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. 1998 Jul;117(3):939–948. doi: 10.1104/pp.117.3.939

Eucalypt NADP-Dependent Isocitrate Dehydrogenase1

cDNA Cloning and Expression in Ectomycorrhizae

Vincent Boiffin 1,2,2, Michael Hodges 2, Susana Gálvez 2, Raffaella Balestrini 3, Paola Bonfante 3, Pierre Gadal 2, Francis Martin 1,*
PMCID: PMC34948  PMID: 9662536

Abstract

NADP-dependent isocitrate dehydrogenase (NADP-ICDH) activity is increased in roots of Eucalyptus globulus subsp. bicostata ex Maiden Kirkp. during colonization by the ectomycorrhizal fungus Pisolithus tinctorius Coker and Couch. To investigate the regulation of the enzyme expression, a cDNA (EgIcdh) encoding the NADP-ICDH was isolated from a cDNA library of E. globulus-P. tinctorius ectomycorrhizae. The putative polypeptide sequence of EgIcdh showed a high amino acid similarity with plant NADP-ICDHs. Because the deduced EgICDH protein lacks an amino-terminal targeting sequence and shows highest similarity to plant cytosolic ICDHs, it probably represents a cytoplasmic isoform. RNA analysis showed that the steady-state level of EgIcdh transcripts was enhanced nearly 2-fold in ectomycorrhizal roots compared with nonmycorrhizal roots. Increased accumulation of NADP-ICDH transcripts occurred as early as 2 d after contact and likely led to the observed increased enzyme activity. Indirect immunofluorescence microscopy indicated that NADP-ICDH was preferentially accumulated in the epidermis and stele parenchyma of nonmycorrhizal and ectomycorrhizal lateral roots. The putative role of cytosolic NADP-ICDH in ectomycorrhizae is discussed.

Ectomycorrhizae are widespread symbiotic associations involving soil fungi and tree roots (Smith and Read, 1997). Symbiosis provides several benefits to both the host plant and the fungal associate. The prospecting and absorbing activities of the extraradical hyphae are committed to facilitating the uptake of soil organic compounds and minerals and responding to the metabolic needs of the plant. However, the fungal hyphae within the root are protected from competition with other soil microbes and, therefore, function as a preferential user of plant photoassimilates. In both symbionts the development of ectomycorrhizae involves the differentiation of structurally specialized tissues with hyphae aggregation and dramatic alterations of root morphogenesis (Peterson and Bonfante, 1994).

There is now considerable evidence that ectomycorrhiza formation and function alter both fungal and plant gene expression, giving rise to novel protein patterns and a highly coordinated metabolic interplay (Martin et al., 1987; Martin and Botton, 1993; Hampp and Schaeffer, 1995; Martin et al., 1997). Ectomycorrhiza development modifies the biosynthesis and distribution of several N- and C-assimilating enzymes, and the nature of these changes depends on the plant and fungal associates (Martin and Botton, 1993; Hampp and Schaeffer, 1995). These changes affect enzymes of the N-assimilation pathways, such as NADP-dependent glutamate dehydrogenase (Martin and Botton, 1993), glycolysis, and the pentose phosphate pathway (Bilger et al., 1989; Schaeffer et al., 1996). As a consequence, the amino acid and carbohydrate contents of mycorrhizal roots are drastically modified (Rieger et al., 1992; Martin and Botton, 1993; Turnbull et al., 1995; Ek, 1997). Knowledge of the regulation of the fungal and root biochemical pathways and comparison of these with those operating in ectomycorrhizae might help in understanding how the symbiosis metabolism is regulated.

In ectomycorrhizal trees, primary N assimilation takes place in roots and their fungal associates (Finlay et al., 1988; Turnbull et al., 1995). The GS/GOGAT cycle is the major N-assimilatory pathway in beech and eucalypt (Eucalyptus globulus) ectomycorrhizae, whereas the NADP-dependent glutamate dehydrogenase/GS pathway is the main assimilatory pathway in spruce-Hebeloma sp. ectomycorrhizae (Martin and Botton, 1993). Irrespective of the pathway used to assimilate inorganic N, accumulation of Gln takes place in fungal and plant cells (Martin et al., 1986; Finlay et al., 1988; Turnbull et al., 1995). The high rate of Gln synthesis requires a continuous supply of 2-OG to be used as C skeletons (up to 30% of the assimilated Glc) (Martin et al., 1986, 1988; F. Martin, V. Boiffin, and P. Pfeffer, unpublished data). Although the synthesis of 2-OG is a major source of C for amino acids in ectomycorrhizal roots, little is known about its regulation in symbiotic tissues.

NADP-ICDH (EC 1.1.1.42) catalyzes the conversion of isocitrate to 2-OG and is mainly present in the cytosol, but mitochondrial, peroxisomal, and plastidial isoenzymes have also been described in higher plants (Gálvez and Gadal, 1995). Its activity is likely to regulate the C flux allocated to N-assimilation pathways (Fieuw et al., 1994; Gallardo et al., 1995; Gálvez and Gadal, 1995). In mitochondria isolated from spinach leaves, the oxidation of malate mainly leads to the export of citrate (Hanning and Heldt, 1993), and it has been suggested that it is converted via cytosolic aconitase and NADP-ICDH to yield the 2-OG necessary for N assimilation (Chen and Gadal, 1990).

