Calcareous impact on arbuscular mycorrhizal fungus development and on lipid peroxidation in monoxenic roots

Calcareous impact on arbuscular mycorrhizal fungus development and on lipid peroxidation in monoxenic roots

Phytochemistry 72 (2011) 2335–2341 Contents lists available at SciVerse ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phyto...

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Phytochemistry 72 (2011) 2335–2341

Contents lists available at SciVerse ScienceDirect

Phytochemistry journal homepage: www.elsevier.com/locate/phytochem

Calcareous impact on arbuscular mycorrhizal fungus development and on lipid peroxidation in monoxenic roots Sonia Labidi b, Maryline Calonne a, Fayçal Ben Jeddi b, Djouher Debiane a, Salah Rezgui b, Frédéric Laruelle a, Benoit Tisserant a, Anne Grandmougin-Ferjani a, Anissa Lounès-Hadj Sahraoui a,⇑ a b

Unité de Chimie Environnementale et Interactions sur le Vivant (UCEIV), Université du littoral Côte d’Opale, 50 Rue Ferdinand Buisson, BP 699, 62228 Calais Cedex, France Unité Cultures Maraîchères et Florales (UCMF), Institut National Agronomique de Tunisie (INAT), 43 Ave Charles Nicolle, 1082 Mahrajène-Tunis, Tunisia

a r t i c l e

i n f o

Article history: Received 24 March 2011 Received in revised form 5 August 2011 Available online 31 August 2011 Keywords: Arbuscular mycorrhiza Glomus irregulare CaCO3 Fatty acids Malondialdehyde Peroxidase

a b s t r a c t The present work underlined the negative effects of increasing CaCO3 concentrations (5, 10 and 20 mM) both on the chicory root growth and the arbuscular mycorrhizal fungus (AMF) Glomus irregulare development in monoxenic system. CaCO3 was found to reduce drastically the main stages of G. irregulare life cycle (spore germination, germinative hyphae elongation, root colonization, extraradical hyphae development and sporulation) but not to inhibit it completely. The root colonization drop was confirmed by the decrease in the arbuscular mycorrhizal fungal marker C16:1x5 amounts in the mycorrhizal chicory roots grown in the presence of CaCO3. Oxidative damage evaluated by lipid peroxidation increase measured by (i) malondialdehyde (MDA) production and (ii) the antioxidant enzyme peroxidase (POD) activities, was highlighted in chicory roots grown in the presence of CaCO3. However, MDA formation was significantly higher in non-mycorrhizal roots as compared to mycorrhizal ones. This study pointed out the ability of arbuscular mycorrhizal symbiosis to enhance plant tolerance to high levels of CaCO3 by preventing lipid peroxidation and so less cell membrane damage. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Calcareous soils are common in semi-arid and arid regions and they are expanded on 30% of the world’s arable soils (Guerinot, 2001). One of the most spread forms of carbonates in the soil is calcium carbonate (CaCO3) which characterised calcareous soils (Leytem and Mikkelsen, 2005). It is well known that high levels of CaCO3 lead to low bioavailability of mineral nutrients (Cartmill et al., 2008), in particular phosphorous (P) (Shen et al., 2004) and iron (Lindsay, 1995). Thereby, these deficiencies affect metabolic processes in the roots and the leaves of the plants (Pestana et al., 2005). Arbuscular mycorrhizal fungi (AMF) are beneficial soil microorganisms living in association with over 80% of the terrestrial plant species (Van der Heijden, 2002). They establish symbiotic interaction with the roots and contribute to improve the water and the mineral plant nutrition. The AMF are known to enhance the uptake of elements with low mobility in the soil (P, Cu. . .) through greater effective root area and penetration of the substrate, and by the activation and excretion of various enzymes by mycorrhizal roots and/or hyphae (Marschner, 1995). Moreover, arbuscular mycorrhi-

