A

Al

Germination des Semences

E

Philippe Grappin
T

grappin@versailles.inra.fr

Embryophytes

(150 ma, crtac)

Pteridospermaphyte

Pteridophyte Rhynia (300 ma)

Coleochaete (540 ma)

(adapt de J. Broutin, 2000)

Fcondation et formation de la graine

Dessin C. Dumas (2000)

A
E

Origine des diverses structures constitutives des semences daprs D. Cme (1982).

Analyse de la qualit physiologique de la semence

UMR INRA INA P-G Biologie des Semences, INRA de Versailles

maturation Dormance

dessication

Leve de Dormance
hydratation

embryogense

ND

hydratation

stockage sec (after-ripening)

Vieillissement

Germination

Fertilization

Growth

Acquisition of
germination capability

Germination

Late M.

MATURATION

E.E.M.

Flowering

Spermaphyte life cycle

La germination est peut se produire ds que la graine est form sur la plante mre
Exemple de la Mangrove

Fertilization

Growth

Acquisition of
germination capability

Germination

Late M.

MATURATION

E.E.M.

Flowering

Spermaphyte life cycle

Fertilization

MATURATION

E.E.M.

Flowering

Dormancy oneset

Late M.

Growth

Acquisition of
germination capability

Germination

Desiccation

Dispersal

orthodox seeds

Fertilization

MATURATION

E.E.M.

Flowering

Dormancy oneset

Late M.

Growth

Acquisition of
germination capability

Germination

H 20
Light
T
02

Desiccation

(storage)

Dispersal

Dormancy release

Aging
Secondary dormancy oneset

time

time

Worlds record for seed longevity - 1300 year old

sacred lotus

lotus fruits germinate.

A high protein repair activity indentified in viable ancient seeds

Shen Miller et al, 1995

R
T
A

Germination,
le premier acte de la vie vgtale
(M. Georges Ville, 1868 Musum dhistoire Naturelle)

dbute par lentre deau dans la graine qui initie une

reprise de lactivit mtabolique et se termine lorsque laxe
embryonaire commence son longation.
(Evanary et al. 1957)

Accomplissement de la germination chez

Nicotiana tabacum

Leubner-Metzger
et al., 1995

enbryon
albumen
tguments

Leubner-Metzger, 2000

La germination
H2 O

mergence
radiculaire

O2

Activit mitotique
Prise deau (% poids frais)

80

Dbut de llongation de laxe embryonnaire

Mobilisation des
reserves

Accumulation de soluts
60

Reprise de la synthse protique

40

Reprise de la transcrition
ABA

20

Reprise de la respiration

Phase 1
Activation du
mtabolisme

GA

expression / leve
de la dormance

Phase 2
Preparation de llongation
cellulaire
Post-germination et
Germination

croissance de la plantule

Potentiel en eau

cell = + c + p
Phase I : entre deau selon |c|
Phase II : entre deau selon || et balance avec
Phase III :

||

|p|

|p|

Synthse dADN durant la germination des graines de tomate (daprs de Castro et al., 2000).
Les noyaux marqus liodure de propidium fluorescent en rouge et les noyaux marqus au FITC
fluorescent en vert, indiquant une incorporation de bromodeoxyuridine (3 h de pulse-labeling en
plus du temps dimbibition annonc) dans lADN en rplication active (phase S). a, pointe de la
radicule de graines sches. b, pointe de la radicule 12 h dimbibition. c, pointe de la radicule 24
h dimbibition. d, pointe de la radicule ; e, mristme apical et f, cotyldons de graines germes
(48h aprs le dbut de limbibition). La barre indique 100 m.

Mtabolisme
nergtique
et
contrle de la
germination

Dormance et contrle mtabolique

Dormance

Modle propos par Cme et Corbineau (1989) :

linhibition spcifique de la glycolyse par oxydation des enzymes de cette voie et la stimulation
de la voie des pentose phosphates favorisent la germination sensu stricto.
Ce contrle mtabolique permettrait daugmenter la production de NADPH, cofacteur ncessaire au bon fonctionnement des
enzymes NADPH-dpendantes telles que les enzymes du systme thiordoxine rductase/ thioredoxines qui joue un rle trs
important dans lactivation de certaine enzymes ncessaire a la germination par rduction/oxydation de ponts disulfures.