We report the isolation of a cDNA clone encoding a cytosolic NADP-ICDH from eucalypt. In addition, we describe NADP-ICDH expression patterns and activities in roots colonized by the ectomycorrhizal Pisolithus tinctorius and discuss the role of cytosolic NADP-ICDH in symbiotic tissues.

MATERIALS AND METHODS

Biological Material and in Vitro Synthesis of Ectomycorrhizae

Seeds of Eucalyptus globulus subsp. bicostata Maid et al. Kirkp. (Kylisa Seeds Co., Weston Creek, Australia) were sterilized with 20% (v/v) NaOCl for 20 min, rinsed with four changes of sterile water, and plated onto low-sugar Pachlewski medium (2.7 mm [NH4]2C4H4O6, 7.3 mm KH2PO4, 2.0 mm MgSO4, 5 mm Glc, 2.9 μm thiamine hydrochloride, and 1 mL of a trace-element stock solution [Kanieltra Hydro Azote Co., France]) in 2.0% (w/v) agar (Hilbert et al., 1991). Isolate 441 of the gasteromycete Pisolithus tinctorius Coker and Couch was also grown on low-sugar Pachlewski medium in 2.0% (w/v) agar. Seven-day-old eucalypt seedlings, with primary roots 1 to 1.5 cm in length, were laid onto the edge of 21-d-old fungal mats to form ectomycorrhizae, and left for 2 to 7 d in a controlled-environment growth chamber with 16 h of light (25°C, 150 mmol m−2 s−1) and 8 h of dark. Petri dishes of free-living mycelium and nonmycorrhizal control seedlings were grown under the same conditions. In some investigations, ectomycorrhiza formation was carried out according to the method of Burgess et al. (1996), with identical results.

Nonmycorrhizal and ectomycorrhizal seedlings, together with the edges of fungal mats, were then sampled, fixed in liquid N2, and stored at −80°C. The amount of fungal material in infected roots was assessed by measuring the concentration of the fungal-specific ergosterol in ectomycorrhizae (Martin et al., 1990). To determine the proportion of fungal biomass in the inoculated roots, a conversion factor was calculated using the ergosterol concentration per milligram fresh weight of the mycelium sampled in the edges of fungal mats. Under our experimental conditions, 1.9 ± 0.3 μg of ergosterol (n = 6) within a root corresponds to 1 mg fresh weight of mycelium. Fungal material in inoculated roots increased linearly after contact, reaching a maximum (25%–30% of mycorrhizal root fresh weight) at the time of dense fungal sheath development (4–7 DAC).

Extraction and Enzyme Assay of the NADP-ICDH

Approximately 0.5 g fresh weight of roots from mycorrhizal and nonmycorrhizal eucalypt seedlings and free-living P. tinctorius were used for each protein extraction. After grinding in liquid N2 with 10% (weight/fresh weight) polyvinylpolypyrrolidone, soluble proteins were extracted as described by Gálvez et al. (1994), except that we added 20% (w/v) glycerol, 1% (w/v) PEG-6000, 2 mm sodium citrate, 2 mm MgCl2, 42 mm β-mercaptoethanol, 1 mm PMSF, 50 μm pepstatin A, and 1.1 mm bestatin. The homogenates were centrifuged twice at 16,000g for 20 min at 4°C. NADP-ICDH activity was measured spectrophotometrically (model DU70, Beckman) by following the reduction of NADP at 340 nm (30°C), as described by Gálvez et al. (1994). The protein content was determined by the Bradford method (Bradford, 1976) using the Bio-Rad protein assay with BSA as a standard, as described by the manufacturer. The extracts were stored at −80°C for further analysis.

Immunochemical Assays

Proteins were concentrated by precipitation in methanol containing 0.1 m NaC2H3O2. Western-blot analysis of NADP-ICDH was carried out using a RTC NADP-ICDH antiserum (Gálvez et al., 1995) at 1:1,000 (v/v) according to the method of Stepien and Martin (1992), except that 5% (w/v) dry milk and a 1:6,000 (v/v) solution of the secondary goat anti-rabbit IgG, conjugated with phosphatase alkaline (Sigma), were used. For nonmycorrhizal roots, 20 μg of protein was loaded. To take into account the increasing amounts of mycelium, and therefore fungal proteins, in mycorrhizal roots (approximately 70% of total proteins in 7-d-old ectomycorrhizae) (Hilbert et al., 1991; Burgess et al., 1995), 60 μg of ectomycorrhizal proteins was loaded in the mycorrhiza lane for samples collected at 7 DAC. Western blots were scanned in 256-gray-scale mode using a desktop scanner (model VistaS8 UMAX scanner, Vista Scientific, Ivyland, PA). The image files were then analyzed using the Image software (version 1.59) from the National Institutes of Health (Bethesda, MD). The absorbance values were used to estimate the relative protein concentrations. For immunotitration of the root NADP-ICDH activity, extracts containing 130 pkat of NADP-ICDH were incubated overnight at 4°C in 1 mL of 0.05 m borate buffer, pH 8.1, containing 0.16 m NaCl and increasing concentrations of RTC NADP-ICDH antiserum. The residual activity of NADP-ICDH was measured in each supernatant after centrifugation of immunoprecipitates at 16,000g for 20 min at 4°C. All experiments were carried out in triplicate.

