Plant Science 181 (2011) 309315

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Plant Science
journal homepage: www.elsevier.com/locate/plantsci

Catalase is a key enzyme in seed recovery from ageing during priming

Serge Kibinza a , Jrmie Bazin a , Christophe Bailly a , Jill M. Farrant b , Francoise Corbineau a ,
Hayat El-Maarouf-Bouteau a,
a
b

UR5 EAC7180 CNRS, UPMC Univ. Paris 06, Bat C 2 me tage, 4, place Jussieu, 75005 Paris, France
Molecular and Cell Biology Department, University of Cape Town, Private Bag, Rondebosch 7701, South Africa

a r t i c l e

i n f o

Article history:
Received 19 February 2011
Received in revised form 2 May 2011
Accepted 6 June 2011
Available online 12 June 2011
Keywords:
Catalase
Reactive oxygen species
Seeds
Ageing
Priming

a b s t r a c t
Ageing induces seed deterioration expressed as the loss of seed vigour and/or viability. Priming treatment,
which consists in soaking of seeds in a solution of low water potential, has been shown to reinvigorate
aged seeds. We investigate the importance of catalase in oxidation protection during accelerated ageing
and repair during subsequent priming treatment of sunower (Helianthus annuus L.) seeds. Seeds equilibrated to 0.29 g H2 O g1 dry matter (DM) were aged at 35 C for different durations and then primed
by incubation for 7 days at 15 C in a solution of polyethylene glycol 8000 at 2 MPa. Accelerated ageing
affected seed germination and priming treatment reversed partially the ageing effect. The inhibition of
catalase by the addition of aminotriazol during priming treatment reduced seed repair indicating that
catalase plays a key role in protection and repair systems during ageing. Ageing was associated with H2 O2
accumulation as showed by biochemical quantication and CeCl3 staining. Catalase was reduced at the
level of gene expression, protein content and afnity. Interestingly, priming induced catalase synthesis
by activating expression and translation of the enzyme. Immunocytolocalization of catalase showed that
the enzyme co-localized with H2 O2 in the cytosol. These results clearly indicate that priming induce the
synthesis of catalase which is involved in seed recovery during priming.
2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Storage of orthodox seeds for prolonged period induces their
deterioration leading ultimately to loss of their viability. The rate
of seed deterioration varies among plant species and seed lots, but
high moisture content and high temperature accelerate this process [13]. The main theory of ageing is the free radical theory
proposed by Harman which postulates that the accumulation of
damage caused by free radicals is the underlying mechanism by
which all living organisms age [4,5]. Numerous studies report the
importance of free radicals in ageing in plant and animals and the
importance of reactive oxygen species (ROS) in seed ageing has
been shown in various species [3,68]. Among these ROS, H2 O2 is
often considered as the most critical because it is stable at biological pH, crosses membranes and can cause severe cell damage due
to the highly aggressive HO that it can generate [9].
Oxidative stress is dened as an imbalance between ROS
production and antioxidant defense against these ROS. The consequence of oxidative stress is an increase in the formation of
oxidized cellular macromolecules. To prevent oxidative damage to
cellular components, cells are armed with various enzymatic and

Abbreviations: CAT, catalase; DM, dry matter; ROS, reactive oxygen species.
Corresponding author. Fax: +33 1 44 27 59 27.
E-mail address: hayat.bouteau@upmc.fr (H. El-Maarouf-Bouteau).
0168-9452/$ see front matter 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.plantsci.2011.06.003

