Special Issue – Regular Paper
Role of Reactive Oxygen Species in the Regulation of
Arabidopsis Seed Dormancy
Juliette Leymarie1, Giedré Vitkauskaité1,2, Hai Ha Hoang1, Emmanuel Gendreau1, Virginie Chazoule1,
Patrice Meimoun1, Françoise Corbineau1, Hayat El-Maarouf-Bouteau1 and Christophe Bailly1,*
1
UR5 EAC7180 CNRS, Université Pierre et Marie Curie- Paris 6, Bat C, 2ème étage, boı̂te 156, 4, place Jussieu, 75252 Paris cedex 05, France
Present address: Vytautas Magnus University, Department of Environmental Sciences. Vileikos st. 8-212, LT-44404 Kaunas, Lithuania
*Corresponding author: E-mail, [email protected]; Fax, + 33-1-44-27-61-51
(Received July 6, 2011; Accepted September 5, 2011)
2
Freshly harvested seeds of Arabidopsis thaliana, Columbia
(Col) accession were dormant when imbibed at 25 C in
the dark. Their dormancy was alleviated by continuous
light during imbibition or by 5 weeks of storage at 20 C
(after-ripening). We investigated the possible role of reactive
oxygen species (ROS) in the regulation of Col seed dormancy. After 24 h of imbibition at 25 C, non-dormant
seeds produced more ROS than dormant seeds, and their
catalase activity was lower. In situ ROS localization revealed
that germination was associated with an accumulation
of superoxide and hydrogen peroxide in the radicle. ROS
production was temporally and spatially regulated: ROS
were first localized within the cytoplasm upon imbibition
of non-dormant seeds, then in the nucleus and finally in
the cell wall, which suggests that ROS play different
roles during germination. Imbibition of dormant and
non-dormant seeds in the presence of ROS scavengers or
donors, which inhibited or stimulated germination, respectively, confirmed the role of ROS in germination. Freshly
harvested seeds of the mutants defective in catalase
(cat2-1) and vitamin E (vte1-1) did not display dormancy;
however, seeds of the NADPH oxidase mutants (rbohD) were
deeply dormant. Expression of a set of genes related to
dormancy upon imbibition in the cat2-1 and vet1-1 seeds
revealed that their non-dormant phenotype was probably
not related to ABA or gibberellin metabolism, but suggested
that ROS could trigger germination through gibberellin
signaling activation.
Keywords: Arabidopsis thaliana Dormancy Germination
Reactive oxygen species Seed.
Abbreviations: CAT, catalase; Col, Columbia; DCFH-DA,
5-(and-6)-chloromethyl-20 ,70 -dichlorofluorescein
diacetate;
DMTU, dimethylthiourea; DPI, diphenyleneiodonium; GR,
glutathione reductase; MV, methylviologen; MN, menadione;
NBT, nitroblue tetrazolium; PVP, polyvinylpolypyrrolidone;
ROS, reactive oxygen species; RT–PCR, reverse transcription–PCR; SOD, superoxide dismutase.
Introduction
Reactive oxygen species (ROS) have long been considered as
only damaging compounds; however, they have recently
emerged as key players in seed physiology (Bailly et al. 2008).
Whereas ROS are ubiquitous and present at all stages of the
seed life, including embryogenesis through germination, an
understanding of the role of ROS as signaling molecules in
seed biology is far from complete. This is due to the existence
of various forms of ROS (e.g. superoxide, hydrogen peroxide and
hydroxyl radical) in seeds (Bailly et al. 2008), the very short life
span of these compounds (Møller et al. 2007), the integration
of individual ROS into several different transduction pathways
(Mittler et al. 2011), their reactivity towards a wide range
of macromolecules that can propagate the oxidative signal
(e.g. proteins, Møller et al. 2007) and their effect on the cellular
redox status (Dietz et al. 2010). It is well known that ROS are
produced to a certain level during seed imbibition. It has been
proposed that the germination is completed only when the
ROS content is within an oxidative window that allows ROS
signaling (Bailly et al. 2008). Above or below the ‘oxidative
window for germination’, low or high amounts of ROS would
not permit progress towards germination. According to this
model, seed dormancy, i.e. the inability of seeds to germinate
in favorable environmental conditions (Finch-Savage and
Leubner-Metzger 2006), is regulated by ROS signaling.
Many hypotheses have been proposed to explain the
beneficial or negative role of ROS in the regulation of seed
germination and dormancy. Alleviation of sunflower seed dormancy during after-ripening is associated with a non-enzymatic
ROS production which triggers oxidation of specific proteins
and mRNA, thus altering cell signaling during subsequent seed
imbibition of after-ripened seeds (Oracz et al. 2007, Bazin et al.
2011). Interaction of ROS with the ABA, gibberellin and
ethylene signaling pathways has been shown in various plant
physiological processes, which might be very relevant to seed
germination, since these hormones are known as major players
in the regulation of seed dormancy (El-Maarouf-Bouteau and
Plant Cell Physiol. 53(1): 96–106 (2012) doi:10.1093/pcp/pcr129, available online at www.pcp.oxfordjournals.org
! The Author 2011. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Plant Cell Physiol. 53(1): 96–106 (2012) doi:10.1093/pcp/pcr129 ! The Author 2011.
Reactive oxygen species and Arabidopsis seed dormancy
Bailly 2008, Finkelstein et al. 2008). Bahin et al. (2011) showed
that the cross-talk between ROS and ABA or gibberellin
metabolism and signaling might control barley seed dormancy.