⇑ Corresponding author. E-mail address: [email protected] (A. Lounès-Hadj Sahraoui). 0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2011.08.016

zal symbioses increase plant tolerance to various biotic and abiotic factors (Smith and Read, 2008). Many studies revealed the fundamental function played by AMF in the enhancement of plant growth under abiotic stresses: salinity (Giri et al., 2007; Abdel Latef and He, 2011), temperature (Charest et al., 1993; Abdel Latef and He, 2010), drought (Augé, 2001; Ruiz-Sánchez et al., 2010) and soil compaction (Miransari et al., 2007, 2008). As well, AMF are implicated in the attenuation of harmful effects of other abiotic stresses such as pollutants: polycyclic aromatic hydrocarbons (Debiane et al., 2008, 2009), fungicides (Campagnac et al., 2010) and heavy metals (Hildebrandt et al., 2007; Sudova et al., 2007; Abdel Latef, 2011). Unfortunately, it was demonstrated that the AMF life cycle may be impeded by many environmental stresses. Several authors described the negative effect of different abiotic stresses on the AMF growth (Debiane et al., 2009; Calonne et al., 2010; Campagnac et al., 2010). But no data are available in the literature concerning CaCO3 impact. Under environmental stresses, all the living organisms produced reactive oxygen species (ROS) like superoxide radical, hydroxyl radical, hydrogen peroxide, alkoxy radical and the singlet oxygen (Elstner, 1982). The ROS initiate oxidative processes such as lipid peroxidation, protein oxidation and nucleic acids damage (Herbinger et al., 2002). In order to protect themselves against oxidative injury, higher plants induce antioxidant systems consisting for example of antioxidant enzymes including superoxide

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dismutase, peroxidase (POD) and catalase (Asada, 1999). One of the most damaging oxidative effects is the peroxidation of membrane lipids, which results in the concomitant production of malondialdehyde (MDA), a secondary end product of polyunsaturated fatty acids (FA) oxidation (Hodges et al., 1999; Cho and Park, 2000; Jouili and El Ferjani, 2003). As a response to environmental stress, cells can modify their membrane lipid composition so as to maintain optimal physical properties (Thompson, 1992). The regulation of the lipid composition and the adjustment of the unsaturation level of membrane FA are extremely important to deal with abiotic stresses and make a contribution to plant survival (Thompson, 1992; Chaffai et al., 2005; Bidar et al., 2008). In several studies, the lipid analysis was limited to the leave tissues. It’s interesting to study root lipids due to the key role of root cell in plant’s interaction with the surrounding medium stresses (Surjus and Durand, 1996). To our knowledge, whereas a number of works concerned the impact of numerous environmental stresses on arbuscular mycorrhizal symbioses, no study was undertaken on the oxidative stress induced by CaCO3. That is why the current study was focused on the investigation of CaCO3 effects both on Glomus irregulare development and on the symbiosis establishment (chicory roots/G. irregulare) in monoxenic conditions. Thus, occurrence of oxidative damage was evaluated through (i) analyzing fatty acids (FA) composition, (ii) measuring the lipid peroxidation biomarker MDA production and (iii) assessing the antioxidant enzyme POD activity. 2. Results 2.1. CaCO3 affected chicory root growth CaCO3 effect on chicory root growth was determined by measuring root dry weight after 8 weeks of incubation in the presence of increasing CaCO3 concentrations (0, 5, 10 and 20 mM). Both noncolonized and colonized root biomasses were significantly reduced in the presence of CaCO3 by comparison to the control. Root dry weight decreases were about 54%, 62% and 72% in non-mycorrhizal roots and about 65%, 73% and 69% in mycorrhizal roots in the presence of 5, 10 and 20 mM CaCO3, respectively (Table 1). 2.2. CaCO3 decreased drastically the arbuscular mycorrhizal G. irregulare development The impact of CaCO3 on the main stages of G. irregulare life cycle was evaluated by measuring several fungal developmental parameters: chicory root colonization, extraradical hyphae length, spore formation and finally spores’ germination as well as germinative hyphae length, in the absence (control) and in the presence of increasing CaCO3 concentrations (5, 10, 20 mM). The results of CaCO3 effect on G. irregulare chicory root colonization are reported in Table 2. After eight weeks incubation, Table 1 Non-mycorrhizal (NM) and mycorrhizal (M) chicory roots dry weight after 8 weeks of incubation in the absence and in the presence of increasing CaCO3 concentrations. CaCO3 (mM)

2.3. CaCO3 disturbed the chicory root fatty acid composition and content In the absence of CaCO3 (control), the FA composition of nonmycorrhizal roots ranged from C16:0 to C18:3. The predominant FA compounds were C16:0 (palmitic acid), C18:2 (linoleic acid) and C18:3 (linolenic acid); they constituted about 95% of the total FA. C18:2 was the main compound (60%) (Table 3). In the presence of CaCO3, whereas FA profile of non-mycorrhizal roots did not change, the total contents increased by 15% due mainly to C16:0 and C18:2 increases (Table 3). In contrast, C18:3 content decreased significantly as compared to the control. Regarding FA composition of mycorrhizal roots, grown on medium without CaCO3, it differed from the non-mycorrhizal ones by the presence of the arbuscular mycorrhizal fungal lipid marker C16:1x5. It represented 32% of the total FA of mycorrhizal roots. C16:0, C18:2 and C18:3 FA represented 19%, 35% and 8% of the total FA, respectively (Table 3). On supplemented medium with 5, 10 and 20 mM of CaCO3, C16:1x5 amount decreased in the mycorrhizal roots by 52%, 44% and 79%, respectively, as compared to the control. Moreover, whereas C18:2 quantity was increased by CaCO3 addition, no significant difference was observed in C18:3 content as compared to the control.

Dry weight/Petri dish (mg) NM

Control 5 10 20

microscopic observations of stained chicory roots showed intraradical hyphae, arbuscules and vesicles in roots grown both on control and on CaCO3 supplemented media (Table 2). The percentages of colonization were significantly (p < 0.05) higher in roots grown on control medium than on CaCO3 supplemented media. Mycorrhizal colonization of the chicory roots were significantly reduced by 36%, 43% and 68% in the presence of 5, 10 and 20 mM CaCO3, respectively (Table 2). The percentages of arbuscular colonization were about 47%, 42%, 36% and 18% in the presence of CaCO3 at 0, 5, 10 and 20 mM, respectively. Vesicles were also from 2 to 3 times less numerous in roots grown on CaCO3 supplemented media, compared to the control (Table 2). Likewise, extraradical hyphae lengths decreased significantly (p < 0.05) on CaCO3 supplemented media. These decreases were about 23%, 73% and 94% in 5, 10 and 20 mM of CaCO3, respectively. Concerning sporulation, while no significant differences were observed in spore number on media containing 0, 5 and 10 mM CaCO3, a drastic drop of 72% occurred on medium supplemented with 20 mM, the highest tested CaCO3 concentration (Table 2). CaCO3 effect on G. irregulare spore germination was evaluated by counting the number of spores that germinated on media containing increasing CaCO3 concentrations (0, 5, 10, 20 mM) after 4 weeks incubation. While 86% of the total spores germinated on control medium, the percentages of germinated spores decreased significantly (p < 0.05) to 58%, 44% and 42% on supplemented media with CaCO3 at 5, 10, 20 mM, respectively (Fig. 1). Regarding the germinative hyphae lengths, whereas they were estimated about 5.1 cm in the absence of CaCO3, they decreased by 68%, 66% and 79% at 5, 10, 20 mM CaCO3, respectively (Fig. 2).

2.4. CaCO3 induced lipid peroxidation in the chicory roots

M a

53.84 ± 8.06 24.84 ± 2.84b 20.28 ± 5.03bc 15.26 ± 1.47c

a

49.84 ± 3.49 17.20 ± 3.58b 13.40 ± 1.29b 15.22 ± 5.36b

Data are presented as means ± Standard error. The means were obtained from five replicates (n = 5). Different letters indicate significant differences between increasing concentrations of CaCO3 according to the LSD test (p < 0.05).

Lipid peroxidation in mycorrhizal and non-mycorrhizal chicory roots, grown in the absence (control) and in the presence of CaCO3, was determined by measuring malondialdedyde (MDA) formation. In the absence of CaCO3, MDA contents were significantly higher in mycorrhizal chicory roots than in non-mycorrhizal ones (Fig. 3). However, in the presence of increasing CaCO3 concentrations, MDA formation increased significantly (p < 0.05) in non-

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Table 2 Mycorrhizal colonization of chicory roots, spore formation and extra-radical hyphae development of G. irregulare after 8 weeks of incubation in the absence and in the presence of increasing CaCO3 concentrations. CaCO3 (mM)

Mycorrhizal colonization (%)

Arbuscules (%)

Vesicles (%)

Extraradical-hyphae length/Petri dish (m)

Spore number/Petri dish

Control 5 10 20

70.55 ± 6.87a 45.00 ± 6.38b 40.00 ± 4.05b 22.22 ± 5.44c

46.67 ± 10.10a 42.22 ± 6.54ab 35.56 ± 3.14b 18.33 ± 5.25c

18.33 ± 4.20a 6.11 ± 2.79b 8.88 ± 4.80b 00.00 ± 0.00c

6.50 ± 0.92a 4.97 ± 1.18b 1.72 ± 0.25c 0.39 ± 0.12d

689 ± 338.44a 780 ± 117.00a 645 ± 264.03a 192 ± 43.32b

Data are presented as means ± Standard error. The means were obtained from five replicates (n = 5). Different letters indicate significant differences between increasing concentrations of CaCO3 according to the LSD test (p < 0.05).

