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

Abstract

SummaryDietary 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.

The genetic basis of foliar terpene yield: Implications for breeding and profitability of Australian essential oil crops

“…For example, Arabidopsis transformed to express an additional copy of hmgr showed a large increase in phytosterols (2.4 fold) (Enfissi et al 2005). The importance of hmgr is confirmed by other studies in Arabidopsis which demonstrate that transforming plants to express a different version of hmgr can lead to large increases in phytosterols as well as cycloartenol by 2-10 fold (Chappell et al 1995;Harker et al 2003). This suggests that hmgr is a key regulatory step in the MVA pathway.…”

Biosynthesis of Phytosterol Esters: Identification of a Sterol O-Acyltransferase in Arabidopsis

“…SEs can be found in lipid bodies in the cytoplasm and are present at a substantial level in seeds. In canola (Brassica napus), for example, phytosterols constitute about 0.5% of seed oil (Sabir et al, 2003), 35% of which is in the form of SEs (Harker et al, 2003). The biochemical process of sterol acylation is believed to play a role in maintaining the free sterol content of cell membranes at their physiological levels (Schaller, 2004).…”

“…However, limited quantities of phytosterols are currently a major barrier in satisfying the demands for such functional foods. Based on a firmly established concept that 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) plays a rate-limiting role in the flux control of sterol biosynthesis (Bach, 1986;Gondet et al, 1992Gondet et al, , 1994Chappell et al, 1995), efforts aimed at increasing phytosterol content have been concentrated on enhancing the activity of HMGR through introducing modified HMGR that is resistant to regulation (Chappell et al, 1995;Re et al, 1995;Schaller et al, 1995;Harker et al, 2003;Hey et al, 2006). Nonetheless, evidence suggesting the existence of other regulatory step(s) in the flux control of sterol biosynthesis can be found in some studies, particularly that of Gondet et al (1994) who studied a steroloverproducing tobacco (Nicotiana tabacum) mutant LAB1-4, with HMGR levels approximately 3-fold higher than normal (Gondet et al, 1994).…”

Multilevel Control of Arabidopsis 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase by Protein Phosphatase 2A

“…Whereas transcriptional modulation of HMGR has been demonstrated in many plant systems, evidence of posttranslational regulation is still scarce (Rodríguez-Concepció n et al, 2011). The membrane domain of plant HMGR exerts negative regulation on the catalytic domain, thus limiting phytosterol biosynthesis (Harker et al, 2003). Arabidopsis HMGR1 can be phosphorylated in cell-free extracts by Brassica oleracea and Spinacea oleracea SnF1-related kinase (SnRK1) activity at a conserved Ser residue (Ser-577 in HMGR1S) (Dale et al, 1995;Sugden et al, 1999).…”

“…In fact, higher phytosterol levels were obtained in tobacco (Nicotiana tabacum) seeds when a variant of Arabidopsis HMGR1S without the phosphorylation site was constitutively expressed (Hey et al, 2006). However, the gain in seed phytosterol content was only one-tenth of that obtained in seeds or leaves that constitutively expressed a single copy of the HMGR catalytic domain (Harker et al, 2003). Thus, present evidence indicates that negative regulation of plant HMGR activity relies more on the presence of the N-terminal domain than on phosphorylation at the conserved Ser residue.…”