Structural organization of rapeseed oil bodies affects their stability and oil extractability

T Chardot, 2015-07-09 Saskatoon thierry,chardot@versailles,inra,fr

http://www-ijpb.versailles.inra.fr/en/bs/equipes/biostructurale/index.htm

Structural organization of rapeseed oil bodies affects their stability and oil extractability Introduction and previous results

• Oil extraction

• Oil bodies

• Reserve accumulation in rapeseed

• Model pressing

• Focus on Amber and Warzanwski

Characterization of OB morphology in mature or developing seeds

• fluorescence and transmission electron microscopy

• pulsed-field gradient NMR

Study of the composition of OB hemi-membrane into

• phospholipids

• tocopherols

• phytosterols

• proteins

Conclusions perspectives

Improvement of the oil extraction yield: Identification of determinant seed features

WP3 : Improvement of the oil extraction yield (CETIOM, IJPB)

CETIOM: optimization of the crushing process (identification of seed features determinant for oil extraction yield)

Energy consumption / ton of crushed rape seeds # 280 kWh (www.creol.fr)

Oil bodies are found in most organisms

Pollen cells , Dangeard, CR Acad Sci 1922, Dinis, Protoplasma 2009

Peanut cotyledon Jacks, Plant Phys, 1967

Yeasts, Sandager, J Biol Chem 2001

Oil bodies play central role in oil metabolism

Oil accumulation

Oil mobilization

Actors of seed oil

accumulation ?

Seed oil mobilization,

Structure ?

Stability

Seed oil Extraction

Oil bodies are stable oil / in water emulsions

Czabany, Jbiol Chem, 2008

Gray et al., JAOCS, 2006

In: Buchanan 2002 Biochemistry and molecular

biology of plants

Smart emulsions / vectors for protein expression (See M Moloney’s and JTC Tzen’s work

Biological material used in our study

96 winter oilseed rape accessions with contrasted oil and protein contents : 54 «00» (low erucic acid, low glucosinolates) 24 «++» (high erucic acid, high glucosinolates), 17 «0+», 1 «+0» Grown in field at Inra RENNES (IGEPP, N Nesi)

Biological material used in our study

High negative correlation between oil and protein content (r = -0.88)

10

15

20

25

30

40 45 50 55 60

lipid content (%)

prot

ein

cont

ent (

%)

Lipid and protein contents measured by NIRS (Inra IGEPP Rennes, N Nesi)

« ++ »

Δ « 00 »

Jolivet et al. Ind Crop Products 2013

Model pressing using texturometer

Savoire et al., OCL 2010

+ : Use of grams of seeds G Global resistance against oil flow

Compressibility of B. napus seed depends on accessions

compr

essibi

lity

inde

x (C

I)

2

3

4

5

6

7

8

20 30 40 50 60

1000

/Gg

A

D

E

G

M

W

r = 0.903

Oil yield (%)

W: Low resistance against oil flow

Higher oil extraction yield in «++» compared to «00» Two contrasted lines: Amber (22% yield, CI 3.3) and Warzanwski (55% yield, CI 7.3) chosen to assess the stability of purified LBs.

A: High resistance against oil flow

Jolivet et al. Ind Crop Products 2013

« ++ »

Δ « 00 »

Amber and Warzanski OB detailed characterization

... use of Amber and Warzanwski for

characterization of OB morphology in mature or developing seeds

• transmission electron microscopy

• pulsed-field gradient NMR

study of the composition of OB hemi-membrane into

• phospholipids: CCM, HPTLC, LC-MS/MS

• tocopherols: GC-MS

• phytosterols: GC-MS

• proteins: immunodetection using specific antibodies, 2D-DIGE

Reserve accumulation in seeds

Seeds were vernalized (nine weeks) to ensure flowering and grown under controlled conditions (growth room)a.

Individual flowers were manually pollinated and tagged.

Amber

10 15 17 20 25 30 DAP

Warzanwski

10 15 20 25 30 DAP

Siliques were collected from 10 to 30 days after pollination (DAP) during embryogenesis and beginning of seed filling.

