Biofortifying Brassica with calcium (Ca) and - IPNC Home Page

XVI International Plant Nutrition Colloquium,Sacramento, CA, USA, August 26-30 2009

Bi f tif i B i ith l i (C )

, , , g

Biofortifying Brassica with calcium (Ca) and magnesium (Mg)1Broadley MR, 2Hammond JP, 3King GJ, 2Bowen HC, 2Hayden RM, 2Spracklen WP, 1Ó Lochlainn S, 4White PJ

1Plant and Crop Sciences Division, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire, LE12 5RD, UK2Warwick HRI, University of Warwick, Wellesbourne, Warwick CV35 9EF, UK3Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, UK4The Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK

Overview

Dietary Ca and Mg deficiencies: the need for biofortificationy g

Brassica as a good target for Ca and Mg biofortificationBrassica as a good target for Ca and Mg biofortification

Potential for genetic improvement of BrassicaPotential for genetic improvement of Brassica

natural genetic variation

induced mutation

Integration and application

induced mutation

Dietary calcium (Ca) and magnesium (Mg) deficiencies

Relatively large amounts of Ca and Mg required in the human diet

Numerous health disorders associated with low Ca and Mg status

Relatively large amounts of Ca and Mg required in the human diet

e.g. Ca… bone-related (osteoporosis, rickets), etc.

e.g. Mg… heart dysfunction, hypertension, diabetes, pre-eclampsia, etc.

Soft-tissue biomarkers of Ca/Mg status unreliable, intake used to identify risk

http://www.eatwell.gov.uk/healthydiet/nutritionessentials/vitaminsandminerals/


White PJ, Broadley MR (2005). Biofortifying crops with essential mineral elements. Trends in Plant Science, 10, 586-593.


UK adult female Reference Nutrient Intake (RNI; ages 19-64)

opul

atio

n

0.8

1.0 Mgor

tion

of p

o

0.4

0.6

Prop

o

0 0

0.2

Intake from all sources (mg d-1)

0 100 200 300 400 5000.0

50.0% <270 mg magnesium d-1


UK adult female Reference Nutrient Intake (RNI; ages 19-64)

opul

atio

n

0.8

1.0 Mgor

tion

of p

o

0.4

0.6

Prop

o

0 0

0.2


0 100 200 300 400 5000.0

2.5% <150 mg magnesium d-1 = Lower Reference Nutrient Intake (LRNI)50.0% <270 mg magnesium d-1


UK adult male Reference Nutrient Intake (RNI; ages 19-64)

opul

atio

n

0.8

1.0 Mgor

tion

of p

o

0.4

0.6

Prop

o

0 0

0.2


0 100 200 300 400 5000.0

50.0% <300 mg magnesium d-1


UK adult male Reference Nutrient Intake (RNI; ages 19-64)

opul

atio

n

0.8

1.0 Mgor

tion

of p

o

0.4

0.6

Prop

o

0 0

0.2


0 100 200 300 400 5000.0

2.5% <190 mg magnesium d-1 = Lower Reference Nutrient Intake (LRNI)50.0% <300 mg magnesium d-1


UK adult female actual intake (RNI; ages 19-64; NDNS, 2003)

opul

atio

n

0.8

1.0 Mgor

tion

of p

o

0.4

0.6

Prop

o

0 0

0.2


0 100 200 300 400 5000.0

14.0% <150 mg magnesium d-1 = 3.2 million UK adult females <LRNI68.0% <270 mg magnesium d-1 = 16.3 million UK adult females <RNI


UK adult male actual intake (RNI; ages 19-64; NDNS, 2003)

opul

atio

n

0.8

1.0 Mgor

tion

of p

o

0.4

0.6

Prop

o

0 0

0.2


0 100 200 300 400 5000.0

45.0% <300 mg magnesium d-1 = 16.3 million UK adult males <RNI12.0% <190 mg magnesium d-1 = 2.7 million UK adult males <LRNI


~6 million UK adults <LRNI for Mg

Calcium RNI = 700 mg d-1; LRNI = 400 mg d-1. [low compared to US]

~3 million UK adults <LRNI for Ca

UK diet high in dairy + mandatory Ca-fortification of non-wholemeal flour

Many more at risk globally?

