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Keywords:

  • sterol;
  • Glomus;
  • Daucus carota (carrot);
  • Cichorium intybus (chicory);
  • monoxenic cultures;
  • Ri T-DNA-transformed roots

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    Characteristic sterols of transformed carrot (Daucus carota) and chicory (Cichorium intybus) roots colonized by different strains of arbuscular mycorrhizal (AM) fungi were identified.
  • • 
    Sterols were extracted, analysed and identified by gas chromatography/mass spectrometry (GC-MS) from monoxenic cultures of mycorrhizal and nonmycorrhizal roots. After colonization by Glomus intraradices, Glomus proliferum and Glomus sp., carrot and chicory roots exhibited a significantly higher 24-methyl/methylene sterol content. A correlation was established between the content of the sum of 24-methyl cholesterol, 24-methylene cholesterol and 24-methyl desmosterol.
  • • 
    This study clearly established that the increment of these characteristic sterols is an appropriate indicator of colonization by AM fungi of transformed roots.
  • • 
    Metabolic origin and specificity of these sterols in mycorrhizal roots was researched. The 24-methyl/methylene sterol increase was observed only when the interaction between fungus and plant was completely established and the fungus was present inside the roots.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Root-staining methods followed by microscopic examination are traditionally used to evaluate the presence of arbuscular mycorrhizal fungi (AMF) and root colonization levels. These methods are tedious, time-consuming (Sylvia et al., 1993) and dependent on the visualization technique employed (Gange et al., 1999).

Several researchers have tried to find biochemical methods for quantifying AMF in colonized roots. The abundance of lipid in spores and vesicles of colonized roots is a potentially useful biochemical marker for evaluation of AMF in host root tissue. Several particular fatty acids have been reported as associated to AMF. They typically contain high contents of the fatty acids C16 : 1 ω5 and 18 : 1 ω7 as well as polyunsaturated 20-carbon fatty acids (Beilby, 1980; Nordby et al., 1981; Pacovsky & Fuller, 1988; Graham et al., 1995). Phospholipid fatty acid C16 : 1 ω5 has proved suitable for estimating the biomass of arbuscular mycorrhizal (AM) mycelium in soil laboratory systems (Olsson et al., 1995, 1998) and in roots (Olsson et al., 1997).

Although little attention has been paid to sterols, few reports have explored the possibility of using fungus-specific sterols as indicators for AM roots. Ergosterol, the predominant sterol of most fungi (Weete, 1989), has been proposed as a potential indicator of AM fungal biomass inside colonized roots (Frey et al., 1992; Antibus & Sinsabaugh, 1993; Fujiyoshi et al., 2000). However the estimated ergosterol content was particularly low. Moreover, ergosterol was not mentioned as either occurring in mycorrhizal roots isolated from pot cultures (Ho, 1977; Nagy et al., 1980; Grandmougin-Ferjani et al., 1995), or in transformed roots colonized by Glomus intraradices grown monoxenically (Fontaine et al., 2001b). These contradictory results suggest that the low ergosterol content detected by some authors may have originated from some mycoparasites (Jeffries & Young, 1994) or saprophytic fungal organisms. 24-methyl and 24-ethyl cholesterol were predominant sterols of spores isolated from pot cultures (Beilby & Kidby, 1980a, 1980b; Beilby, 1980; Grandmougin-Ferjani et al., 1999) and from monoxenic cultures (Declerck et al., 2000; Fontaine et al., 2001b). For these reasons ergosterol should not be considered a suitable indicator for quantifying AM fungal biomass in colonized roots. In a recent issue of New Phytologist, Olsson et al. (2003) concluded that ergosterol cannot be used as biomass indicator for glomalean fungi.

Schmitz et al. (1991) have observed a significant relative increase of 24-methyl cholesterol and 24-methylene cholesterol on mycorrhizal colonization of roots obtained from pot cultures. Our present study established that the increment of 24-methyl/methylene sterols is an appropriate indicator of AM colonization in transformed roots. We also investigated the specificity and origin of this indicator of the mycorrhizal state, using monoxenic cultures of transformed carrot (Daucus carota) and chicory (Cichorium intybus) roots. These cultures have obvious advantages over traditional systems, allowing important production of free spores, extraradical mycelium and colonized roots free of any contaminant microorganism. This aspect is important as AMF from field or pot cultures may harbour hyperparasitic fungi or spore wall-associated microorganisms (Jeffries & Young, 1994; Rousseau et al., 1996; Hijri et al., 2002). Moreover, DNA sequences obtained from AMF spores originating from pot cultures, were reported as probably being from an ascomycete fungus (Redecker et al., 1999), and two ascomycete fungi were recently isolated from AM spores (Hijri et al., 2002).

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In vitro culture of Ri T-DNA-transformed carrot roots colonized or not by G. intraradices and Glomus proliferum

Ri T-DNA-transformed carrot (Daucus carota L.), colonized or not by Glomus intraradices Schenck & Smith (DAOM 197198) or Glomus proliferum Dalpé & Declerck (MUCL 41827), were grown on medium M (Bécard & Fortin, 1988) solidified with 0.25% (w/v) gellan gel (Phytagel: Sigma, St Louis, MO, USA) at 27°C in the dark.

After 4 or 5 months of culture, colonized (typically 60–85% of colonization) and control roots were collected. The root cultures were solubilized for 1 h under agitation in 5 vol sodium citrate (10 mm, pH 6.0) (Doner & Bécard, 1991). Mycorrhizal roots were separated from extraradical fungus by filtration on a 0.5 mm sieve, then roots were washed with distilled water and frozen at −20°C.

Transformation of chicory roots by Ri T-DNA from Agrobacterium rhizogenes and in vitro mycorrhization by G. intraradices, G. proliferum and Glomus sp.

Seeds of chicory (Cichorium intybus L.) were surface-disinfected by soaking alternately in sodium hypochloride (5°C) or sterile water (five baths of 15 min). After rinsing for 1 h with sterile water, seeds were germinated on water agar (1%) at 22°C for 5 days in the dark under a photoperiodic cycle of 18 h light. Aseptic plantlets were then transferred in tubes containing a modified Heller's medium (mg l−1): NaNO3 600, MgSO4 250, NaH2PO4 125, CaCL2 75, KCl 750, AlCl3 0.03, CuSO4 0.03, MnSO4·4H2O 0.075, NiCl2·6H2O 0.01, ZnSO4·7H2O 1, H3BO3 1, KI 0.01, FeEDTA 32,5, sucrose 10 g l−1 pH 5.5. This medium was solidified with 0.25% gellan gel. Plantlets were grown for 30 days at 18°C and the light/dark period was 18/4 h.

Agrobacterium rhizogenes strain 2659 was a gift of Dr Y. Dessaux (CNRS, Orsay, France). This strain, stored in glycerol stocks at −70°C, was grown for 2 days on solid YEB medium (Van Larebeke et al., 1977) and then grown for 14–18 h at 28°C in shaken liquid YEB medium until absorbance was 0.5 at 600 nm. A 1/10 v/v dilution of this bacterial suspension was used for inoculation of plantlets. Aseptic hypocotyls and leaf limb fragments of chicory plantlets were scarified, then dipped for 2 h in the A. rhizogenes suspension and incubated for 2 weeks in the dark at 22°C on solid White's medium. After 3 weeks a few transformed roots, proliferating on wounded sections, were excised and grown into Petri dishes containing White's medium supplemented with 300 mg l−1 carbenicillin. Three subcultures were necessary to obtain transformed roots free of bacteria. Root tips were finally grown on solid M medium (Bécard & Fortin, 1988).

Transformed chicory roots were colonized by different strains of AM fungi able to grow in monoxenic conditions: G. intraradices (DAOM 197198), G. proliferum (MUCL 41827) and Glomus sp. Primary mycorrhizal colonization was achieved by placing spores of AMF with a single transformed root in the same dish, which were then grown according to the procedure previously described.

Sterol extraction, analyses and identification

Before lipid extraction, plant roots (100 mg d. wt) were lyophilized. The freeze-dried roots were extracted according to the method described by Costet-Corio & Benveniste (1988) under dark conditions. Total sterols from total lipid extract were obtained using procedures described by Grandmougin-Ferjani et al. (1999), while free sterol and their esterified forms were purified according to the method described by Fontaine et al. (2001b). Steryl acetates in the final extract were analysed by gas chromatography (GC) using a flame-ionization detector (Perkin Elmer, Autosystem, Norwalk, CT, USA) and a glass capillary column: DB5 (J&W Scientific, Folsom, CA, USA), 30 m × 0.25 mm id, H2 2 ml min−1. The temperature program included a fast rise from 60 to 270°C (30°C min−1), then a slow rise from 270 to 310°C (2°C min−1). Cholesterol (not acetylated) was used as internal standard by introducing a defined amount of cholesterol into every sample just before running it on GC. Each compound was identified by its retention time with regard to free cholesterol, and also by running on GC-MS (Varian, Walnut Creek, CA, USA) at an ionizing potential of 70 eV. Sterols were identified by their specific fragmentation pattern. Data were compared with reference compounds and those reported by Rahier & Benveniste (1989). All the experiments were done in triplicate.