In sunflower, H2O2 produced in the presence of HCN, a
dormancy release compound, has been shown to activate
downstream elements of the ethylene signaling pathway
(Oracz et al. 2009). In Arabidopsis, few data are available.
Nitric oxide (NO), a compound similar to ROS, exhibits a
beneficial effect on Arabidopsis seed dormancy release
(Bethke et al. 2005, Bethke et al. 2006, Bethke et al. 2007).
Liu et al. (2009) proposed that NO could interfere with ABA
metabolism. Liu et al. (2010) suggested that hydrogen peroxide
(H2O2) reduces ABA synthesis and stimulates gibberellin
synthesis, thus releasing dormancy, although the data in this
study were obtained using exogenous H2O2, which does not
reflect the in vivo situation. Recently, Müller et al. (2009a)
demonstrated that rbohB (respiratory burst oxidase
homolog B), a membrane-bound enzyme that produces superoxide, played a role in Arabidopsis seed after-ripening and germination. Similar mechanisms had also been proposed for
various species (Sarath et al. 2007, Oracz et al. 2009). We investigated the putative signaling role of ROS in the regulation of
Arabidopsis seed dormancy.
In this paper we present a comprehensive description of the
ROS status during imbibition of dormant and non-dormant
Arabidopsis seeds. Avoiding assessing germination in the presence of light provides a simple assay to evaluate seed dormancy
of Col. The relationship between in vivo ROS production
and germination was assessed using compounds known to
affect ROS homeostasis and the Arabidopsis mutants,
catalase2-1 (cat2-1; Queval et al. 2007), vitamine E 1-1 (vte1-1;
Sattler et al. 2004) and rbohD (Pogany et al. 2009). The cat2-1
mutant lacks one of the three genes coding for the H2O2dismutating enzyme catalase (CAT), and displays intracellular
redox perturbation and activation of oxidative signaling
pathways under ambient air conditions (Queval et al. 2007).
The vte1-1 mutants do not have tocopherol cyclase activity
and accumulate a redox-active biosynthetic intermediate
(Sattler et al. 2004). Tocopherols are essential compounds to
protect lipids from oxidation, since they scavenge various
ROS and ROS by-products such as lipid peroxyl radicals
(Girotti 1998). The vte1-1 seeds show reduced longevity
(Sattler et al. 2004). NADPH oxidases catalyze the production
of apoplastic superoxide from oxygen and NADPH (Sagi and
Fluhr 2006). In Arabidopsis they are encoded by 10 rboh genes
(AtrbohA–J), among which AtrbohD appears to be the major
active form, and is expressed throughout the plant (Sagi and
Fluhr 2006). Recent data suggest that rbohD is the NADPH
oxidase involved in the transfer of the ROS signal from cell to
cell (Miller et al. 2009). In this work we used a rbohD T-DNA
insertion mutant (Pogany et al. 2009).
In order to decipher the mechanisms involved in ROS signaling during Arabidopsis seed germination, we investigated
the expression of genes known as key players in this process,
which were chosen with the aid of the Arabidopsis eFP Browser
(www.bar.utoronto.ca, Winter et al. 2007, Bassel et al. 2008).
As the balance between ABA and gibberellin levels and sensitivity is a major regulator of seed dormancy, the expression of
genes involved in these pathways has been studied. AtNCED3
and AtNCED9, coding for two 9-cis-epoxycarotenoid dioxygenases, are involved in ABA biosynthesis (Tan et al. 2003,
Lefebvre et al. 2006). AtCCD4 codes for a carotenoid cleavage
dioxygenase (Huang et al. 2009, Brandi et al. 2011).
AtCYP707A2, which codes for an ABA 80 -hydroxylase, is
involved in ABA catabolism (Millar et al. 2006). AtGA3ox1
(GA3 OXIDASE 1) codes for a class of 2-oxoglutarate-dependent
dioxygenases, which catalyzes the conversion of gibberellin
precursors to their bioactive forms (Yamaguchi, 2008), and is
induced during germination (Finch-Savage et al. 2007). ABI5 is
involved in ABA signaling downstream of its perception
(Finkelstein et al. 2008) and AtSLP2 is strongly and quickly
induced by gibberellins (Ogawa et al. 2003). DOG1 (DELAY
OF GERMINATION 1) was identified as a major regulator of
dormancy in various species, although its function remains
unknown (Bentsink et al. 2006, Graeber et al. 2010).
Results
Germination and after-ripening of seeds of the
Columbia accession
Germination of Col seeds was assessed in darkness (Fig. 1).
Freshly harvested seeds completed germination within 4 d at
15 and 20 C but it took 7 d at 10 C (Fig. 1A). At 25 C <10% of
dormant seeds were able to germinate within 10 d, whereas
almost all seeds germinated at this temperature under continuous light (Fig. 1A). Five weeks of dry storage did not markedly
change the patterns of seed germination at 10, 15 and 20 C,
even though germination was slightly faster at 15 and 20 C
following after-ripening (Fig. 1B). In contrast, after-ripened
seeds became able to germinate fully in the dark at 25 C
(Fig. 1B).