Tissue Localization of NADP-ICDH by Indirect Immunofluorescence Microscopy

Twelve sections obtained from six different roots collected on two different sets of nonmycorrhizal and 7-d-old ectomycorrhizal seedlings were fixed in 3% (w/v) paraformaldehyde and 0.1% (v/v) glutaraldehyde in PEM buffer (50 mm Pipes-Na2, 5 mm EGTA, and 5 mm MgSO4, pH 7.0) for 1 h at room temperature. The samples were washed three times in PEM buffer for 15 min, then twice in 0.2 m Na2HPO4 buffer, pH 6.9, with 0.1 m NaBH4 for 10 min, and, finally, three times in Na2HPO4 buffer for 15 min. The fixed tissues were embedded in 8% (w/v) low-melting-point agarose. Thin sections (100 μm) were prepared using a vibratome (Balzers, Bucks, UK). The overnight incubation was carried out at 4°C in a RTC NADP-ICDH antiserum (Gálvez et al., 1995) diluted 1:1000 (v/v) in Na2HPO4 buffer. Control sections were incubated overnight at 4°C in Na2HPO4 buffer lacking antibodies and used to detect the autofluorescence. Sections were then washed three times for 15 min in Na2HPO4 buffer and incubated at room temperature in the dark for 3 h with a goat anti-rabbit IgG conjugated with fluorescein isothiocyanate (Sigma-Aldrich) diluted 1:80 (v/v) in Na2HPO4 buffer. The sections were washed as before and mounted on slides in Slow Fade medium (Molecular Probes, Eugene, OR). The eucalypt NADP-ICDH location was then visualized using a scanning microscope (Optiphot-2 View Scan DVC-250, Nikon) at 494 nm.

cDNA Cloning and Sequencing

A cDNA λ-ZAPII library of E. globulus-P. tinctorius 441 ectomycorrhizae (Tagu et al., 1993) was screened by using the cDNA of a tobacco cytosolic NADP-ICDH (Gálvez et al., 1996). One positive clone, pEgIcdh1, corresponding to a putative truncated NADP-ICDH, was selected. Screening, cloning, and sequencing of this cDNA clone were carried out as described by Gálvez et al. (1996). The RACE technique was used to construct the complete cDNA of EgICDH. The 5′ RACE reaction (100 μL) was initiated using 1 μL of a 5′-enriched cDNA library of E. globulusP. tinctorius 441 ectomycorrhizae (C. Voiblet and F. Martin, unpublished results) as a template for the strand synthesis in the 5′ direction, and the λ-gt11 forward-sequencing primer and idh1 (5′-TCCTTTCTGATGGACCCG-3′) (see Fig. 3) primers.

Figure 3.

Figure 3

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DNA sequence (GenBank accession no. U80912) and deduced amino acid sequence of the full-length eucalypt NADP-ICDH cDNA. The residues marked # and ° correspond to the putative binding sites of l-isocitrate-Mg2+ and NADP+ found in Escherichia coli NADP-ICDH and also present in plant NADP-ICDH. The residues marked * are Trp at the NADP active site that bind adenine (Sankaran et al., 1996). The location of PCR primers idh1 and idh2, used to clone the 5′ cDNA sequence by RACE, are underlined by arrows. The positions of primers idh3R and idh3F, used to amplify the 3′-end-EgIcdh probe, are underlined by discontinuous arrows. The box represents the putative polyadenylation site.

The PCR conditions were: denaturation at 93°C for 1 min, annealing at 55°C for 1.5 min, synthesis at 72°C for 2 min for 40 cycles, and elongation at 72°C for 15 min. PCR products were then diluted 10 times and amplified with the primer idh2 (5′-CATACCAGATTCCAGCAGCC-3′) (see Fig. 3) and the λ-gt11 forward-sequencing primer. The single PCR product obtained was purified and sequenced with a dideoxynucleotide sequencing kit (Prism with Taq FS, Perkin-Elmer/Applied Biosystems) and a DNA sequencer (model ABI373S, Perkin-Elmer/Applied Biosystems). Sequences were edited using Sequencher (Gene Codes, Ann Arbor, MI) and SeqApp (version 1.9, D. Gilbert, anonymous ftp to iubio.bio.indiana.edu). A search for sequence homologs in the National Center for Biotechnology Information databases (March 19, 1998) was carried out using a basic local alignment search tool and worldwide web network services (Altschul et al., 1997). Sequence alignments were carried out using SeqApp/Clustal W.

