catalase is a key enzyme in seed recovery from ageing during priming 2011 plant science

8/17/2019 Catalase is a Key Enzyme in Seed Recovery From Ageing During Priming 2011 Plant Science

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Plant Science 181 (2011) 309–315

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Plant Science

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

Serge Kibinzaa, Jérémie Bazin a, Christophe Bailly a, Jill M. Farrantb, Franç oise Corbineaua,Hayat El-Maarouf-Bouteaua,∗

a UR5 EAC7180 CNRS, UPMCUniv. Paris 06, Bat C 2 ème étage, 4, place Jussieu, 75005 Paris, Franceb Molecular andCell 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 2011Received in revised form 2 May 2011Accepted 6 June 2011Available online 12 June 2011

Keywords:

CatalaseReactive oxygen speciesSeedsAgeingPriming

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 reinvigorateaged seeds. We investigate the importance of catalase in oxidation protection during accelerated ageingand repair during subsequent priming treatment of sunflower (Helianthus annuus L.) seeds. Seeds equi-librated to 0.29 g H2O g−1 dry matter (DM) were aged at 35 ◦C for different durations and then primedby incubation for 7 days at 15 ◦C in a solution of polyethylene glycol 8000 at −2MPa. Accelerated ageingaffected 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 thatcatalase plays akey role in protection and repair systems during ageing. Ageing was associated withH2O2accumulation as showed by biochemical quantification and CeCl3 staining. Catalase was reduced at thelevel of gene expression, protein content and affinity. Interestingly, priming induced catalase synthesisby activating expression and translation of the enzyme. Immunocytolocalization of catalase showed thatthe enzyme co-localized with H2O2 in the cytosol. These results clearly indicate that priming induce thesynthesis 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 theirdeterioration leading ultimately to loss of their viability. The rateof seed deterioration varies among plant species and seed lots, buthigh moisture content and high temperature accelerate this pro-cess [1–3]. 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 bywhich all living organisms age [4,5]. Numerous studies report theimportance of free radicals in ageing in plant and animals and theimportance of reactive oxygen species (ROS) in seed ageing hasbeen shown in various species [3,6–8]. Among these ROS, H2O2 isoften considered as the most critical because it is stable at biolog-ical pH, crosses membranes and can cause severe cell damage dueto the highly aggressive HO• that it can generate [9].

Oxidative stress is defined as an imbalance between ROSproduction and antioxidant defense against these ROS. The con-sequence of oxidative stress is an increase in the formation of oxidized cellular macromolecules. To prevent oxidative damage tocellular components, cells are armed with various enzymatic and

Abbreviations: CAT, catalase; DM, dry matter; ROS, reactive oxygen species.∗ Corresponding author. Fax: +331 44 27 59 27.E-mail address: [email protected](H. El-Maarouf-Bouteau).

non enzymatic mechanisms for detoxification. Among a number of protective enzymes, superoxide dismutases (SODs) remove super-oxide anions (O2•−) by catalysing their conversion into hydrogenperoxide (H2O2), which in turn can be broken down by cata-lase (CAT) to yield oxygen and water. Other antioxidant enzymesare involved in maintaining the redox status of glutathione, acompound that itself participates in ROS removal. Glutathione per-oxidase removes H2O2 by using it to oxidize glutathione (GSH)to disulfide (GSSG), while glutathione reductase regenerates GSHfrom GSSG, with NADPH as a source of reducing power.

Observations made in different species show that oxidativedamage increases with age in seeds [3,6,8] simultaneous withdecreasing efficiency of cellular antioxidant defences [3,6,8,10,11],lending strong support to the free radical theory of ageing. In thiscontext, the importance of antioxidant enzymes has been shownin protection from ROS-induced stress of plant and animals. Trans-genic overexpression of antioxidant enzyme genes hasbeen shownto extend the lifespan of Drosophila [12,13]. Furthermore, syn-theticSOD/CATmimicshavebeenshowntobeparticularlyeffectivein a number of diseases and thereby extending lifespan [14]. Atransgenic mouse strain with a 50-fold increase in CAT enzymeactivity in mitochondria from cardiac and skeletal muscle tissueswas found to have reduced severity of age-dependent arterioscle-rosis and increased genomic stability, as correlated witha decreasein oxidative stress and mitochondrial deletions in heart and mus-cle tissues [15]. In plants, CAT is considered as a primary enzymatic

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doi:10.1016/j.plantsci.2011.06.003

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defense against oxidative stress induced by senescence, chilling,dehydration, osmotic stress, wounding, paraquat, ozone and heavymetals [16–18]. Cutler [15] proposed CAT as a longevity determi-nant enzyme in animals, whether it is the case in seeds, is to bedetermined.

Priming treatment of seeds has been shown to improve thegermination and emergence of many species [19]. Interestingly,priming repairs damage of aged seeds [10,20] or seeds exposed to

abiotic stresses suchas salinity [21], improving germinationperfor-mance. Priming treatment consists of soaking seeds in an osmoticaof low waterpotential to control the amount of water supply to theseed. At the cellular level, few processes have been described to actduring priming some of these being: activation of cell cycle [22,23],endosperm weakening [24,25] and mobilizationof storage proteins[26,27]. The priming-induced increase in the rate of seed germina-tion has been associated with the initiation of germination-relatedprocesses [28–30], repair processes [31–34] and increase in vari-ousfreeradicalscavengingenzymes,suchassuperoxidedismutase,catalase and peroxidase have also been demonstrated [6,27,35]. Itis plausible that the beneficial effect of priming is due to the com-peting effect of a number of these physiological processes but theimportance of each is to be determined.

The aims of the present study were to investigate the impor-tance and the regulation of CAT during ageing and its involvementin repair during priming in sunflower seeds. An accelerated age-ing protocol, used previously for sunflower seeds [3], was used toinduce seed deterioration evaluated by the decrease in germina-tion percentage. The accumulation of H2O2, theCAT substrate,wasrecorded and the regulation of CAT at the level of gene expression,protein synthesis and activity were determined. Priming treatmentwas performedin the presence of 3-amino-l,2,4-triazol,an inhibitorof CAT activity, to highlight the importance of CAT during seedrepair.

2. Materials and methods

2.1. Plant material and treatments

2.1.1. SeedsExperiments were carried out with a sunflower (Helianthus

annuus L.) simplehybrid, called Bellem, grown in field and receivedfrom Monsanto-France (Peyrehorade, France). Seeds harvested in2004 were stored for 3 months at 20◦C and 75% relative humid-ity in order to break their dormancy [36] before ageing treatment.During experiments time, seeds were stored at 15 ◦C and system-atic germination tests were performed to check that seed naturalageing did not occur.

2.1.2. Ageing treatment Ageing treatment was performed according to Kibinza et al. [3].

Seedswereequilibratedfor24hat20 ◦C, in closedflaskswith water

toobtainseedwatercontentof0.29gH2O g−1 drymatter(DM),andthenplacedat35 ◦C for different durations.After ageing treatments,germinationassays were performed on intact seeds,and embryonicaxes (radicle plus gemmula) were isolated and used immediatelyfor cytological experiments or were frozen in liquid nitrogen thenstored at−80 ◦C for protein and RNA extraction.

2.1.3. Priming treatment

Priming treatment was carried out by incubating seeds for 7daysat 15◦C on cotton wool moistened with a solutionof PEG 8000(Sigma) at−2.0MPa according to Bailly et al. [37]. In orderto studythe possible role of CAT activity during seed priming, seeds wereincubated in PEG containing 1 mM of 3-amino-l,2,4-triazol (AT),a catalase inhibitor. To test the efficiency 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 wasreduced from 7.84±0.28 (nmoles/min/mg prot) in primed seedsto 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, onthree replicates of 50 seeds placed in 9-cm diameter Petri dishes

on a layer of cotton wool moistened with deionised water. Germi-nation counts were made daily for 7 days. Germination was scoredas the emergence of radicle from the covering structures. Resultspresented correspond to the means of the germination percentagesobtained after 7 days±SD.

2.3. Subcellular localization and quantification of hydrogen

peroxide

The localization of H2O2 was determined by CeCl3 stainingaccording to Bestwick et al. [38]. Five cubic millimeter sectionsof axes were vacuum infiltrated with 5 m M CeCl3 in 50 mMMOPS buffer (pH 7.2) and then fixed and processed for trans-mission electron microscopy (TEM) using a standard procedure

outlined in Oracz et al. [39]. The blocks were sectioned with glassknives at 120nm using a Reichert Ultractu S (Leica, www.leica-microsystems.com), stained withlead citrate and 2% uranylacetate[40], and viewed with a LEO912 transmission electron microscope(Leo Electron Microscopy, www.stm.zeiss.com).