non enzymatic mechanisms for detoxication. Among a number of

protective enzymes, superoxide dismutases (SODs) remove superoxide anions (O2 ) by catalysing their conversion into hydrogen
peroxide (H2 O2 ), which in turn can be broken down by catalase (CAT) to yield oxygen and water. Other antioxidant enzymes
are involved in maintaining the redox status of glutathione, a
compound that itself participates in ROS removal. Glutathione peroxidase removes H2 O2 by using it to oxidize glutathione (GSH)
to disulde (GSSG), while glutathione reductase regenerates GSH
from GSSG, with NADPH as a source of reducing power.
Observations made in different species show that oxidative
damage increases with age in seeds [3,6,8] simultaneous with
decreasing efciency of cellular antioxidant defences [3,6,8,10,11],
lending strong support to the free radical theory of ageing. In this
context, the importance of antioxidant enzymes has been shown
in protection from ROS-induced stress of plant and animals. Transgenic overexpression of antioxidant enzyme genes has been shown
to extend the lifespan of Drosophila [12,13]. Furthermore, synthetic SOD/CAT mimics have been shown to be particularly effective
in a number of diseases and thereby extending lifespan [14]. A
transgenic mouse strain with a 50-fold increase in CAT enzyme
activity in mitochondria from cardiac and skeletal muscle tissues
was found to have reduced severity of age-dependent arteriosclerosis and increased genomic stability, as correlated with a decrease
in oxidative stress and mitochondrial deletions in heart and muscle tissues [15]. In plants, CAT is considered as a primary enzymatic

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defense against oxidative stress induced by senescence, chilling,

dehydration, osmotic stress, wounding, paraquat, ozone and heavy
metals [1618]. Cutler [15] proposed CAT as a longevity determinant enzyme in animals, whether it is the case in seeds, is to be
determined.
Priming treatment of seeds has been shown to improve the
germination and emergence of many species [19]. Interestingly,
priming repairs damage of aged seeds [10,20] or seeds exposed to
abiotic stresses such as salinity [21], improving germination performance. Priming treatment consists of soaking seeds in an osmotica
of low water potential to control the amount of water supply to the
seed. At the cellular level, few processes have been described to act
during priming some of these being: activation of cell cycle [22,23],
endosperm weakening [24,25] and mobilization of storage proteins
[26,27]. The priming-induced increase in the rate of seed germination has been associated with the initiation of germination-related
processes [2830], repair processes [3134] and increase in various free radical scavenging enzymes, such as superoxide dismutase,
catalase and peroxidase have also been demonstrated [6,27,35]. It
is plausible that the benecial effect of priming is due to the competing effect of a number of these physiological processes but the
importance of each is to be determined.
The aims of the present study were to investigate the importance and the regulation of CAT during ageing and its involvement
in repair during priming in sunower seeds. An accelerated ageing protocol, used previously for sunower seeds [3], was used to
induce seed deterioration evaluated by the decrease in germination percentage. The accumulation of H2 O2 , the CAT substrate, was
recorded and the regulation of CAT at the level of gene expression,
protein synthesis and activity were determined. Priming treatment
was performed in the presence of 3-amino-l,2,4-triazol, an inhibitor
of CAT activity, to highlight the importance of CAT during seed
repair.
2. Materials and methods
2.1. Plant material and treatments
2.1.1. Seeds
Experiments were carried out with a sunower (Helianthus
annuus L.) simple hybrid, called Bellem, grown in eld and received
from Monsanto-France (Peyrehorade, France). Seeds harvested in
2004 were stored for 3 months at 20 C and 75% relative humidity in order to break their dormancy [36] before ageing treatment.
During experiments time, seeds were stored at 15 C and systematic germination tests were performed to check that seed natural
ageing did not occur.
2.1.2. Ageing treatment
Ageing treatment was performed according to Kibinza et al. [3].
Seeds were equilibrated for 24 h at 20 C, in closed asks with water
to obtain seed water content of 0.29 g H2 O g1 dry matter (DM), and
then placed at 35 C for different durations. After ageing treatments,
germination assays were performed on intact seeds, and embryonic
axes (radicle plus gemmula) were isolated and used immediately
for cytological experiments or were frozen in liquid nitrogen then
stored at 80 C for protein and RNA extraction.
2.1.3. Priming treatment
Priming treatment was carried out by incubating seeds for 7
days at 15 C on cotton wool moistened with a solution of PEG 8000
(Sigma) at 2.0 MPa according to Bailly et al. [37]. In order to study
the possible role of CAT activity during seed priming, seeds were
incubated in PEG containing 1 mM of 3-amino-l,2,4-triazol (AT),
a catalase inhibitor. To test the efciency of AT inhibition in our
priming protocol catalase activity has been measured after 7 days