2.5. CaCO3 induced chicory root POD activities

100 90

a

In the absence of CaCO3 (control), no differences in POD activities were detected when comparing non-mycorrhizal and mycorrhizal roots (Fig. 4). In the presence of CaCO3, POD activities were significantly (p < 0.05) induced both in non-colonized and colonized roots as compared to their controls. These activities increased significantly in non-mycorrhizal roots to reach a peak at 10 mM of CaCO3. However, in mycorrhizal roots POD activities were constant with increasing concentrations of CaCO3 (Fig. 4).

Germination (%)

80 70 b

60 50

b

b

40 30 20

3. Discussion

10 0 Control

5 10 CaCO3 (mM)

20

Germinative hyphae length (cm/spore)

Fig. 1. Spore germination frequency of G. irregulare after 4 weeks incubation in the absence (control) and in the presence of increasing CaCO3 concentrations. The means were obtained from 50 replicates. Values sharing different letters are significantly different according to the Chi-Square test (p < 0.05).

7 a

6 5 4 3

b

b b

2 1 0 Control

5

10 CaCO3 (mM)

20

Fig. 2. Germinative hyphae length of G. irregulare after 4 weeks incubation in the absence (control) and in the presence of increasing CaCO3 concentrations. The means were obtained from 50 replicates. Different letters indicate significant differences between treatments according to the LSD test (p < 0.05).

mycorrhizal roots. This increase was about six folds higher at 10 mM of CaCO3 as compared to the control. In contrast, in mycorrhizal roots, MDA contents were similar in the absence and in the presence of increasing concentrations of CaCO3. Only, a slight decrease was observed at 20 mM of CaCO3. MDA contents comparison between mycorrhizal and nonmycorrhizal roots showed significant lower production in mycorrhizal ones (Fig. 3).

Our data demonstrated a negative effect of high CaCO3 levels on both non-mycorrhizal and mycorrhizal chicory root growth. Arbuscular mycorrhizal colonization did not provide a better growth in the presence of CaCO3. This result is not in agreement with many studies that showed benefic effects of arbuscular mycorrhizal symbiosis on plant growth under several other kind of abiotic stresses (Yano-Melo et al., 2003; Cho et al., 2006; Cartmill et al., 2007; Hajiboland et al., 2010). However, in few papers, arbuscular mycorrhizal symbiosis was reported to do not enhance plant growth under environmental stresses (Kaya et al., 2009; Campagnac et al., 2010). A decrease in the growth of different ornamental plant species was observed by Valdez-Aguilar and Reed (2007) in the presence of high concentrations of carbonates (HCO3 ). This inhibition in the root growth could be due to the allocation of a considerable portion of carbon by mycorrhizal roots to maintain the fungus metabolism (Bryla and Eissenstat, 2005). On the other hand, the current work pointed out the CaCO3 detrimental impact on the life cycle of the arbuscular mycorrhizal fungus G. irregulare during the pre-symbiotic (germination) and the symbiotic stages with the transformed chicory (Cichorium intybus L.) roots (root colonization, extraradical hyphal development and sporulation). No data have been described previously in the literature concerning CaCO3 impact on AMF development. Our FA analysis results were coherent with the decrease observed in the chicory root colonization by G. irregulare in the presence of CaCO3 increasing concentrations. In fact, we showed that mycorrhizal roots differed in their FA composition from the non-mycorrhizal ones by the presence of C16:1x5 which is a major FA for most Glomus isolates (Graham et al., 1995; Olsson, 1999; Grandmougin-Ferjani et al., 2005). CaCO3 addition decreased significantly the arbuscular mycorrhizal fungal lipid marker C16:1x5 amount. Similarly, a decrease of host plant colonization by AMF was observed in the presence of other abiotic stresses such as: fungicides (Calonne et al., 2010; Campagnac et al., 2010), polycyclic aromatic hydrocarbons (Debiane et al., 2008, 2009) and salt (Zuccarini and Okurowska, 2008; Hajiboland et al., 2010; Kumar et al., 2010). Juniper and Abbott (1993) suggested that salt stress may decrease AMF colonization via a direct effect on fungal growth. Jahromi et al. (2008) explained the decline of Glomus intraradices sporulation under NaCl stress in a monoxenic culture by the reduction in the

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Table 3 Fatty acid compositions and contents of mycorrhizal (M) and non-mycorrhizal (NM) chicory roots grown during 8 weeks in the absence (control) and in the presence of increasing CaCO3 concentrations. CaCO3(mM)

C16:1x5

C16:0 mg g

0

10 20

% b

M NM M NM M NM M NM

5

1

3.02 ± 0.87 2.67 ± 0.19b’ 4.15 ± 0.66a 3.56 ± 0.29a’ 3.94 ± 0.74ab 3.67 ± 0.47a’ 3.60 ± 0.82ab 3.64 ± 0.10a’

19 21 25 25 23 25 25 24

mg g

C18:0

1

% a

5.45 ± 2.85 – 2.62 ± 0.70b – 3.01 ± 1.53b – 1.13 ± 0.73c –

32 – 15 – 17 – 7 –

mg g

C18:1 1

% a

0.20 ± 0.03 0.25 ± 0.04a’ 0.20 ± 0.12a 0.25 ± 0.04a’ 0.10 ± 0.15a 0.23 ± 0.14a’ 0.07 ± 0.15a 0.19 ± 0.10a’

1 2 1 2 0.66 2 0.37 2

mg g

C18:2 1

% a⁄

0.74 ± 0.25 0.40 ± 0.03a’ 0.56 ± 0.06ab⁄ 0.43 ± 0.03a’ 0.55 ± 0.18ab 0.49 ± 0.16a’ 0.32 ± 0.34b 0.43 ± 0.06a’

5 3 3 3 3 3 2 3

mg g

C18:3 1

% b

5.42 ± 1.19 7.37 ± 0.72b’⁄ 8.50 ± 1.16a 8.99 ± 0.54a’ 8.31 ± 1.47a 9.09 ± 1.27a’ 8.27 ± 1.33a 9.15 ± 0.50a’

35 60 50 62 49 62 58 63

mg g

Total FA 1 a

1.14 ± 0.23 1.65 ± 0.16a’ 1.09 ± 0.10a 1.13 ± 0.08b’ 1.03 ± 0.13a 1.22 ± 0.30b’ 1.02 ± 0.19a 1.14 ± 0.09b’

1

%

mg g

8 14 6 8 6 8 7 8

15.97 ± 5.11a 12.36 ± 1.08b’ 17.14 ± 2.48a⁄ 14.39 ± 0.90a’ 16.96 ± 3.09a 14.73 ± 2.05a’ 14.44 ± 3.17a 14.58 ± 0.62a’

dry wt

Data are presented as means ± Standard error. The means were obtained from five replicates (n = 5). Different letters indicate significant differences (on a same colon) between increasing CaCO3 concentrations for each kind of roots alone (M or NM) according to the LSD test (p < 0.05). Asterisks indicate significant differences between NM and M roots according to the LSD test (p < 0.05). – Not detected.

-1

MDA (µmol.g of proteins)

12

NM

a

M

10 8

b

c

6

c cd

d

cd

4 e

2 0

Control

5

20

10 CaCO3 (mM)

Fig. 3. Malondialdehyde (MDA) concentrations in mycorrhizal (M) and nonmycorrhizal (NM) chicory roots grown during 8 weeks in the absence (control) and in the presence of increasing CaCO3 concentrations. The means were obtained from three replicates. Different letters indicate significant differences between NM and M roots with increasing concentrations of CaCO3 according to the the LSD test (p < 0.05).