H2O2 contents were determined in embryonic axes accordingtothe method of O’Kane et al. [41] and Kibinza et al. [3] based on theextraction of H2O2 using perchloric acid (0.2 N) and spectropho-tometric determination of H2O2 at 590nm by a peroxidase-basedassay. Absorbance of samples was compared with the absorbanceobtained with known amounts of H2O2.

2.4. Enzyme extraction and CAT assay

All extraction procedures were carried out at 4◦

C. Sunfloweraxes (about 1g FW) were ground in 20ml of potassium phos-phate buffer (0.1M, pH 7.8) containing 2mM a-dithiothreitol,0.1mM EDTA, 1.25mM polyethylene glycol 4000, and 20% (p/v)polyvinylpolypyrrolidone, and mixed for 15 min. The homogenatewas centrifuged at 16,000× g for 15min, the supernatant filteredthrough Miracloth, desalted on a PD 10 column (Pharmacia), andused forassays. Catalase (CAT, EC 1.11.1.6) activity was determinedspectrophotometrically following H2O2 consumption at 240 nm[6]. The results were expressed as specific activity, i.e. as nmolH2O2 decomposed min−1 mg−1 protein and correspond to themeans±SD of the valuesobtainedwith nine measurements carriedout on three different extracts (three measurements per extract).Catalaseactivitywas expressedpermilligramofextractableprotein

(specific activity). The protein content of the extracts was deter-mined using the BioRad assay kit with bovine serum albumin asstandard.

2.5. Western blots

Total protein was extracted from sunflower axes isolated fromaged and aged-primed embryos according to Bailly et al. [42]. Pro-teins were then precipitated and the resulting pellet was air-driedand dissolved in 1ml SDS-PAGE sample buffer. 15mg of proteinper lane were loaded onto an 11% acrylamide running gel and a 4%acrylamide stacking gel, and were separated by SDS-PAGE. Afterseparation, the proteins were transferred electrophoretically (20 V,35 min) onto nitrocellulose using a Trans-blot semidry system (Bio-

Rad). Membrane treatment and catalase antibody hybridization

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were performed using polyclonal antibodiy against catalase, anti-CAT6, which detect the 55kDa catalase subunit [43,44] (providedby ProfessorR Eising, Institut für Botanik, Münster, Germany). Eightisoforms, from CAT1 to CAT8, have been identified, in sunflower[45]. CAT isoenzymes, from CAT1 to CAT5, results from the asso-ciation of four 55 and 59kDa subunits in various proportions andCAT isoforms CAT6–CAT8 are formed only by four 55kDa subunitsdiffering in their charge. Because we were interested about global

CAT status, the antibody used in this study for Western blottingand immunolocalization experiments were specific of the 55kDasubunit in order to consider all CAT isoforms.

2.6. Northern blots

Total RNA was extracted according to Verwoerd et al. [46] using25 seed axes per sample and separated (10mg per lane for allextracts) in 1% agarose–formaldehyde gel [47]. RNA loading waschecked using ethidium bromide staining. The RNAs were trans-ferredtonylonfilter(BiodyneB,Pall)bycapillaryactionwithSSC47andfixed by UV crosslinking (Stratalinker, Stratagene). DNAprobeslabelling and hybridization were performed using a CATA1 (bp1522±1710) cDNA probe encoding for sunflower catalase 55kDasubunit (GenBank accession number L28740, [48]).

2.7. Sampling and processing for light microscopy

Axes were cut off from embryos and fixed for 3 h in 3%paraformaldehyde, 1% glutaraldehyde in a 0.1M cacodylate buffer,pH7.2, underintermittent vacuum. They were washedfourtimesinthe same buffer and dehydrated in an ethanol series before embed-ding in LR White resin. Transverse semi-thin sections of 0.5mwere cut with a diamond knife (histo Diatome) and collected onglass slides.

2.8. Immunolocalization of catalase

For light microscopy immunofluorescence, 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 goatserum diluted to 1:30 in PBSB for 20min. They were washed (fourtimes 10min) in PBSB without Tween and incubated overnight at4 ◦C with the catalase antiserum diluted to 1:500 in PBSB. Afterfour washes of 10min in PBSB, they were treated for 1h in thedark at room temperature with a goat anti-rabbit immunoglobulinlabelled with FITC (Biosys) diluted to 1:400 in PBSB. The sectionswere washed six times for 10min in buffer, four times for 5minin distilled water and mounted in Vectashield mounting medium.They were observed with a fluorescence Zeiss microscope (exci-tation filter 450–490 nm and barrier filter 520 nm). The labellingspecificity was checked by omitting the primary specific antibodyand replacing it with buffer.