Overview





induced mutation


induced mutation

Brassica as a good target for Ca and Mg biofortification


Calcium

Karley & White (2009) Current Opinion in Plant Biology 12: 291-298


Magnesium (& K)

Karley & White (2009) Current Opinion in Plant Biology 12: 291-298



2006 production: sica

"

60

80

2006 production:

68.9 Mt yr-1 cabbages / other brassica18.1 Mt yr-1 cauliflowers / broccoli

age

& o

ther

bra

spr

oduc

tion

(Mt)

40

60

cf. 64.4 Mt yr-1 onions and shallots "Cab

ba p

0

20

and

broc

coli"

tion

(Mt)

10

15

Brassica oleracea"C

aulif

low

er

prod

uct

5

10

19971998

19992000

20012002

20032004

20052006

0

http://faostat.fao.org/


Phylogenetic analyses among angiospermsBrassica

Spinachp

Whit PJ & B dl MR (2003) A l f B t 92 487 511Broadley MR et al. (2003). Journal of Experimental Botany, 54, 1431-1446White PJ & Broadley MR (2003). Annals of Botany, 92, 487-511Broadley MR et al. (2004). Journal of Experimental Botany, 55, 321-336White PJ et al. (2004). Journal of Experimental Botany, 55, 1927-1937Watanabe T et al. 2007. New Phytologist, 174, 516-523


Evolutionary conservation of leaf traits shown in variance components analysis

Proportion of variation

C M K N P SCa Mg K N P Se

64 65 50 9 7 5order and above (%) 64 65 50 9 7 5

36 35 50 91 93 95

order and above (%)

within order (%)

Ca and Mg low in commelinoid monocot leaves

C d M hi h i B i d th id

Mg and Mg:Ca high in Caryophyllales orders

Ca and Mg high in Brassicaceae and other rosids

10


hoot

-Ca

. dev

.)

6

8

Can=670Brassica

Stan

dard

ised

s((x

-mea

n)/s

td

2

4 n=429

n=325Spinach

0

2.5

3.0

M

p

ardi

sed

shoo

t-Mg

mea

n)/s

td. d

ev.)

1.0

1.5

2.0Mg

Stan

da((x

-m

0.0

0.5

All species

Brassica

Spinach

Broadley MR et al. 2008. Plant Physiology, 146, 1707-1720

Overview





induced mutation


induced mutation

Potential for genetic improvement of Brassica

Brassica oleracea diversity

Image G King, G Teakle


~450 diversity accessions sampled:

Experiments in glasshouse and field environments (2002-2007)

450 diversity accessions sampled:

sabellica (borecole)italica (broccoli)b t ti ( lifl )botrytis (cauliflower) capitata (cabbage)gongylodes (Kohlrabi)alboglabra (Oriental kale)g ( )sabauda (Savoy cabbage)gemmifera (Brussels sprout)

Potential for genetic improvement of Brassica%

DW

) 3n=429

Leaf

Ca

(%

2

0

2

0.8

Mg

(%D

W)

0.6

0.8

Leaf

M

0.4

capitata

sabauda

alboglabra

gemmifera

sabellica

tronchuda

italica

botrytis

acephala

gongylodes0.0

AGDH DFS and F1s

Broadley MR et al. 2008. Plant Physiology, 146, 1707-1720.


F1 hybrids in field vs glasshouse experiments (2002-2007)…

4 1 0

3

4

0.8

1.0Mg (%DW), R=0.65

perim

ent Ca (%DW), R=0.76

3

0.6

ssho

use

exp

0 2 3 4 50

2

0.0 0.3 0.4 0.5 0.60.0

0.4Gla

Fi ld i t Fi ld i tField experiment Field experiment

…indicates strong genetic component to leaf/shoot Ca and Mg



DW

) 3

90

Leaf

Ca

(%

2

n=90

0

2

0.8

Mg

(%D

W)

0.6

0.8

Leaf

M

0.4

capitata

sabauda

alboglabra

gemmifera

sabellica

tronchuda

italica

botrytis

acephala

gongylodes0.0

AGDH DFS and F1s



V i C M K P Z FVariance component

Ca Mg K P Zn Fe

Genotype (VA) 36.0 37.7 22.2 7.4 18.5 7.1Genotype (VA) 36.0 37.7 22.2 7.4 18.5 7.1

[P]ext 0.2 4.0 2.2 43.1 7.2 3.5

[P]ext / 1.4 1.1 1.4 0.5 2.5 0.0genotype

‘other’ 62.4 57.2 74.2 49.0 71.8 89.4

High heritability in AG population (alboglabra X italica)


C2 C6 C7 C8

2A1B

1B

1B

5D

3D5A

1C

4B

1A

1E5E

3B5B 4A

D

C9

5B1

5F

Quantitative trait loci (QTL)

5E1DQuantitative trait loci (QTL)

for Ca and Mg


2.6A


DW

)

