Regulation of erucic acid accumulation in oilseed rape (Brassica napus L.). Effects of temperature and abscisic acid.

Vegetable oils are an important commodity world-wide with an annual production of about 70 million tonnes. Oilseed rape is one of the four major crops, providing about 10% of the total production. Quality of vegetable oils is determined by the fatty acid composition of the triacylglycerols (TAG) that constitute such oils. These fatty acids comprise a range of chain lengths and desaturated and oxidised residues. A small group of fatty acids dominates the edible oils which are the predominant products, whereas other specific types of fatty acids are used in specialised applications and generally occur in small crops and wild species. One of these fatty acids is erucic acid (22:1), a very long chain monounsaturated fatty acid, which naturally occurs in species of the Brassicaceae. Synthesis of all fatty acids requires a group of enzymes, most of which have been isolated from a number of species. After synthesis of the primary fatty acids, palmitic (16:0), stearic (18:0) and oleic acid (18:1), in the plastids, they are exported as their CoA-esters into the cytoplasm, where modification and incorporation in lipids occurs. This requires another group of enzymes, some of which, like desaturases and acyltransferases, have been isolated and characterised but most of the species specific modifying enzymes have not been isolated yet. However, little is known about the mechanisms that regulate and coordinate the expression and activity of these enzymes. This thesis focuses on the accumulation of 22:1 and the regulation of the elongase synthesising this fatty acid from 18:1. It has been reported that 22:1 levels can be influenced by growth temperature (Canvin, 1965) but little was known about the regulatory mechanism and timing of this influence. We compared the accumulation of oil and 22:1 in seeds of different cultivars at two temperatures, 15 and 25°C. It was shown that the level of 22:1 in seeds increases from 30 to 40 mol% with a temperature decrease from 25 to 15°C in only one cultivar, Reston, whereas Gulle was shown to be insensitive to changes in temperature. In these experiments we also showed that growth temperature exerts its effect only during the time of maximum oil synthesis, not before or after. Similar experiments showed that microspore-derived embryos (MDEs), grown in vitro, followed a similar pattern of oil accumulation and timing of temperature influence as described for seeds on the intact plant. However, the absolute levels of oil and 22:1 were much lower in MDEs than in seeds and the fraction 22:1 of total fatty acids was reduced by about 10 mol% in MDEs. Analysis of the levels of abscisic acid (ABA) in developing seeds together with the low levels of this plant growth regulator in MDEs suggested that ABA may be an important factor in determining the level of 22:1 in the oil of oilseed rape. However, dose response curves for ABA in MDEs grown at 15 and 25°C showed that the sensitivity to ABA is not influenced by culture temperature. At both temperatures a 50% response was observed at about 0.3 µM and the increase in 22:1 was about 10 mol% at saturating ABA levels. We also found that ABA levels in seeds are saturating at both temperatures, implying that ABA cannot be an intermediate in the transduction of a temperature signal. In addition, statistical analysis of temperature and ABA effects in MDEs showed no significant interaction between these two stimuli. This was further confirmed by the fact that, absolute amounts of both oil and 22:1 increased upon addition of ABA, whereas with temperature only affected the fraction of 22:1 and not the total amount of oil in MDEs. We also studied the effects of a group of demethyl-ABA analogs. In this study we found that the T-methyl group is very important for ABA activity. Removal of this group resulted in a 100-fold increase in the amount of the (+)-enantiomer needed to induce a similar increase in 22:1 accumulation as compared to natural (+)-ABA while a complete loss of activity was observed for (-)-7'-demethyl-ABA. The function of the 8'and 9'-methyl groups is less clear. Removal of these groups resulted in a partial reduction in ABA activity, but the effects were different for total fresh weight accumulation and 22:1 levels. This suggests that at least two types of ABA receptors operate in MDEs. Changes in 22:1 level in the oil must be caused by changes in the enzyme activities catalysing oil biosynthesis. Holbrook et al. (1992) had already shown that the addition of ABA to culture medium resulted in an increase in elongase activity, but little was known about the effect of temperature on elongase activity. We elaborated on the effects of ABA application on elongase activity in MDEs at two different temperatures, 15 and 25°C. We found higher total elongase activities in MDEs grown at 15°C, but temperature sensitivity and effect of 18:1 -CoA concentration were not affected. The differences in total activity correlated closely with the differences observed in 22:1 amount in the MDEs, suggesting that the amount of 22:1 is regulated by the total amount of elongase activity. The correlation between elongase activity and the fraction 22:1 in the oil was lower and no correlation was found between acyltransferase activity and the amount of oil accumulated. It was not possible to properly determine the kinetic parameters K m and V max of the elongase complex due to rapidly loss of activity upon isolation from the membrane and to substrate inhibition by 18:1-CoA at relatively low concentrations (about 10 µM). This inhibition by 18:1-CoA is apparently caused by a detergent effect of this compound disrupting the membrane in which the elongase complex is embedded. Based on these observations a model was formulated to describe the regulation of 22:1 accumulation and oil composition in oilseed rape. The total amount of oil is regulated by the activity of fatty acid synthase (FAS), synthesising fatty acids from acetyl-CoA, derived from carbohydrates imported into the embryo. The main product of FAS, oleic acid, is exported from the plastid and enters the cytoplasm as 18:1-CoA. The pool of 18:1-CoA is modified by desaturases and elongase transforming part of it into other fatty acids like 18:2 and 22:1. Subsequently, the fatty acids in this pool are incorporated in TAG. The acyltransferases performing two out of three of these reactions have little or no selectivity for the various fatty acids and therefore the fatty acid composition of the oil largely reflects the fatty acid composition of the acyl-CoA pool. The fraction of 22:1 in this pool is determined by the relative activity of the elongase in comparison to the total flux of fatty acids through the pool. With the knowledge that elongase activity regulates the amount of 22:1, it must be possible to increase the level of 22:1 in the oil beyond the level of about 55% observed so far (Scarth et a/., 1995). Selectivity of the second acyltransferase, excluding 22:1 from the sn-2 position of TAG, which would limit the maximum 22:1 level to 67%, has been circumvented by transformation of oilseed rape with an acyltransferase that can incorporate 22:1 at this position (Lassner et al., 1995). Whether it will be possible to increase the level of 22:1 in the oil of oilseed rape up to 90 or 100% of fatty acids, greatly increasing the value of rapeseed oil for industrial purposes, will depend upon the effects of increased 22:1 levels on lipid metabolism.