Metabolomics

, Volume 8, Issue 4, pp 598–613

Lipid profiling of the model temperate grass, Brachypodium distachyon

Authors

  • M. Nurul Islam
    • School of Biology and Environmental ScienceUniversity College Dublin
  • John P. Chambers
    • School of Biology and Environmental ScienceUniversity College Dublin
    • School of Biology and Environmental ScienceUniversity College Dublin
Original Article

DOI: 10.1007/s11306-011-0352-x

Cite this article as:
Nurul Islam, M., Chambers, J.P. & Ng, C.K. Metabolomics (2012) 8: 598. doi:10.1007/s11306-011-0352-x

Abstract

Lipids are essential metabolites in cells and they fulfil a variety of functions, including structural components of cellular membranes, energy storage, cell signalling, and membrane trafficking. In plants, changes in lipid composition have been observed in diverse responses ranging from abiotic and biotic stress to organogenesis. Knowledge of the lipid composition is an important first step towards understanding the function of lipids in any given biological system. As Brachypodium distachyon is emerging as the model species for temperate grass research, it is therefore fundamentally important to gain insights of its lipid composition. We used HPLC-coupled with tandem mass spectrometry to profile and quantify levels of sphingolipids and glycerophospholipids in shoots and undifferentiated cells in suspension cultures of B. distachyon. A total of 123 lipids belonging to 10 classes were identified and quantified. Our results showed that there are differences in lipid profiles and levels of individual lipid species between shoots and undifferentiated cells in suspension cultures. Additionally, we showed that 4-sphingenine (d18:1Δ4) is the main unsaturated dihydroxy-long chain base (LCB) in B. distachyon, and we were unable to detect d18:1Δ8, which is the main unsaturated dihydroxy-LCB in the model dicotyledonous species, Arabidopsis thaliana. This work serves as the first step towards a comprehensive characterization of the B. distachyon lipidome that will complement future biochemical studies.

Keywords

Brachypodium distachyonLipidomeGlycerophospholipidsSphingolipidsLC–MS/MS

1 Introduction

Lipids are essential metabolites in cells and they fulfil a variety of functions which include structural components of cellular membranes, energy storage, cell signalling, and membrane trafficking (Khalil et al. 2010). In plants, alterations in the composition of lipids have been observed during cold acclimation, heat stress, and nutrient deficiencies. Additionally, changes in lipid composition have also been observed during development of cotton fibres and aging in potato tubers. Changes in lipid composition have also been observed in plants in response to pathogen attack. Lipids can be divided into classes based on their chemical structures: fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids and polyketides. In order to understand the function of lipids in biological systems, it is important to profile the diverse lipid species present in any given organism and quantify their levels. Recent technological advances in liquid chromatography and mass spectrometry have enabled high-throughput, systematic profiling of lipid species, which in current parlance could be called ‘lipidomics’ (Welti et al. 2007; Khalil et al. 2010).

Brachypodium distachyon, a member of the Pooideae, is genetically related to domesticated temperate grass crops like barley (Hordeum vulgare), oat (Avena sativa), and wheat (Triticum aestivum), and perennial ryegrass (Lolium perenne) (Vogel et al. 2006b; Bossolini et al. 2007; Garvin 2007a, b; Opanowicz et al. 2008). B. distachyon is emerging as the new model plant species for temperate grasses because it has a small genome which has been estimated at ~272 Mbp (The International Brachypodium Initiative 2010), a small stature, rapid generation time (6–8 weeks depending on growth conditions), self-fertile, and amenable to transformation by Agrobacterium (Draper et al. 2001; Vogel et al. 2006a; Garvin 2007a, b; Păcurar et al. 2007; Vogel and Hill 2007; Opanowicz et al. 2008; Vain et al. 2008; Alves et al. 2009; Thole et al. 2009). Because B. distachyon is a small plant, it is more economically viable for large scale experiments compared to wheat and barley.

Lipid profiling of plants have demonstrated great diversity in the lipidome from different species (Ohnishi et al. 1985; Hetherington and Drøbak 1992; Uemura and Steponkus 1994; Kawaguchi et al. 2000; Devaiah et al. 2006; Larson and Graham 2006; Wang et al. 2006; Markham and Jaworski 2007; Bamba et al. 2008; Han et al. 2009; Minamioka and Imai 2009; Pata et al. 2010; Burgos et al. 2011; Horn et al. 2011; Okazaki et al. 2011). Given the emergence of B. distachyon as the model for temperate grass research, it is therefore fundamentally important to gain insights of its lipid composition or lipidome. This study aims to use HPLC-coupled to tandem mass spectrometry to profile and quantify levels of glycerophospholipids and sphingolipids, in shoots and undifferentiated cells in suspension cultures, as a first step towards understanding the lipidome of B. distachyon.

2 Materials and methods

2.1 Plant growth

Mature seeds of B. distachyon Bd21 (kindly provided by Dr. David F. Garvin, USDA-ARS, MN, USA) were soaked in distilled water for 2 h before the upper and lower glumes were removed. Seeds were then soaked in 20% household bleach (~1% sodium hypochlorite) for 3 min and washed 3 times with sterile deionized water. They were then placed on moist sterile filter paper in Petri dishes (9 cm Ø) at a density of 20 seeds per plate. Plates were stratified at 4°C for 3 days in the dark before being transferred to a controlled temperature chamber (Sanyo, http://www.sanyo-biomedical.co.uk) at 25°C for 4 days in the dark. Germinated seeds were then transferred to a compost:vermiculite (2:1, v/v) mix (Shamrock multipurpose compost, Ireland) in 5 × 5 cm pots at a density of 1 seedling per pot, and placed in a Microlima 1750 climate controlled growth chamber (Snijders, http://www.snijders-scientific.nl/) under the following conditions: 16/8 h and 24/18°C light/dark; PPFD of 250 μmol m−2 s−1; relative humidity of 70%. Plants were watered daily and shoots harvested for lipid extraction and analysis after 4 weeks.

Seeds of Arabidopsis thaliana Col-0 were sterilized with 20% household bleach (~1% sodium hypochlorite) for 3 min and washed 3 times with sterile deionized water before being placed on ½-strength Murashige and Skoog medium, pH 5.8 (Duchefa, http://www.duchefa.com), supplemented with 1% sucrose and 0.6% plant cell-culture tested agar (Sigma, http://www.sigma-aldrich.com) in Petri dishes (9 cm Ø) at a density of 20 seeds per plate. Seeds were stratified at 4°C for 3 days in the dark before being transferred to a controlled temperature room under the following conditions: 8/16 h and 22/18°C light/dark; PPFD of 250 μmol m−2 s−1. After 10 days, germinated seedlings were transferred to a compost:vermiculite (2:1, v/v) mix (Shamrock multipurpose compost, Ireland) in 5 × 5 cm pots at a density of 1 seedling per pot, and placed in plant propagators under the same conditions. Plants were watered daily and shoots were harvested for lipid extraction and analysis after 6 weeks.

