Submergence tolerance: Molecular mechanisms in water proof rice

1
Submergence tolerance Molecular mechanisms in
water proof rice
ERF transcription factor genes at the Sub1 locus
Sigrid Heuer
2
Outline of presentation

• Submergence tolerance- it works but how?
• Fine mapping of the Sub1 locus and three ERF
  candidate genes
• Why sequencing of QTLs in tolerant donors?
• Details on the ERF transcription factors
• differential expression and tolerant specific
  alleles
• Using sequence information for molecular marker
  development
• and screening for novel tolerance genes
• Assessment of Sub1A and SubC expression during
  plant development and in other stresses
• Conclusions

3
Swarna-sub1 is submergence tolerant
IR42 (susceptible check)
IR-40931(Sub1 donor)
Swarna-Sub1
Swarna
Meanwhile Development of IR64-sub1 and Samba
Mahsuri-sub1 completed, and underway for
Thadokkham 1 (Lao), CR1009/ Savitri (India) and
BR11 (Bangladesh).
4
Fine mapping and sequencing of the Sub1
locus(LOD gt 35 R2 70)
Chr. 9
Sub1
Sub1C
Sub1B
Sub1A
Sub1C
Sub1B
5
Sub1A is absent from the Nipponbare genome
IR40931-26
A303rf
101O9L
Nipponbare
A303rf
101O9L
Sub1A is sometimes present in the sequenced
indica (93-11) genome on chromosome unknown
This gene was only found because the QTL was
sequenced in the tolerant parent
6
Comparison of Pup1 sequence Kasalath and
Nipponbare
Kas Pup1 300 kb, 69 genes NB Pup1 150 kb, 47
genes
It is important to sequence the QTL region in the
tolerant donor
7
The ERF gene family 181 members in
rice, 159 in Arabidopsis
DREB genes only distantly related
8
Sub1A, B and C are putative ERF transcription
factors
(Apetala2/ Ethylene Responsive Element Binding
Protein)
Structure of ERF proteins
guides
binds
protein into nucleus
GCC-box in promoter of target genes
LeERF, predicted AA interacting with DNA (Swiss
model). Tournier et al 2003, FEBS
9
Expression of Sub1 genes is induced by submergence
10
Sub1A and Sub1C have opposite expression pattern
tolerant
intolerant
Kurkaruppan
Goda Heenati
IR64
CO39
submerged (h)
6 30 97 R
6 30 97 R
6 30 97 R
6 30 97 R
SUB1A
SUB1B
SUB1C
11
UbiSub1A-1 plants are submergence tolerant
Julia Bailey-Serres et al, UCR
Sub1C is down-regulated in transgenic plants
con
sub 3d
con
sub 3d
-
Is Sub1A a negative regulator of Sub1C ?
12
Principles of gene expression
exon 1
exon 2
intron
promoter
exon 1
exon 2
intron
pre-mRNA
mRNA
protein
13
Transcription factors regulate downstream gene
expression
Chromosome 1
gene 700
gene 1
AGCCGCC
Chromosome 9
gene 43000
gene 39000
Sub1C
14
The Sub1C upstream region
Start
- 3700 bp

16
6
8
8
9
7
3
5
Sub1C-2
9
8
11
15
16
4
10
13
8
Sub1C-1
1048 bp DELETION
1
2
5
14
12
8
8
9
-511 bp
-1559 bp
- 2700 bp
Cis elements different between tolerant and
intolerant allele

• -7bp RAV1AAT (Binding consensus sequence for
  transcription factor, protein has ap-2 like
  domains)
• -13bp POLLEN1LELAT52 (regulatory elements
  responsible for pollen specific activation
• -38bp SEF3MOTIFGM ( sef 3 binding site)
• -70bp PALBOXPPC
• -217bp Anaerobic consensus
• -1070bp GT1CONSENSUS (binding site in many
  light-regulated genes)
• -1142bp GATABOX (light regulated LHCII
• -2190bp, 2581, 2670 TATA box
• -2218bp 2723 CAAT box
• -2603bp ARR1AT (response regulator)
• -2900bp MYB core (regulates genes responsible
  for water stress)

• -2953bp
• MYCCONSENSUSAT (regulates the transcription of
  BF/DREB1 genes)
• Etiolation induced site
• EBOXBNNAPA (storage-protein gene)
• CACGTGMOTIF (interacts with G box)
• ROOTMOTIFTAPOX1
• -3017bp TAAAGSTKST1 (KST1 gene encodes a K
  influx channel of guard cells)
• -3024bp GT1CONSENSUS (binding site in many
  light-regulated genes)
• -3078bp GT1GMSCAM4 (Pathogen and salt-induced
  gene expression)
• -3101pb CGACGOSAMY3 (coupling element for the
  G box)
• -3361bp GCC box