Isolation of Genomic DNA and Southern-Blot Hybridization

Fungal genomic DNA was extracted as described by Carnero Diaz et al. (1996), except that 10% (weight/fresh weight) polyvinylpolypyrrolidone was added in the extraction buffer for mycelial samples. Plant genomic DNA was extracted from cotyledons using the Floraclean kit (Bio 101, Vista, CA) following the manufacturer's instructions, except that two modifications were included to improve the removal of chlorophylls and phenolic compounds. A phenol/chloroform extraction was performed before the ethanolic precipitation, and DNA was precipitated with 7% (w/v) PEG-8000 and 0.4 m NaCl. The fungal and plant DNAs were digested by HindIII and BamHI. Digested genomic DNA was transferred to a nylon membrane as described by Carnero Diaz et al. (1996). To specifically detect the plant NADP-ICDH RNA and DNA, a 3′-end-specific probe (3′-end-EgIcdh) was amplified from EgIcdh with the following primers: idh3R: 5′-CCAAGTTGGATAACAATGCC-3′ and idh3F: 5′-AGGCGGAAGTGCTTTCCCC-3′ (see Fig. 3). The [α-32P]dCTP labeling of this probe was carried out as described by Carnero Diaz et al. (1996). The prehybridization of the filter was performed at 65°C for 4 h in 0.25 m Na2HPO4, 7% (w/v) SDS, 1 mm EDTA, 6× Denhardt's reagent, and 200 μg mL−1 denatured, fragmented salmon-sperm DNA. After an overnight hybridization, the filter was rinsed twice at 65°C in 0.4 m Na2PO4, 1% (w/v) SDS, and 1 mm EDTA for 20 min.

Isolation of Total RNA and RNA Hybridization

Total RNA was extracted from 500 mg of nonmycorrhizal roots, from 100 mg of free-living fungus, and from 500 mg of 2-, 4-, and 7-DAC ectomycorrhizae. After grinding in liquid N2, total RNA was extracted in the following buffer: 100 mm Tris-HCl, pH 8.4, 4% (v/v) Sarkosyl (BDH, Poole, UK), and 10 mm EDTA. After centrifugation (20,000g for 20 min), 5 g of CsCl was added to 5 mL of supernatant. Total RNA was pelleted by ultracentrifugation at 65,000g for 16 h at 20°C in a rotor (model TFT 70.38, Kontron, Zurich, Switzerland) through a CsCl cushion (0.995 g mL−1 Tris-EDTA buffer). Nonmycorrhizal roots, free-living mycelium, and ectomycorrhizae contained 0.26 ± 0.07 (n = 5), 1.5 ± 0.2 (n = 4), and 1.4 ± 0.3 (n = 3) mg total RNA g−1 fresh weight, respectively. Based on the total RNA-to-ergosterol ratio of free-living mycelium and the ergosterol content of ectomycorrhizae, fungal total RNA was estimated to be 70% of the total RNA in 4-d-old ectomycorrhizae (Nehls and Martin, 1995).

Total RNA was fractionated by gel electrophoresis under denaturing conditions and blotted to nylon membranes (16 h, 20× SSPE) (Sambrook et al., 1989). The radioactive plant-specific 3′-end-EgIcdh probe was prepared with the Ready-To-Go kit (Pharmacia) according to the manufacturer's instructions. RNA gel blots were hybridized with the 3′-end-EgIcdh probe as described by Ruiz-Avila et al. (1991), except that the last washing step was done for 20 min in 1× SSPE, 0.1% (w/v) SDS (61°C). After being stripped, all RNA blots were hybridized with the 5.8 S rDNA clone (GenBank accession no. U66625) (Carnero Diaz et al., 1997) of E. globulus to confirm the presence of undegraded RNA in each lane and to standardize the relative level of plant transcripts in ectomycorrhizal tissues. For nonmycorrhizal roots of 7-, 9-, 11-, and 14-d-old seedlings, 20 μg of total RNA was loaded.

To take into account the increasing amounts of mycelium, and thus fungal transcripts, in mycorrhizal roots (Carnero Diaz et al., 1997), 30, 40, and 60 μg of total ectomycorrhizal RNA was loaded in the mycorrhiza lanes (see Fig. 6A) for ectomycorrhizae collected at 2, 4, and 7 DAC. Hybridization of the RNA blots using the 5.8 S rDNA probe confirmed that equivalent amounts of plant RNAs were loaded in the plant and mycorrhiza lanes (Fig. 6B). RNA blots were exposed to Kodak X-Omat XAR-5 film for various times according to the intensity of the signal. The autoradiograms were scanned in 256-gray-scale mode using a desktop scanner. The image files were then analyzed using the Image software (version 1.59, National Institutes of Health). The absorbance values were used to estimate the relative concentrations of transcripts. All experiments were carried out in triplicate.

Figure 6.