2.2 Cell suspension cultures of B. distachyon Bd21

Spikelet formation in B. distachyon Bd21 was observed 4–6 weeks after transfer to soil and immature embryos were harvested 7–14 days after spikelet formation. Methods for callus induction were according to Vogel and Hill (2007). Embryogenic calli (5-week old) were divided into small pieces of 5–8 mm in size, and 5–6 pieces were transferred to 7 ml of liquid medium (suspension culture media, SCM) containing Linsmaier and Skoog (LS) medium basal salt mix including vitamins (Duchefa, http://www.duchefa.com), pH 5.8, 3% (w/v) maltose, 7.5 mg l−1 2,4-d, and 0.75 mg l−1 kinetin. Liquid cultures were placed on an orbital shaker (at 120 rpm) in the dark at 22°C. Seven millilitres of fresh SCM were added after 3 weeks. The addition of fresh medium at a ratio of 1:1 was performed every 3 weeks until a 100 ml culture volume is attained, at which time, cell suspension cultures were maintained by sub-culturing 50 ml of cell suspension into 50 ml of fresh SCM every 3 weeks for three sub-culturing cycles, after which cells were sub-cultured every 2 weeks. The 2-week sub-culturing regime was maintained for six sub-culturing cycles before the cells were used for lipid extraction and analysis.

2.3 Solvents and standards

LC–MS grade water, methanol, formic acid and ammonium formate were purchased from Fluka (http://www.sigma-aldrich.com). HPLC grade water, tetrahydrofuran (THF) and methanol were purchased from Sigma-Aldrich (http://www.sigma-aldrich.com). All other chemicals were analytical grade unless otherwise noted. Internal standards used for quantitative analysis were purchased from Avanti Polar Lipids (http://www.avantilipids.com) in either solution or powder form: glycerophosphocholine [10.9 μM PC(17:0–20:4)], glycerophosphoethanolamine [13.32 μM PE(17:0–20:4)], glycerophosphoserine [12.16 μM PS(17:0–20:4)], glycerophosphoglycerol [12.4 μM PG(17:0–20:4)], glycerophosphoinositol [12.72 μM PI(17:0–14:1)], diacylglycerophosphatidic acid [15.85 μM PA(17:0–14:1)], long chain base (LCB) [12.5 μM (d17:1)], long chain base phosphate (LCB-P) [12.5 μM (d17:1-P)], ceramide [12.5 μM (d18:1/12:0)], and glucosylceramide [12.5 μM [d18:1/12:0)].

2.4 Lipid extraction

Four-week old B. distachyon shoots and undifferentiated cells in suspension cultures were harvested and flash frozen in liquid nitrogen before freeze-drying. Freeze-dried B. distachyon materials were ground into fine powder using mortar and pestle. 30 mg of powdered material, supplemented 10 μl of internal standards, were transferred into 10 ml glass tube and extracted overnight with 5 ml of 100% methanol containing 10 mM ammonium formate (pH 7.0) at room temperature. After centrifugation at 835g for 15 min, the supernatant was transferred to a 5 ml glass tube using a Pasteur pipette. The extraction procedure was repeated with same solvent for 1 h and the supernatant collected after centrifugation. The remaining lipids were extracted using 5 ml of solvent (chloroform:methanol; 2:1) with vigorous vortexing for 1 min before the addition of 3 ml of water. Phase separation was achieved by centrifugation for 15 min at 835g, and the lipid-enriched chloroform layer was collected. The supernatants were combined after each extraction and dried under nitrogen. The dried crude extract was dissolved in 2 ml of chloroform and washed once with 1 ml of 1 M KCl and twice with 2 ml of water. Finally, the solvent was evaporated under nitrogen and the lipid residue was dissolved in 200 μl of mobile phase (mobile phase A:mobile phase B, 1:1) and again centrifuged for 10 min at 835g before LC–MS/MS analysis.

2.5 LC–MS/MS protocol for lipid profiling

Chromatographic separation of sphingolipids and glycerophospholipids were performed with an HPLC system consisting of a binary pump, auto sampler, column oven (1200 RRLC, Agilent Technologies, http://www.chem.agilent.com). The lipid molecules were separated using a Gemini C18 analytical column (2.0 mm I.D. × 150 mm, particle size 3 μm, Phenomenex, http://www.phenomenex.com). Column oven and auto sampler temperatures were maintained at 45 and 4°C, respectively. The mobile phase consists of solvent A (15 mM ammonium formate (pH 4.0):MeOH:THF, 5:2:3) and solvent B (15 mM ammonium formate (pH 4.0):MeOH:THF, 1:2:7). Elution was performed at a flow rate of 0.25 ml min−1 in a binary gradient mode. The initial composition of mobile phase was 80:20 (A:B), linearly changed to 50:50 (A:B) over 12 min, and linearly changed to 30:70 (A:B) over 25 min, and maintained in this condition for up to 45 min, followed by 8 min of column re-equilibration.

The HPLC was coupled to an Agilent 6520 Q-TOF mass spectrometer (Agilent Technologies, http://www.chem.agilent.com) equipped with an electrospray ion (ESI) source. ESI mass spectra were recorded in positive and negative ionization mode for a mass range from m/z 100 to 1,000. The capillary and fragmentor voltage was set to 4,000 and 170, respectively. Nitrogen was used as the nebulizing gas at a flow rate and temperature of 8 l min−1 and 350°C, respectively. Targeted MS/MS analysis of individual lipids was performed with collision-induced dissociation (CID) voltages ranging from 15 to 48 V with purified nitrogen as the collision gas.

