Zinc and iron speciation in the Zinc and iron speciation in the cereal grain
by:Jan K. Schjoerring
Daniel P. Persson
Thomas H. Hansen
K i ti H L Kristian H. Laursen
and Søren Husted
University of Copenhagen, Faculty of Life Science, Plant and Soil Science Laboratory University of Copenhagen, Faculty of Life Sciences, Plant and Soil Science Laboratory
XVI International Plant Nutrition Colloquium, August 26 - 30 2009, Sacramento
Objectives• To quantify how Zn and Fe are distributed in the
cereal graing• To identify the dominating ligands in cereal
tissue fractions
University of Copenhagen, Faculty of Life Science, Plant and Soil Science Laboratory University of Copenhagen, Faculty of Life Sciences, Plant and Soil Science Laboratory
BackgroundZn and Fe speciation is largely unknown The dominating ligand is considered to be the P-rich g gcompound IP6 (the phytate dogma) Proteins/peptides may also be important, e.g. ferritin,
t ll thi i i ti i ith hi hmetallothioneins or nicotianamine with higher bioavailabilityP and S are indicators of the ligand typeP and S are indicators of the ligand type
Hea
rd e
t al 2
001
University of Copenhagen, Faculty of Life Science, Plant and Soil Science Laboratory University of Copenhagen, Faculty of Life Sciences, Plant and Soil Science Laboratory
PO2-
Myo-inositol (1,2,3,4,5,6) hexakis-phosphatePhytic acid, IP6
CN- C2-
Quantification of Zn, Fe, P and S distribution in rice grain g
Zn140160Fe80
Genotype AGenotype B
6080100120
40
60Genotype BGenotype C
DM
DM
A
PS4000
02040
0
20
ion,
µg
g-1
on, µ
g g-
1
10000
15000
2000
3000
4000
once
ntra
ti
once
ntra
tio
B
0
5000
0
1000
CCo
University of Copenhagen, Faculty of Life Science, Plant and Soil Science Laboratory University of Copenhagen, Faculty of Life Sciences, Plant and Soil Science LaboratoryHansen et al. 2009 Plant Methods 5, 12C
Quantification of Zn, Fe, P and S distribution in rice grain
Zn distributionDry matter distribution
Endosperm
g
Endosperm86%
Bran34%
Endosperm57%
Bran layers, including the aleurone
12%
Embryo2%
Embryo 9%
>40% of both Zn and Fe is localized to the
Bran55%
Endosperm32%
Fe distribution
is localized to the embryo and the bran layers and
therefore lost in polishingEmbryo 13%
University of Copenhagen, Faculty of Life Science, Plant and Soil Science Laboratory University of Copenhagen, Faculty of Life Sciences, Plant and Soil Science Laboratory
Hansen et al. 2009 Plant Methods 5, 12
Nutrient concentration in rice grain as a function of pre ceeding polish timea function of pre-ceeding polish time
E b+ Embryo
3000
3500PhosphorusSulphurm
- Embryo
25
30IronZinc
m
+ Embryo - Embryo
Zn
1500
2000
2500
ntra
tion,
ppm
S15
20
ntra
tion,
ppm Zn
0
500
1000
Con
cen S
P0
5
10
Con
cen
Fe
0 100 200 300 400 5000
Polishing time, s0 100 200 300 400 500
0
Polishing time, s
University of Copenhagen, Faculty of Life Science, Plant and Soil Science Laboratory University of Copenhagen, Faculty of Life Sciences, Plant and Soil Science Laboratory
Multi-elemental speciation analysis by LC ICP MS analysis by LC-ICP-MS
Scavenger column Superdex Peptide
10/300
Superdex 75 10/300SEC column
HPLCpump
UV-DAD
Agilent 7500cx ICP-MSInjector10/300
HR-SEC columnpump
Automatic fraction collection (Agilent 1100 SFC)
P SDemetalizationUltra-Filtration
IP-ICP-MSAgilent 7500cx ICP-MS
C-E
SI-M
S
DI-T
OF-
MSAgilent HPLC-DAD
