Spatial and Temporal Mapping of Key Lipid Species in Brassica napus Seeds

Abstract

The regulation of lipid synthesis in oil seeds is still not fully understood. Oilseed rape (Brassica napus) is the third most productive vegetable oil crop on the global market; therefore, increasing our understanding of lipid accumulation in oilseed rape seeds is of great economic, as well as intellectual, importance. Matrix-assisted laser/desorption ionization-mass spectrometry imaging (MALDI-MSI) is a technique that allows the mapping of metabolites directly onto intact biological tissues, giving a spatial context to metabolism. We have used MALDI-MSI to study the spatial distribution of two major lipid species, triacylglycerols and phosphatidylcholines. A dramatic, heterogenous landscape of molecular species was revealed, demonstrating significantly different lipid compositions between the various tissue types within the seed. The embryonic axis was found to be particularly enriched in palmitic acid, while the seed coat/aleurone layer accumulated vaccenic, linoleic, and α-linoleic acids. Furthermore, the lipid composition of the inner and outer cotyledons differed from each other, a remarkable discovery given the supposed identical functionality of these two tissues. Triacylglycerol and phosphatidylcholine molecular species distribution was analyzed through a developmental time series covering early seed lipid accumulation to seed maturity. The spatial patterning of lipid molecular species did not vary significantly during seed development. Data gathered using MALDI-MSI was verified through gas chromatography analysis of dissected seeds. The distinct lipid distribution profiles observed imply differential regulation of lipid metabolism between the different tissue types of the seed. Further understanding of this differential regulation will enhance efforts to improve oilseed rape productivity and quality.

Figure 1.

NMR visualization of total lipid distribution within oilseed rape seeds at three developmental stages. A to C, Bright-field images of transverse sections of oilseed rape seeds at 20 d after flowering (DAF; A), 27 DAF (B), and 35 DAF (C). Bars = 1 mm. Labels show embryonic axis (EA), outer cotyledon (OC), inner cotyledon (IC), and seed coat/aleurone layer (SC). D to F, Two-dimensional images represent three-dimensional data sets from intact seeds at 20 DAF (D), 27 DAF (E), and 35 DAF (F). Bars = 1.5 mm. The lipid content is color coded (red = maximal signal and blue = minimal signal) and normalized.

Figure 2.

Schematic showing the MALDI-MSI method. A, A laser is rastered over a matrix-coated seed section in a series of spot points with a given x-y coordinate. The example shown is an oilseed rape seed representative section (30 μm thickness). B, The ions produced are directed to a mass spectrometer, where their m/z values are measured. C, Metabolites are identified, and false-color images are generated to depict the ion intensity at each laser spot point. Bar = 1 mm.

Figure 3.

Imaging of selected PC molecular species in oilseed rape seeds at 27 DAF. Bright-field images of seed cross sections at 27 DAF are shown at top left of each part. Bars = 1 mm. The distributions of selected PC molecular species are shown with a fixed mol % to show absolute distribution profiles (A) and an adjusted mol % to show relative distribution profiles (B). Below each image is a color scale bar, with green and red representing low and high levels, respectively. Numbers at either end of the colored bar represent the scale of that image. Each image also is labeled with the total number of acyl carbons and double bonds (e.g. PC-34:1).

Figure 4.

Imaging of selected TAG molecular species in oilseed rape seeds at 27 DAF. Bright-field images of seed cross sections at 27 DAF are shown at top left of each part. Bars = 1 mm. The distributions of selected TAG molecular species are shown with fixed mol % to show absolute distribution profiles (A) and adjusted mol % to show relative distribution profiles (B). Below each image is a color scale bar, with green and red representing low and high levels, respectively. Numbers at either end of the colored bar represent the scale of that image. Each image also is labeled with the total number of acyl carbons and double bonds (e.g. TAG-52:2).

Figure 5.

Imaging and quantification of selected PC molecular species in oilseed rape seeds at three developmental stages. A, Bright-field images of seed cross sections (top of each column; bars = 1 mm) and relative distribution patterns of selected PC species at three time points, 20, 27, and 35 DAF (one developmental stage per column) are shown. Below each image is a color scale bar, with green and red representing low and high levels, respectively. Numbers at either end of the colored bar represent the scale of that image. PC species with total number of acyl carbons and number of double bonds, along with the most likely acyl combination (based on MS data) below, are indicated at left. B and C, Quantification of total molecular species by electrospray ionization-mass spectrometry (ESI-MS; white; n = 3), MALDI-MS of total lipid extracts (TLE; gray; n = 3), and MALDI-MSI (black; n = 3) is compared at three developmental stages. Quantification in lipid extracts was based on internal standards. Values shown are means ± sd.

Figure 6.

Imaging and quantification of selected TAG molecular species in oilseed rape seeds at three developmental stages. A, Bright-field images of seed cross sections (top of each column; bars = 1 mm) and relative distribution patterns of selected TAG species at three time points, 20, 27, and 35 DAF (one developmental stage per column) are shown. Below each image is a color scale bar, with green and red representing low and high levels, respectively. Numbers at either end of the colored bar represent the scale of that image. TAG species with total number of acyl carbons and number of double bonds, along with the most likely acyl combination (based on MS data) below, are indicated at left. B and C, Quantification of total molecular species by ESI-MS (white; n = 3 independent extractions), MALDI-MS of TLE (gray; n = 3 independent extractions), and MALDI-MSI (black; n = 3 sections from three different seeds) is compared at three developmental stages. Quantification in lipid extracts was based on internal standards. Values shown are means ± sd.

Figure 7.

Fatty acid composition of PC in dissected seed tissues at three developmental stages. Tissues analyzed were the seed coat/aleurone layer (SC), embryonic axis (EA), inner cotyledon (IC), and outer cotyledon (OC). Seeds were sampled at 20, 27, and 35 DAF. Values are means ± sd (n = 3). Tables below each graph show the statistical analysis of the fatty acid in the column immediately above: *, P < 0.05; **, P < 0.001; –, no significant difference.

Figure 8.

Fatty acid composition of TAG in dissected seed tissues at three developmental stages. Tissues analyzed were the seed coat/aleurone layer (SC), embryonic axis (EA), inner cotyledon (IC), and outer cotyledon (OC). Seeds were sampled at 20, 27, and 35 DAF. Values are means ± sd (n = 3). Tables below each graph show the statistical analysis of the fatty acid in the column immediately above: *, P < 0.05; **, P < 0.001; –, no significant difference.