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Heqiang Huo, Peetambar Dahal, Sebastian Reyes Chin-Wo,
Claire McCallum1, and Kent J. Bradford
Department of Plant Sciences and Seed Biotechnology Center,
University of California, Davis, CA, USA 1Arcadia Biosciences, Inc., Davis, CA, USA
Environmental and Hormonal Signaling
in Seed Dormancy
This project was supported by the National Research Initiative of the USDA
National Institute of Food and Agriculture grant number #2008-35304-0472
and National Science Foundation grant number 0820451.
SEED DEVELOPMENT Environment Genotype
NON-DORMANT
DORMANT
GERMINATION SEEDLING
Water
Temperature
After-ripening
Light
Chemicals Primary
Secondary
• Seed dormancy is the failure of a viable seed to
germinate when provided with conditions normally
conducive to germination (water, temperature, etc.).
• Seed dormancy as an adaptive mechanism for
successful propagation is generally sensitive to
environmental conditions, particularly temperature, light
and nutrients (i.e., nitrate).
Seed Dormancy
Hormone Balance Is Involved in Seed Dormancy
Finch-Savage and Leubner-Metzger (2006) New Phytologist 171:501–523.
Cadman et al. (2006) Plant Journal 46: 805-822.
Environment
Synthesis and
degradation
Sensitivity
Signaling
Response
Thermoinhibition at High Temperatures
L. sativa cv. Salinas
Argyris et al. 2005. Theor.. Appl. Genet. 111: 1365.
Natural Variation for Thermoinhibition
L. sativa
cv. Salinas
L. serriola
UC96US23
Argyris et al. 2005. Theor. Appl. Genet. 111: 1365.
Recombinant Inbred Line Population
L. sativa
cv. Salinas
L. serriola
UC96US23
X
F1
F21 F22 F23 F299 F298 F2100 . . . . . . .
RIL1 RIL2 RIL3 RIL99 RIL98 RIL100 . . . . . . .
selfed families to
F8 generation
QTL Analysis – Phenotypic Data
• Phenotypes analyzed
in RILs for QTL:
– Germination/dormancy
• Temperature response
• Light and darkness
– Seed traits (SWT, SOC)
– Seedling characteristics
• Root and shoot growth
1A21-2730.0
E35/M59-F-2177.5
E38/M54-F-16419.6LsAO121.1E33/M59-F-08322.2E44/M49-F-24622.8E45/M48-F-20024.4E38/M54-F-09726.2E33/M59-F-12529.81A09-24530.3CLSS2403.b1_F01.ab132.4CLSM3815.b1_N18.ab133.0CLV_S1_Contig177736.5CLSM1367.b1_M06.ab137.3QGG10M24.yg.ab138.8CLSM19714.b1_D09.ab140.2CLS_S3_Contig627241.9CLSS3345.b1_A22.ab142.1CLS_S3_Contig612543.0
LsNCED443.5
CLS_S3_Contig898844.3CLS_S3_Contig264345.5QGB6H05.yg.ab147.1CLPY4291.b1_E17.ab147.5CLS_S3_Contig326649.0CLSM7752.b1_O17.ab149.5CLSM18950.b1_L09.ab152.11A21-09257.4E44/M49-F-07359.1L172263.2E38/M54-F-14964.3E44/M48-F-26365.11A06-22265.9E51/M49-F-43266.21A02-17567.6
1A21-08278.8E38/M54-F-23679.6
E38/M54-F-05082.3E44/M49-F-55683.6E38/M54-F-07584.71A02-23287.01A02-23187.6
E35/M59-F-12296.7
1A21-166101.4
E35/M60-F-109103.9
E45/M48-F-079106.2
E54/M48-F-224111.9
E51/M49-F-206114.4
E35/M59-F-204122.5
1A02-104124.6
Htg
6.1
Htg
6.1
Htg
6.1
6
R 2
= 0
.44
19.6
B
R 2
= 0
.64
29B
R 2
= 0
.70
56B
0 5 10 15 20 25
0 5
10
15
20
25
LOD
High Resolution Mapping of Htg6.1 in Lettuce
Argyris et al. 2011. Theor. Appl. Genet. 122: 95-108.
