Gibberellin signalling: a pathway towards wheat improvement · Wheat Rht-1 dwarfing genes Rice sd1...
Transcript of Gibberellin signalling: a pathway towards wheat improvement · Wheat Rht-1 dwarfing genes Rice sd1...
Gibberellin signalling: a pathway towards wheat improvement
Steve Thomas Plant Biology and Crop Sciences Dept. Rothamsted Research
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
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0.2
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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
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10
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60
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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: