Changes in ascorbate–glutathione pathway enzymes in response to Mycosphaerella fragariae infection in selected strawberry genotypes

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Changes in ascorbate–glutathionepathway enzymes in response toMycosphaerella fragariae infection inselected strawberry genotypesLi Ding a , Marie Thérèse Charles a , Odile Carisse a , Rong Tsao b ,Claudine Dubé a & Shahrokh Khanizadeh aa Horticulture Research and Development Centre, Agriculture andAgri-Food Canada , 430 Gouin Blvd, St-Jean-sur-Richelieu, QC,Canada , J3B 3E6b Agriculture and Agri-Food Canada, Food Research Centre , 93Stone Road West, Guelph, ON, Canada , N1G 5C9Published online: 17 May 2011.

To cite this article: Li Ding , Marie Thérèse Charles , Odile Carisse , Rong Tsao , Claudine Dubé &Shahrokh Khanizadeh (2011) Changes in ascorbate–glutathione pathway enzymes in response toMycosphaerella fragariae infection in selected strawberry genotypes, Archives Of PhytopathologyAnd Plant Protection, 44:8, 712-725, DOI: 10.1080/03235400903266297

To link to this article: http://dx.doi.org/10.1080/03235400903266297

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Changes in ascorbate–glutathione pathway enzymes in response to

Mycosphaerella fragariae infection in selected strawberry genotypes

Li Dinga, Marie Therese Charlesa, Odile Carissea, Rong Tsaob, Claudine Dubea andShahrokh Khanizadeha*

aHorticulture Research and Development Centre, Agriculture and Agri-Food Canada, 430 GouinBlvd, St-Jean-sur-Richelieu, QC, Canada J3B 3E6; bAgriculture and Agri-Food Canada, FoodResearch Centre, 93 Stone Road West, Guelph, ON, Canada N1G 5C9

(Received 29 June 2009; final version received 10 July 2009)

Ten strawberry genotypes, resistant and moderately resistant (Joliette, Seascape,Aromas, FIN005-55 and FIN005-50) and susceptible ones (FIN00132-8,FIN00134-11, FIN00132-14, FIN005-7 and Kent) were used to assess the roleof the antioxidative defence system against Mycosphaerella fragariae infection.The pathogen-induced changes of hydrogen peroxide (H2O2) and antioxidantenzymes ascorbate peroxidase (APX), monodehydroascorbate reductase(MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase(GR) involved in the ascorbate–glutathione (ASC–GSH) cycle were examined inleaves of the selected genotypes. A significant different response was observedamong the genotypes. A marked increase in H2O2 content, APX, MDHAR,DHAR and GR activities were observed in resistant and moderately resistantgenotypes after inoculation by M. fragariae. In contrast, weak changes wereobserved in susceptible genotypes for the aforementioned enzymes andcompounds. It seems that resistant genotypes capable of overproducing H2O2

have a higher capacity to scavenge and reduce the injury to strawberry leaves byregulating the ASC–GSH cycle. The results may be useful in future breedingprogrammes to select those individuals with high scavenging properties to breednew resistant lines.

Keywords: strawberry; Mycosphaerella fragariae; ascorbate–glutathione cycle;antioxidant defence

Introduction

Reactions which occur when a plant is subjected to biotic or abiotic stresses oftenresult in the production of reactive oxygen species (ROS) such as the superoxide ion(O2

.7), hydrogen peroxide (H2O2) and the hydroxy radical (HO.) (Alscher et al.1997; Creissen et al. 1999; Hernandez et al. 1993, 1995). The association of ROS inplant–microbe interactions has been clearly established in recent years (Sandermann2000) and there is strong evidence that the oxidative burst occurs immediately afterpathogen infection which consequently can trigger a hypersensitive response (HR)(Baker and Orlandi 1995; Low and Merida 1996). Characteristic features of the HR

*Corresponding author. Email: [email protected]

