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, 6 (2), 63-76

The Lipidome and Proteome of Oil Bodies From Helianthus Annuus (Common Sunflower)

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The Lipidome and Proteome of Oil Bodies From Helianthus Annuus (Common Sunflower)

Samuel Furse et al. J Chem Biol.

Abstract

In this paper we report the molecular profiling, lipidome and proteome, of the plant organelle known as an oil body (OB). The OB is remarkable in that it is able to perform its biological role (storage of triglycerides) whilst resisting the physical stresses caused by changes during desiccation (dehydration) and germination (rehydration). The molecular profile that confers such extraordinary physical stability on OBs was determined using a combination of (31)P/(1)H nuclear magnetic resonance (NMR), high-resolution mass spectrometry and nominal mass-tandem mass spectrometry for the lipidome, and gel-electrophoresis-chromatography-tandem mass spectrometry for the proteome. The integrity of the procedure for isolating OBs was supported by physical evidence from small-angle neutron-scattering experiments. Suppression of lipase activity was crucial in determining the lipidome. There is conclusive evidence that the latter is dominated by phosphatidylcholine (∼60 %) and phosphatidylinositol (∼20 %), with a variety of other head groups (∼20 %). The fatty acid profile of the surface monolayer comprised palmitic, linoleic and oleic acids (2:1:0.25, (1)H NMR) with only traces of other fatty acids (C24:0, C22:0, C18:0, C18:3, C16:2; by MS). The proteome is rich in oleosins (78 %) with the remainder being made up of caleosins and steroleosins. These data are sufficiently detailed to inform an update of the understood model of this organelle and can be used to inform the use of such components in a range of molecular biological, biotechnological and food industry applications. The techniques used in this study for profiling the lipidome throw a new light on the lipid profile of plant cellular compartments.

Keywords: Lipase inhibition; Lipidome; MS; NMR; Proteome; SANS.

Figures

Fig. 1
Fig. 1
SANS scattering of OB systems containing free protein (a) and not (b). c SANS of OB samples with a lipid-matching concentration of exchangeable deuterium (10 %). The crude preparation (blue) shows a pattern consistent with an elliptical shape approximately two orders of magnitude smaller than the compartmentalised proteins in the purified OB preparation (red)
Fig. 2
Fig. 2
31P NMR spectra of lipidomes of OBs from helianthus OBs. Trace A is a lipid isolate without added enzyme inhibitors. Trace B is from a lipidome isolation containing BPBA (non-selective PLAx inhibitor) and n-butanol. The signal arising from PB is marked with an open diamond. Trace C is from a lipid fraction isolated with PLAx, PLC and PLD inhibitors, and the quantitative standard (lyso-PG, signals arising from the 1-O and 2-O isomers are marked with an asterisk). There is some variation in the shift of the PA signal due to the formation of triethylammonium adducts with the solvent system and appears to be partly concentration-dependent with respect to the lipid
Fig. 3
Fig. 3
31P NMR PLD assay with time points shown. PB remains the dominant species throughout, though the integrations of the signals suggest that the majority of the PB is produced in the first 24 h. The reduction in fraction of all other lipids (especially PA, PC and PI shown here) suggests that PLD acts on a variety of substrates, and/or that other lipases are present
Fig. 4
Fig. 4
a31P NMR showing the comparison of lipidome samples containing PB (unbroken line, unstarred labels) and not containing it (dashed line, starred labels); b1H, 1H TOCSY showing through bond coupling identifying glyceryl–sn-1-3 (panel, left) and butyl (B1-B4) protons (panel, right). Projections shown opposite the x and y axes indicate protons coupled to 31P and are taken from a slice through a 31P,1H HSQC experiment at the 31P chemical shift of 0 · 34 ppm (characteristic of PB in a 1D 31P experiments, (a)). The subscript notations used above are the same as the notations in Supplementary Figure 20
Fig. 5
Fig. 5
Mass spectrum of PB (from PLD assay). m/z 751–5,293 = dilinoleoylphosphatitdylbutanol, m/z 727–5,287 = palmitoyllinoleoylphosphatidylbutanol
Fig. 6
Fig. 6
Schematic diagram of the OB, given the composition described above, and known thermodynamic behaviour [29, 61] of amphiphilic proteins, lipids and triglycerides. A Neutral surface [61] of the monolayer of the OB; b oleosin; c, triglyceride matrix (one molecule shown for clarity); d, phospholipid. Scale: the lipid molecules (d) are 1.8–2.0 nm long [12], and the oleosin helix that extends into the triglyceride matrix is 12 nm long [29]. NB the length of the lipid molecules represents around 0.1 % of the width of the organelle itself

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