Yongfang Wan, Malcolm Hawkesford, Rowan Mitchell and Peter Shewry

Rothamsted Research, UK

Improving the nitrogen responses of UK wheat varieties

Background

• Most varieties exhibit a negative relationship between grain protein and yield.

• Few varieties show reproducible deviations, such as

Cordiale and Marksman with high yield and grain protein.

• These varieties can make most efficient use of nitrogen inputs, have better economic costs and minimise environmental pollution.

• Little is known about the molecular basis for the protein deviation resulting from:

• nitrogen uptake • source metabolism • sink activity

This project will: • identify some candidate transcripts responding

to nitrogen application and /or related to the protein deviation;

• determine the effect of nitrogen levels on protein

composition; • examine interaction between nitrogen, protein

deviation and bread-making quality.

Strategy

Six cultivars (He, Co, Mk, Is,

Ma, Xi)

Three N levels (N100, N200, N350kg/ha)

AA 14 DAA AA 21DAA AA 28 DAA AA 35DAA AA 42DAA mature

Transcriptome Affymetrix and qPCR

Protein composition SE-HPLC;SDS-PAGE

Baking quality

Agronomic traits Grain yield and protein

Field trials

Bioinformatics Hypothesis, Genes, markers

integrated

Grain protein responses to nitrogen in 2009 and 2010 at Rothamsted.

0

0.5

1

1.5

2

2.5

3

Co He Is Ma Mk Xi Co He Is Ma Mk Xi

Gra

in %

N

2009 I 2010

N100

N200

N350

Grain yield responses to nitrogen in 2009 and 2010 at Rothamsted

0

2

4

6

8

10

12

14

Co He Is Ma Mk Xi Co He Is Ma Mk Xi

Gra

in y

ield

(t/

ha@

85

% D

M)

2009 I 2010

N100

N200

N350

105 transcripts are significantly up-regulated by nitrogen.

Transcriptome analysis at 21D in 2009 and 2010 by Affymetrix .

N100 N200 N350 N100 N200 N350

2009 2010 Storage

proteins(13)

Defence genes (6)

photosynthesis genes(5)

Unknown genes (81)

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N1

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N2

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N3

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N1

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N2

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N1

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N2

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N3

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N1

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N2

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N3

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N1

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N2

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N3

50Co

HeIs

MaMk

Xi

Raw

exp

ress

ion

Gliadin transcripts

2009

2010

2009

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N3

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50Co

He IsMa

MkXi

raw

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HMW-glutenin subunit transcripts

2009

2010

2009

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Co He Is Ma Mk XiR

aw e

xpre

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LMW-glutenin subunit transcripts

2009

2010

Response of storage protein transcripts to nitrogen

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1

2

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4

5

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8

9

10

14 21 28 35

REL

ATI

VE

EXP

RES

SIO

N

days after anthesis

N100

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N350

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CO HE IS MA MK XI

N100

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CO HE IS MA MK XI

N100

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2009 2010

A novel γ-gliadin gene is up-regulated by nitrogen.

Expression on Affymetrix

Expression by realtime RT-PCR

1AS

CS-typical γ-gliadin

γ-3 hordein (Hordeum chilense) CS-3

CS-3

CS-1

He-1

T. monococcum

Ae. tauchii

CS-2

He-2

1DS

1BS

Comparison of novel γ-gliadin genes

Xi IS Co Ma MK He

HMW-glutenins

ω- gliadins

LMW-glutenins & α,β,γ gliadins

By

Dx Bx

Ax

Dy

Globulin and Albumin

Effect of nitrogen on protein composition

N100 N200 N350

HMW glutenins ω - gliadins LMW-glutenin & gliadins

Dx2%=Dx2 volume/total HMW glutenins

ω- 5 Dx

Bx By

Dy ω- (1+2)

7.5

12.5

17.5

22.5

27.5

32.5

1Ax1 1Dx5 1Bx17 1By18 1Dy10

N100

N200

N350

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

ω-5a ω-(1+2)a ω-(1+2)b

N100

N200

N350

7.5

12.5

17.5

22.5

27.5

32.5

1Ax1 1Dx5 1Bx17 1By18 1Dy10

N100

N200

N350

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

ω-5a ω-(1+2)a ω-(1+2)b

N100

N200

N350

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

HMW-GS% ω-GLIADIN% LMW-GS+GLIADINS(%)

N100

N200

N350

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

HMW-GS% ω-GLIADIN% LMW-GS+GLIADINS (%)

N100

N200

N350

HMW-glutenin

Omega-gliadin

Group protein

2009 2010

Cordiale

IS IS

32.0

33.0

34.0

35.0

36.0

37.0

38.0

39.0

40.0

40.0 42.0 44.0 46.0 48.0 50.0 52.0

Po

lym

eri

c (F

1+

F2)

Monomeric (F3+F4)

Linear (N100)

Linear (N200)

Linear (N350)

HE

Ma

CO MKMK

IS

HE HE

IS ISXI

N100 N200 N350

Relationship of polymeric and monomeric proteins by SE-HPLC in 2009 mature seeds

2010 baking quality from Rothamsted site

Summary

1: Nitrogen application can increase both grain protein and yield.

2: The storage protein transcripts are significantly up-regulated by nitrogen. 3: The protein compositions respond to nitrogen disproportionally. 4: The baking quality is improved by higher nitrogen levels, but is affected by different sites, and years .

Gene expression Protein composition

Grain protein Grain yield

Nitrogen

Baking quality

environments

Protein deviation

Future

Acknowledgment

Funding:

Partners: Campden BRI, Gemma Chope

HGCA, Dhan Bhandari

Nickerson Seeds RAGT seeds Syngenta seeds KWS-UK

ADM milling Ltd Heygates ltd Warbourtons Rank Hovis McDougal