3. Results

3.1. Seed viability after ageing and priming

Seed viability was assessed by germination ability after 3, 5,7 and 9 days ageing, ageing followed by 7 days of priming andageing followed by 7 days of priming in the presence of 3-amino-l,2,4-triazol which is known for its specific effect in depressingcatalase activity in animal and plant cells [49,50]. Fig. 1 showsthat germination percentage decreased with ageing duration. Thegermination percentage, which was 94% before ageing treatment,reached 32% after 9 days of treatment. A 7 day-priming followingthe ageing treatment improved germination of aged seeds what

ever the duration of ageing (Fig. 1). Interestingly, aminotriazol

0

20

40

60

80

100

120

G e r m i n a t i o n ( % )

a

a a

a

a

aa

b

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Fig. 1. Effects of ageing, priming and CAT inhibitionon seedgermination. Germina-tion percentages obtained with sunflower seeds aged for various durations, 3, 5, 7or 9 days (A3, A5, A7 and A9, respectively), seeds aged at these different durationsand then primed [A (3, 5, 7 or 9)+7] and aged seeds primed in the presence of 3-amino-l,2,4-triazol [A (3, 5, 7 or 9)+7+ AT]. Aged seeds correspond to nondormantseeds containing0. 29g H2O g−1 DMand incubated at 35◦C for different durations.After ageing, seeds were primed on PEG solution (−2MPa) or on PEG supplied byaminotriazol for 7 days before germination tests. Data are means of 4 independentexperiments±SD. Columns having differentletters are significantly different at the

0.05 probability level as determined by thepaired t test.

addition during priming suppressed the improvement of seed ger-mination obtained with priming treatment (Fig. 1). For all ageingduration, aminotriazol application during priming drop the germi-nation percentage to values close to those of aged seeds (Fig. 1).This result indicates that catalase plays a key role in seed recoveryduring priming process. Therefore, we investigate the character-ization of CAT activity regulation during ageing and priming. Wefocused on7 days durationof ageingbecause priming improved sig-nificantly seed germination (Fig. 1). Furthermore, it represents P50whichis an important parameter for consideringseed deteriorationduring storage in orthodox seeds [1].

3.2. H 2O 2 content in aged and primed seeds

Fig. 2 shows that ageing was associated with H2O2 accumu-lation. H2O2 content increased from 5.21mol g DW−1 in controlnon aged seeds(A0) to 8.7mol g DW−1 after 7 days of ageing (A7)(Fig. 2A). Interestingly, priming treatment resulted in a significantdecrease in H2O2 content to 6.71mol g DW−1 (Fig. 2A). Subcellu-lar localization of H2O2 using TEM and CeCl3 staining is evidencedby dark precipitates (Fig. 2B). While non aged seeds indicated thepresence of some H2O2, within the cytosol particularly that sur-rounding lipid bodies (Fig. 2B, A0), 7 days aged seeds displayed alargeincrease(Fig.2B, A7).Priming treatment appeared to diminishthe density and extent of the deposits visualized in the cytoplasm(Fig. 2B, A7+ 7).

3.3. Catalase status during seed ageing and subsequent priming

3.3.1. Catalase activity

AsshowninFig.3A,ageinginducedadecreaseinbothCATactiv-ityand affinity for H2O2. CAT activity decreased by 30%after 7 daysof ageing compared with the activity of non-aged seeds. CAT sub-strate affinity was also reduced at 7 days of ageing as showed bythe 0.5 fold increase of the Km score (Fig. 3A). Priming treatmentconsiderably restored catalase activity of 7 days aged seeds andreduced the Km value to the one of non-aged seeds (Fig. 3A).

3.3.2. Protein and transcript content

Catalase protein content was evaluated by immunoblot after

SDS-PAGE using specific antibody against the 55kDa subunit


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Fig. 2. H2O2 content and localisation during ageing and priming. (A) Biochemicalquantification: H2O2 content in seeds containing 0. 29g H2O g−1 DM but not incu-bated at 35◦C: non aged seeds (A0), 7-day-aged seeds (A7) and 7-day-aged seedsprimed for 7 days (A7+ 7). (B) Subcellular localization of H2O2:H2O2 precipitatesCeCl3 forming electron-dense cesium perhydroxide, visible as black spots by TEM.Pictures show H2O2 precipitatesin all samples. H2O2 precipitatesseem to be local-ized only in cytosol in allcasesand intensified in A7 as indicatedby arrows. cw,cellwall; pb, protein body; ob,oil body.