Archives of Phytopathology and Plant Protection

Vol. 44, No. 8, May 2011, 712–725

ISSN 0323-5408 print/ISSN 1477-2906 online

� 2011 Taylor & Francis

DOI: 10.1080/03235400903266297

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include the formation of dead zone cells around the infection site, the synthesis ofsalicylic acid and the accumulation of antimicrobial agents, such as pathogenesis-related proteins and phytoalexins (Bolwell et al. 1995; Hammound-Kosack andJones 1996; Mehdy et al. 1996). In particular, the presence of H2O2 in the apoplast istoxic for pathogens involved in gene transcription and systemic acquired resistance(SAR), and slows down the spread of invading organisms by cell death around theinfection and a rapid local cross-linking of the cell wall (Hammound-Kosackand Jones 1996). SAR is thought to protect the plant against the systemic spread ofthe invading pathogen and requires the expression of defence genes in healthy plantcells.

Although ROS are important for the initial plant–pathogen interactions, healthyuninfected plant cells must avoid collateral damage from locally released ROS if theplant is to survive, and systemic resistance to develop. Central to the efficientremoval of ROS and maintenance of cellular homeostasis is the ascorbate–glutathione (ASC–GSH) cycle. It is comprised of three interdependent redoxcouples: ascorbate (ASC)/dehydroascorbate (DHA), glutathione (GSH)/oxidisedglutathione (GSSG), and reduced nicotine adenine dinucleotide phosphate(NADPH)/nicotinamide adenine dinucleotide phosphate (NADP) (May et al.1998) and enzymes: ascorbate peroxidase (APX, EC1.11.1.11), monodehydroascor-bate reductase (MDHAR, EC1.6.5.4), dehydroascorbate reductase (DHAR,EC1.8.5.1) and glutathione reductase (GR, EC1.6.4.2) responsible for the redoxcycling ASC, GSH and NADPH (Noctor and Foyer 1998). Operation of the ASC–GSH cycle allows to maintain the reduced, i.e. active forms of GSH and ASC in cellson a suitable level and thus to adjust a cellular redox potential. Moreover, the ASC–GSH cycle participates directly and indirectly in ROS scavenging.

In the ASC–GSH cycle, H2O2 is reduced by APX and ASC, and ASC isoxidized to monodehydroascorbate radical (MDHA). In turn, MDHAR reducesMDHA into ASC using reduced nicotine adenine dinucleotide (NADH) orNADPH as electron donors (Noctor and Foyer 1998). MDHA can also be reducedby photosynthetic electron flow through ferredoxin. Under conditions of limitedsupplies of NADH or NADPH, MDHAs are dismutated into ASC and DHA(Hakam and Simon 1996). DHAR, using GSH as an electron donor, generatesASC from DHA. GR, using NADPH as an electron donor, produces GSH fromGSSG. This enzyme participates not only in H2O2 scavenging, but also in redoxsignaling and activation of the protective mechanisms under stress conditionsthrough compartment-specific regulation of the GSH/GSSG ratio (Foyer et al.1997; Kocsy et al. 2001). The ASC–GSH cycle enzyme activities are present in allleaf and root cell organelles: chloroplasts, plastids, mitochondria and peroxisomes(Mittova et al. 2000).

Leaf spot caused by Mycosphaerella fragariae (Tul.) Lindau (Ramulariatulasnei Sacc.) is one of the most widespread foliar diseases in strawberry crops inCanada (Fall 1951) and the United States (Fulton 1958). The control of thisdisease is done by fungicide, but resistant cultivars have a major effect in the useof pesticide free crop (Maas 1984). Initial symptoms of this disease are small,purple, circular spots that first appear on young leaves; the centre becomes brownwith distinct red borders as the spots enlarge. Lesions produced by M. fragariaediffer depending on host c.v. and environmental conditions during infection, butthe cultivar remains to be the most important factor in controlling the disease(Delhomez et al. 1995; Carisse et al. 2000). Previously, Ehsani-Moghaddam et al.

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(2006) have indicated a positive relationship between superoxide dismutase (SOD)activity and the tolerance of strawberry plants to M. fragariae attack. The aim ofthis work is to study the changes in the level of H2O2 and antioxidant enzymes(APX, MDHAR, DHAR and GR) involved in the ASC–GSH cycle in leaves ofselected strawberry genotypes infected by M. fragariae, and to determine thedifferences of antioxidant and physiological responses among genotypes withvarying levels of resistance to this pathogen under stress conditions. The final goalis to use this information in our breeding programme as a marker to select fordisease resistant lines.

Materials and methods

Plant materials

Ten selected strawberry genotypes including two short day (SD) cultivars Kent andJoliette, two day neutral (DN) cultivars Seascape and Aromas and six advanced DNgenotypes (FIN005-55, FIN005-50, FIN005-7, FIN00132-14, FIN00132-8 andFIN00134-11), with different degrees of susceptibility to M. fragariae from theAgriculture and Agri-Food Canada (AAFC) strawberry breeding programme(Khanizadeh et al. 1996, 2002; Ehsani-Moghaddam et al. 2006). The resistant andmoderately resistant genotypes were Joliette, Seascape, Aromas, FIN005-55 andFIN005-50 while the susceptible genotypes were FIN00132-8, FIN00134-11,FIN00132-14, FIN005-7 and Kent. The plants were propagated by stolon into 400

pots containing mineral soil, peat moss and sand with 1:1:1 volume and were grownfor 2 months at 258C with a 16-h photoperiod in the greenhouse until they wereready for inoculation.

Production of inoculum

The method for production of inoculums of M. fragariae is explained by Delhomezet al. (1995). Inoculums were produced from an isolate of M. fragariae from adiseased strawberry leaf collected at the Agriculture Canada Experimental Farm inL’Acadie, Quebec. The fungus was identified based on morphological characters;symptoms produced by the isolate on strawberry leaves were used to confirm isolatepathogenicity and virulence. The fungus was then transferred to strawberry leaf agar(SLA) and incubated at room temperature (20–258C) for 2 weeks. The SLA wasprepared as follows: 200 g (fresh weight) of pesticide-free strawberry leaves wereblended in 250 ml of distilled water, heated 5 min, and filtered through two layers ofcheesecloth. The solution volume was adjusted to 1 litre. Then, 12 g of agar and 10mg of novobiocin were added, and the solution was autoclaved for 20 min at 100kPa. To increase spore production rapidly, 10 ml of sterile distilled water was pouredonto each plate, and a mycelia suspension was made by rubbing the culture with aglass rod to remove the mycelium. The mycelial suspension was inoculated on SLAplates (2 ml/plate), and the spore-production plates were incubated at roomtemperature (20–258C) for 1 week.

Inoculation

On the day of inoculation, a conidial suspension was prepared from these cultures bypouring 10 ml of sterile distilled water and 0.01% Tween 80 solution into each plate;

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spores were dislodged by gently rubbing the surface of the colony using a glass rod.The final spore suspension was adjusted to 1.14 6 105 spores/ml using ahemacytometer. Twenty plants from each cultivar were transferred to a growthchamber and the spore suspension was sprayed onto both faces of their leaves forinoculation. The control plants were sprayed with 0.01% Tween 80 solution withoutspores. Another 20 plants from each cultivar were kept in a separate growth chamberunder the same conditions and severed as non-inoculated control plants. Bothinoculated and control plants were kept in growth chambers at 258C and 100%humidity with a 16-h photoperiod for 48 h. Forty-eight hours after inoculation, thehumidity was reduced to 85% in both chambers until the end of the study. Triplicatesamples of fully expanded young leaves from both control and inoculated plants werecollected on days 0, 1, 2, 3, 4 and 5 after inoculation randomly from 20 plants. Thesamples were immediately frozen in liquid nitrogen and kept at 7808C until used.

H2O2 assay

H2O2 was measured according to Alexieva et al. (2001). Leaf tissues (0.5 g) werehomogenized an ice bath with 5 ml of 0.1% (w/v) TCA. The homogenate wascentrifuged at 12,000g for 15 min, then 0.5 ml supernatant was mixed with 0.5 ml of10 mM potassium phosphate buffer (pH 7.0) and 1 ml of 1 M potassium iodide (KI).The blank probe consisted of 0.1% TCA in the absence of leaf extract. The reactionsdeveloped for 1 h in darkness and the absorbance was measured at 390 nm. H2O2

concentration was determined using a given standard curve.

Extraction and assay of antioxidant enzymes

Tissue was ground to a fine powder in liquid nitrogen and total proteins wereextracted by homogenising the powdered tissue using 50 mM potassium phosphatebuffer (pH 7.0) containing 1% polyvinylpyrrolidone, and 1 mM EDTA. For assaysof APX, extracts were prepared in the same medium containing 1 mM ASC.Homogenates were centrifuged at 20,000g for 15 min at 48C. The supernatantsobtained were divided into aliquots and stored at 7808C. Protein concentrations inthe enzyme extract were determined with the method of Bradford (1976) usingdefatted BSA as a standard.

APX was assayed as described by Nakano and Asada (1981). The reactionmixture contained 50 mmol/l potassium phosphate (pH 7.0), 0.2 mmol/l EDTA, 0.5mmol/l ascorbic acid and 0.25 mmol/l H2O2. The reaction was started at 258C by theaddition of H2O2 after adding the enzyme extract containing 50 mg of protein. Thedecrease in absorbance at 290 nm for 1 min was recorded and the amount of ASCoxidised was calculated from the extinction coefficient 2.8 mmol/l–1 cm–1.

MDHAR activity was tested with the method of Hossain et al. (1984) with somemodifications by following the decrease in absorbance at 340 nm due to NADHoxidation. A 1.0 ml aliquot of enzyme extract was added to a reaction mixturecontaining 50 mM potassium phosphate (pH 7.6), 0.3 mM NADH and 2.5 mMASC. The reaction was started by adding 0.15 unit of ASC oxidase (EC 1.10.3.3,from Cucurbita species, Sigma, USA) to produce MDHA and an absorptioncoefficient of 6.2 mM71 cm71 was used for calculations. One unit of MDHARactivity was defined as the amount of enzyme that oxidises 1 nmol of NADH per minat 258C.

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DHAR activity was determined by monitoring the increase in absorbance at265 nm due to ASC production, according to Hossain and Asada (1984) with somemodifications. A 1.0 ml aliquot of enzyme extract was added to a reaction mixturecontaining 50 mM potassium phosphate buffer (pH 6.5), 0.1 mM EDTA, 0.5 mMDHA and 2.5 mM GSH. The non-enzymatic reduction of DHA by GSH wassubtracted. An absorption coefficient of 14.6 mM71cm71 was used for calculations.One unit of DHAR activity was defined as the amount of enzyme that produces 1nmol of ASC per min at 258C.

GR activity was measured by following the decrease in absorbance at 340 nmdue to NADPH oxidation with the method of Carlberg and Mannervik (1985). A200 ml aliquot of enzyme extract was added to a reaction mixture containing 1.5 mlof 0.1 M potassium phosphate buffer (pH 7), 150 ml of 20 mM GSSG, 1 ml ofdistilled water and 150 ml of 2 mM NADPH (dissolved in Tris-HCl buffer, pH 7), ina final volume of 3.0 ml. An absorption coefficient of 6.2 mM71cm71 was used forcalculations. One unit of GR activity was defined as the amount of enzyme thatoxidises 1 nmol of NADPH per min at 258C.

Data analysis

The data were subjected to analysis of variance using SAS (1989) and least significantdifference (LSD) was used to separate the mean at 0.05 level.

Results

Detection of H2O2

M. fragariae infection caused a marked increase in H2O2 content as compared tocontrols in all genotypes during the first 2 days, but by day 5 H2O2 content haddropped to those found immediately after inoculation (Figure 1). Variation in H2O2

levels was observed among the tested genotypes mostly on the 2nd day. Joliette andFIN005-50 showed higher H2O2 levels (more than 250% of the control) compared toKent, FIN00132-8 and FIN005-7 (less than 200% of the control) (Table 1).

Effects on the ASC–GSH cycle enzymes

APX

APX activity showed a gradual increase compared to control after the beginning ofinoculation in all the genotypes tested and peaked on the 3rd day, after whichactivities returned to initial levels (Figure 2). On the 3rd day, it is evident that Joliettepresented the highest level of APX activity (282% of the control) as compared toFIN005-50, Seascape and FIN005-55 (239, 222 and 205% of the control,respectively). Kent, the most susceptible, had the lowest level (135% of the control)(Table 1).

MDHAR

Plants of resistant and moderately resistant genotypes exhibited significant increasein MDHAR activity as compared to control during the first 3 days and then declinedafterward (Figure 3). On the 3rd day, MDHAR activity enhancement was found

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maximum in Joliette, FIN005-50 and FIN005-55 (145, 129 and 114% of the control,respectively). In FIN00132-14, FIN00134-11 and FIN005-7, MDHAR levelsremained almost unchanged versus controls during the 5 days after inoculation(Figure 4). In contrast, in Kent, after 3 days of inoculation, MDHAR levelsdecreased to about 83% of that in corresponding control levels (Table 1).

DHAR

In resistant and moderately resistant genotypes, M. fragariae infection resulted insignificant increase of DHAR activity, peak being reached at the 4th day of theinoculation (Figure 4). Joliette showed higher DHAR activity level (309% of the

Figure 1. Hydrogen peroxide content (H2O2, mmol g71 FW) in leaves of selected strawberrygenotypes. Inoculated (—¤—), plants inoculated with M. fragariae; control (—&—), non-inoculated plants. Each point is the average value of three independent measurements + SE.

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control) compared to FIN005-50, Seascape, FIN005-55 and Aromas (258, 230,181% and 178% of the control, respectively) (Table 1). In contrast, M. fragariaeinfection had no effect on DHAR activity in FIN00132-8 and Kent (Figure 5).

GR

No significant changes in GR activity were observed in FIN00132-8, FIN00134-11,FIN00132-14, FIN005-7 and Kent, but GR activity increased significantly forAromas, FIN005-50, FIN005-55, Joliette and Seascape from the 1st day to the 4thday after inoculation, before it began to decrease (Figure 5). On the 4th day, GRactivity in Joliette was 219% over the control, whereas in FIN005-50 and Seascape itreached a maximum of about 193% and 170%, respectively, over the control level(Table 1).

Discussion and conclusion

The present results on the time course of changes in antioxidant activities, inducedin leaves of strawberry genotypes inoculated with M. fragariae, expand ourearlier studies on oxidative processes in the same plant–pathogen interactions(Ehsani-Moghaddam et al. 2006). In our study, foliar H2O2 concentrations in allgenotypes increased significantly after inoculation (Figure 1). Cellular H2O2

concentration is the result of the balance between its production and utilisation(Bowler et al. 1992). A significant increase of SOD activity in both resistant andsusceptible strawberry plants, on the 2nd day of inoculation, was observed in ourprevious study (Ehsani-Moghaddam et al. 2006). However, the activities of APXwere modified byM. fragariae attack on the 1st and 2nd days of inoculation, peakingon the 3rd day (Figure 1) and it seems that H2O2 increase in inoculated leaves may bedue to enhanced SOD activity.