priming and after 7 days priming with AT. The CAT activity was
reduced from 7.84 0.28 (nmoles/min/mg prot) in primed seeds
to 0.95 0.02 in primed seeds in the presence of AT.
2.2. Germination assays
Germination assays were performed at 15 C in darkness, on
three replicates of 50 seeds placed in 9-cm diameter Petri dishes
on a layer of cotton wool moistened with deionised water. Germination counts were made daily for 7 days. Germination was scored
as the emergence of radicle from the covering structures. Results
presented correspond to the means of the germination percentages
obtained after 7 days SD.
2.3. Subcellular localization and quantication of hydrogen
peroxide
The localization of H2 O2 was determined by CeCl3 staining
according to Bestwick et al. [38]. Five cubic millimeter sections
of axes were vacuum inltrated with 5 mM CeCl3 in 50 mM
MOPS buffer (pH 7.2) and then xed and processed for transmission electron microscopy (TEM) using a standard procedure
outlined in Oracz et al. [39]. The blocks were sectioned with glass
knives at 120 nm using a Reichert Ultractu S (Leica, www.leicamicrosystems.com), stained with lead citrate and 2% uranyl acetate
[40], and viewed with a LEO912 transmission electron microscope
(Leo Electron Microscopy, www.stm.zeiss.com).
H2 O2 contents were determined in embryonic axes according to
the method of OKane et al. [41] and Kibinza et al. [3] based on the
extraction of H2 O2 using perchloric acid (0.2 N) and spectrophotometric determination of H2 O2 at 590 nm by a peroxidase-based
assay. Absorbance of samples was compared with the absorbance
obtained with known amounts of H2 O2 .
2.4. Enzyme extraction and CAT assay
All extraction procedures were carried out at 4 C. Sunower
axes (about 1 g FW) were ground in 20 ml of potassium phosphate buffer (0.1 M, pH 7.8) containing 2 mM a-dithiothreitol,
0.1 mM EDTA, 1.25 mM polyethylene glycol 4000, and 20% (p/v)
polyvinylpolypyrrolidone, and mixed for 15 min. The homogenate
was centrifuged at 16,000 g for 15 min, the supernatant ltered
through Miracloth, desalted on a PD 10 column (Pharmacia), and
used for assays. Catalase (CAT, EC 1.11.1.6) activity was determined
spectrophotometrically following H2 O2 consumption at 240 nm
[6]. The results were expressed as specic activity, i.e. as nmol
H2 O2 decomposed min1 mg1 protein and correspond to the
means SD of the values obtained with nine measurements carried
out on three different extracts (three measurements per extract).
Catalase activity was expressed per milligram of extractable protein
(specic activity). The protein content of the extracts was determined using the BioRad assay kit with bovine serum albumin as
standard.
2.5. Western blots
Total protein was extracted from sunower axes isolated from
aged and aged-primed embryos according to Bailly et al. [42]. Proteins were then precipitated and the resulting pellet was air-dried
and dissolved in 1 ml SDS-PAGE sample buffer. 15 mg of protein
per lane were loaded onto an 11% acrylamide running gel and a 4%
acrylamide stacking gel, and were separated by SDS-PAGE. After
separation, the proteins were transferred electrophoretically (20 V,
35 min) onto nitrocellulose using a Trans-blot semidry system (BioRad). Membrane treatment and catalase antibody hybridization

S. Kibinza et al. / Plant Science 181 (2011) 309315

120
a
100

Germination (%)

were performed using polyclonal antibodiy against catalase, antiCAT6, which detect the 55 kDa catalase subunit [43,44] (provided
by Professor R Eising, Institut fr Botanik, Mnster, Germany). Eight
isoforms, from CAT1 to CAT8, have been identied, in sunower
[45]. CAT isoenzymes, from CAT1 to CAT5, results from the association of four 55 and 59 kDa subunits in various proportions and
CAT isoforms CAT6CAT8 are formed only by four 55 kDa subunits
differing in their charge. Because we were interested about global
CAT status, the antibody used in this study for Western blotting
and immunolocalization experiments were specic of the 55 kDa
subunit in order to consider all CAT isoforms.