Fungal sterol analyses were performed on G. intraradices grown in two-compartment Petri dishes. The mycorrhizal roots were confined in one compartment, but the fungus was allowed to grow over the central separation and into the second compartment. After 4 months extraradical mycelium and spores of G. intraradices were collected only from the fungal compartment in order to avoid plant sterol contamination. Fungal material was collected by blending the solidified medium in sodium citrate solution (10 mm) (Doner & Bécard, 1991). Extraradical fungus was collected by filtration on a 53 µm sieve and rinsed with sterile water. Quantitative analysis needed high fungal biomass (approx. 5–10 mg d. wt). Freeze-dried fungal material was extracted and analysed according to the method described by Grandmougin-Ferjani et al. (1999).

Identification and quantification procedure of 24-methyl/methylene sterols

GC did not provide a complete separation of 24-methylcholesterol, 24-methylene cholesterol and 24-methyl desmosterol, and these compounds were detected by GC-MS. These 24-methyl/24-methylene sterols were separated by reverse-phase HPLC (Waters, Milford, CA, USA) according to the method described by Fontaine et al. (2001b). Elution times for 24-methylene cholesteryl acetate and 24-methyl desmosteryl acetate were 27 and 28 min, respectively, while the elution time of Δ5 steryl acetate mixture for 24-ethylcholesterol, 24-methyl cholesterol, stigmasterol and cholesterol were 42, 36, 35 and 32 min, respectively. After separation of each compound by HPLC, the products were collected, quantified by GC and identified by GC-MS.

Culture of pathogenic strains and inoculation of carrot roots

Phytophtora megasperma and Rhizoctonia solani strains were obtained from Station de Pathologie Végétale (INRA, Nice, France) and Station de Pathologie Végétale (INRA, Rennes, France), respectively. Fungi were grown in 100 ml malt liquid media (20 g l−1 malt extract) in 250 ml flasks in a slow-shake culture. Cultures were performed at room temperature (21–24°C) for 3 days for R. solani and 8 days for P. megasperma. For inoculation of carrot roots, fungi were grown on Petri dishes containing malt medium (20 g l−1 malt extract, 15 g l−1 agar) for 8 days at 20°C, and five 1 cm disks of medium containing mycelia of pathogenic fungi were set in each root dish. Root infection by pathogen fungi was verified by staining roots using the same method as that used for determining percentage mycorrhizal infection, described below. Infected roots were grown in the dark at 20°C for 8 days, then infected root media were solubilized for 1 h under agitation in 5 vol sodium citrate (Doner & Bécard, 1991).

Microscopic determination of percentage mycorrhizal infection

The line-intersect method described by Giovannetti & Mosse (1980) was used to assess root colonization after root samples had been cleared in KOH and stained with chlorazol black E (Brundrett et al., 1994).

Analysis of lipid indicators in carrot root tips or in total roots

AM-colonized root extremities 0.5–1 cm were collected in order to evaluate the 24-methyl/methylene sterol content. Root extremities were supposed to contain no or very little intraradical fungus. Given the difficulty of obtaining enough material to perform lipid analysis, it was not possible to estimate the colonization level of these root tips. The degree of colonization was measured only by microscopic observation after staining. The presence of C 16 : 1 ω5 fatty acid in root tips was also investigated. Fatty acids from the total lipid fraction were analysed using the methods described by Declerck et al. (2000).

Chemicals

No commercial sterol standard as 24-methylene cholesterol was available. This compound was isolated from commercial pollen and identified by GC-MS (Barbier, 1970).

Statistical analysis

To compare 24-methyl/methylene sterol contents from transformed control roots, roots colonized by different strains of AM fungi, or stressed roots, anova was carried out using statgraphics release 4.0 (Manugistic Inc., Rockville, MD, USA). The method used to discriminate between the means was the Student–Neuman–Keuls multiple comparison procedure (P < 0.05).