NM

0.06

M

a

0.05

-1

POD activity (nKat.mg of proteins)

0.07

0.04

b

0.02

b

bc

bc

0.03

c d d

0.01 0

Control

5

10 CaCO3 (mM)

20

Fig. 4. Peroxidase (POD) specific activities in mycorrhizal (M) and non-mycorrhizal chicory roots (NM) grown during 8 weeks in the absence (control) and in the presence of increasing CaCO3 concentrations. The means were obtained from three replicates. Different letters indicate significant differences between NM and M roots with increasing concentrations of CaCO3 according to the the LSD test (p < 0.05).

number of branched absorbed structures (BAS) which can progressively form spores in their ramification (Bago et al., 1998). Indeed, in our experimental conditions, direct negative effect of CaCO3 on G. irregulare development has been observed. Increasing CaCO3 concentrations (5, 10 and 20 mM) decreased drastically the spore germination rates and the germinative hyphae elongation. Juniper and Abbott (2006) observed that addition of NaCl, another kind of mineral, to soil inhibited spore germination of 11 AMF species. Estaun (1989) reported also spore germination reduction of Glomus margarita and Glomus mosseae in the presence of NaCl, in solution and agar, respectively. The tendency to decreasing hyphal length as soil salt concentration increases has also been described (Cantrell and Linderman, 2001; Juniper and Abbott, 2006). CaCO3 toxicity to the root and the AMF development was found to be due to an induction of an oxidative damage pointed out through FA analysis, MDA evaluation and POD activity assessment. It is interesting to note that the polyunsaturated FA were disturbed by CaCO3 addition. Whereas C18:2 was increased both in mycorrhizal and non-mycorrhizal chicory roots, C18:3 decrease was observed only in non-mycorrhizal roots. The first hypothesis which could explain the C18:2 amount increase in the roots is the eventual inhibition of desaturase enzymes (Cyril et al., 2002) under CaCO3 stress. The second hypothesis is to consider the C18:2 accumulation as a reaction of the plant cells to adjust their membrane fluidity in response to the disruption caused by the high CaCO3 levels. López-Pérez et al. (2009) observed a similar increase in C18:2 contents in broccoli (Brassica oleracea L.) roots under high concentrations of NaCl allowing them a better adaptation to salt stress. Zhu et al. (2006) allocated the better regeneration of banana meristems, after the freezing process to the increase in C18:2 amounts. In the present study, it seems that the root cells regulate their polyunsaturated FA biosynthesis so as to compensate the lost of the unsaturation level of membrane FA caused by C18:3 degradation. In fact, the reduction of C18:3 amount may suggest an induction of lipid peroxidation related to direct reaction of oxygen free radicals with unsaturated lipids. Under stress conditions, polyunsaturated FA decomposition, especially the linolenic (C18:3) FA (Skórzyn´ska-Polit, 2007), leads to the formation of MDA which reflects the level of membrane peroxidation (Dotan et al., 2004; Debiane et al., 2008). MDA was found to be able to modify proteins and was supposed to be involved in the deterioration of various biological functions through its attachment to proteins and nucleic acids (Yamauchi et al., 2008). Consequently, the estimation of MDA amounts presents an effective mean of assessing oxidative membrane damage (Katsuhara et al., 2005; Shao et al., 2005). In the current study, MDA formation was found to be higher in the nonmycorrhizal roots than in the mycorrhizal ones in the presence of increasing CaCO3 concentrations. The reduction of MDA in

S. Labidi et al. / Phytochemistry 72 (2011) 2335–2341

arbuscular mycorrhizal roots in comparison with non-arbuscular mycorrhizal roots may demonstrate that AMF could alleviate the peroxidation of membrane lipids. The root protection against CaCO3 stress by mycorrhizal colonization is probably related to a reduction of ROS production by inducing ROS scavenging systems. Among the most common mechanism for detoxifying ROS synthesized during stress response, POD induction has been reported in several studies (Abdel Latef and He, 2011). In this study, the oxidative stress caused by the addition of CaCO3 was also confirmed by the assessment of the antioxidant enzyme POD activity which was found to be induced both in the non-mycorrhizal and mycorrhizal roots grown under CaCO3 stress. However, the POD activity induction did not provide enough protection against ROS in non-mycorrhizal roots, as demonstrated by the MDA enhancement. Even, in the absence of higher POD activity induction in mycorrhizal roots, they were more efficient in limiting lipid peroxidation than nonmycorrhizal roots. Other antioxidant enzymatic (such as superoxidase dismutase, catalase. . .) and non-enzymatic (such as ascorbate, b-carotene, a-tocopherol, and reduced glutathione) systems also play important roles in the removal of free radicals (Hodges et al., 1996) and could be implicated in the attenuation of lipid peroxidation. These results demonstrated the involvement of AMF in the protection of chicory roots against carbonates stress by limiting lipid peroxidation, which has been considered as one of the key processes of plant tolerance to abiotic stresses. Very few data were available concerning the mechanism involving in CaCO3 toxicity against AMF. However, similar mechanisms have been reported under several other abiotic stresses like salinity (Juan et al., 2005; He et al., 2007; Koca et al., 2007; Li, 2009; Hajiboland et al., 2010), pollutant (Debiane et al., 2008, 2009), fungicides (Campagnac et al., 2010) and temperature (Abdel Latef and He, 2010). In conclusion, even high levels of CaCO3 affected drastically the main stages of G. irregulare development, it was shown that the AMF was able to fulfill its life cycle. Furthermore, although a protective role of arbuscular mycorrhizal symbiosis against CaCO3 stress was demonstrated at the cellular level, arbuscular mycorrhizal colonization did not allow a better growth of the mycorrhizal chicory roots. CaCO3 probably affected other targets like nucleic acids and proteins, other important cell constituents which will be interesting to study in the future.

