed

Abstract

The first committed step in the conversion of cycloartenol into ⌬ 5 C24-alkyl sterols in plants is catalyzed by an S-adenosylmethionine-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.

Sterol Side Chain Reductase 2 Is a Key Enzyme in the Biosynthesis of Cholesterol, the Common Precursor of Toxic Steroidal Glycoalkaloids in Potato

“…The presence of the alkyl group at C-24 that originates from this reaction is the structural characteristic differentiating C-24 alkylsterol from cholesterol. Therefore, cholesterol levels in plants are hypothesized to be modulated by SMT; this hypothesis is supported by the lower cholesterol level in transgenic plants that overexpress SMT and the higher cholesterol level in SMT knockout plants (Diener et al, 2000;Schaeffer et al, 2000;Sitbon and Jonsson 2001;Holmberg et al, 2002;Arnqvist et al, 2003). However, the considerable amounts of cholesterol and its derivatives, SGAs, in solanaceous plants have suggested that these plants have a mechanism to produce cholesterol that is distinct from other plants.…”

“…In plants, cycloartenol is the first D 24(25) -sterol. If cycloartenol is reduced to cycloartanol by SSR2, the biosynthetic pathway would be efficiently redirected toward cholesterol (Figure 1) (Diener et al, 2000;Schaeffer et al, 2000;Sitbon and Jonsson 2001;Holmberg et al, 2002;Arnqvist et al, 2003). We conducted in vitro assays to examine whether SSR2 has cycloartenol reductase activity and to compare the substrate specificities of SSR1 and SSR2 (Figure 4).…”

Mechanism-based Enzyme Inactivators of Phytosterol Biosynthesis

“…In all plants the critical slow step in phytosterol synthesis is associated with the C-methylation of cycloartenol [24][25][26]. In vascular plants and many fungi such as Pneumocystis carinii, the product of the SMT1 catalyzed reaction can be processed to be the substrate of SMT2 that controls the ratio of 24-methyl to 24-ethyl ∆ 5 -sterols.…”

Lipid Signaling in Plants. Cloning and Expression Analysis of the Obtusifoliol 14α-Demethylase fromSolanum chacoenseBitt., a Pollination- and Fertilization-Induced Gene with Both Obtusifoliol and Lanosterol Demethylase Activity

“…For the upper systemic leaf 6, which corresponds to the position of the leaf assayed in Figure 7, 3 cpm of sterol per milligram of dry weight tissue was recovered from the petiole, which corresponds to 2.4 mg of obtusifoliol per gram of dry weight tissue. Considering that, in plants, obtusifoliol represents from 0.1% to 1% of the total sterol fraction and that total sterols sum up to 2,500 to 4,000 mg per gram of dry weight tissue (Schmitt and Benveniste, 1979;Schaeffer et al, 2001;Holmberg et al, 2002;Schrick et al, 2004), this indicates that a normal physiological concentration of obtusifoliol would be 2.5 to 40 mg per gram of dry weight tissue, suggesting that the observed levels of C14-labeled obtusifoliol detected in leaves 4 and 6 would be in the physiological range of obtusifoliol concentrations.…”

“…Overexpression, down-regulation, and mutant analyses of enzymes involved in sterol biosynthesis before the step leading to sitosterol and campesterol synthesis (SMT1, C24-methyltransferase; CYP51, C14-demethylase; FACKEL, C14-reductase; HYDRA1, D8-D7 sterol isomerase) influence the amount of campesterol and the balance between sitosterol and BRs (Schaller et al, 1998;Jang et al, 2000;Schrick et al, 2000;Kushiro et al, 2001;Sitbon and Jonsson, 2001;Holmberg et al, 2002;Souter et al, 2002). The smt1 mutant also shows a defect in vascular patterning (Carland et al, 2002), and smt1, fk, and hyd1/2 cannot be rescued by exogenous application of BRs.…”