311

a
a

80
b
60

b
c

40
20
0

2.6. Northern blots

Total RNA was extracted according to Verwoerd et al. [46] using
25 seed axes per sample and separated (10 mg per lane for all
extracts) in 1% agaroseformaldehyde gel [47]. RNA loading was
checked using ethidium bromide staining. The RNAs were transferred to nylon lter (Biodyne B, Pall) by capillary action with SSC 47
and xed by UV crosslinking (Stratalinker, Stratagene). DNA probes
labelling and hybridization were performed using a CATA1 (bp
1522 1710) cDNA probe encoding for sunower catalase 55 kDa
subunit (GenBank accession number L28740, [48]).
2.7. Sampling and processing for light microscopy
Axes were cut off from embryos and xed for 3 h in 3%
paraformaldehyde, 1% glutaraldehyde in a 0.1 M cacodylate buffer,
pH 7.2, under intermittent vacuum. They were washed four times in
the same buffer and dehydrated in an ethanol series before embedding in LR White resin. Transverse semi-thin sections of 0.5 m
were cut with a diamond knife (histo Diatome) and collected on
glass slides.
2.8. Immunolocalization of catalase

Fig. 1. Effects of ageing, priming and CAT inhibition on seed germination. Germination percentages obtained with sunower seeds aged for various durations, 3, 5, 7
or 9 days (A3, A5, A7 and A9, respectively), seeds aged at these different durations
and then primed [A (3, 5, 7 or 9) + 7] and aged seeds primed in the presence of 3amino-l,2,4-triazol [A (3, 5, 7 or 9) +7 + AT]. Aged seeds correspond to non dormant
seeds containing 0. 29 g H2 O g1 DM and incubated at 35 C for different durations.
After ageing, seeds were primed on PEG solution (2 MPa) or on PEG supplied by
aminotriazol for 7 days before germination tests. Data are means of 4 independent
experiments SD. Columns having different letters are signicantly different at the
0.05 probability level as determined by the paired t test.

addition during priming suppressed the improvement of seed germination obtained with priming treatment (Fig. 1). For all ageing
duration, aminotriazol application during priming drop the germination percentage to values close to those of aged seeds (Fig. 1).
This result indicates that catalase plays a key role in seed recovery
during priming process. Therefore, we investigate the characterization of CAT activity regulation during ageing and priming. We
focused on 7 days duration of ageing because priming improved signicantly seed germination (Fig. 1). Furthermore, it represents P50
which is an important parameter for considering seed deterioration
during storage in orthodox seeds [1].

For light microscopy immunouorescence, semi-thin sections

were incubated for 5 min with PBS buffer (PBSB, pH 7.4, and 0.1%
BSA) containing 0.1% Tween 20 and transferred to normal goat
serum diluted to 1:30 in PBSB for 20 min. They were washed (four
times 10 min) in PBSB without Tween and incubated overnight at
4 C with the catalase antiserum diluted to 1:500 in PBSB. After
four washes of 10 min in PBSB, they were treated for 1 h in the
dark at room temperature with a goat anti-rabbit immunoglobulin
labelled with FITC (Biosys) diluted to 1:400 in PBSB. The sections
were washed six times for 10 min in buffer, four times for 5 min
in distilled water and mounted in Vectashield mounting medium.
They were observed with a uorescence Zeiss microscope (excitation lter 450490 nm and barrier lter 520 nm). The labelling
specicity was checked by omitting the primary specic antibody
and replacing it with buffer.