ROS metabolism during Columbia seed
germination
Fig. 2 shows the changes in the ability of dormant and
non-dormant Col seeds to produce H2O2, which was measured
by the decrease of scopoletin fluorescence in the incubation
medium, during 1 h of imbibition at 25 C. H2O2 production in
dormant and non-dormant seeds was almost similar and close
to 17 nmol H2O2 h1 g FW1 (Fig. 2). Seed imbibition longer
than 3 h at 25 C in darkness was accompanied by a decreased
production of H2O2 in both dormant and non-dormant seeds
to approximately 10 nmol H2O2 h1 g FW1 after 16 h at this
temperature (Fig. 2). At 24 h of imbibition at 25 C, H2O2 production became significantly higher in non-dormant seeds and
reached about 30 nmol H2O2 h1 g FW1 (Fig. 2), whereas it
remained close to 18 nmol H2O2 h1 g FW1 in dormant seeds
(Fig. 2). At this time point of seed imbibition, no symptom of
Plant Cell Physiol. 53(1): 96–106 (2012) doi:10.1093/pcp/pcr129 ! The Author 2011.
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J. Leymarie et al.
radicle protrusion or testa rupture was visible in either dormant
or non-dormant seeds.
In situ accumulation of O
2 in excised embryos after 24 h
of imbibition at 25 C is shown in Fig. 3. Accumulation of
A
B
Fig. 1 Germination of dormant and non-dormant Col seeds.
(A) Freshly harvested seeds. (B) Seeds after-ripened for 5 weeks at
20 C. Germination was carried out at 10 C (diamonds), 15 C
(squares), 20 C (open circles) and 25 C (crosses) in the dark and at
25 C under continuous light (rectangles). Means ± SD of triplicate
experiments are shown.
formazan in imbibed dormant seeds was very heterogenous:
in some seeds (approximately 50%), any NBT (nitroblue
tetrazolium) precipitation was not detectable, whereas in
some others (approximately 50%) superoxide was visualized
as intense, isolated dark spots, located mainly at the outer
layers of cotyledons and the embryonic axis (Fig. 3A). In contrast, NBT staining was homogenous among the individuals of
the non-dormant seed population: all seeds displayed roughly
the same staining (Fig. 3B). In these seeds, formazan deposits
were mainly localized in the embryonic axis and close to
the vessels in cotyledons. Approximatively 15% of the seeds
displayed staining, with a lower intensity in their embryonic
axis (Fig. 3B).
ROS have also been visualized in seeds by fluorescence
using 5-(and-6)-chloromethyl-20 ,70 -dichlorofluorescein diacetate (DCFH-DA), which produces fluorescent spots within the
cells at the sites of ROS formation (Fig. 3C, D). Dormant
imbibed seeds displayed a weak and homogenous fluorescence
throughout the tissues, suggesting that ROS do not specifically
accumulate in restricted areas (Fig. 3C). In contrast, in
non-dormant seeds imbibed for 24 h, fluorescence was much
more pronounced and localized close to the radicle tip of the
embryonic axis (Fig. 3D) and, to a lesser extent, in the external
part of cotyledons. Fig. 3E–J shows ROS localization at the
cellular level in the radicle tip. In dormant seeds, fluorescence
was weak and localized homogenously within the cytoplasm at
any time of imbibition (Fig. 3E–G). In non-dormant seeds,
fluorescence was intense after 6 h of imbibition (compare
Fig. 3E with H) and indicated a cytoplasmic ROS localization
(Fig. 3H). After 24 h at 25 C, fluorescence appeared as brilliant
and intense localized spots within the cell, suggesting a nuclear
localization (Fig. 3I). After 72 h of imbibition the embryos
excised from non-dormant seeds had grown, which was clear
by the size of the cells (compare Fig. 3J with I). At this stage,
fluorescence revealed that ROS were mainly located in the
cell wall (Fig. 3J).
35
dormant
H2O2 (nmoles h-1 g FW-1)
30
non-dormant
25
20
15
10
5
0
0 (dry)
3
6
16
24
Duration at 25°C (h)
Fig. 2 Production of H2O2 by dormant (red bars) and non-dormant (blue bars) Col seeds. Seeds were incubated for various durations at 25 C
in the dark. Means ± SD of triplicate experiments are shown.
98
Plant Cell Physiol. 53(1): 96–106 (2012) doi:10.1093/pcp/pcr129 ! The Author 2011.
Reactive oxygen species and Arabidopsis seed dormancy
A
B
C
D
E
F
H
G
50 mm
I
J
50 mm
Fig. 3 In situ localization of superoxide anions and ROS in dormant and non-dormant Col seeds. (A and B) NBT (nitroblue tetrazolium) staining
of superoxide anions in dormant and non-dormant seeds imbibed for 24 h at 25 C in the dark, respectively. (C and D) Fluorescence images of
dormant and non-dormant seeds, respectively, treated with DCFH-DA [5-(and-6)-chloromethyl-20 ,70 - dichlorofluorescein diacetate] and viewed
by an apotome microscope. (E–G) and (H–J) Details of the embryonic axis of dormant and non-dormant seeds, respectively, imbibed for 6 (E, H),
24 (F, I) and 72 h (G, J) at 25 C in the dark and stained with DCFH-DA.
Activities of the main antioxidant enzymes, i.e. superoxide
dismutase (SOD), CAT and glutathione reductase (GR), were
assessed in dry and imbibed, dormant and non-dormant seeds.
