Gibberellin signalling: a pathway towards wheat improvement · Wheat Rht-1 dwarfing genes Rice sd1...
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Gibberellin signalling: a pathway towards wheat improvement
Steve Thomas Plant Biology and Crop Sciences Dept. Rothamsted Research
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Gibberellins control plant growth and development
GA1
grd2
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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,… ?
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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
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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)
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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)
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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)
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TILLING in wheat GA20ox1 genes
20ox1A: splice
20ox1A: G->S 20ox1B: Q->stop
20ox1D: G->D
20ox1A: R->S
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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]
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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: