Abstract
Proteins from the plant chromoplast are essential for many physiological processes such as fatty acid biosynthesis. Different protein extraction methods were tested to find the most robust method to obtain oil palm chromoplast proteins for mass spectrometry analysis. Initially, two different solvents were employed to reduce the fruit lipids. Then, two plant cell wall digestive enzymes were used to acquire the protoplasts to increase the protein extraction effectiveness. A two-stage centrifugation-based fractionation approach enhanced the number of identified proteins, particularly the fatty acid biosynthetic enzymes. The effectiveness of each extraction method was assessed using protein yields and 2DE gel profiles. The ideal method was successfully used to establish the 2DE chromoplast proteome maps of low and high oleic acid mesocarps of oil palm. Further nanoLC–MS/MS analysis of the extracted chromoplast proteins led to the identification of 162 proteins, including some of the main enzymes involved in the fatty acid biosynthesis. The established procedures would provide a solid foundation for further functional studies, including fatty acid biosynthetic expression profiling and evaluation of regulatory function.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-015-1576-4) contains supplementary material, which is available to authorized users.
Highlights
Oil palm (Elaeis guineensis Jacques) is the most important plant commodity in Malaysia, covering more than 5 million hectares in 2012
This 1.6-fold increase in protein yield indicated that excess lipid greatly reduced the effectiveness of the protein extraction. two-dimensional gel electrophoresis (2DE) gel profiles of mesocarp proteins with and without solvent washes (Fig. 1) clearly show that more proteins were extracted from delipidated mesocarps
trichloroacetic acid (TCA)/acetone-extracted proteins were not suitable for 2DE analysis since the precipitate could not be dissolved even in urea-containing buffer. These results indicate that sodium dodecyl sulphate (SDS) enhanced protein extraction, but this was countered by poor protein spot resolution in the phenol/ SDS and SDS alone extracts
Summary
Oil palm (Elaeis guineensis Jacques) is the most important plant commodity in Malaysia, covering more than 5 million hectares in 2012. This oil crop is currently the world’s top commodity oil-bearing crop, with about 3.5 tons of oil per hectare produced annually in Malaysian plantations (Basiron 2012; Kirkland 2011; Barcelos et al 2015). Plant fatty acid biosynthesis occurs in the plastids, in both photosynthetic plastids such as leaf chloroplasts and in the non-photosynthetic plastids of flowers and fruits (Stumpf 1969; Joyard et al 2010; Benning 2008, 2009).
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