2.6 LC–MS/MS protocol for lipid quantitation

The HPLC system was coupled online to an Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies, http://www.chem.agilent.com) equipped with a Jet Stream ion source. Data were recorded in positive and negative ionization mode using electrospray ionization with nitrogen as the nebulizing gas. The gas temperature and flow rate was 350°C and 10 l min−1, and the sheath gas temperature and flow rate was 350°C and 12 l min−1, respectively. The ESI needle voltage was adjusted to 4,000 V for positive mode and 3,500 V for negative mode and optimum fragmentor voltage and collision energy were assigned by analysis of reference compounds in selected ion and product ion scanning mode. The reference compounds, PC(18:1–18:0), PI(16:0–18:2), PG(18:0–18:2), PS(18:0–18:2), PE(16:0–18:1), PA(17:0–14:1), sphingosine (d18:1), sphingosine-1-phosphate (d18:1-P), dihydrosphingosine (d18:0), dihydrosphingosine-1-phosphate (d18:0-P), phytosphingosine-1-phosphate (t18:0-P), ceramide (d18:1–c18:0), and glucosylceramide (d18:1–c12:0) were purchased from Avanti Polar Lipids (http://www.avantilipids.com). Phytoceramide (t18:0–c18:0) and phytosphingosine (t18:0) were purchased from Matreya Inc. (http://www.matreya.com). The stock solutions for individual compounds at concentrations of 1 mg ml−1 were prepared in HPLC grade methanol and stored in −20°C. Working solutions of desired concentrations were prepared by diluting the stock solution in methanol for determining multiple-reaction monitoring (MRM) conditions and mass spectrometric structural studies. MRM detection was applied using nitrogen as the collision gas. The chromatographic separation of lipids was carried out using the same HPLC system, column and mobile phase described in the previous section. The gradient condition was optimized based on individual class of lipids (Supplementary Table 1), taking into consideration the diversity and complexity of lipid species Peak areas were used for quantitation by comparison with the peak areas of the added internal standards.

2.7 Identification of sphingolipid long chain bases by LC-FLD

Sphingolipids of 4-week old B. distachyon shoots and 6-week old A. thaliana shoots were hydrolyzed using the method of Markham et al. (2006). Briefly, 50 mg of finely ground freeze-dried samples were spiked with 10 μl internal standards (250 μg ml−1 d16:1 and/or 200 μg ml−1 d20:1, Matreya Inc., http://www.matreya.com) before hydrolyzing with 1 ml of dioxane and 1 ml of 10% (w/v) barium hydroxide octahydrate in water for 16 h at 110°C. At the end of hydrolysis, 2 ml of 2% (w/v) ammonium sulfate was added to precipitate barium ions and to reduce the occurrence of a flocculent precipitate during subsequent derivatizing. Sphingolipid LCBs were extracted with 2 ml of diethylether and centrifuged to separate the aqueous and organic phases. The upper phase was removed to a second tube, dried under nitrogen, and derivatized with o-phthaldialdehyde as previously described (Merrill et al. 2000). The chromatographic separation of LCBs were carried out with a reversed phase HPLC system consists of binary pump, auto sampler, column oven (1200 RRLC, Agilent Technologies, http://www.chem.agilent.com) and fluorescence detector (FP-2020 Plus, Jasco, http://www.jascoinc.com). The analytical column was an Eclipse XBD-C18 column (4.6 mm I.D. × 250 mm, particle size 5 μm, Agilent Technologies, http://www.chem.agilent.com) and the column oven and auto sampler temperatures were maintained at 35 and 4°C, respectively. Elution was carried out at flow rate of 1.5 ml min−1 with 70% solvent A (5 mM potassium phosphate, pH 7), 30% solvent B (100% methanol) for 4 min, increasing to 85% solvent B for 10 min, maintained in this condition for up to 25 min before increasing to 100% solvent B for 30 min with a 5 min 100% solvent B wash before returning to initial conditions and re-equilibrating for 5 min. Separated derivatized LCBs were monitored using the fluorescence detector at excitation and emission wavelengths of 340 and 455 nm, respectively.

2.8 Lipid nomenclature

Lipids were named according to Lipid Maps guidelines (http://www.lipidmaps.org) (Fahy et al. 2005, 2009).

3 Results and discussion

3.1 Lipid profiling of B. distachyon

We used HPLC-coupled to tandem mass spectrometry to profile the lipidome of the B. distachyon community inbred line Bd21. We identified a total of 123 lipid species in a total of 10 classes of sphingolipids (LCBs and LCB-Ps, ceramides, hydroxyceramides and glucosylceramides) and glycerophospholipids (glycerophosphocholines, glycerophosphoethanolamines, glycerophosphoinositides, glycerophosphoglycerols, glycerosphosphoserines and diacylglycerophosphatidic acids). We were unable to detect any glucosyl inositol phosphoceramides (GIPCs) in B. distachyon, and this is likely to be due to the extraction protocol employed. While Markham and Jaworski (2007) were able to extract GIPCs from A. thaliana using an extraction solvent comprising isopropanol/hexane/water (55:20:25 v/v/v), we were unable to do so by employing the same extraction solvent and protocol (data not shown). More recently, Okazaki et al. (2011) did not report the detection of GIPCs based on untargeted lipidomic analysis of A. thaliana using hydrophilic interaction chromatography coupled to ion trap time-of-flight mass spectrometry. It is envisaged that further modification and optimization of the extraction protocol will enable subsequent detection and quantitation of GIPCs in B. distachyon.

Lipids in B. distachyon extracts were identified by targeted MS/MS and retention time using a Q-TOF mass spectrometer (Tables 1, 2). Acquired MS/MS data were compared with previous reports (Bartke et al. 2006; Gao et al. 2006; Markham and Jaworski 2007), as well as comparison of MS/MS spectra of reference compounds obtained from the Lipid Maps database (http://www.lipidmaps.org). MRM tables were constructed for quantitative analysis by selecting the precursor-product ions transitions, and HPLC gradient conditions were optimized for each class of compounds to provide enough separation for identification of peaks associated with individual lipid molecules.
Table 1

Multiple reaction monitoring (MRM) parameters [dwell time (DT), fragmentor voltage (FV), collision energy (CE) and retention time (RT)] used for detection of sphingolipids were determined empirically

Species

[M + H]+ (m/z)

Product ion (m/z)

DT (ms)

FV (V)

CE (V)

RT (min)

LCBs and LCB-Ps

 d18:0

302.3

284.3

120

120

15

5.2

 d18:1

300.3

282.3

120

100

15

4.6

 t18:0

318.3

300.3

120

100

12

4.4

 t18:1

316.3

298.3

120

100

15

3.7

 d18:0-P

382.3

266.3

120

110

20

4.7

 d18:1-P

380.3

264.3

120

100

18

4.2

 t18:0-P

398.3

300.3

120

100

18

4.2

 t18:1-P

396.3

298.3

120

100

18

3.5

 d17:1a

286.3

268.3

120

95

15

3.2

 d17:1-Pa

366.3

250.3

120

110

18

3.0

LCB

FA

[M + H]+ (m/z)

Product ion (m/z)

DT (ms)

FV (V)

CE (V)

RT (min)