Regression analyses were also carried out using the same statistical program to determine the most adequate model to describe the relationships between the data.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Qualitative and quantitative 24-methylene/methyl sterol content of transformed carrot roots

The free and esterified sterol composition of monoxenic Ri T-DNA-transformed carrot roots, colonized or not by G. intraradices, was established (Table 1). Typical plant sterols (Benveniste, 1986; Hartmann, 1998) were detected in control carrot roots. The main Δ5 sterols (4-demethylsterols, the major end-products in most higher plants) identified in free sterols (sterols with a β free hydroxyl group) and sterol esters (sterols esterified by fatty acids) are stigmasterol, 24-ethylcholesterol, 24-methylcholesterol, cholesterol and isofucosterol. The sterol precursors 4α-methyl and 4,4-dimethylsterols were present in only trace amounts. Two new 4-demethysterol compounds, the 24-methylene cholesterol and 24-methyl desmosterol, were detected in free sterols and sterol esters of colonized carrot roots. Under our GC experimental conditions, the 24-methylene cholesterol and 24-methyl desmosterol were not separated from the 24-methyl cholesterol. To quantify these compounds, 4-demethylsterols were separated by reverse HPLC, then quantified by GC and identified by GC-MS. 24-methylene cholesterol and 24-methyldemosterol were identified using a technique based on the mass spectra for the acetate derivatives by comparison with data obtained for reference compounds and those reported by Rahier & Benveniste (1989). The fragmentation pattern of 24-methylene cholesteryl acetate was [m/z 380 (M ± AcOH) 100, 296 (10), 255 (63), 253 (26), 213 (13)]. According to the mass spectra of acetate derivatives [m/z 380 (M ± AcOH) (36), 365 (22), 296 (M ± a-ROH) 100, 281 (45), 255 (15), 253 (50), 213 (22)], this sterol was identified as 24-methyl desmosterol.

Table 1.  Free and esterified sterol composition of mycorrhizal and nonmycorrhizal carrot (Daucus carota) roots by Glomus intraradices grown in vitro
SterolControl rootsColonized roots
Free sterolsSterol estersFree sterolsSterol esters
µg g−1%µg g−1%µg g−1%µg g−1%
  • µg g−1 of freeze-dried root material. Results are means ± SD (n = 3).

  • ‡Not detected.

  • §

    Tr, traces, amounts < 0.5% or 1 µg g−1.

  • *

    Significant difference between means for colonized and control roots (P < 0.05, t-test).

4-demethylsterols
Cholesterol  3 ± 1 2Tr§Tr
24-methyl cholesterol 182 ± 1710 49 ± 324 345 ± 25*16 49 ± 446
24-methylene cholesterol  30 ± 2 1  4 ± 0.4 4
24-methyl desmosterolTrTr  1 ± 0.1 1
Stigmasterol1375 ± 6479 78 ± 6381493 ± 49*71 20 ± 4.5*19
24-epiclerosterol  12 ± 1 1  7 ± 0.1 3  38 ± 3* 2  4 ± 0.5* 4
24-ethyl cholesterol 148 ± 23 9 45 ± 0.122 163 ± 14* 8 16 ± 2*16
Isofucosterol   1 ± 0.4Tr  5 ± 0.1 2   9 ± 2*Tr  1 ± 0.3* 1
4α-methylsterols
Obtusifoliol   2 ± 0.1Tr  2 ± 1 1   1 ± 0.2Tr  2 ± 0.1 4
24-methylene lophenol   4 ± 1Tr  2 ± 0.3 1   2 ± 0.5TrTrTr
Cycloeucalenol   4 ± 0.1Tr  7 ± 1 3   5 ± 1TrTrTr
4,4-dimethylsterols
α-amyrine   2 ± 0.5Tr  1 ± 0.5Tr   2 ± 0.5TrTrTr
β-amyrineTrTr
Lanosterol   1 ± 0.5Tr
24-methylene cycloartanol   3 ± 0.2Tr  1 ± 0.5Tr   9 ± 0.2TrTrTr
Cycloartenol   1 ± 0.5Tr  1 ± 0.4Tr   5 ± 3Tr  2 ± 1 2
EburicolTrTr
Unidentified sterols   1 ± 0.5Tr  4 ± 1 2   3 ± 0.5Tr  1 ± 0.1 1
Total sterols (µg g−1)1735 ± 108 205 ± 15 2116 ± 102 101 ± 13 

After colonization, carrot roots exhibited a higher free 4-demethylsterol content and a significant relative and quantitative increase of 24-methyl cholesterol. A significant increase in the percentage of 24-methyl cholesterol was also observed in esterified sterols.