H2O2 and other ROS are the first defence line against pathogen invasion, eitherdirectly affecting the pathogens or inducing agents which cause subsequent resistance

Table 1. Maximum increase (inoculated/control) of H2O2 content, APX, MDHAR, DHARand GR activity in leaves of selected strawberry genotypes infected by Mycosphaerellafragariae.

CultivarsH2O2 content(%, Day 2)

APX activity(%, Day 3)

MDHARactivity

(%, Day 3)

DHARactivity

(%, Day 4)GR activity(%, Day 4)

AROMAS 237 + 7 d 183 + 6 e 109 + 5 d 178 + 9 d 116 + 5 eFIN00132-14 223 + 6 e 157 + 14 f 99 + 4 ef 150 + 15 f 100 + 4 fFIN00132-8 154 + 2 g 138 + 6 gh 93 + 4 g 108 + 5 g 98 + 4 fFIN00134-11 220 + 9 e 161 + 6 f 105 + 0 e 168 + 5 e 98 + 6 fFIN005-50 271 + 8 b 239 + 12 b 129 + 1 b 258 + 9 b 193 + 9 bFIN005-55 233 + 6 d 205 + 11 d 113 + 5 c 181 + 8 d 129 + 14 dFIN005-7 193 + 6 f 147 + 2 g 100 + 5 f 124 + 10 g 101 + 5 fJOLIETTE 272 + 5 a 282 + 12 a 145 + 5 a 309 + 18 a 219 + 13 aKENT 140 + 2 h 135 + 11 h 83 + 3 h 97 + 2 h 100 + 3 fSEASCAPE 240 + 3 c 122 + 5 c 114 + 4 c 230 + 5 c 170 + 14 c

H2O2, hydrogen peroxide; APX, ascorbate peroxidase; MDHR, monodehydroascorbate reductase; DHR,dehydroascorbate reductase; GR, glutathione reductase.Means within the same column followed by the same letter are not significantly different (p 5 0.05).

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reactions (Peng and Kuc 1992; Vanacker et al. 2000; Aver’yanov et al. 2001).Noticeable differences in production of H2O2 were observed among the genotypesstudied. Greater increase of H2O2 was found in the leaves of resistant genotypesduring the early days of post inoculation when compared to susceptiblegenotypes (Figure 1). It has been reported that ROS, produced by plant cellinteraction with a pathogen, caused host cell death (Levine et al. 1994) and inhibitedfungal spore germination (Peng and Kuc 1992). It may be supposed that the highconcentration of produced H2O2 observed in resistant genotypes can be related totheir greater resistance to M. fragariae.

It is vital for plants to adjust their enzymatic and non-enzymatic antioxidantsystems to control the ROS level in order to avoid oxidative stress (Allen 1995).

Figure 2. Activity of ascorbate peroxidase (APX, nmol mg71 protein min71) in leaves ofselected strawberry genotypes. Inoculated (—¤—), plants inoculated with M. fragariae;control (—&—), non-inoculated plants. Each point is the average value of three independentmeasurements + SE.

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A significant increase in APX was observed in all resistant cultivars (Figure 2). APXis primarily located in both the chloroplasts and cytosol, and as the key enzyme ofthe ascorbate–glutathione pathway, it eliminates peroxides by converting ASC toDHA (Navari-Izzo et al. 1997); it is one of the most important enzymes and plays acrucial role in eliminating toxic H2O2 from plant cells (Foyer et al. 1994). Greaterincrease of APX activity was also found in the leaves of resistant genotypes whencompared to susceptible ones, peak occurring 1 day later than for the H2O2

concentration (Figure 2). Therefore, the increased APX activity observed in thisstudy might have been due to the increased H2O2 production and the resistantgenotypes obviously have greater capacity to metabolise H2O2 to H2O to alleviatethe injury to strawberry leaves.

Figure 3. Activity of monodehydroascorbate reductase (MDHAR, nmol mg71 proteinmin71) in leaves of selected strawberry genotypes. Inoculated (—¤—), plants inoculated withM. fragariae; control (—&—), non-inoculated plants. Each point is the average value of threeindependent measurements + SE.