Ceramides

 d18:0

c16:0

540.5

266.3

100

110

28

11.5

 d18:1

c16:0

538.5

264.3

100

110

28

11.1

 d18:0

c18:0

568.5

266.5

100

110

28

12.6

 d18:1

c18:0

566.5

264.5

100

110

28

12.2

 t18:0

c16:0

556.5

300.3

100

110

25

10.5

 t18:1

c16:0

554.5

298.3

100

110

25

10.1

 t18:0

c18:0

584.5

300.3

100

110

25

11.7

 t18:1

c18:0

582.5

298.3

100

110

25

11.3

 t18:0

c20:0

612.6

300.3

100

110

25

12.7

 t18:1

c20:0

610.6

298.3

100

110

25

12.4

 t18:0

c20:1

610.6

300.3

100

110

25

11.9

 t18:0

c22:0

640.6

300.3

100

110

25

13.6

 t18:1

c22:0

638.6

298.3

100

110

25

13.3

 t18:0

c22:1

638.6

300.3

100

110

25

12.8

 t18:0

c24:0

668.6

300.3

100

110

25

14.6

 t18:1

c24:0

666.6

298.3

100

110

25

14.2

 t18:0

c24:1

666.6

300.3

100

110

25

13.7

 t18:1

c24:1

664.6

298.3

100

110

25

13.4

 t18:0

c26:0

696.6

300.3

100

110

25

15.5

 t18:1

c26:0

694.6

298.3

100

110

25

15.2

 t18:0

c26:1

694.6

300.3

100

110

25

14.6

 t18:1

c26:1

692.6

298.3

100

110

25

14.2

 d18:1a

c12:0

482.5

264.3

100

110

22

8.5

Hydroxyceramides

 t18:0

h18:0

600.6

300.3

100

110

24

11.1

 t18:0

h20:0

628.6

300.3

100

110

24

12.2

 t18:1

h20:0

626.6

298.3

100

110

24

11.8

 t18:1

h20:1

624.6

298.3

100

110

24

11.2

 t18:0

h22:0

656.6

300.3

100

110

24

13.2

 t18:1

h22:0

654.6

298.3

100

110

24

12.8

 t18:0

h22:1

654.6

300.3

100

110

24

12.3

 t18:1

h22:1

652.6

298.3

100

110

24

12.0

 t18:0

h24:0

684.7

300.3

100

110

24

14.1

 t18:1

h24:0

682.7

298.3

100

110

24

13.8

 t18:0

h24:1

682.7

300.3

100

110

24

13.2

 t18:1

h24:1

680.7

298.3

100

110

24

12.9

 t18:0

h26:0

712.7

300.3

100

110

24

15.0

 t18:0

h26:1

710.7

300.3

100

110

24

14.0

 t18:1

h26:0

710.7

298.3

100

110

24

14.7

 t18:1

h26:1

708.7

298.3

100

110

24

12.5

Glucosylceramides

 t18:0

h20:0

790.6

300.3

100

110

25

9.6

 t18:1

h20:0

788.6

298.3

100

110

25

9.1

 t18:0

h22:0

818.7

300.3

100

110

25

11.4

 t18:1

h22:0

816.7

298.3

100

110

25

10.9

 t18:0

h22:1

816.7

300.3

100

110

25

9.9

 t18:1

h22:1

814.7

298.3

100

110

25

9.3

 t18:0

h24:0

846.7

300.3

100

110

25

13.2

 t18:1

h24:0

844.7

298.3

100

110

25

12.7

 t18:0

h24:1

844.7

300.3

100

110

25

11.6

 t18:1

h24:1

842.7

298.3

100

110

25

11.0

 t18:1

h26:0

872.7

298.3

100

110

25

14.4

 t18:1

h26:1

870.7

298.3

100

110

25

12.8

 t18:0

c22:0

802.7

300.3

100

110

25

11.8

 t18:1

c22:0

800.7

298.3

100

110

25

11.4

 t18:0

c26:0

858.7

300.3

100

110

25

16.4

 t18:1

c26:0

856.7

298.3

100

110

25

16.0

 d18:1a

c12:0

644.6

264.3

100

100

22

4.1

aInternal standards

Table 2

Multiple reaction monitoring (MRM) parameters [dwell time (DT), fragmentor voltage (FV), collision energy (CE) and retention time (RT)] used for the detection of glycerophospholipids from B. distachyon were determined empirically

Species

[M + H]+ (m/z)

[M + HCOO] (m/z)

Product ion (m/z)

DT (ms)

FV (V)

CE (V)

RT (min)

Glycerophosphocolines (phosphatidylcholine)

 PC(16:0–16:2)

730.5

774.5

184.1

50

110

30

9.3

 PC(16:0–16:1)

732.5

776.5

184.1

50

110

30

10.0

 PC(16:0–16:0)

734.5

778.5

184.1

50

110

30

10.7

 PC(16:1–18:3)

754.5

798.5

184.1

50

110

30

9.6

 PC(16:0–18:3)

756.5

800.5

184.1

50

110

30

9.8

 PC(16:0–18:2)

758.5

802.6

184.1

50

110

30

10.4

 PC(16:0–18:1)

760.6

804.7

184.1

50

110

30

11.0

 PC(18:3–18:3)

778.5

822.6

184.1

50

110

30

8.8

 PC(18:2–18:3)

780.6

824.6

184.1

50

110

30

9.4

 PC(18:2–18:2)

782.6

826.6

184.1

50

110

30

10.0

 PC(18:1–18:2)

784.6

828.6

184.1

50

110

30

10.6

 PC(18:1–18:1)

786.6

830.6

184.1

50

110

30

11.3

 PC(18:0–18:1)

788.6

832.6

184.1

50

110

30

11.9

 PC(17:0–20:4)a

796.6

184.1

50

110

28

10.9

Species

[M + H]+ (m/z)

[M − H] (m/z)

Product ion (m/z)

DT (ms)

FV (V)

CE (V)

RT (min)

Glycerophosphoethanolamine (phosphatidylethanolamine)

 PE(16:0–18:3)

714.5

712.5

573.5

50

110

30

10.3

 PE(16:0–18:2)

716.5

714.5

575.5

50

110

30

10.8

 PE(16:0–18:1)

718.5

716.5

577.5

50

110

30

11.4

 PE(18:3–18:3)

736.6

734.6

595.6

50

110

30

9.3

 PE(18:2–18:3)

738.6

736.6

597.6

50

110

30

9.9

 PE(18:2–18:2)

740.6

738.6

599.6

50

110

30

10.5

 PE(18:1–18:2)

742.6

740.6

601.6

50

110

30

11.0

 PE(18:0–18:2)

744.6

742.6

603.6

50

110

30

11.6

 PE(24:1–18:2)

826.7

824.7

685.6

50

110

30

13.6

 PE(24:0–18:2)

828.7

826.7

687.6

50

110

30

14.0

 PE(17:0–20:4)a

754.6

613.5

50

110

30

11.3

Species

[M − H] (m/z)

Product ion (m/z)