All activities are expressed as a function of the activities
measured in dry dormant seeds (Fig. 4). The activities of the
three enzymes were not changed substantially by the 5 week
storage. There was a trend for a lower activity of the three
detoxifying enzymes in non-dormant axes after 24 h of imbibition at 25 C, but this decrease was significant only for CAT,
which was markedly stimulated during seed imbibition, and GR
activities (Fig. 4).
Regulation of seed germination by ROS
The possible involvement of ROS in germination of Arabidopsis
seeds was assessed by using compounds that alter seed ROS
content and by mutants affected in ROS metabolism. Table 1
shows the effect of a general free radical scavenger, sodium
benzoate (Hung and Kao 2004), a specific superoxide scavenger,
1,2-dihydroxy-benzene-3,5-disulfonic acid (Tiron; Wise and
Naylor 1987), and a H2O2 scavenger, dimethylthiourea
(DMTU; Levine et al. 1994) on the germination of non-dormant
seeds at 25 C. Sodium benzoate reduced germination at 25 C
by approximately 20%, whereas Tiron and DMTU reduced it
by only approximately 10% (Table 1). Diphenyleneiodonium
(DPI), an inhibitor of NADPH oxidase, reduced germination of
non-dormant seeds at 25 C to approximately 20% (Table 1).
Dormant seeds were treated with methylviologen (MV, 6 h) or
menadione (MN, 16 h), two ROS-generating compounds
known to be effective in releasing sunflower seed dormancy
(Oracz et al. 2007). MV and MN (both 1 mM) improved germination of dormant seeds at 25 C, with MN (approximately
71%) being more efficient than MV (approximately 40%)
(Table 1).
Plant Cell Physiol. 53(1): 96–106 (2012) doi:10.1093/pcp/pcr129 ! The Author 2011.
99
J. Leymarie et al.
250
Enzyme activity (% initial value)
dormant
200
non-dormant
*
150
100
*
50
0
dry
24h 25°C
dry
24h 25°C
CAT
SOD
dry
24h 25°C
GR
Fig. 4 Activities of SOD (superoxide dismutase), CAT (catalase) and GR (glutathione reductase) in dormant and non-dormant Col seeds.
Activities were measured in dry seeds and after 24 h imbibition at 25 C in the dark, in dormant (red bars) and non-dormant (blue bars) seeds.
Means ± SD of triplicates experiments are shown. Asterisks above the bars indicate that activities in non-dormant seeds differed significantly
from those measured in dormant seeds (P < 0.05).
Table 1 Effect of ROS scavengers (Tiron, sodium benzoate and
DMTU), an NADPH inhibitor (DPI) and ROS donors (MV and
MN) on the germination of Col seeds
Germination (%) after 7 d at 25 C
Treatment
Dormant seeds
Non-dormant
seeds
(None) H2O
6±2
90 ± 3
Tiron 0.1 mM
–
77 ± 5
Sodium benzoate 0.1 mM
–
69 ± 2
DMTU 0.1 mM
–
83 ± 2
DPI 0.1 mM
–
21 ± 6
6 h MV 1 mM
40 ± 4
–
16 h MN 1 mM
71 ± 3
–
All assays were performed at 25 C in the dark. Seeds were treated by MV and
MN for the durations indicated in the table and then transferred to water.
DMTU, dimethylthiourea; DPI, diphenyleneiodonium MV, methylviologen;
MN, menadione.
For mutants, the freshly harvested vte1-1 and cat2-1 seeds
germinated almost fully after 7 d at 25 C, whereas germination
of wild-type seeds did not exceed 20% (Fig. 5A). In contrast,
only a small number of rbohD seeds germinated at this
temperature. Five weeks of after-ripening resulted in a faster
germination of the vte1-1 and cat2-1 seeds and almost full germination of wild-type seeds at 25 C (Fig. 5B). However,
germination of rbohD seeds after the same duration of storage
was still 30% (Fig. 5B). Full release of dormancy of rbohD seeds
required at least 15 weeks (data not shown). At 15 C,
after-ripened seeds of all genotypes fully germinated within
2 d (data not shown).
100
Changes in expression of dormancy-related genes
in seeds of mutants vte1-1 and cat2-1
We investigated expression of genes known to be involved in
the regulation of Arabidopsis seed dormancy in the freshly
harvested seeds of vte1-1 and cat2-1 imbibed at 25 C. Gene
expression was evaluated after 24 h imbibition at 25 C in the
dark, using real-time quantitative reverse transcription–PCR
(RT–PCR). In Col seeds, the expression of AtDOG1, AtCCD4,
AtNCED3, AtNCED9 and AtCYP707A2 was similar in dormant
and non-dormant seeds (Fig. 6A). In contrast, the expression of
ABI5 was reduced while AtGA3ox1 and AtSLP2 expression
was highly stimulated when Col seeds were no longer dormant
(Fig. 6A, B). The expression of the genes involved in ABA
metabolism (AtNCED3, AtNCED9 and AtCYP707A2) and signaling (ABI5), and gibberellin metabolism (AtGA3ox1) did not
differ significantly between the germinating vte1-1 or cat2-1
seeds and the dormant wild-type seeds (Fig. 6A). AtCCD4
showed the same pattern of expression (Fig. 6A). However,
the expression of DOG1 was greater in imbibed vte1-1
seeds, and was more dramatic in the cat2-1 seeds (Fig. 6A).
The expression of ABI5 remained unchanged (Fig. 6A). As in
Col non-dormant seeds, AtSLP2 was up-regulated in the vte1-1
and cat2-1 seeds, although a high variability was observed
among biological replicates (Fig. 6B).