Figure 6

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Steady-state level of EgICDH transcripts in nonmycorrhizal eucalypt roots (R), ectomycorrhizae (M2, M4, and M7, corresponding to 2, 4, and 7 DAC), and free-living P. tinctorius 441 (F) determined by RNA analysis. A, The filter was hybridized with the 32P-labeled 3′-end-EgIcdh probe. B, The same filter was rehybridized with the plant-specific 5.8 S rDNA probe. C, Ratio of EgIcdh:plant 5.8 S rDNA signal intensity determined in nonmycorrhizal and ectomycorrhizal roots during the time-course experiment. Shown are ectomycorrhizae (black bars) and control nonmycorrhizal roots (white bars) at 2, 4, and 7 DAC. Letters indicate significantly different (P < 0.01) values based on the parametric Scheffé test from the analysis of variance procedure from three independent replicates.

RESULTS

Root NADP-ICDH Activity in Ectomycorrhizae

The NADP-ICDH activity of nonmycorrhizal roots of E. globulus, 7-d-old ectomycorrhizae of E. globulus-P. tinctorius, and free-living P. tinctorius was 4.0 ± 1.1, 7.5 ± 2.1, and 3.8 ± 0.8 nkat g−1 fresh weight (n = 6) (Table I), respectively. In ectomycorrhizae, the total NADP-ICDH activity increased nearly 2-fold in comparison with nonmycorrhizal roots and free-living mycelium (P < 0.01, Student's t test). The plant NADP-ICDH was detected in root (Fig. 1A, lane R) and 7-DAC ectomycorrhizal (Fig. 1A, lane M) extracts by western blotting using an antiserum raised against the RTC NADP-ICDH (Gálvez et al., 1995). This antiserum did not cross-react with the fungal NADP-ICDH (Fig. 1A, lane F). The molecular masses of the eucalypt and the truncated RTC NADP-ICDH polypeptides were 41.7 and 40.8 kD, respectively. In ectomycorrhizae, the amount of NADP-ICDH, measured by densitometry of western blots, increased nearly 2-fold in comparison with that of nonmycorrhizal roots.

Table I.

NADP-ICDH activity of the root in 7-DAC ectomycorrhizae

Portion Root Fresh Wt Plant NADP-ICDH Activity Root-Related Activity in Mycorrhiza
% of total % nkat g−1 fresh wt
Root 100 100 4.0  ± 1.1
Mycorrhiza 70 82 8.8  ± 2.4
Fungus 0 0 0
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The percentage of plant fresh weight in ectomycorrhizae was obtained by subtracting the fungal fresh weight (estimated by the ergosterol assay; Martin et al., 1990) from the total ectomycorrhiza fresh weight. The percentage of root NADP-ICDH activity in ectomycorrhizae was estimated by immunotitration (Fig. 1B). The NADP-ICDH activity of plant cells in ectomycorrhiza was thus calculated by using these two factors (7.5 nkat g−1 fresh weight × 0.82 × 100/70).

Figure 1.

Figure 1

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Immunochemical assays of the root NADP-ICDH. A, Detection of the plant NADP-ICDH by western-blot analysis using an antiserum raised against RTC NADP-ICDH. Lane R, Soluble proteins were extracted from nonmycorrhizal eucalypt roots (20 μg of proteins was loaded); lane M, 7-d-old ectomycorrhizae (60 μg of proteins [20 μg of plant proteins, see Methods] was loaded); lane F, free-living P. tinctorius (20 μg of proteins was loaded); and lane T, purified truncated (the first 36 residues are lacking at the N terminus) RTC NADP-ICDH used as a positive control. B, Proportion of root NADP-ICDH activity in the ectomycorrhiza protein extract assayed using an anticatalytic immunoprecipitation with RTC NADP-ICDH (1:100) antiserum. These experiments were performed with fungal (⋄), ectomycorrhizal (•), and root (□) protein extracts. Letters indicate significantly different (P < 0.05) values based on the parametric Scheffé test from the analysis of variance procedure from three independent replicates.

To quantify the proportion of root NADP-ICDH activity in ectomycorrhizal tissues, an anticatalytic immunoprecipitation was performed on protein extracts with the RTC NADP-ICDH antibody (Fig. 1B). The root activity of the NADP-ICDH was completely inhibited in the presence of 100 μL of immunoserum, whereas the activity was not affected in the fungal extract. The root activity of the NADP-ICDH represented the major part (82%, i.e. 6.15 nkat g−1 fresh weight) of the total ectomycorrhizal activity, although a residual enzyme activity persisted in the 7-DAC ectomycorrhizal extract. This activity probably corresponded to the activity of the hyphae present in the symbiotic tissues and not to a novel, mycorrhiza-specific root NADP-ICDH isoform not recognized by the antibodies.

In 7-DAC ectomycorrhizae of E. globulus-P. tinctorius, plant tissues represented about 70% of the fresh weight of the symbiotic organ (Hilbert et al., 1991). Taking into account the dilution factor (0.70) of root biomass by fungal material and the proportion of root NADP-ICDH activity (82%) in ectomycorrhiza, the plant enzyme activity was about 8.8 nkat g−1 fresh weight of root cells in ectomycorrhizae (Table I). The host enzyme was therefore stimulated nearly 2-fold in the mycorrhizal roots compared with the nonmycorrhizal control roots. The up-regulation of the NADP-ICDH specific activity (in nanokatals per gram of protein) was probably higher, because the plant protein content in ectomycorrhizae was dramatically decreased during ectomycorrhiza development, as shown previously by two-dimensional PAGE (Hilbert et al., 1991; Burgess et al., 1996). In 7-DAC ectomycorrhizae, plant proteins and transcripts represent less than 30% of total ectomycorrhizal proteins and transcripts as a result of the formation of the ectomycorrhizal sheath and Hartig net (Laurent, 1995; Carnero Diaz et al., 1997).