DT (ms)

FV (V)

CE (V)

RT (min)

Glycerophosphoinositides (phosphatidylinositol)

 PI(16:1–18:3)

829.5

241.1

75

110

35

8.6

 PI(16:0–18:3)

831.5

241.1

75

110

35

9.5

 PI(16:0–18:2)

833.5

241.1

75

110

35

10.2

 PI(16:0–18:1)

835.5

241.1

75

110

35

10.9

 PI(18:3–18:3)

853.5

241.1

75

110

35

8.4

 PI(18:2–18:3)

855.5

241.1

75

110

35

9.1

 PI(18:2–18:2)

857.5

241.1

75

110

35

9.8

 PI(18:1–18:2)

859.5

241.1

75

110

35

10.6

 PI(18:2–18:0)

861.5

241.1

75

110

35

11.5

 PI(17:0–14:1)a

793.5

269.2

75

120

33

9.4

Glycerophosphoglycerols (phosphatidylglycerol)

 PG(16:0–16:1)

719.5

255.2

75

110

25

10.5

 PG(16:0–16:0)

721.5

255.2

75

110

25

11.4

 PG(18:3–16:1)

741.5

253.2

75

110

25

9.4

 PG(16:0–18:3)

743.5

255.2

75

110

25

10.4

 PG(16:0–18:2)

745.5

255.2

75

110

25

11.0

 PG(18:1–16:0)

747.5

255.2

75

110

25

11.7

 PG(18:0–16:0)

749.5

255.2

75

110

25

12.6

 PG(17:0–20:4)a

783.6

303.3

75

110

25

11.6

Diacylglycerophosphatidic acid (phosphatidic acid)

 PA(14:0–18:3)

641.4

227.2

75

110

25

9.5

 PA(14:0–18:2)

643.4

227.2

75

110

25

10.3

 PA(16:0–16:1)

645.4

153.1

75

110

25

11.1

 PA(16:0–16:0)

647.4

153.1

75

110

25

11.9

 PA(16:2–18:3)

665.5

153.1

75

110

25

9.1

 PA(16:1–18:3)

667.5

153.1

75

110

25

10.0

 PA(16:0–18:3)

669.5

153.1

75

110

25

10.8

 PA(16:0–18:2)

671.5

153.1

75

110

25

11.5

 PA(18:3–18:3)

691.5

153.1

75

110

25

9.7

 PA(18:2–18:3)

693.5

153.1

75

110

25

10.4

 PA(18:2–18:2)

695.5

153.1

75

110

25

11.1

 PA(18:2–18:1)

697.5

153.1

75

110

25

11.8

 PA(18:1–18:1)

699.5

153.1

75

110

25

12.7

 PA(17:0–14:1)a

631.4

269.2

75

110

25

10.7

Glycerophosphoserines (phosphatidylserine)

 PS(16:0–18:3)

756.5

669.5

75

110

28

9.5

 PS(16:0–18:2)

758.5

671.5

75

110

28

10.2

 PS(18:3–20:0)

812.7

725.6

75

110

28

12.0

 PS(18:2–20:0)

814.7

727.6

75

110

28

12.6

 PS(22:0–18:3)

840.6

753.6

75

110

28

13.1

 PS(22:0–18:2)

842.6

755.6

75

110

28

13.6

 PS(24:1–18:3)

866.7

779.6

75

110

28

13.1

 PS(24:0–18:3)

868.7

781.6

75

110

28

14.1

 PS(24:0–18:2)

870.7

783.6

75

110

28

14.6

 PS(17:0–20:4)a

796.6

709.5

75

110

26

10.8

aInternal standards

3.2 Elucidating the identity of d18:1 in B. distachyon

Sphingolipid profiling indicated the presence of d18:1 (Table 1). Previous work by Markham and colleagues (Markham et al. 2006; Markham and Jaworski 2007; Michaelson et al. 2009), showed that dihydroxy-LCB desaturated at carbon position 4 (d18:1Δ4) is absent in Arabidopsis thaliana, leading them to question the relevance of bioactivity of d18:1Δ4 and d18:1Δ4-P observed in guard cells of A. thaliana (Coursol et al. 2003). The predominant d18:1 species in A. thaliana is d18:1∆8 (Markham et al. 2006; Markham and Jaworski 2007; Michaelson et al. 2009). Given the observation that the mass over charge (m/z) values for both d18:1∆4 or d18:1∆8, and their diagnostic daughter ions following collision-induced fragmentation are identical, it is not possible to distinguish between these two LCBs using LC–MS/MS. Currently the only method to distinguish between d18:1∆4 and d18:1∆8 relies on HPLC separation of o-phthaldialdehyde derivatized LCBs followed by fluorescence detection. Figure 1a shows the HPLC separation of o-phthaldialdehyde derivatized LCB standards d16:1, t18:0, d18:1Δ4, d18:0, and d20:1. Figure 1b shows the HPLC separation of o-phthaldialdehyde derivatized LCBs from shoots of A. thaliana. The results are consistent with the observations of Markham and colleagues (Markham et al. 2006; Markham and Jaworski 2007; Michaelson et al. 2009) that d18:1Δ4 is absent in shoots of A. thaliana.
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Fig. 1

HPLC analyses of o-phthaldialdehyde derivatives of LCBs from shoots of 6-week old A. thaliana and shoots of 4-week old B. distachyon.a Standard LCB mixture (d16:1, t18:0, d18:1Δ4, d18:0, and d20:1), bA. thaliana extract, cA. thaliana extract spiked with d18:1Δ4, dB. distachyon extract, and eB. distachyon extract spiked with d18:1Δ4. Chromatograms are representative of n = 5

As d18:1Δ4 and d18:1Δ8 are structurally similar, it may be difficult to sufficiently separate these two LCBs to enable their identification. We optimized a gradient protocol to separate these two LCBs by spiking extracts of A. thaliana with d18:1Δ4. Although d18:1∆4 and d18:1∆8 have the same molecular mass, the retention time of their o-phthaldialdehyde derivatives are sufficiently different to allow HPLC separation (Fig. 1c). To elucidate the identity of the d18:1 LCB in B. distachyon, extracts were derivatized with o-phthaldialdehyde and separated using HPLC. Analysis of the retention time indicated that the B. distachyon d18:1 LCB is desaturated at carbon position 4 (d18:1Δ4) (Fig. 1d). To confirm this, B. distachyon extracts were spiked with d18:1Δ4 and HPLC separation of o-phthaldialdehyde derivatives showed the presence of only one peak corresponding to d18:1Δ4 (Fig. 1e) as opposed to two peaks in A. thaliana (Fig. 1c).