Rle de la thiordoxine
dans la germination du bl
(Besse et al., 1996).

Mtabolisme des ARN et germination

Niveaux dARN polyadnyls des embryons de bl en germinations (Rushton et Bray, 1987).

Contrle
de
la germination
Analyses Physiologiques

Dormance
Incapacit germinative dans des conditions sub-optimales de germination

13C + fluridone 10 M
13C
27C + fluridone 10 M
27C

% Germination

Cvi, a proper model for comparative proteomics in Arabidopsis

7m

100
80
60

After-ripening

40

control

20

Dormancy release

% Germination

10

100
80

15

20

4j

25

Imbibed dormant seeds

60

environment

Stratification

40

1j

control

0
0

10

Seed sensitivity
to germination

2j

20

% Germination

http://www.mpiz-koeln.mpg.de

30

15

25 M30
10

20

100

NO 2

80

NO 3 7 mM

60

GA 3
100 M

40

control

20
0
0

10

15

20

25

30

http://www.mpiz-koeln.mpg.de

Imbibed non dormant seeds

Days after sowing

Ali-Rachedi et al, 2004

Changes in endogenous ABA level during imbibition

in A. thaliana Cvi seeds

Freshly harversted (D)

ABA pmol/g dry seeds

6 months after-ripened (ND) seeds

500
400
WTD

300
200
WT ND
After ripening

100

stratification
WTD+flu

0
0

21

Days after sowing

The Arabidopsis cytochrome P450 CYP707A
encodes
Ali Rachedi et al. 2004

ABA 8-hydroxylases: key enzymes in ABA

catabolism.
Kushiro et al, (2004) Embo J 23 1647-1656

This catabolism seemed more important in ND than in D seeds.

In both species, dormancy was actively controlled in imbibed seeds by ABA

production.

In both species, most of the treatments efficient in dormancy breaking

supressed this synthesis.

Imbibition of D seeds, through ABA metabolism, is clearly a pivotal point of

seed dormancy control.

what happens during dormancy maintenance in term of gene expression?

what are the regulated genes in response to ABA?

Un contrle hormonale de la rsistance des enveloppes de

lembryon la perce radiculaire

Figure 1-8 : Forces requises pour traverser lalbumen de graine de

tomates sauvages imbibes sur eau () ou sur 1O M dABA () et
de graine gib1 imbibes sur eau () (Toorop et al., 2000). Aucune des
graines mesures na accomplie la germination, la flche indique les
premires graines sauvages sur eau ayant germ.

Fig 1-9 : Empreintes de graines de tomates en germination

(24h dimbibition) et germes, hybrides avec une sonde
ribonuclique LeMAN2 (Nonogaki et al., 2000).

Contrle hormonal de la germination chez N.

tabacum

Leubner, 2000

Le rle des GAs dans la germination

Structure de GA1 et rsum des
rponses aux gibbrellines
(daprs Hooley, 1994,; Davies, 1995)).

CROISSANCE CELLULAIRE

DEVELPPEMENT DE LA
FLEUR ET DU FRUIT

- longation des tissus de la

tige
- forme de la feuille et
croissance des
ptales
- croissance du tube pollinique
- germination de la graine

-initiation florale
- dveloppement de
lanthre
- pigmentation de la
corolle
- dveloppement du fruit

MOBILISATION DES RESERVES

PAR LES CELLULES A
ALEURONES
- synthse et scrtion de diverses
hydrolases
- forme de la feuille et croissance des
ptales
- libration de myo-inositol, de
phosphore et dions minraux

Rponse aux GAs est module par

concentration en GA dans les cellules / aptitude de la cellules rpondre aux GAs
Richards et al. 2001

Un transcriptome rgul par les GAs

ga1-3

Germination kinetic

Ogawa et al. Plant Cell 2003

Rgulation de la synthse des GA dans la graine

par la lumire
Yamaguchi et al., Plant Cell, 1998

Les 2 gnes GA4 et GA4H dArabidopsis codent pour une GA3Bhydroxylase qui catalyse ltape final de la biosynthse de GA actives

Sequence Alignment of GA 3b-Hydroxylases.