1A21-2730.0
E35/M59-F-2177.5
E38/M54-F-16419.6LsAO121.1E33/M59-F-08322.2E44/M49-F-24622.8E45/M48-F-20024.4E38/M54-F-09726.2E33/M59-F-12529.81A09-24530.3CLSS2403.b1_F01.ab132.4CLSM3815.b1_N18.ab133.0CLV_S1_Contig177736.5CLSM1367.b1_M06.ab137.3QGG10M24.yg.ab138.8CLSM19714.b1_D09.ab140.2CLS_S3_Contig627241.9CLSS3345.b1_A22.ab142.1CLS_S3_Contig612543.0
LsNCED443.5
CLS_S3_Contig898844.3CLS_S3_Contig264345.5QGB6H05.yg.ab147.1CLPY4291.b1_E17.ab147.5CLS_S3_Contig326649.0CLSM7752.b1_O17.ab149.5CLSM18950.b1_L09.ab152.11A21-09257.4E44/M49-F-07359.1L172263.2E38/M54-F-14964.3E44/M48-F-26365.11A06-22265.9E51/M49-F-43266.21A02-17567.6
1A21-08278.8E38/M54-F-23679.6
E38/M54-F-05082.3E44/M49-F-55683.6E38/M54-F-07584.71A02-23287.01A02-23187.6
E35/M59-F-12296.7
1A21-166101.4
E35/M60-F-109103.9
E45/M48-F-079106.2
E54/M48-F-224111.9
E51/M49-F-206114.4
E35/M59-F-204122.5
1A02-104124.6
Htg
6.1
Htg
6.1
Htg
6.1
6
R 2
= 0
.44
19.6
B
R 2
= 0
.64
29B
R 2
= 0
.70
56B
0 5 10 15 20 25
0 5
10
15
20
25
LOD
Chromosome 6
NCED Is in the ABA Biosynthetic Pathway
Yamaguchi et al. (2007) In Bradford and Nonogaki, Seed
Development, Dormancy and Germination, Blackwell, pp. 224-247.
• NCED genes encode
9-cis-epoxycarotinoid
dioxygenases that form
xanthoxin in the ABA
biosynthetic pathway.
• The closest homologs to
LsNCED4 in Arabidopsis are
AtNCED6 and AtNCED9,
which are expressed during
seed development.
• AtNCED6 and AtNCED9 are
involved in seed dormancy,
while AtNCED9 is involved
specifically in thermoinhibition
(Lefebvre et al. 2006; Toh et
al. 2008).
LsNCED4 Expression and ABA Content
Argyris et al. (2008) Plant Physiol. 148: 926-947.
LsNCED4 expression and ABA content remain elevated only in seeds of
the Salinas variety imbibed at 35°C, which exhibit thermoinhibition.
Functional Complementation of UC96US23
Salinas seeds
LsNCED4 expression high
T > 30ºC
ABA biosynthesis
Thermoinhibition
of germination
UC96US23 seeds
LsNCED4 expression low
ABA biosynthesis low
No thermoinhibition of
germination
Functional Complementation of UC96US23
Salinas seeds
LsNCED4 expression high
T > 30ºC
ABA biosynthesis
Thermoinhibition
of germination
UC96US23 seeds
pSal::LsNCED4 expression high
ABA biosynthesis
Thermoinhibition of
germination
Transform pSal::LsNCED4
into UC96US23
Silencing of Salinas LsNCED4
Salinas seeds
LsNCED4 expression high
T > 30ºC
ABA biosynthesis
Thermoinhibition
of germination
UC96US23 seeds
LsNCED4 expression low
ABA biosynthesis low
No thermoinhibition of
germination
Silencing of Salinas LsNCED4
Salinas seeds
LsNCED4 expression low
T > 30ºC
ABA biosynthesis low
No thermoinhibition
of germination
UC96US23 seeds
LsNCED4 expression low
ABA biosynthesis low
No thermoinhibition of
germination
RNAi of
LsNCED4
LsNCED4 Expression Is Required for Thermoinhibition
Both of these approaches worked as predicted,
indicating that LsNCED4 expression is required for
the induction of thermoinhibition (unpublished data
not shown).
0
20
40
60
80
100
Rela
tive
ger
min
atio
n at
35°C
(%)
Progeny lines from original F3 mutant plants
TILLING Mutants of LsNCED4
1 1146 Thr
Ala
1 1777 1186
Glu
Gly
1 1777 1510
Missense 1 Missense 2 Stop
In cooperation with Arcadia Biosciences, an induced mutant population of lettuce cv. Desert Storm
was TILLED to identify mutations in LsNCED4. Three were identified, one stop codon and two
missense mutations.