Reserve accumulation in seeds Seeds were characterized for their fresh weight, protein and total fatty acid contents.

Fresh weight of W (opened symbols) seeds decreased earlier / A (filled symbols) seeds.

W (opened symbols) mature seeds weight < A (filled symbols) mature seeds.

Evolution of protein and FA content similar with a lag phase for lipid accumulation.

OB morphology TEM

EM: S. Chat and C. Longin (MIMA2 platform, Jouy en Josas)

0

0.5

1

1.5

2

2.5

0 10 20 30 40 50 60 70 80

Dap

Diam

eter

(µm

)

Amb_hyp_confocal Amb_hyp_TEM Amb_cot_confocal Amb_cot_TEM

0

0.5

1

1.5

2

2.5

0 10 20 30 40 50 60 70 80

Dap

Diam

eter

(µm

)

Warz_hyp_confocal Warz_hyp_TEM Warz_cot_confocal Warz_cot_TEM

Amber

Warzanwski

0

0.5

1

1.5

2

2.5

0 10 20 30 40 50 60 70 80

Dap

Diam

eter

(µm

)

Amb_hyp_confocal Amb_hyp_TEM Amb_cot_confocal Amb_cot_TEM

0

0.5

1

1.5

2

2.5

0 10 20 30 40 50 60 70 80

Dap

Diam

eter

(µm

)

Warz_hyp_confocal Warz_hyp_TEM Warz_cot_confocal Warz_cot_TEM

Amber

Warzanwskihyp W>A cot W=A

S Starch N nucleus OB Oil bodies, Chl Chloroplast

TEM

Accumulation of fatty acids and proteins in rapeseed.

S1 S2 S4 Slo1

Clo1

10 20 30 40 50 DAF

S1, S5 Slo1

mRNA

OB Proteins Clo1

S2 S3 S4

S3 S5

Proteins

Fatty acids

Cru4 Cru2/3

Embryo-genesis

Seed filling

Storage Proteins

Gallardo et al CRC book, in press

OB morphology solide state NMR Pulsed Field Gradient NMRa analyzed the restricted diffusion properties of TGs inside OBs.

Schematic representation of TAG diffusion in OB (r = a)

Two pulses are separated by a long diffusion time (∆).

The echo intensity is correlated to diffusion coefficient D. I/I0 = exp(-kD)

The diffusion coefficient of TGs embedded in OBs is low compared with its value in oil.

The product D∆ is proportional to the mean square displacement of molecules (⟨r2⟩ ~ D∆)

Free diffusion is observed when molecules never meet physical barriers restricting their displacements during ∆.

Gromova M, Guillermo A, Bayle PA, Bardet M (2015) In vivo measurement of the size of oil bodies in plant seeds using a simple and robust pulsed field gradient NMR method. Eur Biophys J 44:121–129

OB morphology solide state NMR The product DmeasD is plotted versus the diffusion time D.

oil

seeds

Dmeas ~ 1.1 10-11 m2s-1

A plateau value is reached at long diffusion time in the case of seeds and not for oil. In case of confinement in spherical domains of radius a, the limit value of DmeasD is a2/5.

OB morphology solide state NMR

W 0.88 µm diameter

A 0.62 µm diameter

OB Diameter (µm)

Surface (µm2)

Volume (µm3)

A 0.62 1.2 0.12

W 0.88 2.4 0.36

Perspectives Studies on non mature seeds and on variants. Link with oil extraction

Measurements: only possible using electron microscopy on mature seeds

OB hemi-membrane composition into phospholipids

a LC-MS/MS analysis of PLs was carried out in collaboration with S. Nicolaÿ and A. Solgady (SAMM, IPSIT, Châtenay Malabry)

Extraction of lipids (Folch), separation by TLC.

PLs = ~ 2% total lipid content

Phospholipid extraction (solid phase extraction) and HPTLC.

