FIGURE 1.2 Carbohydrate catabolism in relation to lipid biosynthesis in photosynthetic developing seeds of B. napus L. Reactions having a substantial effect on the flow of carbon into seed oil are indicated with an asterisk. Glycolysis operates in the cytosol and plastid. Cytosolic triose phosphate (TP) in the form of dihydroxyacetone phosphate provides substrate for cytosolic sn-glycerol-3-phosphate dehydrogenase (G3PDH) to produce the glycerol backbone for triacylglycerol (TAG) bioassembly in the endoplasmic reticulum (ER). The oxida-tive pentose phosphate pathway (OPPP) also operates in the cytosol and plastid. Arabidopsis knockouts for cytosolic glucose-6-phosphate dehydrogenase (Glu6DH) have been shown to accumulate more seed oil, suggesting that more glucose-6-phosphate (Glu6P) is available for lipid production (Waako et al. 2007). The nonoxidative reactions of the OPPP combined with ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) action (without the Calvin cycle) drive the conversion of fructose-6-phosphate (Fru6P) into 3-phosphoglyceric acid (PGA) (Schwender et al. 2004a). This process bypasses the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase, recycling half of the CO2 produced by plastidial pyruvate dehydrogenase action. As a result, 20% more acetyl-CoA is available for fatty acid (FA) synthesis with 40% less loss of carbon as CO2. Photosynthesis provides reducing power (NADPH, nicotinamide adenine dinucleotide phosphate) and O2 for adenos-ine triphosphate (ATP) production for FA biosynthesis. Acetyl-CoA is produced in both the plastid and mitochondrion through the catalytic action of the pyruvate dehydrogenase (PDH) complex. Flux analysis has demonstrated that mitochondrial metabolism provides acetyl-CoA for FA elongation and not FA biosynthesis in the plastid (Schwender et al. 2006). Citrate (Cit) derived from acetyl-CoA through the TCA (tricarboxylic acid) cycle reactions is transported out of the mitochondrion and is converted to cytosolic acetyl-CoA through the catalytic action of ATP:citrate lyase. Antisense repression of mitochondrial pyruvate dehydrogenase kinase (PDHK), however, has been shown to result in more mitochondrial (continued on page 11)

suppression hybridization to identify differentially expressed genes in seeds of two near-isogenic B. napus L. lines differing in seed oil content. Interestingly, PDHK was identified as one of the few down-regulated genes in the high-oil line. Thus, the decrease in PDHK expression is consistent with results on antisense repression of the gene.

In plants, glycolysis occurs in both the cytosol and plastid, with both compartments linked via membrane transporters in the plastidial envelope (Plaxton and Podesta 2006). Disruption of the gene encoding the prsubunit of heteromeric plastidic pyru-vate kinase complex in Arabidopsis has been shown to lead to reduced plastidial pyruvate kinase activity and a 60% reduction in seed oil content (Andre et al. 2007). The seed oil phenotype, however, was completely restored through expression of the cDNA encoding the prsubunit of the complex. Thus, pyruvate kinase was critical in the formation of seed oil, with the enzyme of the plastidial pathway representing the preferred route for producing precursors of FA biosynthesis. Analysis of differential gene expression in two near-isogenic lines of B. napus differing in seed oil content indicated that expression of pyruvate kinase along with the activity of the enzyme was increased in the high-oil line (Li et al. 2006).

Wakao et al. (2008) suggested that cytosolic glucose-6-phosphate dehydrogenase had a role in supplying NADPH (nicotinamide adenine dinucleotide phosphate), via the oxidative pentose phosphate pathway, for oil accumulation in developing seeds in which photosynthesis may be limited. Studies with single and double mutants disrupted in two cytosolic forms of the enzyme indicated that loss of cytosolic glucose-6-phosphate dehydrogenase (G6PDH) activity affects developing seed metabolism by increasing carbon substrates for synthesis of oil instead of decreasing the supply of NADPH for FA synthesis. Seeds of the double mutant had higher seed oil content and increased seed weight compared to the wild type. It was concluded that a highly dynamic metabolic network compensates for the inactivation of one or both G6PDHs.

Given that inactivation of PDHK or G6PDH has been shown to result in enhanced seed oil content, these enzymes may thus represent promising targets for enhancing seed oil content using a targeting-induced local lesions in genomes (TILLING) approach. TILLING involves screening for mutations in a known target of interest (McCallum et al. 2000). In the case of an enzyme, a single-base mutation in the encoding gene could potentially lead to reduction or even elimination of enzyme activity. The application of TILLING to a major oilseed crop (Slade and Knauf 2005), such as B. napus, could potentially constitute a nongenetic engineering approach to enhance seed oil content through inactivation or partial inactivation of PDHK or G6PDH.

FIGURE 1.2 (continued from page 10) PDH complex activity, leading to the production of more acetyl-CoA, resulting in enhanced accumulation of seed oil (Zou et al. 1999, Marillia et al. 2003). The precise mechanism by which mitochondrial acetyl-CoA promotes enhanced seed oil accumulation remains to be elucidated. Additional metabolites: Fru 1, 6BP, fructose-1, 6-bisphosphate (Fru1,6BP); OAA, oxaloacetate; PEP, phosphoenolpyruvate. Additional information for the depicted scheme is available in the work of Ruuska et al. (2004).

Conversely, mutations might also be identified that lead to enzyme activation, such as the one associated with the phenylalanine insertion at position 469 of DGAT1-2 from Z. mays (Zheng et al. 2008). Therefore, a TILLING strategy could potentially be useful for activation of DGAT1 to produce seed with enhanced oil content.

Recent advances in metabolite analysis and pathway flux are likely to lead to novel strategies for enhancing seed oil content. Stable isotope labeling methods have been developed to probe in vivo intermediary metabolism (Schwender et al. 2004b). The flow of carbon in developing seeds of B. napus has been studied extensively through 13C-labeling experiments on cultured embryos (Schwender and Ohlrogge 2002, Schwender et al. 2003, 2004a, 2006). The experiments provided additional insights into seed metabolism, such as the involvement of amino acids in the production of cytosolic acetyl-CoA for FA elongation. In addition, it was demonstrated that mitochondrial metabolism did not provide acetyl-CoA for plastidial FA biosynthesis (Schwender and Ohlrogge 2002, Schwender et al. 2006). This observation, however, makes it difficult to interpret the proposed role of the mitochondrial PDH complex in providing acetyl moieties for use in plastidial FA biosynthesis (Marillia et al. 2003). Thus, it appears that the contribution of mitochondrial respiration to lipid biosynthesis is more complex than outlined in some metabolic flux models.

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