These characteristic 24-methyl/methylene sterol compounds are good candidates for quantifying the proportion of colonized roots. A complete separation of 4-demethylsterols is laborious and based on the combination of HPLC, GC analysis and then GC-MS identification. It might be sufficient, as suggested by Schmitz et al. (1991), to determine relative percentages of the sum of 24-methyl cholesterol, 24-methylene cholesterol and 24-methyl desmosterol from total sterols. A correlation was established between the content of the sum of these compounds and percentages of carrot root colonization by G. intraradices (Fig. 1). Traditional microscopic measurements were used to quantify root colonization (Giovanetti & Mosse, 1980). Statistical analysis showed a significant exponential relationship between 24-methyl/methylene sterols and colonization measurements of carrot roots (r2 = 0.87, P < 0.05). The fatty acid 16 : 1 ω5 was used previously to estimate the AM fungal biomass (Olsson et al., 1997). Figure 2 shows the high linear correlation between this fatty acid signature and 24-methyl/methylene sterol contents. Statistical analysis shows a significant relationship between these data (r2 = 0.92, P < 0.05).

image

Figure 1. Correlation between mycorrhizal colonization of transformed carrot (Daucus carota) roots and 24-methyl/methylene sterol contents.

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image

Figure 2. Relationship between the arbuscular mycorrhizal (AM) fungal fatty acid signature (C16 : 1 ω5) and 24-methyl/methylene sterols (P < 0.05) in transformed carrot (Daucus carota) roots colonized by Glomus intraradices.

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Quantitative sterol content of G. intraradices grown in vitro

Major fungal sterols were found to be 24-methyl cholesterol and 24-ethyl cholesterol (Table 2). The other 4-demethylsterols identified were cholesterol, 24-methylcholesta-5, 22 dienol, ergosta-7, 24(241) dienol and 24-ethylidene cholesterol. Traces of 24-methylene cholesterol and 24-methyl desmosterol were observed. No ergosterol was detected. Low quantities of 4,4-dimethylsterols were also detected: α-, β-amyrine and lanosterol.

Table 2.  Sterol composition of Glomus intraradices grown in vitro
SterolTotal sterols
µg g−1%
  1. Results are means ± SD (n = 3). †µg g−1 of freeze-dried root material. ‡Tr, traces, amounts < 0.5% or 1 µg g−1. §Amounts of these metabolic intermediates were so low that they were not systematically detected.

4-demethylsterols
Cholesterol  40 ± 2 3
24-methyl cholesterol 710 ± 555
24-methylene cholesterolTr§Tr§
24-methyl desmosterolTr§Tr§
24-ethylcholesta-5,22-dien-3β-ol  20 ± 3 2
24-ethyl cholesterol 360 ± 1028
24-ethylidene cholesterol  50 ± 1 4
Ergosta-7,24 (241)-dien-3β-ol  50 ± 20 4
4,4-dimethylsterols
α-amyrine  30 ± 10 2
β-amyrineTrTr
Lanosterol  20 ± 0.5 2
Unidentified sterolsTrTr
Total sterols (µg g−1)1280 ± 51 

Because of low sterol contents, the stereochemistry at C24 of fungal sterols could not be determined. But it is well established that ergosterol and other fungal sterols have a side chain with a 24β-oriented methyl group (Nes & Venkatramesh, 1994). 24-ethylsterols from higher plants generally have a 24α configuration whereas 24-methylsterols are mixtures of both 24α and 24β epimers. In Table 1, 24-ethyl cholesterol and stigmasterol detected in carrot roots specifically correspond to 24α epimer. Mycorrhizal roots contained a mixture of 24α (plant origin) and 24β (fungal origin) epimers of 24-ethyl cholesterol not separated under our analytical conditions.

