Proteomic profiling of fatty acid biosynthetic enzymes from oil palm chromoplast

Plant fatty acid metabolism has proven to be amenable to manipulation by conventional breeding, genetic and metabolic engineering to enhance the fatty acid profile. This can be done by engineering palm fruit to synthesise more oleic acid at the expense of palmitic acid. This would produce an oil with greater perceived nutritional quality and higher market value. Although the biochemistry of fatty acid biosynthesis in plants is well described, crosstalk between transcriptional and metabolic controls in regulating fatty acid composition remains poorly understood. Our hypothesis is that phosphorylation is one of the main regulators of acetyl-CoA carboxylase, fatty acid synthase complex and stearoyl-ACP-desaturase in increasing the oleic acid level between oil palm (Elaeis guineensis Jacq. var. Tenera) low and high oleic acid varieties. This study utilised advanced proteomic techniques to isolate, detect and identify chromoplast-based phosphorylated proteins associated with the fatty acid biosynthesis pathway. Sub-organelle isolation using differential centrifugation enriched the chromoplast fraction that contained the fatty acid biosynthetic enzymes before their protein extraction. Gel-based and non-gel based mass spectrometry techniques were then employed to separate and improve the identification of key fatty acid biosynthetic enzymes. Protein expression was analysed using isobaric labelling strategy. Five key enzymes, namely the β-ketoacyl-ACP reductase (EC 1.1.1.100), β-hydroxyacyl-ACP dehydrogenase (EC 4.2.1.58 and 4.2.1.59), 3-enoyl-ACP reductase (EC 1.3.19), β-ketoacyl-ACP synthase (EC 2.3.1.41) and stearoyl-ACP desaturase (EC 1.14.99.6) were identified using GeLC-MS/MS strategy. An additional two subunits of acetyl-CoA carboxylase (EC 6.4.1.2) were identified from the 2DLC-MS/MS strategy. The expression of β-hydroxyacyl-ACP dehydrogenase and β-ketoacyl-ACP synthase was up-regulated in the high oleic acid variety. In contrast, 3-enoyl-ACP reductase was down-regulated in the high oleic acid variety. The existence of other differentially regulated metabolic enzymes associated with fatty acid biosynthesis suggested that the control of fatty acid production, particularly the synthesis of oleic acid, involves more than just the main fatty acid biosynthetic enzymes. Subsequently, the role of phosphorylation in regulating these fatty acid biosynthetic enzymes was investigated using a novel combination of neutral loss-triggered MS3 and Selected Reaction Monitoring. Acetyl-CoA carboxylase and 3-enoyl-ACP reductase were postulated to be phosphorylated in both low oleic acid and high oleic acid-producing oil palms during the fruit maturation stage of 20th week after anthesis. However, other fatty acid biosynthetic enzymes from these oil palm varieties did not show any indication of phosphorylation despite the prediction of phosphoserine-containing peptides. The location of the phosphorylated serine residues in the protein domains of acetyl-CoA carboxylase and 3-enoyl-ACP reductase suggested that phosphorylation could have regulated their enzyme activities. This study has produced a robust method to capture and identify chromoplast-based enzymes that are related to plant fatty acid biosynthesis. The differences in their protein expression levels suggested that fatty acid biosynthetic enzymes were differentially regulated and phosphorylation might be involved in this regulation, at least in the enzyme activity. The outcomes reported in this thesis have significantly improved the knowledge of the possible regulation mechanisms in plant fatty acid biosynthesis. The logical extension of this work in future efforts will be to determine the biological significance of this differential protein expression and to understand the exact role of phosphorylation in the regulation of these enzymes