There appears to be species differences with regards to the identity of the d18:1 LCB. d18:1Δ4 is notably absent from A. thaliana and Glycine max (soybean) (Markham et al. 2006), while d18:1Δ4 is present in Lycopersicon esculentum (tomato) (Markham et al. 2006), Commelina communis (Ng et al. 2001; Michaelson et al. 2009), and B. distachyon. The functional significance for the observed variance between species remains to be determined.

3.3 Quantitative analysis of sphingolipids

Quantitative analyses of LCBs showed that t18:0 (4-hydroxysphinganine or phytosphingosine) is the predominant LCB in B. distachyon shoots, followed by d18:0 (sphinganine or dihydrosphingosine), t18:1 (4-hydroxy-8-sphinganine), and d18:1Δ4 (4-sphingenine or sphingosine) (Fig. 2a). t18:0 is also the predominant LCB in undifferentiated cells in suspension cultures, followed by similar levels of d18:0, d18:1 and t18:1 (Fig. 2a). The levels of LCBs (nmol g−1 dw) are similar in A. thaliana (Markham and Jaworski 2007) and shoots of B. distachyon.
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Fig. 2

Quantitative analyses of LCBs and LCB-Ps from 4-week old B. distachyon shoots and undifferentiated cells of B. distachyon in suspension cultures. a LCBs and b LCB-Ps. Values are means ± SE (n = 5)

Interestingly, the levels of t18:0, were about 6.5-fold higher in undifferentiated cells in suspension cultures (ca. 13 nmol g−1 dw) compared to shoots of B. distachyon (ca. 2 nmol g−1 dw). In contrast, the predominant phosphorylated-LCB (LCB-P) in B. distachyon shoots is t18:1-P (ca. 0.014 nmol g−1 dw) followed by similar levels of d18:0-P, d18:1-P, and t18:0-P (Fig. 2b). Analysis of the LCB-P profile of undifferentiated cells in suspension cultures showed that t18:0-P (ca. 0.08 nmol g−1 dw) is the predominant LCB-P (as opposed to t18:1-P in shoots), followed by d18:1-P, d18:0-P and t18:1-P (Fig. 2b). The level of t18:0-P in undifferentiated cells in suspension cultures is about 10-fold higher compared to shoots (Fig. 2b). We did not detect the presence of d18:2 in B. distachyon, although very low levels of d18:2 were detected in A. thaliana Col-0 (Michaelson et al. 2009). Additionally, d18:2 were also detected in flowers of A. thaliana Col-0 (Michaelson et al. 2009). It remains to be determined if flowers of B. distachyon also contain d18:2.

The predominance of the t18:0 LCB is also seen in the ceramide fraction of the isolated sphingolipids from shoots and undifferentiated cells in suspension cultures of B. distachyon. This is in contrast to A. thaliana where t18:1 is the predominant LCB in ceramides (Markham and Jaworski 2007). t18:0 is also the predominant LCB moiety of ceramides isolated from seed bran, endosperm and leafy stems of rice (Oryza sativa), another member of the Poaceae (Fujino et al. 1985; Ohnishi et al. 1985). For all fatty acid chain lengths detected (c16 to c26, saturated and unsaturated), t18:0 is the most abundant LCB backbone, followed by t18:1 (Fig. 3a). d18:1 and d18:0 were detected but only as minor constituents, by comparison to t18:0 or t18:1, of ceramide with short chain fatty acids (c16 and c18) in shoots and undifferentiated cells in suspension cultures. The most prevalent ceramide in shoots is t18:0–c22:0 followed by similar levels of t18:0–c24:0 and t18:0–c24:1, and t18:0–c26:0 (Fig. 3a). In contrast, t18:0–c24:0 is the predominant ceramide species in undifferentiated cells in suspension cultures. These results suggest that the fatty acid moieties of ceramides from B. distachyon (shoots and cells in suspension cells) are very long chain fatty acids (VLCFAs). The predominance of VLCFAs was also observed in ceramides from seed bran, endosperm and leafy stems of rice (O. sativa) (Fujino et al. 1985; Ohnishi et al. 1985). Ceramide containing c20:1 fatty acids is present in very minor amounts (Fig. 3a) in both shoots and undifferentiated cells in suspension cultures. Interestingly, all four LCBs (d18:0, d18:1, t18:0, and t18:1) were found in ceramides with relatively short fatty acid chain lengths (c16:0 and c18:0). The LCBs d18:0 and d18:1, were notably absent in ceramide species with fatty acid chain lengths ≥c20 in both shoots and undifferentiated cells in suspension cultures (Fig. 3a). Higher levels of ceramides (ca. 1.8-fold) were observed in undifferentiated cells in suspension cultures compared to shoots (Fig. 3a).
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Fig. 3

Quantitative analyses of sphingolipids from 4-week old B. distachyon shoots and undifferentiated cells of B. distachyon in suspension cultures. a Ceramides, b hydroxyceramides, and c glucosylceramides. Values are means ± SE (n = 5)

Examination of the hydroxyceramide species indicated the absence of either the d18:0 or d18:1 LCB backbones in both shoots and undifferentiated cells in suspension cultures of B. distachyon. t18:0 is the predominant LCB backbone across the range of hydroxy-fatty acids detected (h18 to h26, both saturated and unsaturated) in both shoots and undifferentiated cells in suspension cultures (Fig. 3b). The most prevalent hydroxyceramide species in shoots is t18:0–h24:0, which occurs at amounts about 5 times the next most abundant species, t18:1–h24:0. Similar profiles of hydroxyceramides were observed in undifferentiated cells in suspension cultures. Hydroxyceramides with h18:0 and h20:1 fatty acids appear to be very minor components (Fig. 3b). The relative abundance of hydroxyceramides in undifferentiated cells in suspension cultures were about 3-fold higher compared to shoots (Fig. 3b).

Analysis of the glucosylceramide fraction in both shoots and undifferentiated cells in suspension cultures of B. distachyon revealed the absence of d18:0 or d18:1 and almost negligible levels of t18:0. t18:1 appears to be the predominant LCB backbone in glucosylceramides in B. distachyon (Fig. 3c). t18:1 also appears to be the major LCB in glucosylceramides from other members of the Poaceae, e.g., leafy stems of rice (O. sativa), leaves of maize (Zea mays), oat (Avena sativa), wheat (Triticum aestivum), winter rye (Secale cereale), and wheat germ (Ohnishi et al. 1985; Uemura and Steponkus 1994; Imai et al. 1997; Takakuwa et al. 2005). The predominant glucosylceramide in both shoots and undifferentiated cells in suspension cultures are t18:1–h22:0, t18:1–h24:0, t18:1–h24:1, t18:1–h20:0, and t18:1–h22:1 (Fig. 3c). It is worth noting that even the most abundant glucosylceramides species, t18:1–h22:0 is detected at a level ca. 8 nmol g−1 dw, well below the most abundant ceramide species (Fig. 3a, c). This is in contrast to A. thaliana where d18:1–h16:0 is one of the predominant glucosylceramide species (Markham and Jaworski 2007). Interestingly, the levels of glucosylceramides in both shoots and undifferentiated cells in suspension cultures were comparable (Fig. 3c), and this is in contrast to ceramides and hydroxyceramides where levels were about 1.8- and 3-fold higher in undifferentiated cells than shoots, respectively (Fig. 3a, b).