24-methyl/methylene sterol signature in transformed colonized roots

The observed increase in methyl/methylene sterol content in carrot roots colonized by G. intraradices was searched in chicory and carrot roots colonized by different species. The clone of chicory roots was then colonized with three different strains of AM fungi known to develop in monoxenic conditions: G. intraradices, G. proliferum and Glomus sp. Sterol analyses were performed on mycorrhizal and nonmycorrhizal chicory roots, and also with carrot roots colonized by G. proliferum (Fig. 3). After colonization by these different AM fungi, carrot and chicory roots were found to have a significantly higher 24-methyl/methylene sterol content. 24-methylene cholesterol and 24-methyl desmosterol were detected by GC-MS in all colonized roots.

image

Figure 3. 24-methyl/methylene sterols measured in transformed carrot (Daucus carota) and chicory (Chicorium intybus) roots colonized or not by different strains of arbuscular mycorrhizal (AM) fungus (n = 3). Data on percentage of 24-methyl/methylene sterols in carrot roots were subjected to anova using the test of Student–Newman–Keuls to identify differences caused by the mycorrhiza formation with different fungal species. In the same way, data obtained from chicory roots were subjected to the same test of multiple comparison procedure. Bars with the same letters are not significantly different (P = 0.05).

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Specificity of 24-methyl/methylene sterol content in carrot roots

To demonstrate that the methyl/methylene sterol increase was characteristic of AM colonized roots, we searched for a similar increase in roots infected by pathogenic fungi. The specificity of the 24-methyl/methylene sterol content in colonized carrot roots was investigated. Nonmycorrhizal transformed carrot roots were infected by two fungal pathogenic strains, R. solani and P. megasperma. Very high infection levels (up to 80%) were detected in roots only 1 week after inoculation. Infection by the two pathogenic fungi was controlled after 1 week by visualizing the intraradical fungi after staining with Chlorazol black E. After approx. 3 months of culture, carrot roots colonized by G. intraradices exhibited an average of 60–85% of colonization. AM fungal development is particularly slow compared to the development of pathogenic fungi, so we chose to compare the sterol content of carrot roots with similar infection or colonization levels (Fig. 4). No traces of 24-methylene cholesterol and no increase of 24-methyl/methylene sterols were detected in pathogen-infected carrot roots.

image

Figure 4. Occurrence of 24-methyl/methylene sterols in carrot (Daucus carota) infected by phytopathogenic strains or colonized by Glomus intraradices (n = 3). Bars with the same letters are not significantly different (P = 0.05).

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Metabolic origin of 24-methyl/methylene sterol increase

Under our GC conditions it was not possible to discriminate between plant and fungal sterols extracted from roots. 24-methyl cholesterol is a major end product of the sterol pathway in plant tissue (Hartmann, 1998) and represents 11% of total sterols in carrot roots. Fontaine et al. (2001b) demonstrated the ability of the AM fungus G. intraradices to synthesize 24-methylcholesterol. It is a predominant sterol in extraradical mycelium and spores of G. intraradices (Table 2). Moreover the 24-methylene cholesterol and 24-methyl desmosterol are biosynthetic precursors of 24-methyl cholesterol in plant cells (Benveniste, 1986). Traces of both metabolic intermediates were sometimes observed in the sterol composition of G. intraradices (Table 2). 24-methyl/methylene sterols are present in the two partners of the symbiosis. Table 3 shows the presence of 24-methyl/methylene sterols and fatty acid C16 : 1 ω5 in different parts of colonized carrot roots (apex or total root) harvested after 2 or 4 months of growth. We checked the presence of intraradical AM fungi by a classical staining technique, and observed that these lipid compounds were always present when the fungus was present in the root.

Table 3.  Presence of intraradical mycelium and signature lipids in apex and total mycorrhizal root of carrot (Daucus carota) (n = 3)
Age of harvested roots (months)Part of rootDegree of colonizationPresence of lipid indicators
C16 : 1 ω5 (%)24-methyl/ methylene sterols (%)
  • –, no colonization to ++, high mycorrhizal colonization.

  • ‡Tr, traces, degree of colonization was estimated after apex staining.