15
The GCC-box is shifted in the tolerant Sub1C-1
allele
Intolerant GCTCCCGGCAGCCGCCGCCTCC Tolerant
GCTCCTGGCAGCAGCCGCCTCC

• Can Sub1A bind to the GCC box of Sub1C ?
• -------- possibly
• Is different position of the GCC box important
  for expression level?
• -------- possibly

16
The Sub1A upstream region
1.4 kb repeat region
Start
MYC
CAAT box
TATA box
-1461bp
Sub1A-1
-408bp
-932bp
-114bp
-1530bp
Sub1A-2
Etiolation- and light induced element
(overlapping)
RY- repeat (seed specific expression/ ABA
response)
anaerobic consensus and Dof-core target site
also 5 SNPs not part of known cis elements
Hypothesis If any of these elements are
importance for Sub1A expressionthey must be
present in the two other tolerant varieties
(Goda Heenati and Kurkaruppan)
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No regulatory domains common to all tolerant
varieties
1.4 kb repeat region
Start
CAAT box
TATA box
-1461bp
Sub1A-1 IR40931
-408bp
-932bp
-114bp
-1530bp
Sub1A-2
Goda Heenati
Kurkaruppan
- 470 bp(from ATG)

• Only minimal promoter region common
• to all tolerant accessions
• No SNPs in that part of the gene

Where is the domain responsible for high Sub1A
expression in tolerant and low expression in
intolerant accessions ?
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The Sub1A intron A regulatory domain?
promoter
intron
Sub1A
3 SNPs
5 UTR
3 UTR 1 SNP
MITE transposon
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Double stranded RNA induce gene silencing
Mathieu and Bender (2004), Journal of Cell
Science
20
MITE present in tolerant and intolerant Sub1A
allele
Intolerant Teqing
Tolerant IR40931-26
low expression
high expression
Is this missmatch effecting gene silencing ?
21
Sub1A and Sub1C have tolerant specific alleles
22
Sub1A and Sub1C have alleles specific to tolerant
varieties
23
Tolerant and intolerant Sub1A and Sub1C alleles
are distinct by putative MAPK phosphorylation
sites
putative MAPK sites
ERF DNA binding domain 56 AA
acidic domain 22 AA
SNP186Ser tolerantPro intolerant
NLS
Sub1A
SNP175Pro tolerantThr intolerant
Sub1C
SNP based markers distinguish Sub1A and Sub1C
tolerant and intolerant alleles.
24
Sub1A and Sub1C have tolerant specific
alleles AND are differentially expressed during
submergence
1. Using sequence information for MAS and
identification of novel tolerance genes
2. Expression analysis of Sub1A and Sub1C
25
SNP markers for Sub1A
tolerant ATGTGTGGAGGAGAAGTGATCCCCGCCGACAT
GCCGGCGGCGCCGTTCACGCCACGCCAC 60 susceptible
------------------------------ATGCCGGCGGCGCCGTTCAC
GCCACGCCAC 30

tolerant
GGCGACGGCGAGACATGGGTTGACAGAAAGAGGAGGAACAAGAAGAAGAG
GAAGCGCGGC 120 susceptible
GGCGACGGCGAGACATGGGTTGACAGAAAGAGGAGGAACAAGAAGAAGAG
GAAGCGCGGC 90

tolerant
GCCGACGAAGAATGGGAGGCCGCCTTCCAGGAGTTCATGGCTGCTGACGA
CGACGACGAC 180 susceptible
GCCGACGAAGAATGGGAGGCCGCCTTCCAGGAGTTCATGGCTGCTGACGA
CGACGACGAC 150

tolerant GGCGGCGGACTCGTGTTAAGT
AGTAAATCTTTGGTGTTGAGGTCACCAGGTGAAAATGAT 240
susceptible GGCGGCGGACTCGTGTTAAGTAGTAAATCTTTG
GTGTTGAGGTCACCAGGTGAAAATGAT 210

tolerant GCAGGCCGGGGCGCCGCCGCC
ACCATGTCCATGCCGCTGGACCCCGTGACCGAGGAGGCC 300
susceptible GCAGGCCGGGGCGCCGCCGCCACCATGTCCATG
CCGCTGGACCCCGTGACCGAGGAGGCC 270