Fig. 2 shows that ageing was associated with H2 O2 accumulation. H2 O2 content increased from 5.21 mol g DW1 in control
non aged seeds (A0) to 8.7 mol g DW1 after 7 days of ageing (A7)
(Fig. 2A). Interestingly, priming treatment resulted in a signicant
decrease in H2 O2 content to 6.71 mol g DW1 (Fig. 2A). Subcellular localization of H2 O2 using TEM and CeCl3 staining is evidenced
by dark precipitates (Fig. 2B). While non aged seeds indicated the
presence of some H2 O2, within the cytosol particularly that surrounding lipid bodies (Fig. 2B, A0), 7 days aged seeds displayed a
large increase (Fig. 2B, A7). Priming treatment appeared to diminish
the density and extent of the deposits visualized in the cytoplasm
(Fig. 2B, A7 + 7).

3. Results

3.3. Catalase status during seed ageing and subsequent priming

3.1. Seed viability after ageing and priming

3.3.1. Catalase activity

As shown in Fig. 3A, ageing induced a decrease in both CAT activity and afnity for H2 O2 . CAT activity decreased by 30% after 7 days
of ageing compared with the activity of non-aged seeds. CAT substrate afnity was also reduced at 7 days of ageing as showed by
the 0.5 fold increase of the Km score (Fig. 3A). Priming treatment
considerably restored catalase activity of 7 days aged seeds and
reduced the Km value to the one of non-aged seeds (Fig. 3A).

Seed viability was assessed by germination ability after 3, 5,

7 and 9 days ageing, ageing followed by 7 days of priming and
ageing followed by 7 days of priming in the presence of 3-aminol,2,4-triazol which is known for its specic effect in depressing
catalase activity in animal and plant cells [49,50]. Fig. 1 shows
that germination percentage decreased with ageing duration. The
germination percentage, which was 94% before ageing treatment,
reached 32% after 9 days of treatment. A 7 day-priming following
the ageing treatment improved germination of aged seeds what
ever the duration of ageing (Fig. 1). Interestingly, aminotriazol

3.2. H2 O2 content in aged and primed seeds

3.3.2. Protein and transcript content

Catalase protein content was evaluated by immunoblot after
SDS-PAGE using specic antibody against the 55 kDa subunit

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Fig. 3. CAT status during ageing and subsequent priming. (A) CAT activity (histograms) and CAT afnity (curve) of A7 and A7 + 7 comparing to A0 and C. C, dry
non-treated seeds. A0, non aged seeds containing 0. 29 g H2 O g1 DM, A7, 7-dayaged seeds and A7 + 7, 7 day aged seeds primed for 7 days. Data are means of 4
independent experiments SD. (B) Immunoblot showing catalase levels in proteins
extracted from C, A0, A7 and A7 + 7 seeds using CAT 6 antiserum. (C) Northern blot
of total RNA isolated from C, A0, A7 and A7 + 7 seeds hybridized with a CAT probe
and as a loading control an rRNA probe.

Fig. 2. H2 O2 content and localisation during ageing and priming. (A) Biochemical
quantication: H2 O2 content in seeds containing 0. 29 g H2 O g1 DM but not incubated at 35 C: non aged seeds (A0), 7-day-aged seeds (A7) and 7-day-aged seeds
primed for 7 days (A7 + 7). (B) Subcellular localization of H2 O2 :H2 O2 precipitates
CeCl3 forming electron-dense cesium perhydroxide, visible as black spots by TEM.
Pictures show H2 O2 precipitates in all samples. H2 O2 precipitates seem to be localized only in cytosol in all cases and intensied in A7 as indicated by arrows. cw, cell
wall; pb, protein body; ob, oil body.