In Vitro Functional Analysis of the Recombinant GA4H Protein.

(A) Diagram showing conversion of GA9 and GA20 to GA4 and GA1 by 3b-hydroxylation.
(B) Functional analysis of the GA4H protein. Lysates of E. coli containing the MBP (as a
control) or MBPGA4H were incubated with 14C-GA9. The reaction mixture was separated on
a silica gel by thinlayer chromatography. The positions of authentic GA4 and GA9 are
indicated by arrowheads.

Yamaguchi et al. Plant Cell, 1998,

GA4 est accumul dans les siliques et en dbut dimbibition

Lexpression de GA4H est spcifique de la graine en germination

-GA4 et GA4H sont induits par une

irradiation rouge claire d1 heure dans la
graine imbibe.
- Laccumulation de lARN GA4H est
maintenu jusqu la germination.

GA4 et GA4H sont rprims par le rouge lointain

En context phyB-1 GA4H nest pas induit par le rouge claire

Modle simplifi de la germination

des graines de tabac

Daprs Leubner-Metzger et Meinz (1999)

La rupture des tgument et de lalbumen sont des vnements spars

chez Nicotiana tabacum.

Identification de gnes
impliqus dans la germination

Lapproche de gntique directe a vite montr ses limites

pour caractriser de nouveaux gnes impliqus dans le
contrle de la germination

Toujours les mmes mutants isols

(GA, ABA dficients /insensibles)
Approche QTL met en vidence un
dterminisme gntique complexe
(M. Koornneef)

Slection sur le
critre dune
sgrgation
anormale de la
rsistance la
kanamycine

T2

Ks
1/3

Kr

2/3

Non
germes
1/3

Exemple de crible gntique sur la collection de

mutants dinsertion dArabidopsis de lINRA de

B. Dubreucq

Versailles

Dubreucq et al., 1996

Approche globale pour caractriser de nouvelles fonctions

impliques dans le contrle de la germination
Caracterisation
physiologique et
biochimique

Gnomique
fonctionnelle
Gntique
inverse

Hypothse de travail
gnes candidats

Analyse spatio-temporel
de
lactivit gnique

(a) Germinated Arabidopsis seed

under a dissection microscope.
(b) Scanning electron
microscopy of germinated
Arabidopsis seeds.
(c) Close up view of the surface
cells of the radicle.
(d) Close up view of the surface
cells of the endosperm.
(e) Scanning microscopy of
germinated seed immediately
after radicle emergence.
Scale bars 100 lmfor (a), (b)
and (e); 10 lmfor (c) and (d).

Arabidopsis seed structure following germination.

P-P Liu et al. Plant J, 2005, 41, 936944

Germination time course of wild-type Arabidopsis (Columbia-0) seeds. Completion of germination, which
was defined as penetration of radicle through the endosperm layer (endosperm rupture; closed symbol),
was recorded together with the occurrence of testa rupture (open symbol). Imbibition time indicates time
of incubation at 22C. Each data point represents the average of three replicates; vertical bars indicate
SD.

Variable tissue-specific GUS

expression in germinating and
germinated seeds of the enhancertrap Arabidopsis lines.
(ac) GUS expression in the whole
seed (except for testa), the embryo
and the endosperm in the same
seeds, respectively.
(dg) GUS expression in the whole
axis, hypocotyl, hypocotyl plus
cotyledons and cotyledon tips
(arrows) in germinating seeds,
respectively.
(h) GUS expression in whole radicle
of germinated seed.
Scale bars 10 lm.

P-P Liu et al. Plant J, 2005, 41, 936944

(ac) Embryo-specific GUS expression.

(dg) Endosperm-specific GUS expression.
GUS activity is detected only in the
micropylar half of the endosperm (with
testa; d), but not in the lateral half
of the endosperm (e), radicle (f) and
hypocotyls plus cotyledons (g).
(h) GUS and tetrazolium (TZ) staining of
scratched seeds. Part of testa was
removed before staining to enhance the
penetration of the substrate
Scale bars 10 lm.