tolerant GAGCCGGCGGTGGCTGAGAAG
CCTCGCCGGCGCCGGCCGAGGCGGAGCTACGAGTACCAC 360
susceptible GAGCCGGCGGTGGCTGAGAAGCCTCGCCGGCGC
CGGCCGAGGCGGAGCTACGAGTACCAC 330

tolerant GGCATCCGGCAGCGGCCGTGG
GGGCGGTGGTCGTCGGAGATCCGCGACCCCGTCAAGGGC 420
susceptible GGCATCCGGCAGCGGCCGTGGGGGCGGTGGTCG
TCGGAGATCCGCGACCCCGTCAAGGGC 390

tolerant GTCCGCCTCTGGCTCGGCACC
TTCGACACCGCCGTCGAAGCCGCGCTCGCCTACGACGCC 480
susceptible GTCCGCCTCTGGCTCGGCACCTTCGACACCGCC
GTCGAAGCCGCGCTCGCCTACGACGCC 450

tolerant GAGGCCCGCCGCATCCACGGC
TGGAAAGCCCGGACAAACTTCCCACCCGCCGATCTTTCT 540
susceptible GAGGCCCGCCGCATCCACGGCTGGAAAGCCCGG
ACAAACTTCCCACCCGCCGATCTTTCT 510

tolerant TCGCCGCCGCCGCCGTCGCAG
CCGCTCTGCTTCTTGCTCAACGACAACGGCCTCATCACA 600
susceptible TCGCCGCCGCCGCCGCCGCAGCCGCTCTGCTTC
TTGCTCAACGACAACGGCCTCATCACA 570

tolerant ATCGGAGAAGCGCCGACCGAC
GACGCCGCGTCGACGTCGACGTCGACGACGGAGGCGTCC 660
susceptible ATCGGAGAAGCGCCGACCGACGACGCCGCGTCG
ACGTCGACGTCGACGACGGAGGCGTCC 630

tolerant GGCGACGCGCGCATACAACTG
GAGTGCTGCTCGGACGACGTGATGGACAGCCTCCTCGCC 720
susceptible GGCGACGCGCGCATACAGCTGGAGTGCTGCTCG
GACGACGTGATGGACAGCCTCCTCGCC 690

tolerant GGCTACGACGTGGCCAGCGGC
GACGACATATGGACATGGACATCTGGAGCCTCCTCCACC 780
susceptible GGCTACGACGTGGCCAGCGGCGACGACATATGG
ACATGGACATCTGGAGCCTCCTCCACC 750

tolerant TCTGTTAACCAAGAGATCAAG
ACCCCATCGATCCACCAAAACATATCATATGCAGGTGCC 840
susceptible TCTGTTAACCAAGAGATCAAGACCCCATCGATC
CACCAAAACATATCATATGCAGGTGCC 810

tolerant CGCCCCATGACTTGTCACTTT
AAGAATCATAAAAACACTTTTGTACAAATGGAGTGCTCA 900
susceptible CGCCCCATGACTTGTCACTTTAAGAATCATAAA
AACACTTTTGTACAAATGGAGTGCTCA 870

tolerant ACCATGCTAAACTTACTCAAA
GGCCACAAACAATAA 936
susceptible ACCATGCTAAACTTACTCAAAGGCCACAAACAA
TAA 906

tolerant ATGTGTGGAGGAGAAGTGATCCCCGCCGACAT
GCCGGCGGCGCCGTTCACGCCACGCCAC 60 susceptible
------------------------------ATGCCGGCGGCGCCGTTCAC
GCCACGCCAC 30

tolerant
GGCGACGGCGAGACATGGGTTGACAGAAAGAGGAGGAACAAGAAGAAGAG
GAAGCGCGGC 120 susceptible
GGCGACGGCGAGACATGGGTTGACAGAAAGAGGAGGAACAAGAAGAAGAG
GAAGCGCGGC 90

tolerant
GCCGACGAAGAATGGGAGGCCGCCTTCCAGGAGTTCATGGCTGCTGACGA
CGACGACGAC 180 susceptible
GCCGACGAAGAATGGGAGGCCGCCTTCCAGGAGTTCATGGCTGCTGACGA
CGACGACGAC 150

tolerant GGCGGCGGACTCGTGTTAAGT
AGTAAATCTTTGGTGTTGAGGTCACCAGGTGAAAATGAT 240
susceptible GGCGGCGGACTCGTGTTAAGTAGTAAATCTTTG
GTGTTGAGGTCACCAGGTGAAAATGAT 210

tolerant GCAGGCCGGGGCGCCGCCGCC
ACCATGTCCATGCCGCTGGACCCCGTGACCGAGGAGGCC 300
susceptible GCAGGCCGGGGCGCCGCCGCCACCATGTCCATG
CCGCTGGACCCCGTGACCGAGGAGGCC 270