Discussion
Among the numerous Arabidopsis accessions, seeds of the
ecotype Col are often considered as low or non-dormant,
whereas seeds of Landsberg erecta (Ler) and Cape Verde islands
(Cvi) accessions are considered as dormant (Koornneef et al.
Plant Cell Physiol. 53(1): 96–106 (2012) doi:10.1093/pcp/pcr129 ! The Author 2011.
Reactive oxygen species and Arabidopsis seed dormancy
100
A
80
vte1-1
60
cat2-1
40
Germination (%)
20
wt
rbohD
0
0
100
2
B
4
6
8
10
cat2-1
vte1-1
80
wt
60
40
rbohD
20
0
0
2
4
6
8
10
Duration at 25°C (days)
Fig. 5 Dormancy of the cat2-1, vte1-1 and rbohD seeds. (A) Freshly
harvested seeds. (B) Seeds after-ripened for 5 weeks at 20 C. Col
(diamonds), cat2-1 (squares), vte1-1 (open squares) and rbohD
(crosses) seeds were germinated at 25 C in the dark. Means ± SD of
triplicate experiments are shown.
A
2004, Preston et al. 2009). Germination of freshly harvested
Arabidopsis seeds is generally assessed at 22–24 C under
continuous light. In these conditions the germination of Col
seeds is indeed markedly stimulated, as shown in Fig. 1: they are
able to germinate almost fully at 25 C, whereas the speed is low.
However, when freshly harvested Col seeds are germinated
at the same temperature in the dark, their germination is
prevented and limited to approximately 20% (Fig. 1A). This
suggests that light overcomes Col dormancy during imbibition
at temperatures higher than 20 C and that it can be considered
as a dormancy release factor (Finch-Savage and LeubnerMetzger 2006). The dormancy is not expressed between 10
and 20 C and disappears after a dry storage period of approximately 5 weeks at 20 C (Fig. 1). Dormancy is well known to be
a relative phenomenon that is controlled by environmental
factors during seed imbibition, and its expression can vary
greatly with hydration level, temperature, oxygen availability
or light (Bewley and Black 1994). Using the simple, suboptimal
condition, i.e. germination at 25 C in the dark, allowed the
study of seed dormancy regulation with wild-type Col, and
the mutants available for this accession. Moreover, the dark
condition excludes any effect by mechanisms that might be
associated with photosensitivity.
Using this system we investigated the role of ROS in the
regulation of Arabidopsis seed germination. Dry after-ripening
was not associated with marked changes in the ability of
Col seeds to produce H2O2 (Fig. 2). Bahin et al. (2011) demonstrated that after-ripening of barley grains was not associated
with accumulation of H2O2. It should be noted that Arabidopsis
and barley seeds both display physiological seed coat dormancy.
B
7
12
Transcript abundance
Col dormant
6
Col non-dormant
10
cat2-1 dormant
5
vte1-1 dormant
8
4
6
3
4
2
2
1
0
0
DOG1
CCD4
NCED3
NCED9 CYP707A2
ABI5
GA3ox
SLP2
Fig. 6 Transcript abundance of (A) DOG1, CCD4, NCED3, NCED9, CYP707A2, ABI5 and GA3ox, and (B) SLP2 analyzed by real-time RT–PCR
[arbitrary units with the value 1 assigned to the Col dormant sample and expression relative to UBQ5 (UBIQUITIN5) and EMB1345 (EMBRYO
DEFECTIVE 1345) as reference genes in seeds imbibed for 24 h at 25 C in the dark]. Blue bars, Col dormant seeds; dark red bars, Col non-dormant
seeds; yellow bars, fresh cat2-1 seeds; green bars, fresh vte1-1 seeds. Means ± SD of triplicate experiments are shown.
Plant Cell Physiol. 53(1): 96–106 (2012) doi:10.1093/pcp/pcr129 ! The Author 2011.
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J. Leymarie et al.
In contrast, the ROS content of the sunflower embryonic axis
increases markedly during after-ripening; however, sunflower
seeds display embryo dormancy (Oracz et al. 2007). This
suggests that patterns of ROS accumulation in the dry state,
which is associated with after-ripening, might depend on the
type of dormancy. H2O2 production in Arabidopsis Col seeds
decreased 16 h after imbibition, followed by an increase 24 h
after imbibition. ROS content increases during the early stages
of seed imbibition, as a consequence of the resumption of
respiration (Bailly 2004). This increase might occur very early
(i.e. before 3 h) or be masked by an efficient activation of
scavenging or detoxifying mechanisms, such as CAT (Fig. 4).