Immunolocalization of the Root NADP-ICDH

The tissue and subcellular localization of eucalypt root NADP-ICDH was examined using the antiserum raised against the RTC NADP-ICDH (Gálvez et al., 1995). By using indirect immunofluorescence microscopy, the polypeptide was found in most root tissues, but it was preferentially accumulated in the vascular and epidermal tissues of the root (Fig. 2). In the parenchyma cells of the vascular tissues, the cytosol was strongly labeled (Fig. 2C). In the cortical cells, the fluorescence was found on the thin cytoplasmic layer between the plasma membrane and the large vacuole (Fig. 2B). In root apices, the fluorescein isothiocyanate-labeled NADP-ICDH was also present in epidermal and vascular tissues, but was absent in the quiescent meristematic center (Fig. 2A). The tissue localization of the NADP-ICDH was identical in nonmycorrhizal and ectomycorrhizal roots (data not shown), indicating that mycorrhiza development did not alter the compartmentation of this enzyme. No green fluorescence was observed in control root sections treated without the primary antibody. A nonspecific yellow-orange autofluorescence was observed (Fig. 2D).

Figure 2.

Figure 2

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Immunocytochemical detection of NADP-ICDH in eucalypt roots by fluorescent detection. A, Longitudinal section of a lateral root tip. The labeling is present in the cytoplasm of root-cap cells and differentiating cells. Note the absence of labeling in a zone corresponding to the quiescent center. Magnification, ×750. B, Cross-section of a lateral root showing outer cell layers of the cortex and the epidermis with an intense labeling in the epidermal cell cytosol. Magnification, ×750. C, Longitudinal sections of a lateral root showing an intense labeling in stele parenchyma cells. Magnification, ×770. D, Control cross-sections of a lateral root incubated in Na2HPO4 buffer lacking antibodies. The orange signal is caused by the autofluorescence of the cell walls. Magnification, ×740. Bars = 10 μm. c, Collenchyma tissue; e, epidermis; q, quiescent center; and v, vascular tissue.

Cloning and Characterization of a Eucalypt ICDH cDNA

A cDNA library of E. globulus-P. tinctorius ectomycorrhizae (Tagu et al., 1993) was screened by using a cDNA sequence encoding the cytosolic NADP-ICDH of tobacco (Gálvez et al., 1996) as a heterologous probe. About 300,000 phages were initially screened and only one positive clone was obtained. The deduced amino acid sequence (220 residues) of this 894-bp cDNA clone, pEgIcdh1, shared a high homology with the C-terminal region of plant NADP-ICDHs. The nucleotide sequence of pEgIcdh1 provided the necessary information to synthesize two primers, idh1 and idh2 (Fig. 3), to amplify the sequence 5′ of pEgIcdh1 from the ectomycorrhiza cDNA library. An amplification product of 1.0 kb was sequenced and found to encode the N-terminal EgICDH amino acids. The nucleotide information from pEgIcdh1 and the amplification product was used to synthesize the full-length cDNA, EgIcdh (GenBank accession no. U80912).

The size of the full-length cDNA sequence represented 1645 bp, with a 5′-untranslated region of 159 nucleotides, an open reading frame of 1248 nucleotides, and a 3′ noncoding region of 237 nucleotides (Fig. 3). The deduced amino acid sequence possessed 416 residues and the molecular mass of the putative polypeptide was 46.7 kD, with a calculated pI of 6.5. The alignment of the deduced amino acid sequence with NADP-ICDH sequences from other eukaryotes (not shown) revealed the highly conserved residues involved in the binding site of l-isocitrate-Mg2+ and NADP (as initially shown in E. coli NADP-ICDH) (Fig. 3). The Trp residues of the porcine NADP-ICDH identified to be involved in adenine fixation of the NADP active site (Sankaran et al., 1996) were also present (Fig. 3). The presence of these residues strongly suggested that EgIcdh encoded NADP-ICDH.

The phylogenic tree (Fig. 4) obtained with the PAUP program (Swofford, 1993) confirmed that the putative protein encoded by EgIcdh belonged to the family of the plant NADP-ICDHs. The eucalypt NADP-ICDH shared 92% identity with the cytosolic tobacco NADP-ICDH (GenBank accession no. P50218) (Gálvez et al., 1991). Because the EgICDH protein lacks an N-terminal targeting sequence and has the highest similarity to the cytoplasmic isoform of tobacco, it probably represents a cytoplasmic isoform.

Figure 4.

