Triacylglycerol biosynthesis and gene expression in microspore-derived cell suspension cultures of oilseed rape

developing seeds of oilseed rape is fundamental to the rational development of biotechnological strategies for The effect of sucrose concentration on triacylglycerol increasing seed oil content and manipulating oil composibiosynthesis and associated gene expression was tion. In oilseeds, the biosynthesis of TAG in developing examined in a microspore-derived cell suspension culseeds is catalysed by the sequential addition of fatty acyl ture of oilseed rape (Brassica napus L. cv. Jet Neuf). groups to a glycerol backbone derived from sn-glycerol The triacylglycerol content of the cells increased about 3-phosphate (Stymne and Stobart, 1987; Stobart and 5-fold on a fresh weight basis when the sucrose conStymne, 1990). Diacylglycerol acyltransferase (DGAT; centration in the growth medium was raised from 2% EC 2.3.1.20) catalyses the acylation of sn-1, to 22% (w/v). The specific activity of microsomal 2-diacylglycerol to yield TAG (Stymne and Stobart, diacylglycerol acyltransferase (EC 2.3.1.20) and its 1987). In developing seeds of Brassica napus L., maximum activity per unit fresh weight increased about 2.5-fold DGAT activity occurs during the rapid phase of oil and 6-fold, respectively, when sucrose concentration accumulation (Tzen et al., 1993; Weselake et al., 1993a). was increased from 2% to 14%. mRNA encoding the The enzyme may also exert significant flux control in major oleosin also appeared to increase in abundance TAG bioassembly (Perry and Harwood, 1993a, b). The over the 2–14% sucrose concentration range when enzymes of TAG bioassembly are associated with the RNA fractions were analysed by the reverse transcripendoplasmic reticulum (ER) (Stobart et al., 1986). tion-polymerase chain reaction. The sucrose-mediated The apparently hydrophobic and labile nature of these increases in diacylglycerol acyltransferase activity and enzymes appears to have impeded progress on their oleosin mRNA indicated that the cell suspension could purification and characterization (Gibson et al., 1994; be a useful research tool for the identification of Little et al., 1994; Eccleston and Harwood, 1995; Kocsis cDNAs encoding triacylglycerol biosynthetic enzymes et al., 1996). TAG accumulates in developing seeds of B. and associated proteins. napus as small droplets, termed oil bodies, which are incapsulated by a single layer of phospholipid (Murphy,