Gibberellin signalling: a pathway towards wheat improvement

Steve Thomas Plant Biology and Crop Sciences Dept. Rothamsted Research

steve.thomas@rothamsted.ac.uk

Gibberellins control plant growth and development

GA1

grd2

Gibberellin signalling allows plants to adapt to the environment

HO

O C

O

COOH GA4

Light

Germination

Stem/Hypocotyl Elongation

Temperature Drought Salt

Fruit Growth

Flowering Leaf Growth

Root Growth

Seed Development

Nutrients, Hypoxia Gravity, Pathogens, Mechanical damage, Etc,… ?

Gibberellin biosynthesis and signalling mutants have improved agronomic traits

Wheat Rht-1 dwarfing genes Rice sd1 dwarfing genes

Monna et al. 2002, Sasaki et al. 2002 and Spielmeyer et al. 2002

Peng et al. 1999 and Pearce et al. 2011

Gibberellin signalling pathway

GGPP CPP

CPS

ent-KAURENE

KS

ent-KAURENOIC ACID KO

GA53

KAO

GA20 GA20ox

GA1 GA3ox

GA8 GA2ox

Bio-active Bio-inactive

GA12

GA13ox (SD1)

Are current wheat dwarfing genes optimal?

Rht-1 semi-dwarfing alleles can increase grain yield, but reduce stem elongation

Adverse pleiotropic effects…. Reduced grain size Reduced seedling vigour Fertility defects?

Rht-1 Rht-B1c Rht-1 Rht-B1b

Flintham et al., 1997 Ellis et al., 2004

Lack of diversity in wheat Rht-1 dwarfing genes

Chandler and Harding (2013) J. Ex. Bot.

A recent forward genetics-based screen

Rht-1

Rht-B1d Rht-B1b Rht-D1b

(Rht1) (Rht2)

Rht-B1c Rht-D1c

(Rht3) (Rht10)

Two classes of Rht-1 mutants

Why do the Rht-1 mutations cause dwarfism?

Peng et al. 1999

Rht-1 Rht-B1b Rht-D1b

Early GA-response genes

GID1 GA X GRAS

DELLA

Re-initiation of translation?

STOP codons introduced

GRAS DELLA

Regulatory Domain

Functional Domain

Molecular characterisation of Rht-B1 and Rht-D1 mutations

B1s B1b D1b D1d B1e B1c D1c

Rht-B1a, Rht-D1a

Rht-B1b, B1s

Rht-B1e

Rht-D1b, D1c, D1d

Most Rht-1 dwarfing mutations introduce premature stop codons

Pearce et al. Plant Physiol. 2011, Wu et al. Plant Physiol. 2011, Li et al. New Phytologist 2012

transposon insertion into Rht-B1

cDNA

gDNA

Partially splicing leaves in-frame insertion

Early GA-response genes

GID1 GA X

Rht-B1c

Rht-D1 gene copy number and expression is elevated in Rht-D1c

Rht-D1a Rht-D1b Rht-D1c Rht-D1d

D1a D1b D1c D1d D1c D1d

Pearce et al. Plant Physiol. 2011

Rht-B1 dwarfing mutations prevent interaction with the GID1-GA receptor complex

Rht-B1c

Early GA-response genes

GID1 GA X GRAS

Rht-B1b

Early GA-response genes

GID1 GA

X DELLA GRAS

Pearce et al. Plant Physiol. 2011

Identification of novel Rht-1 dwarfing mutations by TILLING in Hexaploid wheat.