University of Copenhagen, Faculty of Life Science, Plant and Soil Science Laboratory University of Copenhagen, Faculty of Life Sciences, Plant and Soil Science Laboratory
RPC
MA
LD
Analytical challenges: The S sensitivity in ICP MS is very lowsensitivity in ICP-MS is very low
32S-isotopes
• The major isotope (32S) is a prohibited mass due to e.g. the double oxygen interference
94.9%
yg
• S is usually measured in standard mode as the low abundant 34Smode as the low abundant 34S isotope
• S has a high ionization potential (10.36 eV) 32 33 34 35 36
0.8%4.3%
0.02%
University of Copenhagen, Faculty of Life Science, Plant and Soil Science Laboratory University of Copenhagen, Faculty of Life Sciences, Plant and Soil Science Laboratory
Increasing the sensitivity of S by O2/He addition to the octopole
The reaction is• The reaction is thermodynamically favourable: S+ + O2 SO+ + O
ΔHf = -6.2 kJ mol-1(Bandura et al. 2002)
32 + 32 48 16
Fe FeO+
56 72 • Can Fe, P and Zn also be analysed as oxides?
56 72
P PO+
31 47
Zn Zn+ + ZnO+
66 8266
University of Copenhagen, Faculty of Life Science, Plant and Soil Science Laboratory University of Copenhagen, Faculty of Life Sciences, Plant and Soil Science Laboratory
Octopole Collision and Reaction Cell
Signal-to-noise ratio and LOD was significantly improved
• S/N markedly yimproved for 48SO+
compared to 34S in standard modeo 8
10 34S (std mode)48SO (O2 mode
500
60072FeO (O2 mode)57Fe (std mode)
standard mode (LOD~3 ppb)
to n
oise
rati
4
6
/N ra
tio
200
300
400
72
Sig
nal
2
4S/
0
100
200 • LOD for 72FeO+
was improved >20 times compared to
[S] (ppb)0 10 20 30 40 50 60
0
[Fe] (ppb)0 2 4 6 8 10 12
p57Fe in standard mode (~0.5 ppb)
University of Copenhagen, Faculty of Life Science, Plant and Soil Science Laboratory University of Copenhagen, Faculty of Life Sciences, Plant and Soil Science Laboratory
Persson et al. 2009 Metallomics 1, 418-426
The method was validated against three metalloproteins with known S:metal p
ratio
Z t i I Z t i II F t i
Enzyme/Protein
Carbonic anhydrase
(bovine Metallothionein 2A
(rabbit liver)Myoglobin
(horse heart)
Zn-protein I Zn-protein II Fe protein
erythrocytes)Molecular
weight(Da)
28 983 6581 16 954
Histidines 11 0 11
Methionines 4 1 2
Cysteines 0 20 0Theoretical
S:metal ratio 4 3 2
Analysed
University of Copenhagen, Faculty of Life Science, Plant and Soil Science Laboratory University of Copenhagen, Faculty of Life Sciences, Plant and Soil Science Laboratory
AnalysedS:metal ratio 4.08 ± 0.02 3.06 ± 0.06 1.91 ± 0.05
Persson et al. 2009
Speciation pattern of barley embryo sample in
32 kDa
6.6 kDa1.4 kDa
17 kDa
Th i ti f F d Z i
barley embryo sample in oxygen mode
800
1200
1600 0.46 kDa1.4 kDa
Fe
• The speciation of Fe and Zn is clearly de-coupled
Z l t l i l k
400
3000
4000
5000
72FeO+
12.3 kDa
ts s
-1 P• Zn elutes as onle single peak
(3.0 kDa) overlapping with the LMW S-peak (ratio of S:Zn peak 43±6 2 S atoms per Zn)
800
n in
tens
ity (c
ount
s s-1
)
1000
2000
3000
47PO+
0.5 kDa
nsity
, cou
nt
43±6.2 S atoms per Zn)
• The major Fe peak overlaps with P as a major (12 3 kDa) and a1600
200
400
600Ion
66Zn+
3 kDa
Ion
inte
n
Zn
P as a major (12.3 kDa) and a minor (0.5 kDa) complex
• Which P peak contains phytic400
800
1200
1600
48SO+
S
University of Copenhagen, Faculty of Life Science, Plant and Soil Science Laboratory University of Copenhagen, Faculty of Life Sciences, Plant and Soil Science Laboratory
• Which P-peak contains phytic acid?
