Structural organization of rapeseed oil bodies affects ... · Grown in field at Inra RENNES (IGEPP,...
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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)