Using lipidomics to reveal details of lipid accumulation in developing seeds from oilseed rape (Brassica napus L.)

Abstract

With dwindling available agricultural land, concurrent with increased demand for oil, there is much current interest in raising oil crop productivity. We have been addressing this issue by studying the regulation of oil accumulation in oilseed rape (Brassica napus L). As part of this research we have carried out a detailed lipidomic analysis of developing seeds. The molecular species distribution in individual lipid classes revealed quite distinct patterns and showed where metabolic connections were important. As the seeds developed, the molecular species distributions changed, especially in the period of early (20days after flowering, DAF) to mid phase (27DAF) of oil accumulation. The patterns of molecular species of diacylglycerol, phosphatidylcholine and acyl-CoAs were used to predict the possible relative contributions of diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase to triacylglycerol production. Our calculations suggest that DGAT may hold a more important role in influencing the molecular composition of TAG. Enzyme selectivity had an important influence on the final molecular species patterns. Our data contribute significantly to our understanding of lipid accumulation in the world's third most important oil crop.

Keywords: Brassica napus (L); Developing oilseed rape; Lipid accumulation; Lipidomics; Regulation of synthesis.

Fig. 1

Analysis of acyl glycerols at a mid-point (27 DAF) of lipid accumulation in oilseed rape. Diacylglycerol (A) and triacylglycerol (B) molecular species were analysed by multiple reaction monitoring (MRM) using a triple quadrupole instrument after separation of the lipid classes by reverse phase HPLC as described in Materials and methods. Four individual biological samples are depicted, each of which was analysed with technical triplicates (means ± SD shown). Major molecular species (> 1% total) are detailed here, with further information on minor species listed in Supplementary Tables 3 and 4. The fatty acid abbreviation in parenthesis refers to the MRM transition (neutral loss of a specified fatty acid) used for the measurement. At the mid-point of lipid accumulation (27 DAF) the amounts of diacylglycerol and triacylglycerol were 0.66 ± 0.24 and 72.5 ± 0.6 μg fatty acid/mg FW embryo, respectively, as measured by TLC/GLC.

Fig. 2

Analysis of phosphoglycerides at a mid-point (27 DAF) of lipid accumulation in oilseed rape. Phosphatidate (A), phosphatidylcholine (B) and phosphatidylethanolamine (C) molecular species were separated by hydrophilic interaction liquid chromatography (HILIC) and analysed by multiple reaction monitoring as detailed in Materials and methods. Four individual biological samples are shown, each of which was analysed with technical triplicates (means ± SD shown). Individual molecular species of 1% or greater abundance are detailed with full information of all species detected given in Supplementary Tables 5–7. The relative amounts of the PA, PC and PE at 27 DAF were 0.32 ± 0.14, 2.37 ± 0.17 and 0.38 ± 0.03 μg fatty acid/mg FW embryo, respectively, as measured by TLC/GLC.

Fig. 3

Changes in the percentages of major molecular species of triacylglycerol during oil accumulation in oilseed rape. Triacylglycerol molecular species were analysed by multiple reaction monitoring (MRM) using a triple quadrupole instrument after separation of lipid species by reverse phase liquid chromatography (RPLC) as described in Materials and methods. Three developmental time points were analysed, 20, 27 and 35 days after flowering (DAF), representing early, mid and late stages of oil accumulation respectively in B. napus cv. Westar. Means ± s.d. (n = 5) are shown. *P < 0.05, **P < 0.01, ***P < 0.001. Major molecular species (> 1% total) are detailed here, with further information on minor species listed in Supplementary Table 8. The amounts of triacylglycerol at the three stages were 15.17 ± 0.35, 72.55 ± 0.58 and 146.98 ± 0.84 μg fatty acid/mg FW, respectively, as measured by TLC/GLC.

Fig. 4

Changes in the percentages of major molecular species of diacylglycerol during oil accumulation in oilseed rape. Diacylglycerol molecular species were analysed by multiple reaction monitoring (MRM) using a triple quadrupole instrument after separation of lipid species by RPLC as described in Materials and methods. Three developmental time points were analysed, 20, 27 and 35 days after flowering (DAF), representing early, mid and late stages of oil accumulation, respectively, in B. napus cv. Westar. Means ± s.d. (n = 5) are shown. *P < 0.05, **P < 0.01, ***P < 0.001. Major molecular species (> 1% total) are detailed here, with further information on minor species listed in Supplementary Table 9. The amounts of diacylglycerol at the three stages were 0.23 ± 0.01, 0.66 ± 0.24 and 1.23 ± 0.14 μg fatty acid/mg FW, respectively, as measured by TLC/GLC.

Fig. 5

Changes in the percentages of major molecular species of phosphatidic acid during oil accumulation in oilseed rape. Phosphatidic acid molecular species were analysed by multiple reaction monitoring (MRM) using a triple quadrupole instrument after separation of lipid species by HILIC as described in Materials and methods. Three developmental time points were analysed, 20, 27 and 35 days after flowering (DAF), representing early, mid and late stages of oil accumulation, respectively, in B. napus cv. Westar. Means ± s.d. (n = 5) are shown. *P < 0.05, **P < 0.01, ***P < 0.001. Major molecular species (> 1% total) are detailed here, with further information on minor species listed in Supplementary Table 10. The amounts of phosphatidic acid at the three stages were 0.34 ± 0.25, 0.32 ± 0.14 and 0.11 ± 0.07 μg fatty acid/mg FW, respectively, as measured by TLC/GLC.

Fig. 6

Changes in the percentages of major molecular species of phosphatidylcholine during oil accumulation in oilseed rape. Phosphatidylcholine molecular species were analysed by multiple reaction monitoring (MRM) using a triple quadrupole instrument after separation of the lipid classes by HILIC as described in Materials and methods. Three developmental time points were analysed, 20, 27 and 35 days after flowering (DAF), representing early, mid and late stages of oil accumulation, respectively, in B. napus cv. Westar. Means ± s.d. (n = 5) are shown. *P < 0.05, **P < 0.01, ***P < 0.001. Major molecular species (> 1% total) are detailed here, with further information on minor species listed in Supplementary Table 11. The amounts of phosphatidylcholine at the three stages were 1.37 ± 0.12, 2.37 ± 0.03 and 3.65 ± 0.08 μg fatty acid/mg FW, respectively, as measured by TLC/GLC.

Fig. 7

Changes in the percentages of major molecular species of phosphatidylethanolamine during oil accumulation in oilseed rape. Phosphatidylethanolamine molecular species were analysed by multiple reaction monitoring (MRM) using a triple quadrupole instrument after separation of the lipid classes by HILIC as described in Materials and methods. Three developmental time points were analysed, 20, 27 and 35 days after flowering (DAF), representing early, mid and late stages of oil accumulation, respectively, in B. napus cv. Westar. Means ± s.d. (n = 5) are shown. *P < 0.05, **P < 0.01, ***P < 0.001. Major molecular species (> 1% total) are detailed here, with further information on minor species listed in Supplementary Table 12. The amounts of phosphatidylethanolamine at the three stages were 0.42 ± 0.07, 0.38 ± 0.03 and 0.58 ± 0.04 μg fatty acid/mg FW, respectively, as measured by TLC/GLC.

Fig. 8

Distribution of acyl-CoA species during the rapid period of oil accumulation in oilseed rape. Means ± S.D. (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001. The inset shows the total amount of acyl-CoAs at the three development times.