Plant Biotechnology Journal (2006) 4, pp. 219–229 doi: 10.1111/j.1467-7652.2005.00174.x © 2005 Blackwell Publishing Ltd 219 Blackwell Publishing, Ltd. Oxford, UK PBI Plant Biotechnology Journal 1467-7644 © 2005 Blackwell Publishing Ltd ? 2005 2 ? Original Article Enhanced seed phytosterol accumulation Sandra J. Hey et al. Enhanced seed phytosterol accumulation through expression of a modified HMG-CoA reductase Sandra J. Hey 1 , Stephen J. Powers 2 , Michael H. Beale 1 , Nathaniel D. Hawkins 1 , Jane L. Ward 1 and Nigel G. Halford 1, * 1 Crop Performance and Improvement, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK 2 Agriculture and the Environment, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK Summary The regulation of phytosterol biosynthesis in seeds is of interest to biotechnologists because of the efficacy of dietary phytosterols in reducing blood cholesterol in humans. Mevalonate synthesis via 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) is a key step in phytosterol biosynthesis. HMG-CoA reductase is inactivated by phosphorylation by SNF1-related protein kinase 1 (SnRK1). With the aim of increasing seed phytosterol levels, transgenic tobacco plants were produced expressing a full-length Arabidopsis (Arabidopsis thaliana) HMG-CoA reductase gene (HMG1) coding sequence, a modified HMG1 sequence encoding a protein lacking the target serine residue for phosphorylation by SnRK1, or a chimaeric sequence encoding the N-terminal domain of the Arabidopsis HMG1 enzyme fused with the catalytic domain of yeast HMG-CoA reductase, which lacks an SnRK1 target site. All three transgenes (35S-AtHMG1, 35S-AtHMG1m and 35S-AtScHMG1) were under the control of a cauliflower mosaic virus 35S RNA promoter. Levels of seed phytosterols were up to 2.44-fold higher in plants transformed with the 35S-AtHMG1m gene than in the wild-type, and were significantly higher than in plants expressing 35S-AtHMG1 or 35S-AtScHMG1. In contrast, levels of phytosterols in leaves of plants transformed with the 35S-AtHMG1m gene were unchanged, suggesting that regulation of HMG-CoA reductase by SnRK1 is an important factor in seeds but not in leaves. A total of 11 independent transgenic lines expressing 35S-AtHMG1m or 35S-AtScHMG1 also showed an altered flower phenotype, comprising a compact floret, prolonged flowering, short, pale petals, a protruding style, short stamens, late anther development, little or no pollen production, premature flower abscission and poor seed set. Because of this phenotype, the modified HMG-CoA reductase gene would have to be expressed seed specifically if it were to be engineered into a crop plant for biotechnological purposes. Received 17 November 2005; revised 6 October 2005; accepted 7 October 2005 *Correspondence (fax (44) (0) 1582 763 010; e-mail nigel.halford@bbsrc.ac.uk) Keywords: Arabidopsis, isoprenoids, metabolic engineering, metabolite signalling, phosphorylation, phytosterols, SnRK1, tobacco. Introduction The isoprenoids are a large family of compounds derived from the five-carbon unit, isopentenyl diphosphate (IPP). In plants, they include phytosterols, some plant hormones, components of photosynthetic pigments, phytoalexins and a variety of other specialized compounds, including the fat-soluble vitamins E and K. The phytosterols have attracted particular attention from biotechnologists because the predominant naturally occurring phytosterols are structurally related to cholesterol (Figure 1). These compounds, which include β-sitosterol, campesterol and stigmasterol, competitively inhibit the uptake of cholesterol from the small intestine in humans (Westrate and Meijer, 1998). In animals and yeast, IPP is derived from acetyl-CoA via a bio- synthetic pathway in which mevalonic acid is an intermediate (Figure 1). The NADPH-dependent reduction of 3-hydroxy-3- methylglutaryl-coenzyme A (HMG-CoA) to mevalonic acid is the overall rate-limiting step for the whole isoprenoid biosynthetic pathway, and HMG-CoA reductase is the enzyme which