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was placed in the middle of each Petri dish (5 cm) containing the medium without (Control) or with the different concentrations of CaCO3 (5, 10 and 20 mM of CaCO3). Fifty spores per treatment were used. The roots and the spores’ cultures were incubated at 27 °C in the dark. 4.2. CaCO3 treatments CaCO3 was added to the media to obtain the concentrations of 5, 10 and 20 mM. The control contained M medium without CaCO3 (0 g L 1). After the addition of CaCO3, media were sterilized (121 °C, 30 min). The bottles were shaken to prevent CaCO3 precipitation. 25 and 10 ml aliquots of media were poured respectively into each standard Petri dish (9 cm) and (5 cm). 4.3. Determination of chicory root dry weight Colonized and non-colonized chicory roots were collected from the medium by solubilising the solidified media during 15 min under agitation in one vol Tris buffer (Tris HCl 50 mM pH 7.5 EDTA 10 mM) at room temperature, and collected by filtration on a 0.5 mm sieve. Roots were rinsed with sterile water and then lyophilised during 48 h and weighed to determine their dry weight. 4.4. Determination of G. irregulare development Arbuscular mycorrhizal colonization parameters were determined after 8 weeks of culture. Extraradical hyphae length was measured using the gridline method and data were integrated in the formula of Tennant (1975). The number of spores formed was counted using the method described by Declerck et al. (2001). Chicory roots were collected as described in the Section. 4.3. One aliquot of these roots was cleared in KOH and stained with 0.05% trypan blue in lactic acid as described by Phillips and Hayman (1970) and modified by Koske and Gemma (1989) to quantify arbuscular mycorrhizal colonization using the gridline intersect method (McGonigle et al., 1990). The other aliquot of roots was frozen at 80 °C for lipids, MDA and POD activity measurements. Concerning spores’ culture, after 4 weeks of incubation, spore germination rate was determined by counting the number of germinated spores. The germinative hyphal length was measured using the same method for extraradical hyphae length, by observation under a low power microscope at 10–40 magnification.

4. Experimental

4.5. FA extraction, analysis and identification

4.1. Plant and fungal material

Before FA extraction, chicory roots were lyophilised during 48 h. The freeze-dried plant material (20 mg dry weight) was saponified with 3 ml of 6% (w/v) KOH in methanol at 85 °C for 2 h. After extraction of the unsaponifiable fraction with hexane, the saponifiable fraction was adjusted to pH 1 with HCl (6 N). FA, after addition of one volume of distilled water, were extracted (3) with three volumes of hexane and evaporated under N2. FA were methylated using 1 ml of BF3/Methanol (14%) at 70 °C for 3 min and reaction was stopped in ice (Morrison and Smith, 1964). The FA methyl esters were extracted (3) with three volumes of hexane after the addition of 1 ml of distilled water. These extracts were evaporated under N2 and transferred in chromatography vials. Final extracts were analysed with the use of a PerkinElmer Autosystem gas chromatograph (GC) equipped with a flame-ionization detector (Norwalk, CT) and a EC-1000 (Alltech) capillary column (30 m  0.53 mm i.d.) with hydrogen as carrier gas (3.6 ml min 1). The temperature program included a fast rise from 50 °C to 150 °C at 15 °C min 1 and then a rise from 150 °C to 220 °C at 5 °C min 1. FA quantification was made by using heptadecanoic acid methyl ester (C17:0) as an internal standard and by introduc-