PC = major PL (~ 60%)

PL classes and species separated and analyzed by LC-MS/MSa

• normal phase column (elution order: PA, PI, PE, PS, PC) • negative ionization ([M-H]-, [M+CH3COOH]- for PC) • molecular structure determined according to lipid maps from MS1 • fragmentation to identify acyl chains • no absolute quantification possible • comparaison of species content between Amber and Warzanwski

5 PL classes identified: PC, PE, PI, PS, PA

65 molecular species: 34 (16:0-18:n), 36 (18:n-18:n), 38 (18:n-20:n), 40 (18:n-22:n), 42 (18:n-24:n)

OB hemi-membrane composition into phospholipids PC

-

0.4

0.8

1.2

1.634

:4

34:3

34:2

34:1

36:5

36:4

36:3

36:2

38:3

40:4

40:3

Tota

l ion

cou

nt (T

IC x

106 )

PE

-

0.4

0.8

1.2

34:3

34:2

34:1

36:5

36:4

36:3

36:2

38:4

38:3

38:2

40:4

40:3

40:2

Tota

l ion

cou

nt (T

IC x

106 )

PI

-

1

2

3

34:3

34:2

34:1

34:0

36:6

36:5

36:4

36:3

36:2

36:1

38:3

40:4

40:3

40:2

Tota

l ion

cou

nt (T

IC x

106 )

PS

-

20

40

60

80

34:3

34:2

34:1

36:5

36:4

36:3

36:2

38:4

38:3

38:2

40:4

40:3

40:2

42:2

Tota

l ion

cou

nt (T

IC x

103 )

PA

-

100

200

300

34:3

34:2

34:1

36:6

36:5

36:4

36:3

36:2

38:4

38:3

38:2

40:4

40:3

Tota

l ion

cou

nt (T

IC x

103 )

PC

-

0.4

0.8

1.2

1.634

:4

34:3

34:2

34:1

36:5

36:4

36:3

36:2

38:3

40:4

40:3

Tota

l ion

cou

nt (T

IC x

106 )

PE

-

0.4

0.8

1.2

34:3

34:2

34:1

36:5

36:4

36:3

36:2

38:4

38:3

38:2

40:4

40:3

40:2

Tota

l ion

cou

nt (T

IC x

106 )

PI

-

1

2

3

34:3

34:2

34:1

34:0

36:6

36:5

36:4

36:3

36:2

36:1

38:3

40:4

40:3

40:2

Tota

l ion

cou

nt (T

IC x

106 )

PS

-

20

40

60

80

34:3

34:2

34:1

36:5

36:4

36:3

36:2

38:4

38:3

38:2

40:4

40:3

40:2

42:2

Tota

l ion

cou

nt (T

IC x

103 )

PA

-

100

200

300

34:3

34:2

34:1

36:6

36:5

36:4

36:3

36:2

38:4

38:3

38:2

40:4

40:3

Tota

l ion

cou

nt (T

IC x

103 )

PS A > PS W (x 1.8)

unsaturation A < W

38, 40 and 42 species A < W

Amber

Warzanwski

Hemi-membrane composition into tocopherols and phytosterols

Sterol biosynthetic pathway in higher plants

Rapeseed oil is rich in tocopherols (vitamine E) and phytosterols

Phytosterols decrease membrane fluidity (specially β-sitosterol). The introduction of a double bond at C22 (as in stigmasterol and brassicasterol) could increas fluidity

SMT1

SMT2 SMT: cycloartenol methyltransferase; DIM: sterol reductase

SMT1

SMT2 SMT: cycloartenol methyltransferase; DIM: sterol reductase

SMT1

SMT2 SMT: cycloartenol methyltransferase; DIM: sterol reductase

/crinosterol

OB hemi-membrane composition into tocopherols and phytosterols

22

25.8 26.0 26.2 26.4 26.6 26.8 27.0 27.2 27.4 27.6 27.8 28.0 28.2 28.4 28.6

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

28.22

27.43

26.8825.95

26.68

27.10 28.4727.9427.7327.2526.10 26.26 26.4825.85

Time (min)