Figure 4

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Phylogeny of the deduced coding sequence of NADP-ICDH. The eucalypt NADP-ICDH (EgIcdh) was compared with sequences representative of plant, fungal, and bacterial NADP-ICDHs: tobacco (NtIcdh; Gálvez et al., 1996), potato (StIcdh; Fieuw et al., 1994), soybean (GmIcdh; GenBank accession no. L12157), alfalfa (MsIcdh; GenBank accession no. M93672), Saccharomyces cerevisiae (SmIcdh, the mitochondrial isoform [SwissProt accession no. P21954] and ScIcdh, the cytosolic isoform [SwissProt accession no. P41939]), Aspergillus niger (AnIcdh; DNA Data Bank of Japan accession no. AB000261), and E. coli (EcIcdh; GenBank accession no. AE000123). The cladogram was constructed using the multiple-alignment program Clustal W and the phylogeny program PAUP (Swofford, 1993). The numerical values shown along the stem of each supported clade represent the length of the branch, whereas numerical values in parentheses are those deriving from 1000 replicates of heuristic parsimony bootstrap analysis. Sequences of signal peptides of mitochondrial and chloroplastic ICDH were deleted before alignment.

Southern-blot analysis was performed at high stringency, both to confirm the plant origin of the EgIcdh cDNA and to estimate the total copy number of the EgIcdh gene. Restricted genomic DNA revealed a single plant DNA band of 4.8 kb (Fig. 5, lane 1) or 5.1 kb (Fig. 5, lane 2) in BamHI and HindIII digests, respectively. This indicated that the EgIcdh cDNA was encoded by a single-copy gene in the eucalypt genome. The probe did not recognize the fungal genomic DNA (Fig. 5, lanes 3 and 4).

Figure 5.

Figure 5

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Genomic Southern-blot analysis of restricted genomic DNA from eucalypt and P. tinctorius hybridized to a 32P-labeled 3′-end-EgIcdh probe. Ten micrograms of plant (lanes 1 and 2) and fungal (lanes 3 and 4) DNA was digested to completion with BamHI (lanes 1 and 3) or HindIII (lanes 2 and 4), and separated by electrophoresis on a 1% agarose gel. λ phage digested by the restriction enzymes HindIII and EcoRI were used as a DNA ladder.

Steady-State Level of EgIcdh mRNAs in Ectomycorrhizae

We wanted to determine whether the expression of the root NADP-ICDH changes during ectomycorrhiza development. To this end, the steady-state level of EgIcdh mRNAs was assessed in nonmycorrhizal roots of eucalypt and in ectomycorrhizal roots at different stages of the symbiosis development. Total RNA extracted from nonmycorrhizal control roots, ectomycorrhizal roots, and free-living mycelium was analyzed by RNA blotting using the eucalypt-specific 3′-end-EgIcdh probe (Fig. 6A). A single transcript of about 1600 nucleotides was detected in control root and ectomycorrhiza RNA extracts, but not in the fungal RNA extracts. The concentration of the EgIcdh transcripts remained stable in roots during the growth of nonmycorrhizal seedlings (Fig. 6C). In contrast, the formation of ectomycorrhizae by P. tinctorius led to an enhanced accumulation of EgIcdh transcripts (Fig. 6A). Control hybridization with the eucalypt-specific 5.8 S rDNA (Carnero Diaz et al., 1997) was used to normalize the amount of plant RNA (Fig. 6B). The signal intensity corresponding to EgIcdh mRNAs was 2.5-fold higher in roots of seedlings colonized by P. tinctorius (Fig. 6C). RNA-blot data approximated the enzyme-activity data. The increased level of EgIcdh mRNAs was already observed 2 DAC, when the mycelium sheath was forming around the roots. After 7 DAC, the steady-state level of EgIcdh transcripts decreased slightly (Fig. 6C).

DISCUSSION

To gain a better understanding of the C and N interactions in ectomycorrhizae, we investigated the regulation of the NADP-ICDH in eucalypt roots colonized by the ectomycorrhizal P. tinctorius. A constant synthesis of 2-OG is needed to sustain the rapid accumulation of glutamate and Gln taking place in symbiotic tissues (Martin et al., 1986; Finlay et al., 1988; Turnbull et al., 1995), and it is believed that NADP-ICDH yields the 2-OG necessary for N assimilation (Chen and Gadal, 1990). NADP-ICDH activity was found in root, mycorrhizal, and fungal extracts. An anticatalytic immunoassay using antibodies raised against the RTC NADP-ICDH (Gálvez et al., 1995) made it possible to specifically estimate the proportion of root enzyme in the intermingling plant and fungal tissues forming the mycorrhiza. Root NADP-ICDH accounted for 82% of the total enzyme activity in symbiotic tissues (Fig. 1B), and the root-related activity was stimulated nearly 2-fold in ectomycorrhizae (Table I).