Germinating seeds produced approximately twice more H2O2
than dormant seeds 24 h after imbibition (Fig. 2). It is possible
that this increase in H2O2 was partly related to the slight
decrease of CAT activity, which was demonstrated for nondormant seeds imbibed for 24 h (Fig. 4). However, it is also
likely that these changes were related to an activation of the
mechanisms involved in ROS production, since our method
of H2O2 measurement evaluates the ability to produce this
compound. The absence of marked changes in SOD and GR
activities in dormant and non-dormant seeds reinforces
this hypothesis, as well as in situ localization of O
2 and ROS
(Fig. 3). NBT staining revealed that despite a constant and
unchanged SOD activity (Fig. 4), O
2 accumulates specifically
in the radicle of the embryo in non-dormant seeds, whereas
dormant seeds accumulate this compound irregularly (Fig. 3A,
B). ROS signaling requires cell-to-cell propagation which probably involves NADPH oxidases that create an auto-propagative
wave (Miller et al. 2009, Mittler et al. 2011). The localization of
superoxide, the product of NADPH oxidase, in the whole radicle
of non-dormant seeds would suggest that this propagation
system might be efficient in germinating seeds. In contrast,
the localization of formazan blue as isolated spots in the
embryonic tissues of dormant seeds suggests that the propagation of the ROS signal is impaired. DCFH-DA staining also revealed intensive ROS production specifically in the radicle of
non-dormant seeds (Fig. 3C, D). ROS localized to and remained
in the cytoplasm of radicle cells of dormant seeds during
imbibition at 25 C (Fig. 3E–G). In contrast, ROS in the cells of
non-dormant seeds imbibed at 25 C were first localized in the
cytoplasm, then found in the nuclei 24 h after imbibition, and
finally detected in the cell wall after germination (Fig. 3H–J).
These data suggest that ROS metabolism is tightly associated
with the completion of germination in Arabidopsis seeds.
The timely increase of ROS production in different subcellular compartments suggests various roles for ROS in cell
signaling during germination. In the cytoplasm, ROS may
modulate the redox status (Dietz et al. 2010) and trigger
oxidative modifications of proteins (Oracz et al. 2009) and
stored mRNAs (Bazin et al. 2011), which in turn regulates cell
signaling to trigger germination. To date there are only sparse
data about the production and the role of ROS in nuclei,
particularly in plants. However, it was demonstrated that
ROS production occurred within plant cell nuclei
102
(Ashtamker et al. 2007) and that they also contained antioxidant redox systems (Pulido et al. 2009, Vivancos et al. 2010).
Furthermore, there is evidence that NADPH oxidase is also
localized in the nucleus (Ushio-Fukai 2006, Chandrakuntal
et al. 2010). In the nucleus, ROS could control gene expression
through the regulation of transcription factors, either directly
or by modifying the nucleus redox status, or by its interplay
with calcium homeostasis (Mazar et al. 2010). In addition it has
recently been proposed that the nucleus redox status, which is
controlled by ROS and glutathione, would modulate cell cycle
activity (Vivancos et al. 2010). Finally, the apoplastic localization of ROS evidenced in germinated seeds is undoubtedly
related to the role of these compounds in cell elongation,
which has already been demonstrated in various plant materials
(Schweikert et al. 2000, Liszkay et al. 2004, Carol and
Dolan 2006, Shin et al. 2011) including germinating seeds
(Müller et al. 2009b).
The role of ROS in Arabidopsis seed germination is highlighted by the data obtained by pharmacological and genetic
approaches. Chemicals modulating ROS content altered
germination of dormant and non-dormant Arabidopsis seeds.
ROS scavengers had only a slight effect on germination of
non-dormant seeds, except for sodium benzoate, a general
free radical scavenger, which decreased germination at 25 C
by approximately 20% (Table 1). The most apparent effect
was produced by the NADPH oxidase inhibitor DPI which
almost fully suppressed germination at this temperature.
On the other hand, as was observed in sunflower (Oracz
et al. 2007, Oracz et al. 2009) and barley (Bahin et al. 2011)
seeds, the ROS-generating compounds MV and MN partially
released dormancy of Arabidopsis seeds, with MN, a major
stimulant of mitochondrial ROS production, being more
efficient than MV (Table 1). Germination of the cat2-1,
vte1-1 and rbohD seeds displayed marked phenotypes of
dormancy. The cat2-1 and vte1-1 seeds were able to germinate
at 25 C after harvest (Fig. 5), suggesting that the alteration of
seed antioxidant properties through CAT and tocopherols
is associated with a reduced level of dormancy. In contrast,
rbohD seeds failed to germinate at 25 C, even after 5 weeks
of after-ripening (Fig. 5), and therefore displayed a marked
phenotype of dormancy. Together with the data obtained
with DPI, our results suggest NAPDH oxidase as a key player
in germination of Arabidopsis seeds. The role of NADPH
oxidase in seed germination was pointed out in switchgrass
(Sarath et al. 2007), sunflower (Oracz et al. 2009) and barley
(Ishibashi et al. 2010). Müller et al. (2009a) also proposed that
an NADPH oxidase, AtrbohB, was involved in Arabidopsis seed
after-ripening. Interestingly, among the rboh genes, rbohD has
been shown to be involved in ABA and ethylene signaling, two
hormones known to play a role in seed dormancy (Kwak et al.
2003, Jakubowicz et al. 2010). It should also be noted that
rbohD is specifically required for the initiation and propagation
of the rapid ROS systemic signal (Miller et al. 2009, Wong et al.
2009), which again underlines the role of the dynamic ROS
signaling in seed germination.
Plant Cell Physiol. 53(1): 96–106 (2012) doi:10.1093/pcp/pcr129 ! The Author 2011.
Reactive oxygen species and Arabidopsis seed dormancy
We chose to study gene expression related to Arabidopsis
seed dormancy using the non-dormant mutants vte1-1 and
cat2-1, which exhibited clear phenotypes during imbibition at
25 C. Surprisingly, the expression of the genes related to ABA
metabolism (CCD4, NCED3, NCED9 and CYP707A2) was not
altered by dormancy alleviation in the mutants (Fig. 6).
