Summary• Metabolic control analysis allows the study of metabolic regulation. We applied both singleand double-manipulation top-down control analysis to examine the control of lipid accumulation in developing oilseed rape (Brassica napus) embryos.• The biosynthetic pathway was conceptually divided into two blocks of reactions (fatty acid biosynthesis (Block A), lipid assembly (Block B)) connected by a single system intermediate, the acyl-coenzyme A (acyl-CoA) pool. Single manipulation used exogenous oleate. Triclosan was used to inhibit specifically Block A, whereas diazepam selectively manipulated flux through Block B.• Exogenous oleate inhibited the radiolabelling of fatty acids from [1-14 C]acetate, but stimulated that from [U-14 C]glycerol into acyl lipids. The calculation of group flux control coefficients showed that c. 70% of the metabolic control was in the lipid assembly block of reactions. Monte Carlo simulations gave an estimation of the error of the resulting group flux control coefficients as 0.27 ± 0.06 for Block A and 0.73 ± 0.06 for Block B.• The two methods of control analysis gave very similar results and showed that Block B reactions were more important under our conditions. This contrasts notably with data from oil palm or olive fruit cultures and is important for efforts to increase oilseed rape lipid yields.

In plants, fatty acid and complex lipid synthesis requires the correct spatial and temporal activity of many gene products. Quantitative northern analysis showed that mRNA for the biotin carboxylase subunit of heteromeric acetyl-coenzyme A carboxylase, fatty acid synthase components (3-oxoacyl-acyl carrier protein [ACP] reductase, enoyl-ACP reductase, and acyl-ACP thioesterase), and stearoyl-ACP desaturase accumulate in a coordinate manner during Brassica napus embryogenesis. The mRNAs were present in a constant molar stoichiometric ratio. Transcript abundance of mRNAs for the catalytic proteins was found to be similar, whereas the number of ACP transcripts was approximately 7-fold higher. The peak of mRNA accumulation of all products was between 20 and 29 d after flowering; by 42 d after flowering, the steady-state levels of all transcripts fell to about 5% of their peak levels, which suggests that the mRNAs have similar stability and kinetics of synthesis. Biotin carboxylase was found to accumulate to a maximum of 59 fmol mg Ϫ1 total RNA in embryos, which is in general agreement with the value of 170 fmol mg Fatty acids are synthesized by a common biochemical pathway in all organisms. In plants, de novo synthesis takes place in plastids, using two enzyme systems: acetyl-CoA carboxylase (ACCase) and fatty acid synthase (FAS). The type II FAS, of plants, is composed of separate soluble enzymes that each carry out a single enzymatic reaction (Caughey and Kekwick, 1982; Hoj and Mikkelsen, 1982;Shimakata and Stumpf, 1982) with the growing acyl chains attached to acyl carrier protein (ACP). This is in contrast to the arrangement in animals and yeast where the enzyme activities and ACP are located on one or two multifunctional polypeptides (type I FAS).Acetyl-CoA is carboxylated to malonyl-CoA in plastids of non-graminaceous plants by a heteromeric ACCase, which is encoded by four subunits (Sasaki et al., 1993(Sasaki et al., , 1995. The ␣-carboxyltransferase (␣-CT) polypeptide is encoded in the plastid genome (Sasaki et al., 1993), whereas the ␤-carboxyltransferase (␤-CT), biotin carboxyl carrier protein (BCCP) and biotin carboxylase (BC) subunits are nuclear encoded. MalonylCoA undergoes a thiol-exchange reaction carried out by malonyl-CoA:ACP transacylase (MCAT) to form malonyl-ACP, before condensation with acetyl-CoA. The initial condensation reaction, catalyzed by ketoacyl-ACP synthase (KAS) III, is unique in that malonyl-ACP reacts with acetyl-CoA. Subsequent condensations add two carbon units from malonyl-ACP to the saturated acyl-ACP chain by the action of KAS isoforms I and II. The 3-oxo group is sequentially reduced to a methylene group by the action of 3-oxoacyl-ACP reductase (KR), hydroxyacyl-ACP dehydrase (DH), and enoyl-ACP reductase (ENR) via hydroxyl and enoyl intermediates, before a further condensation reaction takes place.When the acyl chain is 16 or 18 carbons long, several possible reactions occur in plastids. The saturated acyl-ACP may have a double bond introduced, between carbons 9 and 10, by...

We have generated antisense plants of Brassica napus, targeting either the cytoplasmic Type I acetyl-CoA carboxylase or the p-ketoacyl-acyl carrier protein reductase of the Type I1 dissociable fatty acid synthase, in order to investigate the importance of these components in regulating lipid metabolism in plants. Using a range of down-regulated plants, it has become clear that down-regulation of these genes also causes down-regulation of other components of lipid metabolism, at the activity, translational and transcriptional levels. These plants exhibit effects, not immediately predicted, on oil yield and carbon resource allocation in seeds.

Brassica napus cv Westar plants were transformed with 3-oxoacyl-ACP reductase (KR) in antisense orientation, driven by either the cauliflower mosaic virus 35S promoter or a seed-specific acyl carrier protein promoter to determine the effects on plant productivity and on the activity of other fatty acid synthase (FAS) components. In plants with altered KR activity, total seed yield was reduced in all cases. In less severely affected plant lines, seeds had a normal appearance and composition but the yield of seeds was reduced by approximately 50%. In more severely affected lines, reductions in both seed fatty acid content and the number of seeds produced per plant were evident, resulting in a 90% reduction in fatty acid synthesized per plant. These phenotypes were independent of the promoter used. In severely affected lines, a large proportion of seeds showed precocious germination, and these had a reduced oleate content and increased levels of polyunsaturated 18-carbon fatty acids, compared with normal seeds of the same line. This reduction in 18:1 fatty acids was mimicked on imbibition of seeds with a normal appearance, indicating a preferential use of oleate moieties in precocious germination events. The reduction in activity of KR was mirrored for a second fatty acid synthase component, enoyl-ACP reductase, indicating a mechanism to maintain the ratio of fatty acid synthase components throughout embryogenesis.

In young expanding leaves of Brassica napus, the demand for fatty acids is met by de novo biosynthesis of fatty acid synthase components, as demonstrated by 3-oxoacyl-ACP reductase. Using a novel radio-chemical assay for 3-oxoacyl-ACP reductase and specific antibodies, we have demonstrated a direct relationship between the increase in activity and synthesis of polypeptide. The maximum rate of fatty acid synthesis was between 4 and 7 days post-emergence, but slowed after this point even though 3-oxoacyl-ACP reductase activity was high. Leaf area continued to expand in a linear fashion after reductions in both enzyme activity and the rate of fatty acid synthesis. ß