Rel

ativ

e ab

unda

nce

γ-tocopherolMW 416

α-tocopherolMW 430

β-sitosterolMW 414

campesterolMW 400

brassicasterol/crinosterol

MW 398

C-22 desaturase

Using GC-MS (EI), determination of content of tocopherols and phytosterols in A and W OB lipids

OB hemi-membrane composition into tocopherols and phytosterols

Warzanwski contained less campesterol increase in C22 desaturase more brassicasterol increase in membrane fluidity

A contained more tocopherols (x 2) than W but OB surface is more important

more phytosterols (x 3) Amber Warzanwski

γ-tocopherol

α-tocopherol campesterol

β-sitosterol

brassicasterol

2.316.20.75.139826.88brassicasterol/crinosterol

1.522.10.838.840027.43campesterol

3.161.71.256.141428.22β-sitosterol

2.942.92.329.443026.68α-tocopherol

2.957.12.370.641625.95γ-tocopherol

SDMoySDMoy(Da)(min)

WarzanwskiAmberMWRTcompound

2.316.20.75.139826.88brassicasterol/crinosterol

1.522.10.838.840027.43campesterol

3.161.71.256.141428.22β-sitosterol

2.942.92.329.443026.68α-tocopherol

2.957.12.370.641625.95γ-tocopherol

SDMoySDMoy(Da)(min)

WarzanwskiAmberMWRTcompound

OB hemi-membrane composition into proteins Protein abundance was compared using semi-quantitative immunoblotting (IB)

and spectral counting (PAI protein abundance index)

Results were normalized against the seed lipid content and expressed as ratio A/W.

Amber is enriched in BnS1, BnS2 and BnS4 (H-oleosins)

A/W IB PAI BnS1 1.00 1.18

BnS2 1.41 1.03

BnS4 1.78 1.10

BnS3 0.85 0.89

BnS5 0.77 0.79

BnS5 Nter ………………………… Cter BnS3 Nter ………………………… Cter BnS1 Nter … 18 aa insertion ….. Cter BnS2 Nter … 18 aa insertion ….. Cter BnS4 Nter … 18 aa insertion ….. Cter

L forms

H forms

Conclusions

The use of the two contrasted rapeseed, Amber and Warzanwski, was a powerful tool to link OB structure and oil extractability.

Amber OBs are smaller than Warzanwski OBs but the necessary hemi-membrane surface to store the same lipid quantity is more important. Logically Amber OBs are enriched in proteins, tocopherols and phytosterols.

The differences observed in crushing ability, oil extraction yield and OB stability could be explained by the differences found in compositions of OB hemi-membrane rather than by the quantity of hemi-membrane constituents.

Characteristics of Amber OBs (which are very stable):

•enrichment in H-oleosins and Slo better coverage of OB surface numerous protein-protein interactions

• enrichment in PS more interactions with proteins

• depletion in poly-unsaturated PLs more rigid structure

• enrichment in phytosterols decrease in membrane mobility.

Boulard C, Bardet M, Chardot T, Dubreucq B, Gromova M, et al. (2015) The structural organization of seed oil bodies could explain the contrasted oil extractability observed in two rapeseed genotypes. Planta 242: 53-68.

Thanks to

26

Seeds (Nathalie Nesi, Rennes)

Plant breeding (Hervé Ferry)

Cytology (Martine Miquel, Bertrand Dubreucq, Olivier Grandjean, Halima Morin, Daniel Zaharia)

TEM (Marine Froissard, Sophie Chat and Christine Longin, Jouy)

NMR (Michel Bardet, Marina Gromova, Armel Guillermo, CEA Grenoble)

2D-DIGE (Gwendal Cueff, Céline Boursier, Châtenay)

Proteomics (Thierry Balliau, PAPPSO)

HPTLC (Michel Canonge.)

LC-MS et GC-MS (Stéphanie Yen-Nicolaÿ, Audrey Solgadi, Châtenay)