Micropylar region-localized GUS expression in germinating Arabidopsis seeds.

Summary of tissue-specific GUS expression patterns in germinating

and germinated seeds of the Arabidopsis enhancer-trap lines.

Identification of the T-DNA insertion site in the TMH1 enhancertrap line.

(b) PCR product amplified with the GSP and TSP from wild type (WT) and the enhancer-trap line (TMH1) genomic DNA.
(c) PCR products amplified using At5g02750 and At5g02760 gene-specific primers with wild-type Arabidopsis genomic
DNA (gDNA) and reverse transcription products (RT) of total RNA extracted from germinated (36 h) wild-type seeds.

The T-DNA insertion sites identified

in the enhancer-trap lines and their
GUS expression patterns. The
genomic DNA region (10 kb) in the
vicinity of the T-DNA insertion sites
(arrows) containing the candidate
trapped genes is shown in the table.

Analyse du transcriptome
de
la graine au cours de la germination

- A control of gene expression and of protein accumulation related to

physiological changes (dormancy release, aging) occurs in dry seeds

Cluster
IV IV
Cluster

Cluster V

(209 protines)

(137 protines)

3 months

6 onths

9 months

2D electrophoresis

12 months

Separation
proteins

K. Chibani

12

12

A. thaliana

After-ripening
aging
ga1V0 / ga1V2

ARNm
J. Bove

zea4V0 vs zea4V2

100000

zea4V0 / zea4V2

ga1V2

Hybridation

zea4V2
zea4V2

10000

cDNA-arrays

1000

100

10
10

V0

V2

ga1V0

100

1000

10000

100000

zea4V0
zea4V0

N. plumbaginifolia

Identification de gnes impliqus dans la dormance des

graines de Nicotiana plumbaginifolia
D

ND

ND D ND

D ND

S 8 16 24 S 8 16 24 S 8 16 24 S 8 16 24 S 8 16 24 S 8 16 24 S 8 16 24 S 8 16 24

Fragments dADNc dtects

15 000

Induits chez les graines D

544

Induits chez les graines ND

476

Clons et squencs

412

Similaires des squences

des bases TrEMBL et SwissProt ([E]<10-4)

137

Fonctions assignes

85

Transcriptomic analysis of dormancy maintenance and release

in N. plumbaginifolia using cDNA-AFLP
Detected cDNA fragments

15 000

Induced in D seeds

544

Induced in ND seeds

476

Resistance, protection against aging

Transports

Dormant

Non dormant

(%)

(%)

4,8

4,8

Metabolism 11,9

4,8

Signaling

7,1

Transcription

7,1

Proteolysis

7,1

6
8,3
13,1

Protein synthesis

1,2

Vesicular trafficking

1,2

14,3
8,3

Dormancy release is related to upregulation of protein biosynthesis genes at the

RNA level

Bove et al, 2005

De Diego et al, 2006
Collaboration E Cerventes (CSIC, Salamanca)

Conclusion 2
-La traduction est indispensable la germination (Rajjou et al., 2004)
- ABA rgule les gnes de contrle de la traduction (Hoth et al., 2002)

LABA maintient la dormance des graine via une rpression au niveau

ARN de lactivit de biosynthse des protines.

Protein biosynthesis in germinated seed

- Protein translation is required in imbibed
seeds for germination (Rajjou et al, 2004)
-amanitine

H 2O

cycloheximide

- RNA encoding translation factors are up-accumulated during dormancy

release (Bove et al, 2005; Cadman et al, 2006)
D dry

pI
MM
(kDa)

89.3

4
.
5

4
.
9

5
.
2

5
.
3

5
.
6

5
.
8

5 6
.
9

6 7
. .
3 2

8
.
3

9
.
1

ND dry

5.8
9

5.8
9

79.3
68.7

Up accumulation of

46.6

translation proteins in ND
seeds ?