Retention time (sec.)
0 200 400 600 800 1000 1200 1400
400
Retention time, s Persson et al. 2009 Metallomics 1, 418-426
Dephosphorylation of fractionated P species by phytase incubation
IP6
- phytate identification by IP-ICP-MS
Fe4(IP6)18
tens
ity (C
PS
)
IP5
IP4
IP
12.3 kDa
0 5 kDa
Fe4(IP6)18
47P
O+
in IP10.5 kDa
2h
8h
oP
Time
start
University of Copenhagen, Faculty of Life Science, Plant and Soil Science Laboratory University of Copenhagen, Faculty of Life Sciences, Plant and Soil Science Laboratory
Retention time
Time Persson et al. 2009 Metallomics 1, 418-426
Mass balance- Enzymatic extractions
Extraction with phytase doubled Fe extractability but
%)
100
120
Zn
FeExtraction with protease XIV
doubled Fe extractability, but did not increase
that of Zn Zn
ed o
f tot
al (%
60
80P
S
Extraction with protease XIVincreased Zn extractability
5 times, but did not increase that of Fe
P
Extra
cte
20
40 P extractability increased with phytase (86%),
but not with protease
S
TRIS Phytase Protease XIV0
S extractability increased with protease (96%),
but not with phytaseFe
University of Copenhagen, Faculty of Life Science, Plant and Soil Science Laboratory University of Copenhagen, Faculty of Life Sciences, Plant and Soil Science Laboratory
FePersson et al. 2009 Metallomics 1, 418-426
Size exclusion chromatography f i d
30000
of rice endospermnt
s s-1
)
20000
25000
30000
FeP
s s-1
) 30000
40000
66Zn48SO+
Sunts
s-1
ount
s s-
1
inte
nsity
(cou
n
10000
15000
inte
nsity
(cou
nt
10000
20000
Zn
nsity
, co
nsity
, co
0 00 00 00 00 00 00 00 00 00 00
Ion
0
5000
0 00 00 00 00 00 00 00 00 00 00Io
n
0
Ion
inte
n
Ion
inte
n
Retention time (s)
0 200 400 600 8001000
12001400
16001800
2000
Retention time (s)
0 200 400 600 8001000
12001400
16001800
2000
Retention time, s Retention time, s
University of Copenhagen, Faculty of Life Science, Plant and Soil Science Laboratory University of Copenhagen, Faculty of Life Sciences, Plant and Soil Science Laboratory
Fe associated with phytate but not Zn
unts
s-1
)
8000
TSN endosperm with phytaseTSN endosperm without phytase
nts
s-1 )
1.2e+5
1.4e+5
1.6e+5 TSN endosperm with phytaseTSN endosperm without phytase
tens
ity (c
o
4000
6000
ensi
ty (c
oun
6.0e+4
8.0e+4
1.0e+5 ZnFe
FeO
+ io
n in
2000
4000
6 Zn
ion
inte
0.0
2.0e+4
4.0e+4
Retention time (s)0 200 400 600 800 1000 1200 1400
72F 0
Retention time (s)0 200 400 600 800 1000 1200 1400 1600
66
University of Copenhagen, Faculty of Life Science, Plant and Soil Science Laboratory University of Copenhagen, Faculty of Life Sciences, Plant and Soil Science Laboratory
Extraction of unprepared rice versus k d i ( d 200 20 500 kD ) cooked rice (superdex 200: 20- 500 kDa)
35000
TSN TRIS/NaCl
unts
s-1)
20000
25000
3000072FeO+
66Zn
TSN_TRIS/NaCl
25000
TSN_TRIS/NaCl_Boiled
Ion
inte
nsity
(co
5000
10000
15000
(cou
nts s
-1)
15000
20000
Retention time (s)
0 200 400 600 800 1000 1200 1400 1600
0
Ion
inte
nsity
(5000
10000
Retention time (s)
Retention time (s)
0 200 400 600 800 1000 1200 1400 1600
0