As these genes are associated with dormancy (Finkelstein
et al. 2008), their unchanged expression in our experimental
system might be related to the absence of light, since most of
the transcriptomic studies for Arabidopsis seed germination
were carried out under continuous light. Cadman et al.
(2006) also did not detect major differences in the expression
of these genes in dormant and after-ripening seeds imbibed in
the dark, except for AtCCD4 whose expression was significantly
lower in non-dormant imbibed seeds. Liu et al. (2010) proposed
that H2O2 up-regulates ABA catabolism in Arabidopsis, which is
obviously not the case here. In contrast, in barley, Bahin et al.
(2011) showed that H2O2 induced HvNCED but did not affect
ABA sensitivity, although it alleviated seed dormancy. We
found here that the mutations in the antioxidant system did
not change the expression of ABI5 and GA3ox1, compared
with Col dormant seeds, whereas ABI5 and GA3ox1 were
down-regulated and up-regulated in non-dormant Col seeds,
respectively (Fig. 6A). With regards to ABI5, this indicates that
the non-dormant phenotype of vte1-1 and cat2-1 is probably
not associated with an inhibition of ABA signaling. Concerning
gibberellin metabolism, it was suggested that exogenous H2O2
induces expression of GA20ox and GA3ox in Arabidopsis and
barley, thus stimulating gibberellin biosynthesis (Liu et al. 2010,
Bahin et al. 2011). If there is activation of gibberellin synthesis
in the vte1-1 and cat2-1 mutants, this is not through the transcriptional regulation of AtGA3ox1 but possibly through other
genes regulating gibberellin synthesis. Alternatively, gibberellin
signaling might be enhanced in these mutants since AtSLP2,
a protease induced by gibberellin (Ogawa et al. 2003,
Arabidopsis eFP Browser), is up-regulated as it is in nondormant Col seeds (Fig 6B). The relationship between ABA,
gibberellin and ROS in the regulation of dormancy is probably
highly complex, although our data do not support interaction
between ROS and ABA. The activation of gibberellin signaling
by ROS has been described in barley (Bahin et al. 2011);
however, a detailed kinetic analysis is required to understand
properly the interactions which modulate the ABA/gibberellin
balance. The expression of DOG1 was slightly lower in nondormant wild-type seeds than in dormant wild-type seeds
(Fig. 6A) but it was stimulated in the vte1-1 and, more
markedly, in the cat2-1 seeds (Fig. 6A). In Arabidopsis, deeper
dormancy has been associated with the accumulation of DOG1
transcript, which disappears during seed imbibition (Bentskink
et al. 2006). However, other studies demonstrated that the expression of DOG1 was not always strictly related to seed dormancy (Finch-Savage et al. 2007, Laserna et al. 2008). In
Lepidium sativum, DOG1 was shown to be induced by ABA
(Graeber et al. 2009). In our system, germination of the vte1-1
and particularly cat2-1 seeds occurred with a high level of DOG1
transcripts (24 h after imbibition), and without an apparent
relationship to ABA synthesis. The induction of DOG1 expression by ROS is an interesting finding that should be considered
for elucidating the function of DOG1 in seed dormancy.
In summary, our findings reveal that ROS play a regulatory
role in Arabidopsis seed germination and primary dormancy.
We propose that their production triggers a set of sequential
cellular events, in various subcellular compartments, whose
completion is associated with the realization of germination.
Our molecular data highlight that seed dormancy can be
regulated by various, complex regulatory networks, since
dormancy alleviation by ROS does not seem to involve ABA
or gibberellin metabolism, although gibberellin signaling might
be activated. This work also underlines that dormancy is a
relative process and that multiple mechanisms are associated
with it. Assessing Arabidopsis dormancy in the absence of light
is a convenient experimental system devoid of any interference
with seed photosensitivity; however, it may lead to a reconsideration of the results obtained when germinating seeds in
the light.
Materials and Methods
Plant material and germination tests
Arabidopsis (Arabidopsis thaliana) seeds (Col) were used in this
study. The cat2-1 mutant (accession No. SALK_057998.55.00.x)
is characterized by a T-DNA insertion in the CAT2 gene
(At4g35090) in the third exon from the 50 end (Queval et al.
2007). The vte1-1 mutant was isolated from M3 ethyl
methanesulfonate-mutagenized A. thaliana in an HPLC-based
screen for mutants with altered leaf tocopherol profiles
(Sattler et al. 2003). The Arabidopsis T-DNA insertion line
SALK_070610 (rbohD; seventh exon insertion) was obtained
from the Salk collection (Pogany et al. 2009). Seeds were first
sown in a mixture of soil and vermiculite (9 : 1) and placed in
a growth chamber at 21 C under a photoperiod of 16 h
light/8 h dark. The light intensity was between 150 and
200 mmol m2 s1. After 12–15 d, germinated seeds were transferred on soil/perlite/vermiculite (2 : 1 : 1) and plants were
grown in the same conditions. Siliques were harvested at
maturity, dried at room temperature for 10 d and seeds were
collected. Freshly harvested seeds were either stored at 30 C
to preserve their dormancy or after-ripened at 20 C
(approximately 30% relative humidity) in the dark.