2.3

2.4

2.5

Shoo

t-Ca

(%

2.1

2.2

2.3

******

***

**C

A12GD33

AGSL118

AGSL119

AGSL120

AGSL121

AGSL122

AGSL123

AGSL127

AGSL129

AGSL134

AGSL137

AGSL138

AGSL141

AGSL158

AGSL160

AGSL161

AGSL165

AGSL168a

AGSL168b

AGSL169

AGSL1730.0

2.0C8 C9 C8 C9 C8 C9 C8 C9C8 C9 C8 C9 C8 C9 C8 C9

(%D

W) 0.6

A A A A A A A A A A A A A A A A A AG A A

* * * * ***

B

Shoo

t-Mg

0.5***

Broadley MR et al. 2008. Plant Physiology 146, 1707-1720A12

GD33

AGSL118

AGSL119

AGSL120

AGSL121

AGSL122

AGSL123

AGSL127

AGSL129

AGSL134

AGSL137

AGSL138

AGSL141

AGSL158

AGSL160

AGSL161

AGSL165

AGSL168a

AGSL168b

AGSL169

AGSL1730.0

0.4

2.6A


DW

)

2.3

2.4

2.5

Shoo

t-Ca

(%

2.1

2.2

2.3

******

***

**C

A12GD33

AGSL118

AGSL119

AGSL120

AGSL121

AGSL122

AGSL123

AGSL127

AGSL129

AGSL134

AGSL137

AGSL138

AGSL141

AGSL158

AGSL160

AGSL161

AGSL165

AGSL168a

AGSL168b

AGSL169

AGSL1730.0

2.0C8 C9 C8 C9 C8 C9 C8 C9C8 C9 C8 C9 C8 C9 C8 C9

(%D

W) 0.6

A A A A A A A A A A A A A A A A A AG A A

* * * * ***

B

Shoo

t-Mg

0.5***

Broadley MR et al. 2008. Plant Physiology 146, 1707-1720A12

GD33

AGSL118

AGSL119

AGSL120

AGSL121

AGSL122

AGSL123

AGSL127

AGSL129

AGSL134

AGSL137

AGSL138

AGSL141

AGSL158

AGSL160

AGSL161

AGSL165

AGSL168a

AGSL168b

AGSL169

AGSL1730.0

0.4

C2 C6 C7 C8

2A1B

1B

1B

5D

3D5A

1C

4B

1A

1E5E

3B5B 4A

D

C9

5B1

5F

Collinear with 106 genes on

5E1DCollinear with 106 genes on

Arabidopsis chromosome 5



AGSLs introgressed into maternal A12 ‘rapid’ cycling background (B1F2s)


Brassica napus diversity set

Potential for genetic improvement of BrassicaD

W)

4

DW

)

1.0

Ca Mg

ncen

trat

ion

(% D

3

ncen

trat

ion

(% D

0.6

0.8

♀ ♀♀ ♀

Ca Mg

n=130 n=130

ot o

r lea

f Ca

con

1

2

ot o

r lea

f Mg

con

0.4

0.6♀

♂

♀

♂♂ ♂

Shoo

0

1

Shoo

0.0

racea

DFS

cea A

GDH

napus D

FS

a rapa I

RRI

racea

DFS

cea A

GDH

napus D

FS

a rapa I

RRI

Brassic

a oler

a

Brassic

a oler

ace

Brassic

a nap

Brassic

a r

Brassic

a oler

a

Brassic

a oler

ace

Brassic

a nap

Brassic

a r

Brassica napus diversity set


Brassica rapa much more rapid-cycling, and it is our current major focus:

Brassica rapa, R-o-18, staged sowing

7 11 16 19 23 27 31 36 39 43 47 51 55

10 cm

7 11 16 19 23 27 31 36 39 43 47 51 55

Days after sowing (composite image)


R500 annual Yellow Sarson seed oil (R500) (ssp. tricolaris), USA (8 wks)

R-o-18 (8 wks)

IMB211 Rapid cycling Chinese cabbage (ssp. pekinensis), Japan (6 wks)

Potential for genetic improvement of Brassica(%

DW

)

4

(% D

W)