tolerant GAGCCGGCGGTGGCTGAGAAG
CCTCGCCGGCGCCGGCCGAGGCGGAGCTACGAGTACCAC 360
susceptible GAGCCGGCGGTGGCTGAGAAGCCTCGCCGGCGC
CGGCCGAGGCGGAGCTACGAGTACCAC 330

tolerant GGCATCCGGCAGCGGCCGTGG
GGGCGGTGGTCGTCGGAGATCCGCGACCCCGTCAAGGGC 420
susceptible GGCATCCGGCAGCGGCCGTGGGGGCGGTGGTCG
TCGGAGATCCGCGACCCCGTCAAGGGC 390

tolerant GTCCGCCTCTGGCTCGGCACC
TTCGACACCGCCGTCGAAGCCGCGCTCGCCTACGACGCC 480
susceptible GTCCGCCTCTGGCTCGGCACCTTCGACACCGCC
GTCGAAGCCGCGCTCGCCTACGACGCC 450

tolerant GAGGCCCGCCGCATCCACGGC
TGGAAAGCCCGGACAAACTTCCCACCCGCCGATCTTTCT 540
susceptible GAGGCCCGCCGCATCCACGGCTGGAAAGCCCGG
ACAAACTTCCCACCCGCCGATCTTTCT 510

tolerant TCGCCGCCGCCGCCGTCGCAG
CCGCTCTGCTTCTTGCTCAACGACAACGGCCTCATCACA 600
susceptible TCGCCGCCGCCGCCGCCGCAGCCGCTCTGCTTC
TTGCTCAACGACAACGGCCTCATCACA 570

tolerant ATCGGAGAAGCGCCGACCGAC
GACGCCGCGTCGACGTCGACGTCGACGACGGAGGCGTCC 660
susceptible ATCGGAGAAGCGCCGACCGACGACGCCGCGTCG
ACGTCGACGTCGACGACGGAGGCGTCC 630

tolerant GGCGACGCGCGCATACAACTG
GAGTGCTGCTCGGACGACGTGATGGACAGCCTCCTCGCC 720
susceptible GGCGACGCGCGCATACAGCTGGAGTGCTGCTCG
GACGACGTGATGGACAGCCTCCTCGCC 690

tolerant GGCTACGACGTGGCCAGCGGC
GACGACATATGGACATGGACATCTGGAGCCTCCTCCACC 780
susceptible GGCTACGACGTGGCCAGCGGCGACGACATATGG
ACATGGACATCTGGAGCCTCCTCCACC 750

tolerant TCTGTTAACCAAGAGATCAAG
ACCCCATCGATCCACCAAAACATATCATATGCAGGTGCC 840
susceptible TCTGTTAACCAAGAGATCAAGACCCCATCGATC
CACCAAAACATATCATATGCAGGTGCC 810

tolerant CGCCCCATGACTTGTCACTTT
AAGAATCATAAAAACACTTTTGTACAAATGGAGTGCTCA 900
susceptible CGCCCCATGACTTGTCACTTTAAGAATCATAAA
AACACTTTTGTACAAATGGAGTGCTCA 870

tolerant ACCATGCTAAACTTACTCAAA
GGCCACAAACAATAA 936
susceptible ACCATGCTAAACTTACTCAAAGGCCACAAACAA
TAA 906

SNP
CCGTCGC CCGCCGC
CCGTCGC tolerant (S186) CCGCCGC intolerant
(P186)
ACAACTG tolerant ACAGCTG intolerant
26
Sub1A allele specific SNP markers

• CAPS marker for the silent SNP

intolerant tolerant
ACAACTG T ACAGCTG S
AluI restriction site only in intolerant allele

