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 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.

Figure 1

Schematic representation of the sterol pathway post-squalene. Solid lines indicate that one enzymatic step is involved in the conversion and dashed lines indicate the involvement of more than one enzyme.

Figure 2

Binary plant transformation vector pNH7 and pNH19 holding the CERV-Ntsmt1-1-nopaline synthase (NOS) and ACP-Ntsmt1-1-NOS expression cassettes, respectively. LB, Left border; RB, right border. Arrows indicate the orientation of the Ntsmt1-1 and the hygromycine resistance genes. The size bar indicates 1,000 bp.

Figure 3

The SMT1 activity of T₀ transgenic and control lines. Transgenic lines are indicated in black staples and control in white staples. The average activities of four independent tobacco SR1 control lines are given and the sds are indicated with error bars. A, The SMT1 activity in expanding leaves transformed with pNH7 (CERV-Ntsmt1-1-nos). B, The SMT1 activity in seeds collected 17 d after anthesis of tobacco transformed with pNH7. C, The SMT1 activity in seeds collected 17 d after anthesis of tobacco transformed with pNH19 (ACP-Ntsmt1-1-nos).

Figure 4

A, Endogenous HMGR activity in the seed of T₁ transgenic tobacco lines NH7:27 (CERV-Ntsmt1-1-nos) and NH19:27 (ACP-Ntsmt1-1-nos) collected 14 d after anthesis. The average HMGR activity of SR1 tobacco was calculated from the activities of seed samples of four individual plants. The error bar corresponds to the sd. B, Real-time PCR to detect the Ntsmt1-1 and the Nthmgr transcript levels in transgenic seed of NH7:27 and NH19:27.