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

kjbradford@ucdavis.edu

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