Anti-RTC NADP-ICDH antibodies were used for direct localization of the root NADP-ICDH. By indirect immunofluorescence microscopy, the NADP-ICDH was detected in most eucalypt root tissues, except a region likely corresponding to the quiescent meristematic center (Fig. 2A). The enzyme appears to be preferentially accumulated in epidermal cells (Fig. 2B) and vascular tissues (Fig. 2C). This tissue localization is not affected by the ectomycorrhiza formation. The presence of the NADP-ICDH transcript and protein in the epidermal and vascular tissues has been reported previously in tobacco (Gálvez et al., 1996; S. Gálvez, O. Roche, and M. Hodges, unpublished results). The N-assimilating enzymes, GS and NADH-dependent GOGAT, are also preferentially localized in the vascular tissues in various species (Edwards et al., 1990; Kamachi et al., 1992; Hayakawa et al., 1994; Dubois et al., 1996), suggesting that the epidermal and vascular tissues are the site of intense amino acid synthesis.

To determine whether the up-regulation of NADP-ICDH activity resulted from an increased gene expression, a full-length cDNA, EgIcdh, which encodes the eucalypt NADP-ICDH, was cloned and characterized (Fig. 3). The putative amino acid sequence of EgIcdh showed a high amino acid identity with plant NADP-ICDH (89–92%). However, alignment of the amino acid sequence data from fungal ICDHs (e.g. yeast and A. niger) showed these ICDH sequences to be more distantly related (65%–72% similarity) to the eucalypt sequence (Fig. 4). The plant origin was confirmed by Southern-blot and RNA analyses (Figs. 5 and 6). Because the EgICDH protein lacks an N-terminal targeting sequence and has the highest similarity to the cytoplasmic ICDH isoform of tobacco, it probably represents a cytoplasmic isoform. Preliminary analysis of immunogold-stained sections by electron microscopy confirmed the cytosolic localization of the EgICDH protein in eucalypt roots (V. Boiffin, R. Balestrini, P. Bonfante, and F. Martin, unpublished results).

Ectomycorrhiza development was accompanied by an increased concentration of EgIcdh transcripts (Fig. 6). Similarly, NADP-ICDH activity increased when P. tinctorius colonized eucalypt seedlings. The up-regulation of NADP-ICDH transcripts was observed as early as 2 DAC, when roots were colonized by the aggregating fungal mycelium. This enhanced accumulation of NADP-ICDH transcripts likely led to the observed enhanced polypeptide concentration (Fig. 1A) and the observed stimulation of the host NADP-ICDH activity in mycorrhiza (Table I). This higher cytosolic NADP-ICDH activity in ectomycorrhizae might increase cytosolic 2-OG synthesis. This would allow the rapid conversion of Gln translocated from the fungal partner to glutamate (via the GS/GOGAT cycle) for amino acid biosynthesis (Martin and Botton, 1993; Turnbull et al., 1995; F. Martin, V. Boiffin, and P. Pfeffer, unpublished data). An enhanced NADP-ICDH activity has been correlated to increased synthesis of amino acids in tomato (Gallardo et al., 1995), potato (Fieuw et al., 1994), Norway spruce (Wallenda et al., 1996), and tobacco (Scheible et al., 1997).

Our results showed that the plant NADP-ICDH is regulated temporally, but not spatially, during ectomycorrhiza formation. The enhanced activity results from an increased concentration of the NADP-ICDH protein, which is correlated with a higher transcript level. Such a regulation of gene expression could be under the control of metabolic sensors and/or diffusible factors produced by the mycosymbiont. In addition to these symbiosis (development)-related processes, the containment of the root tissues by the rapidly aggregating mycelium may alter the source-sink relationships previously established by the host plant with the surrounding growth medium. These changes require a rapid adjustment of the C and N fluxes within the colonized roots. Regulation of the NADP-ICDH may be the result of either of these complex mechanisms. Studies of other enzymes involved in C and N interactions (e.g. PEP carboxylase) and N assimilation (e.g. GS, GOGAT) are currently ongoing in E. globulus-P. tinctorius ectomycorrhizae to determine whether the expression of these enzymes and NADP-ICDH is co-regulated during symbiosis development.

ACKNOWLEDGMENTS

We thank Evelyne Bismuth (Université Paris-Sud, Orsay) for technical assistance in the NADP-ICDH assay, and Phil Murphy, Murielle Mourer, and Pascal Laurent (Institut National de la Recherche Agronomique [INRA], Champenoux, France) for helpful discussions about RNA and DNA extractions, mycorrhiza synthesis, and immunochemical assays, respectively. We also acknowledge Catherine Voiblet (INRA, Champenoux) for providing the ectomycorrhiza λ-gt11 cDNA library. We are very grateful to Denis Tagu (INRA, Champenoux) and Philipp Pfeffer (U.S. Department of Agriculture, Wyndmoor, PA) for stimulating discussions and critical reading of the manuscript.

Abbreviations:

DAC

days after contact

GS

Gln synthetase

NADP-ICDH

NADP-dependent isocitrate dehydrogenase

2-OG

2-oxoglutarate

RACE

rapid amplification of cDNA ends

RTC

recombinant tobacco cytosolic

Footnotes

1

This work was supported by research grants from the Eureka-Eurosilva program (“Changes in Gene Expression during Ectomycorrhiza Differentiation and Function”) and the Groupement de Recherche et d'Etude des Génomes. V.B. was supported by a doctoral scholarship from the Ministère de l'Enseignement Supérieur et de la Recherche.

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