2Apex 012
Total root±Tr13
4Apex+ 713
Total root++1918

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This investigation clearly established that 24-methyl/methylene sterol content increases in AM roots grown in vitro. Here we present evidence that these sterols increase in monoxenic cultures of transformed roots of two different plants, carrot and chicory colonized by different strains of AM fungi: G. intraradices, G. proliferum and Glomus sp. After intense colonization carrot and chicory roots exhibited a higher relative and quantitative 24-methyl cholesterol content. In the same way Nagy et al. (1980) reported that campesterol (24-methyl cholesterol) was found at higher percentages of free sterols in colonized roots of several Citrus species, but no quantitative change of free sterols was established. Mycorrhizal roots of Zea mays were also reported to contain more campesterol than nonmycorrhizal roots (Ho, 1977). Two recent investigations (Schmitz et al., 1991; Grandmougin-Ferjani et al., 1995) reported a significant increase of 24-methyl/methylene sterols for mycorrhizal roots of diverse plants grown in pot culture. An additional compound was detected in colonized roots and was identified as 24-methylene cholesterol. This sterol was proposed as a specific indicator for VA mycorrhizas (Schmitz et al., 1991). Our study showed the presence of this sterol (traces or low amounts) and an additional 24-methyl sterol, the 24-methyl desmosterol in colonized roots.

Our study has established that the increase of 24-methyl/methylene sterols is an appropriate indicator of AM-colonized transformed roots and can be used to quantify root colonization. Several biochemical methods have been reported for the quantification of AM root colonization. The chitin content has been used by Hepper (1977) to determine the level of root fungal colonization. This chitin assay was based on the assumption that chitin content is invariant with the state of development of the fungus. Grandmaison et al. (1988) and Jabaji-Hare et al. (1990) demonstrated that this was not the case. Furthermore, this compound is also present in other soil organisms (insects, arthropods) and could lead to high background values obtained from field experiments. In most AMF, high contents of particular fatty acids such as C16 : 1 ω5, C18 : 1 ω7 and polyunsaturated 20-carbon fatty acids have been found (Beilby, 1980; Nordby et al., 1981; Pacovsky & Fuller, 1988; Graham et al., 1995). Polyunsaturated 20-carbon fatty acids are rare in fungi and are not present in significant amounts in bacteria (Lösel, 1988; Graham et al., 1995). C16 : 1 ω5 and C18 : 1 ω7 are normally not detected in fungi (Lösel, 1988; Müller et al., 1994; Jansa et al., 1999), but are present in some bacterial genera (Walker, 1969; Nichols et al., 1986). Thus these fatty acids are not AMF-specific. C16 : 1 ω5 can be used as an indicator of AMF in different experimental systems with a high sensitivity on conditions that several controls with nonmycorrhizal plants and detection of noise background of C16 : 1 ω5 in soil are performed (Olsson, 1999). The C16 : 1 ω5 is associated with two important class of lipid in AMF: neutral lipids, the most abundant lipid in AMF; and phospholipids, important membrane constituents (Jabaji-Hare, 1988; Fontaine et al., 2001a). Phospholipid fatty acid C16 : 1 ω5 was used to evaluate AMF biomass in soil and roots. The background of this fatty acid in soil mainly originated from bacteria (Olsson, 1999) and is probably nonexistent in a monoxenic culture system. Total or neutral fatty acid C16 : 1 ω5 can be used to monitor levels of energy storage in AMF in soil and in roots. No background was provided from bacteria because these organisms produce very little neutral lipid. Moreover, neutral fatty acid C16 : 1 ω5 is more sensitive than phospholipid fatty acid C16 : 1 ω5 as indicator of AM mycelia (Olsson, 1999). The AM fungal colonization of the roots was demonstrated to correlate well with the content of phospholipid and neutral lipid fatty acid C16 : 1 ω5 in roots (Olsson et al., 1997). In comparison with the other biochemical methods used to evaluate the root colonization based on chitin analysis or fatty acid signatures, our sterol method offers several advantages. First, the occurrence of 24-methylcholesterol and its precursors (24-methylenecholesterol and 24-methyldesmosterol) in fungi is not usual (Lösel, 1988; Weete, 1989). These sterols were mainly identified in primitive organisms such as Chytridiomycota and Hyphochytridiomycota (Weete, 1989), in some zygomycetes and some pathogenic higher fungi (Grandmougin-Ferjani et al., 1999; Fontaine et al., 2001b). Bacteria are assumed to contain no sterols (Ourisson & Rohmer, 1982). Our sterol method of evaluating colonization was performed on a monoxenic root system, but could be developed for roots grown in field soil with a low background of 24-methyl/methylene sterols. One other advantage is that the 24-methyl/methylene sterol indicator is specific to AM colonization. The sterol analysis of AM-colonized roots could reveal the presence of pathogens or saprophytic fungi by the detection of ergosterol, the most common fungal sterol (Weete, 1989). As for the C16 : 1 ω5 fatty acid, 24-methyl/methylene sterols could be analysed with high sensitivity. The same lipid extraction can allow us to obtain the fatty acid and sterol content. So a combination of both lipid indicators can easily be used for routine estimations. Another advantage is that 24-methyl/methylene sterols such as the phospholipid fatty acid C16 : 1 ω5 could be used as biomass indicators of AMF (Olsson, 1999). Our results clearly indicate an increase of these free sterols, known to be membrane compounds. Quantitative sterol data obtained from colonized roots and extraradical mycelium from the same Petri dishes should give us, in the future, an opportunity to calculate the biomass of intraradical mycelium in order to evaluate the intra- and extraradical distribution of AMF.

