- Top of page
- Materials and Methods
- Results and Discussion
The production of edible as well as some nonedible oils in agricultural crops is of great economic importance (Murphy, 1994). The commercial interest has been enhanced in recent years because of the development of transgenic oil crops (Harwood, 1998). Such crops have been produced in order to provide speciality oils for industrial purposes (Murphy, 1994) as well as to modify traditional crops so that their storage products have more desirable properties (Ohlrogge, 1994; Voelker & Kinney, 2001).
Of the main oil crops in the world today, the oilpalm is the most important plant grown exclusively for oil. Furthermore, oilpalm is extremely productive, producing c. 5–10 t oil ha−1 compared with a yield of 1–3 t oil ha−1 for typical oilseeds (Gunstone et al., 2007). The oilpalm is a tropical crop and is of great importance for Indonesia and, particularly, for Malaysia which exports over 90% of its palm oil products (Ahmad, 2003). It is also the world's major source of edible oil (Gunstone, 2006).
Despite the economic importance of oil crops, as well as the great advances in our knowledge of their basic biochemistry and molecular biology (Harwood, 1996; Ohlrogge & Jaworski, 1997; Murphy, 2005), we still know relatively little about the regulation and/or control of lipid synthesis and accumulation (Ohlrogge & Jaworski, 1997). In fact, it has been remarked that efforts to change metabolism through the manipulation of single genes, often thought, misguidedly, to encode ‘rate-limiting’ steps, has produced disappointing results (Stitt & Sonnewald, 1995). Nevertheless, within plant lipid metabolism there have been a few experiments that provide some information about regulation and control. For example, the importance of acetyl-CoA carboxylase in controlling lipid synthesis in leaves was indicated by the levels of malonyl-CoA and other acyl-thioesters (Post-Beittenmiller et al., 1992; Ohlrogge & Jaworski, 1997) and confirmed by direct measurement of its flux control coefficient (Page et al., 1994). Increasing the activity of this enzyme by genetically transforming potato can also lead to enhanced fatty acid production, as would be predicted (Klaus et al., 2004). In developing oil crops, the relative importance of fatty acid synthesis compared with lipid accumulation has been highlighted by the effect of exogenous fatty acids on lipid synthesis in embryos of Cuphea (Bafor et al., 1990) and other crops (Bao & Ohlrogge, 1999). Moreover, stable isotope methods have been applied to the study of lipid metabolic pathways in oil seeds (Pollard & Ohlrogge, 1999) and such techniques can provide additional quantitative information about fluxes through alternative pathways of central carbon metabolism (see Ratcliffe & Shacher-Hill, 2001) including the sources of acetyl-CoA for fatty acid formation (Schwender & Ohlrogge, 2002).
In further efforts to pinpoint particular enzyme steps that may be important for regulating lipid biosynthesis, two genetic approaches have been used. First, quantitative trait loci (QTLs) that control seed oil and fatty acid composition have been examined in several important crops such as Brassica napus (Burns et al., 2003) or soybean (Csanadi et al., 2001) as well as in the experimental model plant Arabidopsis (Hobbs et al., 2004). Second, cDNA microarrays which compare wild-type with the low-lipid wrinkled 1 mutation of Arabidopsis have examined over 3500 genes in order to discover which may exert significant control (Ruuska et al., 2002).
Because of the paucity of quantitative data that would allow a more precise description and, hence, a better understanding of the regulation and control of lipid synthesis, we decided to use the technique of metabolic control analysis (MCA). With this method, it is possible to obtain a quantitative measure of the relative importance of particular parts of a metabolic pathway in controlling metabolic fluxes or metabolic intermediate concentrations under defined conditions (Fell, 1997). It is important to realize that MCA recognizes that for an enzyme, or block of reactions, even to approach being rate-limiting is extremely rare, and that all steps/parts of a metabolic pathway can contribute to control of flux, with their contributions often altering as (physiological) conditions change (Kacser & Burns, 1973; Heinrich & Rapoport, 1974). There are two important variants of control analysis, top-down (or modular) and bottom-up. In top-down control analysis (TDCA) a metabolic pathway is conceptually simplified by being divided into blocks with a unique chosen intermediate connecting the blocks of reactions. The advantage of this approach is that it provides an immediate overview of the distribution of flux control over a complex metabolic pathway (Quant, 1993) and can then be refined to allow further detailed analysis of each block. In earlier papers (Ramli et al., 2002a,b), we applied this method for the first time to lipid synthesis, performing a single-manipulation TDCA to gain an initial, broad overview of the control over total lipid biosynthesis fluxes in oilpalm and olive tissues. To achieve this, we conceptually divided the biosynthetic pathway (our analytical system) into two groups, or blocks, of reactions (those of fatty acid synthesis in the plastid (Block A) or of complex lipid assembly in the endoplasmic reticulum (Block B)) linked by a unique intermediate, cytosolic acyl-CoA. Oleate was then introduced to the experimental system to manipulate the level of the common intermediate directly and the responses of the system variables (the fluxes (JA; JB) through the blocks and the acyl-CoA levels (X)) were measured empirically.
In this paper we have applied a different method of analysis, that of double-manipulation TDCA. With this technique, inhibitors specific to each of the Blocks (of reactions) were used to manipulate the system fluxes independently. This is (to our knowledge) the first time such a method has been applied to lipid biosynthesis in any tissue. The results from the double-manipulation TDCA were found to be in good agreement with those found previously for single manipulation (Ramli et al., 2002b) and with a more thorough analysis of the latter using Monte Carlo simulations (MCS) and reported in this paper. These data show that more control is exerted by the fatty acid biosynthesis group of reactions (Block A) but that, nevertheless, substantial control resides with the complex lipid assembly group of reactions. Taken together, these data provide new knowledge and quantitative insight into the way in which lipid synthesis is regulated in plants and provide specific information about control of pathways in the world's major oil crop.