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MDHAR and DHAR are enzymes responsible for ASC regeneration in planttissues. For resistant genotypes and moderately resistant ones, MDHAR activityincreased with the peak level reached on the 3rd day, but for FIN00132-14,FIN00134-11, and FIN005-7 it was similar to control and even decreased forFIN00132-8 and Kent (Figure 3). The function of MDHAR is to limit the formationof MDHA, a free radical intermediate produced by APX catalysis, through theenzymatic disproportion, thus generating DHA (Arrigoni 1994; Asada 1999).However, DHA accumulation is generally considered as a negative event for cellmetabolism (Arrigoni 1994; De Gara et al. 2000). It was suggested that the increasein DHAR activity is induced when cellular ASC contents are significantly decreased

Figure 4. Activity of dehydroascorbate reductase (DHAR, nmol mg71 protein min71) inleaves of selected strawberry. Inoculated (—¤—), plants inoculated with M. fragariae; control(—&—), non-inoculated plants. Each point is the average value of three independentmeasurements + SE.

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(Arrigoni 1994), which may be required to sustain cycling of oxidised ASC when theflux through the ASC–GSH cycle is higher. In our study, M. fragariae infectionenhanced the DHAR activity of the resistant and moderately resistant genotypes andreached the highest level on the 4th day after the inoculation, but had no significanteffect on FIN00132-8 and Kent (Figure 4). Therefore, the regeneration of ASC fromDHA by DHAR or from MDHA by MDHAR plays a key role in the resistantgenotypes.

The reduction of DHA to ASC by DHAR is at the expense of GSH as an electrondonor, with the consequent production of GSSG (Foyer andMullineaux 1998; Uranoet al. 2000). GR activity contributes to the maintenance of the GSH pool in thereduced state, which is used by DHAR to reduce DHA to ASC (Carlberg and

Figure 5. Activity of glutathione reductase (GR, nmol mg71 protein min71) in leaves ofselected strawberry. Inoculated (—¤—), plants inoculated with M. fragariae; control (—&—),non-inoculated plants. Each point is the average value of three independentmeasurements + SE.

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Mannervik 1985; Stevens et al. 1997; Asada 1999). In our study, GR activity onlyshowed increase in Joliette, FIN005-50, Seascape and FIN005-55, and reached thehighest level on the 4th day of the inoculation, as did DHAR activity (Figure 5). GRmay play an important role in the control of endogenous H2O2 content through anoxido-reduction cycle involving GSH and ASC (Edwards et al. 1990; Foyer andMullineaux 1998; Bray et al. 2000). Our results indicate that in resistant genotypes, GRmight have an important role in the antioxidant defence system of leaves againstinfection.

In summary, in resistant genotypes, increasing activity of SOD seems to inducean overproduction of H2O2, which is partially counterbalanced by the increase inAPX activity. That might contribute to ASC oxidation, which was paralleled by anincreased capacity for ASC regeneration via MDHAR, DHAR and GR activities.All antioxidant enzymes, through the ASC–GSH cycle, may act in conjunction tometabolise H2O2 to H2O to alleviate the injury to strawberry leaves. Our resultshighlight the capacity of resistant genotypes to withstand conditions by regulatingthe ASC–GSH cycle. The results obtained in this investigation underline theimportant role of some antioxidant enzymes and compounds and, the possible use inprotecting cellular apparatus during infection, which could be used as a marker toselect lines resistant to M. fragariae in a breeding programme.

Acknowledgements

The authors acknowledge the support that received from ABIP-100 project entitled:Nutraceuticals Emerging from Agricultural Technologies Network (NEATNet), ChinaScholarship Council (CSC), Natural Sciences and Engineering Research Council (NSERC)and International Scientific Cooperation Bureau (ISCB) of Agriculture and AgriFood Canada(AAFC).

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