(Fig. 3B). Ageing resulted in a decrease in catalase content relative to control and unaged seeds and subsequent priming resulted
in a considerable increase in this protein (Fig. 3B). CAT transcripts
were assessed by Northern blot analysis (Fig. 3C). Ageing reduced

CATA 1 transcripts to an undetectable level but priming restored

the transcript content to levels typical of non aged seeds (Fig. 3C).
3.3.3. In situ catalase detection
Catalase polyclonal antiserum allowed the detection and
the localization of the protein within the seed cells using
light microscopy. Immunouorescence, using uorescein isothiocyanate (FITC) as a uorochrome, revealed a marked green
uorescence within cells of non aged seeds (Fig. 4A). No uorescence was observed in controls performed in the absence of specic
catalase antiserum (Fig. 4B). Aged seeds displayed a very weak
uorescence in accordance with the reduction of protein content
evaluated by Western blot (Fig. 4C). Priming reinforced green uorescence suggesting increased presence of the protein in the cytosol
relative to the aged condition (Fig. 4D). The patterning of uores-

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313

Fig. 4. In situ detection of catalase in sunower seeds. Semi-thin transverse sections of non aged seeds, A0 (A), 7-day-aged seeds, A7 (C) and 7-day-aged seeds and primed,
A7 + 7 (D) are presented. Non aged seeds appear highly uorescent comparing to aged seeds and primed seeds display an intense uorescence comparable to non aged seeds
(arrows). No uorescence is detected on control seeds performed in the absence of specifc catalase antiserum (B). FITC immunofuorescence green labelling showed that
catalase is located in cytosol. cw, cell wall; pb, protein body; ob, oil body, IS, intercellular space. Bar = 5 m. (For interpretation of the references to color in text, the reader is
referred to the web version of the article.)

cence showed that catalase was clustered mainly in the cytosolic

spaces (Fig. 4) which effectively coincide with H2 O2 localisation
(Fig. 2B).
4. Discussion
Ageing induces the deterioration of cell integrity and functional
performance in all organisms leading to cell death. In seeds, ageing
is associated with loss of seed vigour and then viability evaluated by
the ability to germinate. In sunower seeds, germination decreased
signicantly with ageing duration at 35 C (Fig. 1), and sustained
ageing treatment for 14 days resulted in a complete loss of seed
viability (data not shown). We demonstrated that catalase is determinant in seed recovery and subsequent germination since the
inhibition of CAT by aminotriazol during priming treatment abolished the benecial effect of priming on seed germination (Fig. 1).
This result conrms that seed viability is dependent on oxidative
stress resistance. According to the free-radical theory of ageing, ROS

produced by respiration contribute to ageing of all organisms [4]. In

our model, ageing effectively leads to the accumulation of hydrogen peroxide in seed cell cytoplasm (Fig. 2). This increase of H2 O2
content might result from the production of H2 O2 by the mitochondrion since sunower seed water content during ageing was
0. 29 g g1 DM, which allowed active respiration [3], and seeds are
devoid of photosynthetic activity.
ROS accumulation induces oxidative stress when the imbalance
between ROS production and antioxidant defense against these
ROS take place. We show that during ageing CAT decreases at
the level of gene expression, protein content and protein afnity
(Fig. 3). Semi-thin transverse sections showed that catalase was
not detected in aged cells (Fig. 4). The decrease in protein content could be explained by their degradation due to ageing induced
oxidations. Endogenous production of ROS induces modication of
proteins, such as fragmentation and increased sensitivity to proteolysis [51]. Catalase activity was shown to be itself inhibited by ROS
such as superoxide [52]. It has been shown that catalases are oxi-

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dized and subjected to increased proteolytic degradation caused by