Rht-1

Rht-B1d Rht-B1b Rht-D1b

(Rht1) (Rht2)

Rht-B1c Rht-D1c

(Rht3) (Rht10)

Lack of diversity in wheat Rht-1 dwarfing genes

Pearce et al. Plant Physiol. 2011

Rht-1 Homoeologues have similar expression profiles

Three distinct regions of DELLAs are required for the binding to the GID1-GA complex

Murase et al. Nature 2008

SLR1 AAGEEEDVDELLAALGYKVRSSDMADVAQKLEQLEMAMGMGGVSAPGAADDGFVSHLATDTVHYNPSDLSSWVESMLSELN 111

Rht-D1 AAGEGEEVDELLAALGYKVRASDMADVAQKLEQLEMAMGMGGVGAGAAPDDSFATHLATDTVHYNPTDLSSWVESMLSELN 110

RGA EDGGGNMDDELLAVLGYKVRSSEMAEVALKLEQLETMMSN-------VQEDGLS-HLATDTVHYNPSELYSWLDNMLSELN 110

* : *****.******:*:**:** ****** *. . :*.: ***********::* **::.******

Rht-B1e Rht-B1b Rht-D1b

Rht-B1c

An in vitro assay to assess whether Rht-1 mutations are likely to cause GA-insensitivity

1 2 3 4

10mM 3AT + GA3

25mM 3AT + GA3

50mM 3AT + GA3

10mM 3AT - GA3

25mM 3AT - GA3

50mM 3AT - GA3

SC-Leu/Trp

1 2 3 4

1 Empty bait/ Rht-D1 NT

2 GID1/Rht-D1 NT

3 GID1/Rht-A1 NT

4 Empty bait/ Rht-A1 NT

Identification of novel Rht-1 dwarfing mutations by TILLING in Hexaploid wheat.

Strategy: Screen the TILLING population using Rht-1 homoeologue-specific PCR

followed by HRM analysis for the detection of mutations.

- Rht-1 homoeologue-specific PCR established

- Screened approx. 1200 lines for mutations in all three Rht-1 homoeologues

- 12 missense mutations in Rht-A1.

- 11 missense mutations in Rht-D1.

- 13 putative mutations in Rht-B1.

Rht-A1 AAGEGEEVDELLAALGYKVRASDMADVAQKLEQLEMAMGMGGVGAGAAPDDSFATHLATDTVHYNPTDLSSWVESMLSELNA

Rht-D1 AAGEGEEVDELLAALGYKVRASDMADVAQKLEQLEMAMGMGGVGAGAAPDDSFATHLATDTVHYNPTDLSSWVESMLSELNA

Rht-B1e Rht-B1b Rht-D1b

Sln1-d

Gibberellin signalling pathway in wheat – multiple paralogs of key genes

GGPP CPP

CPS (1)

ent-KAURENE

KS (1)

ent-KAURENOIC ACID KO (1)

GA53

KAO (1)

GA20 GA20ox (4)

GA1 GA3ox (2)

GA8 GA2ox (8)

Bio-active Bio-inactive

Multigene families

GA12

GA13ox (2)

Rht (1)

GID1 (1) GID2 (1)

(SD1 in rice)

Differential expression within paralogous groups identifies possible targets

0

0.2

0.4

0.6

0.8

1

GA20ox1 GA20ox2 GA20ox3 GA20ox4 GA3ox1 GA3ox2

Peduncle Endosperm

Rel

ativ

e ex

pre

ssio

n le

vel (

fro

m

RN

Ase

q)

TILLING in wheat GA20ox1 genes

20ox1A: splice

20ox1A: G->S 20ox1B: Q->stop

20ox1D: G->D

20ox1A: R->S

Mutations in wheat 20ox1 genes reduce height

WT Cadenza

ga20ox1a/b/d triple

heterozygote

ga20ox1a/b/d triple

homozygote

0

10

20

30

40

50

60

70

80

90

100

wt heterozygote tripleH

eigh

t [c

m]

Acknowledgements

Andy Phillips Peter Hedden Rht-1 mutation characterisation Stephen Pearce Robert Saville (JIC) Paul Nicholson (JIC) Margaret Boulton (JIC) Peter Chandler (CSIRO) Rht-1 TILLING Archana Patil Chen Liang Cuneyt Ucarli 20ox1 TILLING Katie Tearall Carlos Banyon Barbora Gallova

Funding: