Zinc and iron speciation in the cereal...

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


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


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%


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


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


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%


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


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)


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


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


• 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


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


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


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


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


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


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


gwww.meta-phor.eu

Zinc and iron speciation in the cereal...

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