What is the metabolic origin of the 24-methyl/methylene sterol increase in AM roots? In higher plants 24-methylcholesterol is a major sterol compound and 24-methylsterols are mixture of both 24α and 24β epimers. 24-methylene cholesterol and 24-methyl desmosterol are metabolic precursors of 24-methyl cholesterol in plant sterol pathways (Benveniste, 1986). Occurrence of 24-alkyl sterols in fungi is relatively unusual. Ergosterol is by far the predominant sterol in most fungi (Weete, 1989), and fungal sterols usually present a C24 β configuration (Nes & Venkatramesh, 1994). AM fungi were found to contain 24 alkyl sterols as main sterols (Grandmougin-Ferjani et al., 1999), and were shown to be able to synthesize these compounds (Fontaine et al., 2001b). Because of the low amounts of sterols extracted from G. intraradices, the stereochemistry at C24 could not be determinate. Thus 24-methyl/methylene sterols are present in the two partners of the symbiosis. The correlation between the AM fungal fatty acid C16 : 1 ω5 and 24-methyl/methylene sterol content demonstrated that the increase of 24-methyl/methylene sterols is probably caused by fungal production. But we cannot strictly exclude a metabolic response of roots to colonization. The proof would be provided by an increase of the C24 β configuration of 24-methyl sterols in colonized roots by NMR spectroscopy analysis. All our studies were performed on whole AM root tissues. Arbuscules lie deep within root tissue and are difficult to extract. Our attempts to isolate large amounts of purified arbuscules and intraradical hyphae (Saito, 1995) in order to analyse sterol composition failed. In an alternative approach we tried to find the origin of the increase of 24-methyl/methylene sterols in AM roots by analysing the apex of AM roots containing, or not containing, the fungus. 24-methyl/methylene sterol increase was observed only when the interaction between both organisms was fully established and the intraradical fungus was present in the apex of roots. This increase mainly concerns the 24-methyl cholesterol (90%) and 9% of stigmasterol. These points strengthen the idea that the increased content of 24-methyl/methylene sterols could have a fungal origin.

Whatever the origin of the 24-methyl/methylene sterols, the increase of the free 4-demethylsterol amount and the enhancement of 24-methyl cholesterol metabolic intermediates in AM roots lead to the hypothesis that sterol metabolism is particularly stimulated. Fungal hyphae penetrate cortical cells and differentiate to form highly branched hyphae termed arbuscules. The plant plasma membrane extends to surround the arbuscules and form a peri-arbuscular membrane. The formation of arbuscules and peri-arbuscular membrane required a massive membrane synthesis, and therefore active synthesis of free sterols, one of the major compounds present in plant or fungus plasma membranes (Hartmann & Benveniste, 1987; Weete, 1989). Little is known about the lipid composition of these specialized membranes, consistent with the difficulties in isolating them today. However, high ATPase activity, which is modulated by sterols (Grandmougin-Ferjani et al., 1997), was detected on the peri-arbuscular membrane and in intercellular hyphae (Harisson, 1999). In this type of interaction colonized roots are subject to intense metabolic exchanges. Pfeffer et al. (1999) demonstrated that triacylglycerols were synthesized by the intraradical fungus and then moved to external mycelium, suggesting high lipid metabolic activities within the AM roots.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We wish to thank Pr Guillaume Bécard (Université de Toulouse, France) for providing us Ri T-DNA-transformed carrot roots colonized by G. intraradices, and Dr Stéphane Declerck (Université catholique de Louvain, Belgique) for providing us with monoxenic strains of G. proliferum and Glomus sp. Financial support of this work, provided by the région Nord Pas-de-Calais, is gratefully acknowledged.

References

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  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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