46

57.7

35.8

45

80.76

80.
76

46
45

EF-2 elongation factor

29.3
25.2

17.6
14.0

Chibani et al, 2006

Control of protein synthesis and dormancy release

20000

cpm/g de protEins

[35S]-Met incorporation

Protein de novo synthesis activity is similar in D and ND hydrated seeds

15000
10000
5000
0

D 1d

Control

ND 1d

After-ripening reprograms the pattern of protein synthesis upon imbibition

Mm
(kDa)

ND 1 day

D 1 day

pI

4.5 4.9 5.3

89.3
89.3
79.3

272

68.7
57.7

6
NI

46.6
35.8

659
NI

NI

4.5 4.9

7.2 9.1

89.3
79.3

5.3

7.2 9.1

272

68.7
57.7

30
30
31
692

31

46.6
35.8

29.3

29.3

25.2

25.2

NI

145 111

480
17.6

17.6

14.0

14.0

117

Autoradiography (35S-Met)

213

ND seeds translate specific

set of genes required for
germination

901

Chibani et al, 2006

Collaboration D Job (CNRS, Lyon)

Contrle des GA sur le transcriptome de la graine

ga1-3

wt

Ogawa et al. Plant Cell 2003

Ogawa et al. Plant Cell 2003

Un transcriptome rgul par les GAs

ga1-3

Germination kinetic

Ogawa et al. Plant Cell 2003

GA4

GA4

(A)GA up-regulated genes

(B)GA down-regulated genes

GA4

GA4

Cellular distribution of GA biosynthesis and response

Cellular Localization of GA-Regulated Transcripts

(A)
(B)
(C)
(D)
(E)

Expression profiles of GA-upregulated genes used for in situ

hybridization experiments.
Scheme of an Arabidopsis mature embryo. CO, cotyledon;
HY, hypocotyl; RA, radicle; SA, shoot apical meristem.
In situ hybridization analysis of the GA biosynthesis gene
AtGA3ox1 in wild-type seeds imbibed for 24 h.
In situ hybridization analysis of GA-upregulated genes :AtXTH5 (At5g13870), AtCP1 (At4g36880), and PDF1
(At2g42840).
Magnified views highlighting AtCP1 transcript accumulation
in the aleurone and the epidermis after GA4 treatment.

GA-Regulated Cellular and Metabolic Pathways during Seed Germination.

(A) Genes implicated in cell elongation (top) and cell division (bottom).
(B) GA biosynthesis genes. Gene identities are described in the legend to Figure 2.
(C) Ethylene biosynthesis and response genes. ACO (At2g19590), ERS1 (At2g40940), and HLS1 (At4g37580).
(D) Genes implicated in auxin transport. AUX1 (At2g38120), PIN2/EIR1 (At5g57090), and PIN7 (At1g23080).
(E) Genes encoding (putative) transcription regulators whose expression was regulated by GA4 within 6 h. ATHB-16 (homeodomain-Leu zipper protein;
At4g40060), AtMYB34/ATR1 (At5g60890), Dof (Dof zinc finger protein; At2g28510), SCL3 (SCARECROW-LIKE3; At1g50420), AtMYB66/WER (At5g14750),
PIF4 (At2g43010), Zinc finger1 (At1g27730), and Zinc finger2 (At2g28200).

Contrle de la rponse aux GAs

Reprsentation des mutants gai et

rga altrs dans la rponse aux
gibbrellines.

Rle des protines GAI/RGA/SLR

Silverstone AL et al. (2001)

Rle de NpRGL2?
Hypothse dun rgulateur positif de la rponse aux GA

NpRGA2, a positive regulator seed coat imposed

dormancy
Light
ABA

After-ripening

GA
Exogenous ABA

NpRGA2
micr.
rad.

end.
coty.
rad.

end.

Testa rupture

rad.

coty.

Endosperm
weakening

te.
coty.

te.
te.

micr.
rad.

ext. te.
int. te.

te.

te.

micr.

end.

end.

0.1 mm

0.1 mm

0.1 mm

Validation of the cDNA-AFLP approach

Godin et al. (in preparation)

ABI5, impliqu dans la perception de lABA,

rprime la germination en absence de GA

Relation perception ABA RGL2

dans le contrle de la
germination

Rle de GPA1 dans le contrle

hormonal de la germination
dArabidopsis
(Ullah et al., 2002).