University of Copenhagen, Faculty of Life Science, Plant and Soil Science Laboratory University of Copenhagen, Faculty of Life Sciences, Plant and Soil Science Laboratory
Retention time (s)
Endosperm Fe speciation in rice overexpressing
nicotiamine synthetase8000
10000OsNAS3D-1WT
+ (cou
nts s
-1)
Fe
Transfo nicotiamine synthetase
2000
4000
6000
on in
tens
ity; 72
FeO
+ Fe
• Activation-tagging mutant lines in which expression of a rice NAS gene, OsNAS3, was increased by
WT
ormant
0
O+ (c
ount
s s-1
)
4000
6000
Io
P
a rice NAS gene, OsNAS3, was increased by introducing 35S enhancer elements - Prof. Gynheung An, Pohang Univ., South Korea
• WT and OsNAS3-D1 had an equal amount of Fe bound in a medium molecular weight complex co-
Ion
inte
nsity
; 47PO
0
2000
4000 bound in a medium molecular weight complex co-eluting with P in a Fe: IP6 oligomer complex
• OsNAS3D-1 had significantly more (7.0±0.3 times) Fe bound in a low molecular weight complexTh l l l i ht F k did t l t
6000
8000
O+ (c
ount
s s-1
)
S
• The low molecular weight Fe peak did not co-elute with neither P nor S. Fe was thus not bound to IP6, or to cysteine/methionine containing ligands (peptides or proteins)
2000
4000
6000
Ion
inte
nsity
; 48SO • Mass calibration indicated an apparent molecular
mass of approximately 1300±400 Da for the Fe complex, suggesting a cluster with Fe bound to the –NH+ and/or –COO- functional groups of several NA
University of Copenhagen, Faculty of Life Science, Plant and Soil Science Laboratory University of Copenhagen, Faculty of Life Sciences, Plant and Soil Science LaboratoryRetention time (s)
0 200 400 600 800 1000 1200 14000
g pligands
Lee et al. 2009 PNAS ( in press)
Conclusions & perspectivesConclusions & perspectives• The speciation of Fe and Zn in rice and barley grain were clearly de-
coupledcoupled – Fe is predominantly speciated with IP6 polymers – Zn with LMW peptidesp p
• The phytate dogma is challenged: Neither the speciation analysis nor the enzymatic extraction indicated Zn:IP6 bindingLi d h i l i t id h l i• Ligand exchange is always an issue to consider when analysing coordination complexes…..
• LC-ICP-MS is a powerful tool to study the elemental speciation of grain tissues
• Ideal tool for testing e.g. the various transgenic approaches used to incerase bioavailability of Fe and Zn in the cereal grains
University of Copenhagen, Faculty of Life Science, Plant and Soil Science Laboratory University of Copenhagen, Faculty of Life Sciences, Plant and Soil Science Laboratory
incerase bioavailability of Fe and Zn in the cereal grains
Acknowledgements • Daniel P. Persson• Søren Husted
Acknowledgements
• Søren Husted• Thomas H. Hansen• Kristian H. Laursen
B t B• Bente Broeng• Bente Postvang
www.phime.eu + DANIDA
• Prof. Gynheung An, Pohang Univ Sci & T h l D t I t t
Danish Mi i t fTechnol, Dept Integrat
Biosci & Biotechnol, Pohang South Korea
t h
Ministry of Science
University of Copenhagen, Faculty of Life Science, Plant and Soil Science Laboratory University of Copenhagen, Faculty of Life Sciences, Plant and Soil Science Laboratory
gwww.meta-phor.eu