• Dominant marker for the functional SNP

CCGTCGCAGCCGCTCTGCTTC T CCGCCGCAGCCGCTCTGCTTC
S
IR40931
IR64
H2O
IR64/IR40931 (BC1F2)
T T S T T S T T T T T T S T
27
Sub1C allele specific SNP markers
T T S S S S S S T S S S
28
Allele-specific markers for Sub1A and Sub1C
Upstream/ promoter region
GnS2 Alu1 (A?G)
IYT4 NlaIII (T?C )
Sub1a
IYT1 BsaA1 (T?G)
5 UTR
3 UTR
AEX1 (T?C) Pro ? Ser
ART2 (14bp indel)
ART4 DraI (T?G)
ART3 (7bp indel)
Sub1c
ART5 (15bp indel)
5 UTR
3 UTR
ERF3_173 Cac8I (or 9 bp indel)
29
SSR markers across the Sub1 region
RM23668 (0.6 Mb)
Chromosome 9
RM23778 (3.9)
RM23679 (0.8)
RM316 (1.0)
0 Mb
RM23788 (4.2)
Centromere (2.7 Mb)
RM8303 (2.3)
RM23805 (4.5)
RM23770 (3.7)
RM23772 (3.8)
RM23778 (3.9)
RM5899 (5.1)
RM23788 (4.2)
RM23805 (4.5)
RM23831, RM23833 (5.4)
10.4 Mb
Sub1 region
RM23835 (5.5)
RM464 (6.5)
RM23887 (6.5)
RM444 (dom.), RM23852 (5.9)
RM23917 (7.3)
RM23922 (7.4)
RM23928 (7.5)
RM23865 (6.2)
RM219 (7.8)
RM23958, RM23957 (7.9)
Sub1 locus
RM23869 (6.3)
RM464 (dom.) (6.5)
SC9/RM23887 (6.5)
RM24005 (9.2)
SC3/RM8300 (6.6)
RM24011 (9.3)
RM24046 (10.1)
RM6920 (7.0)
RM23911 (7.1)
RM24070 (10.4)
RM23915, RM23916 (7.2)
22.6 Mb
RM23917 (7.3)
30
Using the markers for breeding
recipient parent x Sub1 donor
BC1F1
1 2 3 4 5 6 7 8 9 10 11 12
1 2 3 4 5 6 7 8 9 10 11 12
MAS can replace phenotyping
Sub1 locus
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Using the markers to move beyond Sub1 Screening
of 178 landraces for novel tolerance genes
Determination of tolerance and Sub1 haplotype
Find tolerant accessions that are not Sub1A-1
/ Sub1C-1
32
Faro 27 is tolerant but not Sub1 Sub2
Faro 27 semi dwarf complex cross from IRRI
released as a variety in Nigeria in 1982
33
However Some accessions with tolerant Sub1A and
Sub1C alleles are intolerant
Is the expression of Sub1A and Sub1C more
important than the allele ?
34
RT-PCR analysis of representative accessions
Sub1A
35 cycles 37 cycles
203 bp
Sub1C
173 bp

• 1 1 1 1 1 1 2 1 1 1 1 1 2
  2
• 1 1 1 1 1 2 3 2 1 1 1 1 2
  2 2

Allele Sub1A Sub1C
Tolerant Intolerant
in white elongating type
35
?
Since Sub1 works in many different
backgrounds these results are unexpected and need
in-depth analyses

• Assessing the role of Sub1C in cross over
  plants
• One Sub1A-2 / Sub1C-1 plant identified in 1200

• Detailed expression studies

36
Sub1A and Sub1C expression during plant
development
internodes
nodes
panicles
leaves
imat. med mature
Sub1 A
s c s c s c s c s c s c s c s
c
GH T
173 bp
173 bp
IR42 S
Sub1 C
GH T
203 bp
203 bp
IR42 S
actin GH IR42
37
Sub1A is not expressed in nodes of intolerant
IR42
Sub1A
Sub1C
IR42
GH
IR42
GH
s c
s c
s c
s c
Kende et al. (1998) Plant Phys.
38
Sub1A response to other abiotic stresses
Preliminary results
39
Conclusions
Sequencing the QTL in tolerant parent is
important Transfer of the Sub1 locus and Sub1A
over-expression confers tolerance to intolerant
plants Sub1A and Sub1C are widely expressed and
show opposite expression pattern in tolerant and
intolerant accessions under submergence Sub1A
expression is most likely not mediated by the
promoter region but possibly by intra-genic
regions (MITE) Faro27 is a putative source of
novel tolerance genes
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Plant Cell August (2006) Preview online
41
Special thanks to
Dave Mackill
Abdel Ismail
Trang Thi Minh Dang
Gina Vergara
Gina Vergara
Tala Mac Fugen
Varoy Alvaro Pamplona
Septi Endang Septiningsih
Namrata Singh
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Thank you for your attention !
43
Strategy to survival
high chlorophyll a and b level
high soluble carbohydrates
T
I
Fukao et al. Plant Cell, 2006
44
efficient ethanolic fermentation
PDC
relative low ethylene level
T
S
T
S
I
ADH
T
Fukao et al. Plant Cell, 2006
45
Ethylene induces Sub1A and Sub1C
GA3 induces Sub1C
Fukao et al. Plant Cell, 2006
46
Upstream regulators of Sub1A, B, C
Chromosome 9
tolerant
intolerant