cadmium [53]. On the other hand, other protein modications such
as site-specic amino acid modications, altered electrical charge,
oxidation of Fe-S centers or cross-linking reactions are induced
by ROS. CAT being heme-containing proteins, the iron oxidation
could explain the decrease of the afnity of non degraded CAT
protein in aged seeds. Redox regulation of the activity of other
heme-dependent enzyme as human cystathionine -synthase has
been reported [54]. The iron oxidation seems to trigger conformational movement that modulates the active site structure in heme
peroxidase [55].
CAT transcripts are also reduced to an undetectable level at
7 days of ageing (Fig. 3). This could be due to the degradation
of oxidized RNA after ageing-induced ROS accumulation. In fact,
the content of total RNA extracted from aged seeds was 2.6 fold
lower than that of non-aged seeds. This reduction indicates also
the absence of CAT gene activation even if ROS induce a number
of transcription factors that regulate the expression of antioxidant
genes as the antioxidant responsive elements (ARE) which are
found in the promoter region of the three maize CAT genes [56].
The absence of CAT transcript accumulation during seed ageing
indicates that the expression machinery is affected probably due
to oxidative stress and temperature treatment. De novo synthesis of CAT transcripts and proteins resumed in aged primed seeds
(Figs. 3 and 4). Priming treatment used in this study allowed 0. 31 g
H2 O g1 DM of seed water content [10] which is comparable to that
of ageing treatment (0. 29 g H2 O g1 DM) but the temperature was
15 C versus 35 C. It was reported that heat stress decreases protein
accumulation and alters composition of maize kernels [57]. In aleurone layer cells of barley, heat stress induces damage to membrane
system of endoplasmic reticulum, which leads to arrest of protein
synthesis [58]. Thus the lack of CAT replacement by de novo synthesis by temperature treatment leads to the accumulation of H2 O2
showed during ageing.
In situ CAT localization, during priming, showed that it was
clustered mainly in the cytosolic spaces (Fig. 4) which coincide with H2 O2 localisation (Fig. 2B). Similar observation was
reported for the stress-inducible CAT isoform (CAT3) supposed to
be induced by high substrate levels [59]. The induction of CAT and
its co-localization with H2 O2 insure effective detoxication of ROS
responsible for seed deterioration.
In addition to protein and nucleic acid syntheses, priming treatment allows different metabolic processes as DNA replication or
ethylene synthesis [33,6062]. It was proposed that priming provided the additional repair time such that individuals could recover
the capacity to germinate under standard conditions [20]. Transcriptomic analysis comparing primed and germinated Brassica
oleracea seeds has showed that the majority of genes are upregulated in both states [30]. At the protein level, osmopriming
is associated by only a subset of events comparing to germination
in Arabidopsis thaliana seeds [27]. Interestingly, among polypeptides whose level specically increased during hydropriming in A.
thaliana seeds, a CAT isoform has been identied [27].
Priming treatment did not improve the germination of all non
germinated 7 days-aged seeds (Fig. 1). We suggest, in accordance
with Butler et al. [20], that priming repair is possible if accumulated damage is not irreversible. Irreversible damage at the cellular
level was designated as the point of non return which is the point
when the cell becomes irreversibly committed to die [63]. Seeds
that had been irreversibly damaged, whatever the ageing period,
were not repaired by priming. Seed population can be classied in
three categories according to their ageing responses: (i) germinating seeds, (ii) seeds which are unable to germinate without priming
treatment and (iii) seeds unable to germinate even after priming treatment. These categories correspond to (i) seeds that had
incurred soft damage due to the low accumulating ROS, (ii) seeds

that had accumulate important but not irreversible damage and are
not able to repair fully without a priming treatment and (iii) seeds
that had accumulate severe ROS inducing damage which become
beyond repair. We suggest that within the seed population, individuals contains different initial ROS content and/or antioxidant
status depending on their development conditions on the mother
plant, harvesting, transport or initial period of conservation (i.e.
before ageing experiments). Additional ageing induced-ROS leads
in the third category of seeds to exceed a threshold responsible
for redox dysregulation and cell homeostasis loss. The increase of
half-cell reduction potential was shown in response to ageing in
Pisum sativum seeds [8]. This increase was proposed to be related
to a programmed cell death (PCD)/DNA fragmentation suggesting
an active and genetically regulated cell death in response to ageing
in seeds. Interestingly, catalase was also associated to the reduction
of hypersensitive response induction of PCD [64]. If PCD is the common way for seeds to loose their viability during ageing, catalase
involvement seems to be highly signicant.
Although the involvement of protective enzymes like SOD or
GR is likely to be probable, our data demonstrated that CAT is a key
enzyme for seed repair against ageing ROS-induced damage during
priming treatment.

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