0 8

1.0

Ca Mg

conc

entr

atio

n (

2

3

g co

ncen

trat

ion

(

0.6

0.8

♀

♂

♀♀

♂♀

♂

Shoo

t or l

eaf C

a

1

2

Shoo

t or l

eaf M

g

0.4

♂ ♂♂

S

0

S

0.0

a olerac

ea D

FS

olerac

ea A

GDHsic

a nap

us DFS

ssica

rapa I

RRI

a olerac

ea D

FS

olerac

ea A

GDHsic

a nap

us DFS

ssica

rapa I

RRI

Brassic

a

Brassic

a ol

Brassic

Brass

Brassic

a

Brassic

a ol

Brassic

Brass


Overview





induced mutation


induced mutation

Potential for genetic improvement of BrassicaM d lli di t i ifi t i t t di l C t tModelling… predicts significant resistance to radial Ca-transport:

White PJ (1998). Annals of Botany, 81, 173-183; White PJ (2001). Journal of Experimental Botany, 52, 891-899; White PJ et al. (2002). New Phytologist, 153, 201-207.

Potential for genetic improvement of BrassicaC l i h t id tifi d f t t t t D S lt l b

9000

10000

Calcium-phenotype identified among fast-neutron mutants, Dave Salt lab:

g kg

-1 D

W)

7000

8000

9000

ot M

g (m

g

5000

6000

7000

Col-0

Shoo

3000

4000

5000esb1

Shoot Ca (mg kg-1 DW)

500010000

1500020000

2500030000

3500040000

450003000

(No Mg phenotype) Shoot Ca (mg kg DW)

Baxter I et al. (2009) PLoS Genetics, 5, e1000492.

PiiMS Database, www.ionomicshub.org

( g p yp )


Increased suberin deposition in esb1

Baxter I et al. (2009) PLoS Genetics, 5, e1000492.

Potential for genetic improvement of BrassicaHACC

(Annexin)DACC(TPC1)

KOR(SKOR,GORK)

VICC(GLR,CNGC)

Ca2+

(Annexin)(TPC1)(SKOR,GORK) (GLR,CNGC)

ACA8

Ca2+ Ca2+ Ca2+ Ca2+

cytoplasmACA4

CAX

SV Channel

HACC

Ca2+

Ca2+

H+

Ca2+

Ca2+

vacuole

V-typeATPase

ACA1IP3

IP6

H+

H+

Ca2+

Ca2+

plastid

ECA1

cADPRCa2+

NAADP-R

IP3-R CADPR-R

ECA1ACA1

endoplasmic reticulum

Vacuolar Ca accumulation via P2B-ATPases (ECA/ACA), Ca2+/H+ antiporters (CAX)

endoplasmic reticulum


CAX1-related phenotypes

Ca2+ : Mg2+ = 0 04 mol : mol

e.g. cax1 on faux-serpentine media

Bradshaw HD (2005). New Phytologist 167: 81–88.

Ca : Mg 0.04 mol : mol

e.g. overexpression of a modified CAX1 (sCAX1) increases bioavailable Ca

[carrot, lettuce, tomato, potato…]

Park et al., 2004; Park et al., 2005a,b; Kim et al., 2006; Morris et al., 2008; Park et al., 2009


)1000

Col-0

g kg

-1 D

W

600

800 cax1

cax3

ot M

g (m

g

400

cax1cax3

Shoo

0

200

Shoot Ca (mg kg-1 DW)

0500

10001500

20002500

30000

Shoot Ca (mg kg DW)

(Ca and Mg phenotype in cax1cax3) PiiMS Database, www.ionomicshub.org

Overview





induced mutation


induced mutation


BB B. nigra

Brassica genetic scheme (U, 1935)

BB B. nigra

AABB

(n=8)

AABB B. junceaCCBBB. carinata(n=18)(n=17)

AACCB. oleracea B. rapa

AACCAACC

B. napus

cabbagebroccolikale

t

turnipChinese cabbagepak choi

(n=9) (n=10)(n=19)

etc… oilseed rape / canolaswede

Integration and applicationB i ‘Chiif ’ l t i 2009 ( 500 Mb)

A2A1 A4A3 A7A5 A8A6 A10A9

Brassica rapa ‘Chiifu’ sequence complete in 2009 (~500 Mb)

BraCAX1

BraCAX3

KBrHOO4D08 anchored B. rapa ‘Chiifu’ BACÓ Lochlainn S et al. unpublished


TILLing population available (http://revgenuk.jic.ac.uk/)

Multiple alignment novel R-o-18 CAX ith kCAX sequences with known Brassica and Arabidopsis CAX fragments

AtCAX1

BrChiifuCAX1BrR-O-18CAX1-2BrR-O-18CAX1-1

AtCAX2

AtCAX6AtCAX5

Ó Lochlainn S et al. unpublished

0.1AtCAX4

AtCAX3


New project (2009-2013) aims to integrate Brassica phenotypic data (natural variants, eQTLs) with Arabidopsis IonomeHub