Germination assays were performed by placing seeds in
9 cm Petri dishes (100 seeds per dish, three replicates) on a
filter paper on the top of a layer of cotton wool moistened
with deionized water, or with the solutions indicated in the
Results. Except where indicated in the Results, all assays were
performed in darkness. Germination was assessed daily up
to 10 d. A seed was considered as germinated when the radicle
protruded through the envelopes. The germination percentages of seeds maintained continuously in the dark for
10 d were similar to those obtained after daily assessment of
Plant Cell Physiol. 53(1): 96–106 (2012) doi:10.1093/pcp/pcr129 ! The Author 2011.
103
J. Leymarie et al.
germination in the light, i.e. the presence of light during germination counts was not sufficient to stimulate germination.
In situ localization of superoxide anion
Embryos were dissected from imbibed seeds and incubated in
6 mM NBT (Sigma) in 10 mM Tris–HCl buffer (pH 7.4) at 20 C
for 1 h. Embryos were rinsed three times with deionized water,
and superoxide anion was visualized as precipitates of dark blue
insoluble formazan compounds (Beyer and Fridovich 1987).
The specificity of superoxide production, and of other
ROS measurements, was assessed using either SOD or CAT in
the incubation medium, as described in Bailly and Kranner
(2011).
In situ localization of hydrogen peroxide
Intracellular production of ROS was visualized by using
DCFH-DA (Molecular Probes) (Oracz et al. 2009) together
with an apotome microscope (Zeiss Imager Z1). Seed coat
was removed carefully and embryos were incubated in
20 mM potassium phosphate buffer (pH 6.0) containing
50 mM DCFH-DA for 15 min at 20 C in the dark. Samples
were rinsed for 15 min in the potassium phosphate buffer
solution. Images were acquired (excitation, 488 nm; emission,
525 nm) with the apotome microscope using a 10 or 20
numerical aperture objective. Z-series were performed with
a Z-step of 1.5 mm, and maximum projections of Z planes are
displayed. For each plant, a maximal projection of 15 planes was
performed. To compare the intensity of DFCH-DA labeling, axes
from different experimental conditions were fixed at the same
time and analyzed under the apotome microscope using
the same settings.
Measurement of hydrogen peroxide production
A 30 mg aliquot of seeds was incubated in 250 ml of potassium
phosphate buffer (20 mM, pH 6.0) containing 5 mM scopoletin
(Sigma) and 1 U ml1 (final concentration) horseradish peroxidase (Boehringer Mannheim) in darkness at 25 C on a shaker,
as described by Schöpfer et al. (2001). H2O2 production was
evaluated by the decrease in fluorescence (excitation, 346 nm;
emission, 455 nm) of the incubation medium and was transformed into molar H2O2 concentration using a linear calibration curve. Results correspond to the mean ± SD of three
replicates and are expressed per mg of the initial fresh weight
of dry seeds.
Measurement of antioxidant enzyme activities
Protein extraction was carried out as described by Bailly et al.
(1996). A 100 mg aliquot of seeds was ground and extracted in
potassium phosphate buffer (0.1 M, pH 7.8) containing 2 mM
dithiotreitol, 0.1 mM EDTA and 1.25 mM polyethylene glycol
4000 with traces of insoluble polyvinylpolypyrrolidone (PVP).
After centrifugation (15 min, 13,000 g), the extracts were
desalted on Sephadex PD10 columns (GE Healthcare) and
used for enzyme assays.
104
SOD (EC 1.15.11), CAT (EC 1.11.1.6) and GR (EC 1.6.4.2)
activities were determined as previously described by Bailly
et al. (1996). Enzyme activities are expressed as a percentage
of the values measured in dormant seeds and correspond to
the means of three biological replicates ± SD. Protein content of
the extracts was determined using the BioRad assay kit
with bovine serum albumin as the calibration standard.
Real-time quantitative RT–PCR
A 30 mg aliquot of of seeds was ground in liquid nitrogen with
insoluble PVP, and total RNA was extracted with a hot phenol
procedure according to Verwoerd et al. (1989). Total RNA
(1 mg) was treated with DNase I (Sigma), reverse transcribed
with Revertaid Reverse Transcriptase (Fermentas) in a 25 ml
reaction volume and amplified with Mastercycler ep Realplex
(Eppendorf ) using 5 ml of 30-fold diluted cDNA solution.
Primers were designed with primer3 software, except that for
HvNCED3 we used the primer sequence from Seo et al. (2004).
They were obtained from Eurogentec and the primer sequences
are shown in Supplementary Table S1. Real-time PCRs were
performed with the MaximaTM SYBR Green qPCR Master Mix
(Fermentas) and 0.23 mM of each primer (Fermentas) in a 15 ml
reaction. Critical thresholds (Cts) were calculated using the
Realplex 2.0 software (Eppendorf ). For each plate and each
gene, a standard curve made with dilutions of cDNA pools
was used to calculate the reaction efficiencies, and the relative
expression was calculated according to Hellemans et al. (2007)
with UBQ5 (UBIQUITIN5, AT3G62250) and EMB1345 (EMBRYO
DEFECTIVE 1345, AT2G26060) as reference genes. An arbitrary
value of 1 was assigned to the Col dormant seed samples,
which were used as control samples for normalization (Pfaffl
2001). Results presented are the means ± SD of four biological
replicates.
Supplementary data
Supplementary data are available at PCP online.
Acknowledgments
The authors wish to thanks Graham Noctor (Université
Paris XI), Michel Havaux (CEA Cadarache) and A. Savouré
(UPMC, Paris) for providing mutant seeds.
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