WT
TILLING Mutants of LsNCED4
Messing et al. (2010) Plant Cell 22: 2970-2980.
By sequence homology with
maize VP14, the Stop and MS1
mutations are near a histidine
residue that is important for
binding an iron atom that is
involved in the active site of the
enzyme. The MS2 mutation is
not near the active site.
MS2
Stop, MS1
GA20
High temperature
Zeaxanthin
Violaxanthin
GA1
Germination
LsNCED4
GRAS/DELLA
Ent-Kaurene
Xanthoxin
ABA
ABI3, 4, 5
SNF4
LsABA8ox4
Phaseic acid
LsGA3ox1
LsGA3ox2
LsZEP
LsGA20ox1
LsGA20ox2
LsGA2ox1
Inactive GAs
SAM ACC EthyleneLsACS1 ACO
CTR1
High temperature
Abscisic
aldehyde
SDR
GGDP
LsCPS1
LsKS1
ABA3
Ent-Kaurenoic
acid
KO
GA12-aldehyde
KAO
Hormone-Related Gene Expression
• In general, high temperature
promotes expression of
genes in the ABA biosynthetic
and response pathways and
inhibits expression of genes
in the later stages of the GA
biosynthetic pathway.
• Expression of ethylene
biosynthetic genes is also
inhibited.
• High temperature also
promotes expression of
germination repressors in the
ABA , GA and ethylene action
pathways.
Argyris et al. (2008) Plant Physiol. 148: 926-947.
Lettuce GeneChip® for Transcriptome Analysis
L. sativa
76,043 ESTs
L. serriola
52,034 ESTs
L. saligna
28,851 ESTs
L. perennis
28,066 ESTs
L. virosa
28,335 ESTs
35,747 unigenes Represented on chip
L. sativa
29,417 unigenes
L. serriola
22,327 unigenes
L. saligna
11,990 unigenes
L. perennis
12,661 unigenes
L. virosa
12,733 unigenes
• Affymetrix platform:
6.6 million 5 μm
features
• Average of 173
probes/unigene in 2
bp overlapping tiling
path
• Identified and
mapped over
15,000 new SNP
markers in lettuce
• Used for high
density mapping
Van Deynze, Michelmore, Van Leeuwen et al.
Germination Time Courses at 20 and 35ºC
• mRNA was extracted from
dry Salinas and UC96US23
seeds and from seeds
imbibed at 20 or 35ºC in
the light for 24 or 48 h.
• Samples were hybridized to
the lettuce microarray and
~13,000 genes differentially
expressed above
background levels were
analyzed.
Argyris et al. (2008) Plant Physiol. 148: 926-947.
GA, ABA and Ethylene Pathways Respond to Temp
GA, ABA and Ethylene Pathways Respond to Temp
2011 PNAS 108: 20236-20241
Deep dormancy Shallow dormancy
Conclusions
• The Htg6.1 QTL in lettuce is due to lack of induction of
expression of LsNCED4 in UC96US23 seeds in response
to high imbibition temperature.
• Gene functional assays, RNAi silencing, mutations and
gene transfers confirm that LsNCED4 is necessary and
sufficient for the induction of thermoinhibition in lettuce
seeds.
• Markers, NILs and mutants are available for introgression
of the alleles into lettuce cultivars to alleviate
thermoinhibition in warm season plantings.
• A general picture is emerging of how environmental cues
are transduced into hormonal signals regulating dormancy.
Acknowledgements
Heqiang (Alfred) Huo Peetambar Dahal
Jason Argyris
Claire McCallum Sebastian Reyes-Chin-Wo
Richard Michelmore
http://cgpdb.ucdavis.edu/
This project was supported by
the National Research Initiative
of the USDA National Institute
of Food and Agriculture, grant
number #2008-35304-0472.
NSF grant number 0820451
Allen Van Deynze Fei-Yian Yoong
UC Davis Seed Biotechnology Center
sbc.ucdavis.edu
Plant Breeding Academy
• A professional education program
to advance the knowledge, skills
and careers of current or potential
plant breeders.
• Class II of the European PBA
began in October 2011 in Gent,
Belgium; sessions held in 5 EU
locations and Davis, CA, USA
• Class IV of Davis PBA begins in
Fall 2012
• Asian PBA to begin in Fall 2012 in
Chang Mai, Thailand
http://pba.ucdavis.edu