Ri T-DNA transformed chicory roots (C. intybus L.), colonized or not by G. irregulare Blaszk., Wubet, Renker and Buscot (DAOM197198), were grown on a modified M medium (Bécard and Fortin, 1988) [solidified with 0.05% (w/v) gellan gel (phytagel, Sigma, St Louis, MO, USA)]. Cultures were inoculated from standardized root inoculum of 2-month-old monoxenic cultures of Ri T-DNA transformed chicory roots, colonized or not by G. irregulare, and using a 10-mm cork borer as described by Verdin et al. (2006). A disk of culture medium containing roots from monoxenic cultures (non-colonized or colonized chicory roots) was placed in the middle of each Petri dish containing the medium without (Control) or with the different concentrations of CaCO3. After 8 weeks of culture, roots used as inoculum were discarded and were not taken into account in the different parameters analysed. At the same time, another culture consisted only in G. irregulare spores was undertaken to carry out germination test. Spores were extracted from a 2-month-old monoxenic culture of Ri T-DNA transformed chicory roots, colonized by G. irregulare. One spore

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ing a defined amount of this compound into every sample just before running on GC. Their identification relied on the retention times of a wide range of standards. Overall, 37 different references FA were used as standards (lipids standards: fatty acid methyl ester mixtures C4–C24:1, Sigma Aldrich). 4.6. Preparation of crude cell-free extracts Frozen tissues from roots samples (100 mg) were ground and then suspended in 1 ml of phosphate buffered saline (10 mM). After centrifugation (3 min/10000 g), supernatants were divided in 250 lL-aliquots to determine MDA concentration and POD activity. 4.6.1. Determination of MDA concentration A high performance liquid chromatography MDA assay was used to evaluate the MDA production (Shirali et al., 1994). Two hundred microlitres of root aliquot were mixed with 1 ml of 0.1 N HCl and extracted twice with 3 ml of ethylacetate. The mixture was shaken for 5 min and centrifuged at 3000g for 10 min. The organic layers were collected and evaporated under the stream nitrogen. After evaporation, the extract was suspended in 100 lL of methanol. The HPLC system of Jasco PU-980 pump, was equipped with a Nucleosil column (C18, 150  4.6 mm, 5 lm particle size), a Rheodyne 7725 automated injector, a UV detector (detection wavelength = 532 nm) and a Shimadzu CR3A integrator (Vasse Industries, Lille, France). The mobile phase was a blend of 50 mM KH2PO4 and methanol 60/40 (v/v) adjusted to pH 6.8 (KOH 1 M). Tetraethoxypropane (Sigma, Saint Quentin Fallavier, France) was used as the standard, and thiobarbituric acid (TBA) as the reagent for MDA assay. One hundred microlitres of either standard solutions or methanol extracts were injected in the HPLC system and the MDA–TBA adducts were detected. 4.6.2. Determination of POD activity and total proteins concentrations POD activity was carried out in supernatants using the method described by Mitchell et al. (1994). Total proteins concentrations were determined in supernatants using the Total Protein Kit, Micro Lowry, Peterson’s Modification (Sigma–Aldrich, Saint Louis, Missouri, USA). 4.7. Statistical analysis Effects of CaCO3 concentrations on the measured parameters were tested with a PROC ANOVA procedure of SAS (9.1) version with the LSD (Least Significant Difference 0.05) means comparison option. CaCO3 effects on spores’ germination rate were evaluated with the Chi-square (p 6 0.05) test. The percentage data (mycorrhizal colonization) were arcsine transformed. Acknowledgments This work was supported by the Tunisian Ministry of High Education and Research which partially financed S. Labidi PhD thesis. We are grateful to Natacha Bourdon from UCEIV (ULCO) for her technical support. References Abdel Latef, A.A., 2011. Influence of arbuscular mycorrhizal fungi and copper on growth, accumulation of osmolyte, mineral nutrition and antioxidant enzyme activity of pepper (Capsium annuum L.). Mycorrhiza. doi: 10. 1007/s00572-0100360-0. Abdel Latef, A.A., He, C.X., 2010. Arbuscular mycorrhizal influence on growth, phytosynthetic pigments, osmotic adjustment and oxidative stress in tomato plants subjected to low temperature stress. Acta Physiol. Plant. 33, 1217–1225.

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