An Investigation into the Components of Triricinoleic Acid Production in the Developing Castor Bean Endoplasmic Reticulum

Ricinoleic acid (12-hydroxyoleic acid) has a wide range of industrial uses. Its current source is the castor plant (Ricinus communis) which contains up to 90% ricinoleic acid in its seed storage lipid. R. communis has significant limitations as an agricultural source of ricinoleic acid; it produces potent allergens, requires hand harvesting and only grows effectively in limited climatic zones. A solution to these limitations is to identify the components of the seed storage lipid biosynthetic pathway and transfer them to an agronomic host such as oil seed rape. The developing seed ER is the major compartment of storage oil biosynthesis, whereas during germination these storage compounds are broken down to support the germinating seedling. In this study, a quantitative gel-based proteomic approach has been used to identify the proteins elevated in the developing seed ER compared to the germinating seed ER. On identification of the protein components of storage lipid biosynthesis in the developing seed, their influence on oil quality will be assessed. The use of yeast may be useful in this regard as the influence of transformed gene products on oil production can be measured within days of transformation. A protocol for the analysis of lipid production, including triricinolein production, in a yeast model has been established. Developing ER preparations were made from seed material harvested between 25 to 30 days after flowering; a stage where lipid biosynthesis is at its maximum. ER samples prepared from 3-day germinated seed were used in a differential screen to identify a subset of proteins elevated in the developing seed. Four independent replicates of developing and germinating ER were prepared and analysed using 2-Dimensional Difference In-Gel Electrophoresis (2D DIGE). Spots elevated by ≥10%, with the criteria that they were present in all four gels with a student t-test value of p<0.02, were deemed significant and selected for picking and mass spectrometry analysis. Protein sequence data and peptide mass fingerprints were generated and used to search a complete R. communis protein database. Prior to the 2D DIGE analysis, all stages of the proteomic methodology were validated and if necessary optimised for the R. communis samples, from sample preparation through fluorescent labelling, isoelectric focussing, reproducibility of the analytical gels to the ability to identify and effectively pick spots from high loading preparative gels. 91 protein spots were identified as significantly elevated in the developing preparations and 15 spots as significantly elevated in the germinating preparations. On analysis with mass spectrometry a total of 54 developing spots and 10 germinating spots gave confident identities. The majority of the developing spots identified were protein chaperones, folding proteins, and storage proteins. No components of lipid biosynthesis were identified in the 2D DIGE analysis, likely due to their membrane bound nature and the loss of these proteins due to poor solubility in 2D electrophoresis buffers or their precipitation during isoelectric focussing. The oleaginous yeast Yarrowia lipolytica was identified as a suitable candidate for in-vivo assays of the effect of R. communis lipid biosynthesis gene products on oil composition. This yeast can utilise hydrophobic substrates for growth including ricinoleic acid, makes significant amounts of storage oil, has a complete genome sequence available and mature genetic tools facilitating its transformation. A protocol for its growth on hydrophobic substrates, lipid extraction and analysis of triricinolein production has been established. 2D DIGE provides a statistically rigourous method for identifying and quantifying elevated proteins in differential screens of plant seed ER. For the identification of the components of lipid biosynthesis in the developing ER, attention now turns to the membranes. Gel-free mass spectrometry based approaches provide the best chance of identifying these proteins and will complement the proteomic analysis presented here.