“Getting the Brassicaceae genomes/ionomes talking” [cyberinfrastrcture]Getting the Brassicaceae genomes/ionomes talking [cyberinfrastrcture]


UK adult female actual intake (RNI; ages 19-64; NDNS, 2003)

popu

latio

n

0.8

1.0or

tion

of p

0.4

0.6

Prop

0.0

0.2 Mg


0 100 200 300 400 500


Integration and application1,700,000

+1 portion of curly kale (UK Food Standards Agency)

popu

latio

n

0.8

1.0or

tion

of p

0.4

0.6

Prop

0.0

0.2 Mg


0 100 200 300 400 500


Integration and application500,000

+1 portion of Chinese cabbage (UK Food Standards Agency)

popu

latio

n

0.8

1.0or

tion

of p

0.4

0.6

Prop

0.0

0.2 Mg


0 100 200 300 400 500


Integration and application2,900,000

+1 portion of our “best” Brassica rapa

popu

latio

n

0.8

1.0or

tion

of p

0.4

0.6

Prop

0.0

0.2 Mg


0 100 200 300 400 500



Or even a pint of best!!

popu

latio

n

0.8

1.0or

tion

of p

0.4

0.6

Prop

0.0

0.2 Mg


0 100 200 300 400 500

XVI International Plant Nutrition Colloquium,Sacramento, CA, USA, August 26-30 2009

Bi f tif i B i ith l i (C )

, , , g

Biofortifying Brassica with calcium (Ca) and magnesium (Mg)1Broadley MR, 2Hammond JP, 3King GJ, 2Bowen HC, 2Hayden RM, 2Spracklen WP, 1Ó Lochlainn S 4White PJ

1Plant and Crop Sciences Division, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire, LE12 5RD, UK2Warwick HRI, University of Warwick, Wellesbourne, Warwick CV35 9EF, UK3Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, UK4The Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK

Brassica as a good target for Ca and Mg biofortification100

1

%D

W) Ca

44 elements in plant leaves

0.01

com

posi

tion

(%

Mg

0.0001

leaf

min

eral

c

0.000001Plan

t

Sources:Broadley MR Bowen HC Cotterill HL Hammond JP Meacham MC Mead A White PJ (2004) Phylogenetic variation in the shoot mineral concentration of angiosperms Journal of Experimental Botany 55 321 336

0.000000010.00000001 0.000001 0.0001 0.01 1 100

Human mineral composition (%DW)

Watanabe T, Broadley MR, Jansen S, White PJ, Takada J, Satake K, Takamatsu T, Jaya Tuah S, Osaki M. (2007). Evolutionary control of leaf element composition in plants. New Phytologist, 174, 516-523.

Epstein E. (1972). Mineral nutrition of plants: principles and perspectives. New York, USA: John Wiley and Sons, Inc.

Emsley J. (1998). The elements, 3rd ed. Oxford, UK: Clarendon Press

Broadley MR, Bowen HC, Cotterill HL, Hammond JP, Meacham MC, Mead A, White PJ. (2004). Phylogenetic variation in the shoot mineral concentration of angiosperms. Journal of Experimental Botany, 55, 321-336.

Hodson MJ, White PJ, Mead A, Broadley MR. (2005). Phylogenetic variation in the silicon composition of plants. Annals of Botany, 96, 1027-1046.

Marschner H. (1995). Mineral Nutrition of Higher Plants, 2nd Edition. Academic Press, London, UK.

Kay CE, Tourangeau PC, Gordon CC. (1975). Fluoride levels in indigenous animals and plants collected from uncontaminated ecosystems. Fluoride, 8, 125-133.


“Cabbage and other brassica” “Cauliflower and broccoli”

on to

nnes

)

30

40

on to

nnes

)

8

10

odcu

tion

(mill

io

20

odcu

tion

(mill

io

4

6

hinandia tio

n c ofpan

aineesia and

ania rica any

Nam

2006

pro

0

10

hinandia ric

apain Italy nce

xico anddom stan any key

2006

pro

0

2

Chin Ind

Russian Federatio

Korea, Republic o

JapaUkrain

IndonesPolan

Roman

United States of Americ

Germany

Viet Nam

Chin Ind

United States of Americ Spai Ita

yFranc

MexicPolan

United Kingdom

Pakista

Germany

Turkey

http://faostat.fao.org/

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Biofortifying Brassica with calcium (Ca) and - IPNC Home Page