3.4 Quantitative analysis of glycerophospholipids

Quantitative analysis of the various classes of glycerophospholipids present in shoots of B. distachyon showed that the predominant glycerophosphocholines are PC(16:0–18:3) and PC(16:0–18:2), at ca. 575 and 545 nmol g−1 dw, respectively. The next most abundant glycerophosphocholines are PC(18:3–18:3), PC(18:2–18:3), PC(18:2–18:2), and PC(18:1–18:2) (Fig. 4a). Minor glycerophosphocholines are PC(16:0–16:0), PC(18:0–18:1), PC(16:0–16:2), PC(16:0–16:1), and PC(16:1–18:3) (Fig. 3a). PC(16:0–18:2) is the predominant glycerophosphocholine (ca. 164 nmol g−1 dw) in undifferentiated cells in suspension cultures, followed by PC(16:0–18:3) at ca. 99 nmol g−1 dw (Fig. 4a). The levels of glycerophosphocholines were about 6.5-fold lower in undifferentiated cells in suspension cultures compared to shoots (Fig. 4a).
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Fig. 4

Quantitative analyses of glycerophospholipids from 4-week old B. distachyon shoots and undifferentiated cells of B. distachyon in suspension cultures. a Glycerophosphocholines, b glycerophosphoethanolamines, c glycerophosphoglycerols, d glycerophosphoinositols, e glycerophosphoserines, and f diacylglycerophosphatidic acids. Values are means ± SE (n = 5)

Glycerophosphoethanolamines (Fig. 4b) are present at lower amounts compared to glycerophosphocholines (Fig. 4a). The predominant glycerophosphoethanolamine species in shoots and undifferentiated cells in suspension cultures of B. distachyon is PE(16:0–18:2) at ca. 154 and 190 nmol g−1 dw, respectively. The next most abundant glycerophosphoethanolamine species in shoots are PE(16:0–18:3) and PE(18:2–18:2), at ca. 154 and 131 nmol g−1 dw, respectively. In contrast, the next most abundant glycerophosphoethanolamine species in undifferentiated cells in suspension cultures are PE(18:2–18:2) and PE(16:0–18:3), at ca. 112 and 58 nmol g−1 dw, respectively The least abundant glycerophosphoethanolamine species in shoot and undifferentiated cells are PE(24:0–18:2) and PE(24:1–18:2) (Fig. 4b). In contrast to glycerophosphocholines (Fig. 4a), similar levels of glycerophosphoethanolamine were observed in both shoots and undifferentiated cells in suspension cultures (Fig. 4b).

The glycerophosphoglycerol profiles are markedly different in shoots and undifferentiated cells in suspension cultures in B. distachyon (Fig. 4c). Additionally, glycerophosphoglycerols are about 17-fold higher in shoots compared to undifferentiated cells in suspension cultures (Fig. 4c). The levels of the most abundant glycerophosphoglycerols in shoots (Fig. 4c) are similar to those observed for glycerophosphoethanolamines (Fig. 4b). Similar levels of the glycerophosphoglycerols (ca. 113–124 nmol g−1 dw), PG(18:3–16:1), PG(16:0–16:0), and PG(16:0–18:3) were observed in shoots (Fig. 4c). In undifferentiated cells in suspension cultures, the most abundant glycerophosphoglycerol is PG(16:0–18:2) at ca. 12 nmol g−1 dw, followed by PG(16:0–16:0) at 11 nmol g−1 dw, and PG(16:0–18:3) at 8 nmol g−1 dw (Fig. 4c).

Analysis of the glycerophosphosinositol profiles of shoots and undifferentiated cells in suspension cultures of B. distachyon showed the predominance of two glycerophosphoinositol species, PI(16:0–18:3) and PI(16:0–18:2) (Fig. 4d). The relative abundance of glycerophosphoinositols in shoots is about 3.4-fold higher compared to undifferentiated cells in suspension cultures (Fig. 4d). The most abundant glycerophosphoinositol in B. distachyon shoots is PI(16:0–18:3) at ca. 109 nmol g−1 dw (Fig. 4d), followed by PI(16:0–18:2), which is about 2-fold lower at ca. 63 nmol g−1 dw (Fig. 4d). In contrast, similar levels of PI(16:0–18:2) and PI(16:0–18:3) ca. 27 and 25 nmol g−1 dw, respectively, were observed in undifferentiated cells in suspension cultures (Fig. 4d). The other glycerophosphoinositols are present at relatively low levels ranging from ca. 1.2 to 6.5 nmol g−1 dw (Fig. 4d) in shoots, and ca. 0.006 to 1.7 nmol g−1 dw in undifferentiated cells in suspension cultures (Fig. 4d).

We observed that PS(22:0–18:3) and PS(22:0–18:2), ca. 266 and 229 nmol g−1 dw, respectively, are the predominant glycerophosphoserines in shoots of B. distachyon (Fig. 4e). These two glycerophosphoserines are also the predominant species in undifferentiated cells in suspension cultures (Fig. 4e). The relative abundance of glycerophosphoserines in shoots is about 5-fold higher compared undifferentiated cells in suspension cultures (Fig. 4e).

Levels of the most abundant diacylglycerophosphatidic acids (Fig. 4f) are similar to the most abundant glycerophosphoserines in shoots of B. distachyon (Fig. 4e). The predominant diacylglycerophosphatidic acids in shoots are PA(16:0–18:3), PA(18:2–18:3), PA(18:2–18:2), PA(16:0–18:2), PA(18:3–18:3) with levels ranging from ca. 134 to 272 nmol g−1 dw (Fig. 4f). The diacylglycerophosphatidic acids, PA(18:2–18:1) and PA(16:1–18:3) are about 7.5–8.5-fold lower at ca. 37 and 32 nmol g−1 dw, respectively (Fig. 4f). In contrast, the predominant diacylglycerophosphatidic acid in undifferentiated cells in suspension cultures is PA(18:2–18:2) at ca. 56 nmol g−1 dw, followed by PA(16:0–18:2) at ca. 39 nmol g−1 dw, similar levels of PA(16:0–18:3) and PA(18:2–18:3), at ca. 27 and 26 nmol g−1 dw, respectively (Fig. 4f). The relative levels of diacylglycerophosphatidic acids in shoots are about 6.7-fold higher in shoots compared to undifferentiated cells in suspension cultures (Fig. 4f).