(Fig. 3B). Ageing resulted in a decrease in catalase content rela-tive to control and unaged seeds and subsequent priming resultedin a considerable increase in this protein (Fig. 3B). CAT transcripts

were assessed by Northern blot analysis (Fig. 3C). Ageing reduced

Fig. 3. CAT status during ageing and subsequent priming. (A) CAT activity (his-tograms) and CAT affinity (curve) of A7 and A7+7 comparing to A0 and C. C, drynon-treated seeds. A0, non aged seeds containing 0. 29g H2O g−1 DM, A7, 7-day-aged seeds and A7+7, 7 day aged seeds primed for 7 days. Data are means of 4independent experiments±SD. (B)Immunoblot showing catalase levels in proteinsextracted from C, A0, A7 and A7+7 seeds using CAT 6 antiserum. (C) Northern blotof total RNA isolated from C, A0, A7 and A7+7 seeds hybridized with a CAT probeand as a loading control an rRNA probe.

CATA 1 transcripts to an undetectable level but priming restoredthe transcript content to levels typical of non aged seeds (Fig. 3C).

3.3.3. In situ catalase detection

Catalase polyclonal antiserum allowed the detection andthe localization of the protein within the seed cells usinglight microscopy. Immunofluorescence, using fluorescein isoth-iocyanate (FITC) as a fluorochrome, revealed a marked greenfluorescence within cells of non aged seeds (Fig. 4A). No fluores-cence wasobserved in controls performed in theabsence of specificcatalase antiserum (Fig. 4B). Aged seeds displayed a very weakfluorescence in accordance with the reduction of protein contentevaluated by Western blot (Fig. 4C). Priming reinforced green fluo-rescence suggestingincreased presence ofthe protein in thecytosol

relative to the aged condition (Fig. 4D). The patterning of fluores-


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Fig. 4. In situ detection of catalase in sunflowerseeds. 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. Nonagedseedsappear highlyfluorescent comparingto aged seedsand primedseeds display an intense fluorescencecomparableto nonagedseeds(arrows). No fluorescence is detected on control seeds performed in the absence of specifc catalase antiserum (B). FITC immunofuorescence green labelling showed thatcatalase is located in cytosol. cw,cell wall; pb, protein body; ob, oilbody,IS, intercellular space.Bar =5 m. (For interpretation of thereferencesto color in text, thereader isreferred to theweb version of thearticle.)

cence showed that catalase was clustered mainly in the cytosolicspaces (Fig. 4) which effectively coincide with H2O2 localisation(Fig. 2B).

4. Discussion

Ageing induces the deterioration of cell integrity and functionalperformance in all organisms leading to cell death. In seeds, ageingis associated with loss of seed vigourand thenviability evaluatedbytheability to germinate. In sunflower seeds,germination decreasedsignificantly with ageing duration at 35 ◦C (Fig. 1), and sustainedageing treatment for 14 days resulted in a complete loss of seedviability (data not shown). We demonstrated that catalase is deter-minant in seed recovery and subsequent germination since theinhibition of CAT by aminotriazol during priming treatment abol-ished the beneficial effect of priming on seed germination (Fig. 1).This result confirms that seed viability is dependent on oxidative

stressresistance. Accordingto the free-radical theoryof ageing, ROS

produced by respiration contributeto ageingof allorganisms [4]. Inour model, ageing effectively leads to the accumulation of hydro-gen peroxide in seed cell cytoplasm (Fig. 2). This increase of H2O2

content might result from the production of H2O2 by the mito-chondrion since sunflower seed water content during ageing was0. 29gg−1 DM, which allowed active respiration [3], and seeds aredevoid of photosynthetic activity.

ROS accumulation induces oxidative stress when the imbalancebetween ROS production and antioxidant defense against theseROS take place. We show that during ageing CAT decreases atthe level of gene expression, protein content and protein affinity(Fig. 3). Semi-thin transverse sections showed that catalase wasnot detected in aged cells (Fig. 4). The decrease in protein con-tent could be explained by their degradation due to ageing inducedoxidations. Endogenous production of ROS induces modification of proteins, such as fragmentation and increased sensitivity to prote-olysis [51]. Catalase activity was shown to be itselfinhibited by ROS

such as superoxide [52]. It has been shown that catalases are oxi-


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