Our results clearly indicate that there are differences in (1) the lipid profiles and (2) levels of individual lipid species between shoots and undifferentiated cells in suspension cultures of B. distachyon (Figs. 2, 3, and 4). The relative levels of LCBs (Fig. 2a) LCB-Ps (Fig. 2b), ceramides (Fig. 3a) and hydroxyceramides (Fig. 3b) appear to be higher in undifferentiated cells in suspension cultures compared to shoots, whereas the reverse is true for glycerophospholipids (Fig. 4a–f). Numerous studies have indicated that sphingolipids are important in plant growth and development (see reviews by Spassieva and Hille 2003; Sperling and Heinz 2003; Dunn et al. 2004; Lynch and Dunn 2004; Lynch et al. 2009; Pata et al. 2010). Our results clearly demonstrated comprehensive differences in the lipid profiles and levels in the highly differentiated shoot compared to the undifferentiated cells in suspension cultures of B. distachyon. This is hardly surprising given previous observations of fundamental differences in the transcriptomes of undifferentiated cells in suspension cultures of A. thaliana compared to differentiated tissues (Yamada et al. 2003). Park et al. (2010) showed using transcript profiling and lipidomic analysis of ceramide subspecies in mouse embryogenic stem cells and embryoid bodies that there is good agreement between changes in the ceramide lipidome and relative transcript levels of ceramide synthases during embryogenesis. There are also indications that glycerophospholipids may participate in regulating cellular proliferation and differentiation in mammalian retinal stem cells, and that the observed difference in glycerophospholipids are correlated with the activities of long-chain acyl-CoA synthetases (Li et al. 2007). Having characterized the lipidome of undifferentiated cells in suspension cultures of B. distachyon, it will be therefore be of interest to determine whether changes in the lipidome of B. distachyon following differentiation is mirrored by changes in the gene expression profiles.

There appears to be differences in the levels of glycerophospholipids in B. distachyon compared to the dicotyledonous model species, A. thaliana. Total glycerophosphoinositols from B. distachyon shoots were ca. 26-fold lower compared to A. thaliana. Total glycerophosphoglycerols, glycerophosphoethanolamines, and glycerophosphocholines from B. distachyon shoots were lower by ca. 40-, 19-, and 8-fold, respectively compared to A. thaliana (Welti et al. 2002). In contrast, comparable levels of total diacylglycerophosphatidic acids were observed between B. distachyon shoots and A. thaliana while the total glycerophosphoserines were about 1.5-fold higher in B. distachyon shoots compared to A. thaliana (Welti et al. 2002). Some of the observed differences may be due to the use by Welti et al. (2002) of the product ion of m/z 241 for quantitation of glycerosphosphoinositol and m/z 153 for glycerophosphoglycerol and diacylglycerophosphatidic acid. These product ions are not specific to particular glycerophospholipid classes and may result in over-estimation. This is further exacerbated by the use of direct infusion without HPLC separation by Welti et al. (2002). In contrast, we have used (1) HPLC separation prior to MS quantitation of B. distachyon extracts, and (2) lipid-class specific product ions for MRM quantitation.

Allwood et al. (2006) reported that levels of glycerophosphoglycerol are altered in B. distachyon (accessions ABR1 and ABR5) in response to the fungal pathogen, Magnaporthe grisea. They observed reductions in levels of glycerophosphoglycerol species, PG(16:0–16:1), PG(16:1–18:3), PG(16:0–18:3), and PG(16:0–18:2). Changes in the levels of diacylglycerophosphatidic acids were also observed in B. distachyon ABR1 and ABR5 in response to M. grisea. However, the observed alterations are not consistent across both accessions. In B. distachyon ABR1, levels of PA(16:0–18:3) and PA(16:0–18:2) were increased in response to M. grisea whereas levels of PA(18:2–18:3) were reduced. In contrast, levels of PA(16:0–18:3) were reduced while PA(16:0–18:2) and PA(18:2–18:3) were increased following infection with M. grisea (Allwood et al. 2006). Given the observation of the relative importance of glycerosphospholipids in responses of B. distachyon to infection by the fungal pathogen M. grisea (Allwood et al. 2006), future work should focused on comprehensive examination of the changes in the lipidome of B. distachyon in response to other important fungal pathogens of temperate grasses, e.g., Fusarium graminearum.

4 Conclusions

We have identified and quantified the levels of 123 lipids from 10 classes of sphingolipids and glycerophospholipids, gaining an insight into the sphingolipidome and glycerophospholipidome of B. distachyon, the model temperate grass (monocotyledon) species. Interestingly, we showed that d18:1Δ4 is the predominant desaturated dihydroxy LCB as opposed to the situation in A. thaliana (the model dicotyledonous species) where d18:1Δ8 predominates. Knockout mutants of the sphingolipid Δ4-desaturase (DES1-like) gene of A. thaliana do not appear to display any observable phenotype, leading the authors to question a role for d18:1Δ4 and d18:1Δ4-P in A. thaliana (Michaelson et al. 2009). As d18:1Δ4 is the predominant dihydroxy LCB in tomato and B. distachyon, it is envisaged that characterizing knockout mutants of the DES1-like gene in B. distachyon would enable the role(s) of d18:1Δ4 to be determined. Whether there are species differences in the position of desaturation in the dihydroxy LCB in plants remains to be determined, and systematic analysis of representative plants from different phylogenetic groups is likely to lead to further insights. Additionally, similarities in the predominance of particular LCBs and VLCFAs in ceramides and glucosylceramides between B. distachyon and other members of the Poaceae lend credence to the use of B. distachyon as the lipidomic model for the Pocaeae. Finally, it will be of interest to determine if differences in the lipid profiles and levels of individual lipid species are mirrored by differences in expression levels of genes encoding enzymes involved in lipid metabolism and/or activities of enzymes involved in lipid metabolism between shoots and undifferentiated cells in suspension cultures.

Acknowledgments

This work is supported by Science Foundation Ireland (SFI) Research Frontiers Programme and Equipment Grants (06/SFI/RFP/GEN034, 06/SFI/RFP/GEN034ES, 08/SFI/RFP/EOB1087) to C.K-Y.N. and an Irish Research Council for Science, Engineering, and Technology (IRCSET) Postgraduate Scholarship to J.P.C.

Supplementary material

11306_2011_352_MOESM1_ESM.doc (50 kb)
Supplementary material 1 (DOC 50 kb)

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© Springer Science+Business Media, LLC 2011