| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
Plant Physiol. (1998) 117: 939-948
Eucalypt NADP-Dependent Isocitrate Dehydrogenase1
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
ABSTRACT |
|---|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
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 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 In ectomycorrhizal trees, primary N assimilation takes place in roots
and their fungal associates (Finlay et al., 1988 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 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.
Biological Material and in Vitro Synthesis of Ectomycorrhizae
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
). 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).
; 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.
; 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.
). 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).
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
). 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
s1) 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.
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
-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
-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 -gt11
forward-sequencing primer and idh1 (5-TCCTTTCTGATGGACCCG-3) (see Fig.
3) primers.
|
-CATACCAGATTCCAGCAGCC-3) (see Fig. 3) and the -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
[
-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 mL1 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 mL1 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 g1 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).
). 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.
Root NADP-ICDH Activity in Ectomycorrhizae
Immunolocalization of the Root NADP-ICDH
Cloning and Characterization of a Eucalypt ICDH cDNA
),
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.
View larger version (18K):
[in a new window]
Figure 6.
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
Top
Abstract
Introduction
Methods
Results
Discussion
References
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.
View this table:
Table I.
NADP-ICDH activity of the root in 7-DAC
ectomycorrhizae
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
g1 fresh weight × 0.82 × 100/70).
View larger version (28K):
[in a new window]
Figure 1.
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 ``Materials and 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.
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.
). 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 g1 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).
). 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).
View larger version (140K):
[in a new window]
Figure 2.
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.
) 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).
-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.
Steady-State Level of EgIcdh mRNAs in
Ectomycorrhizae
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 Received October 27, 1997;
accepted April 8, 1998.
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.
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
Altschul SF,
Madden TL,
Schaffer AA,
Zhang JH,
Zhang Z,
Miller W,
Lipman DJ
(1997)
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
Nucleic Acids Res
25:
3389-3402
Bilger I,
Guillot V,
Martin F,
Le Tacon F
(1989)
Assessment of the contribution of glycolysis and the pentose phosphate pathway to glucose respiration in ectomycorrhiza and non-mycorrhizal roots of Norway spruce (Picea abies L. Karsten).
Ann Sci For
46:
724s-727s
Bradford MM
(1976)
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem
72:
248-254
[CrossRef][Web of Science][Medline]
Burgess T,
Dell B,
Malajczuk N
(1996)
In vitro synthesis of Pisolithus-Eucalyptus ectomycorrhizae: synchronization of lateral tip emergence and ectomycorrhizal development.
Mycorrhiza
6:
189-196
Burgess T,
Laurent P,
Dell B,
Malacjczuk N,
Martin F
(1995)
Effects of fungal-isolate infectivity on the biosynthesis of symbiosis-related polypeptides in differentiating eucalypt ectomycorrhizas.
Planta
195:
407-417
Carnero Diaz E,
Martin F,
Tagu D
(1996)
Eucalypt
Carnero Diaz E,
Tagu D,
Martin F
(1997)
Ribosomal DNA internal transcribed spacers to estimate the proportion of Pisolithus tinctorius and Eucalyptus globulus RNAs in ectomycorrhizas.
Appl Environ Microbiol
63:
840-843
[Abstract]
Chen RD,
Gadal P
(1990)
Do the mitochondria provide the 2-oxoglutarate needed for glutamate synthesis in higher plant chloroplasts?
Plant Physiol Biochem
28:
141-145
[Web of Science]
Dubois F,
Brugière N,
Sangwan RS,
Hirel B
(1996)
Localization of tobacco cytosolic glutamine synthetase enzymes and the corresponding transcripts shows organ- and cell-specific patterns of protein synthesis and gene expression.
Plant Mol Biol
31:
803-817
[CrossRef][Web of Science][Medline]
Edwards JW,
Walker EL,
Coruzzi GM
(1990)
Cell specific expression in transgenic plants reveals nonoverlapping roles for chloroplast and cytosolic glutamine synthetase.
Proc Natl Acad Sci USA
87:
3459-3463
Ek H
(1997)
The influence of nitrogen fertilization on the carbon economy of Paxillus involutus in ectomycorrhizal association with Betula pendula.
New Phytol
135:
133-142
[CrossRef]
Fieuw S,
Müller-Röber B,
Gálvez S,
Willmitzer L
(1994)
Cloning and expression analysis of the cytosolic NADP+-dependent isocitrate dehydrogenase from potato. Implications for nitrogen metabolism.
Plant Physiol
107:
905-913
[Abstract]
Finlay RD,
Ek H,
Odham G,
Södeström B
(1988)
Mycelial uptake, translocation and assimilation of nitrogen from 15N-labelled ammonium by Pinus sylvestris plants infected with four different ectomycorrhizal fungi.
New Phytol
110:
59-66
[CrossRef]
Gallardo F,
Gálvez S,
Gadal P,
Cànovas FM
(1995)
Changes in NADP+-linked isocitrate dehydrogenase during tomato fruit ripening: characterization of the predominant cytosolic enzyme from green and ripe pericarp.
Planta
196:
148-154
Gálvez S,
Bismuth E,
Sarda C,
Gadal P
(1994)
Purification and characterization of chloroplastic NADP-isocitrate dehydrogenase from mixotrophic tobacco cells. Comparison with the cytosolic isoenzyme.
Plant Physiol
105:
593-600
[Abstract]
Gálvez S,
Gadal P
(1995)
On the function of the NADP-dependent isocitrate dehydrogenase in living organisms.
Plant Sci
105:
1-14
Gálvez S,
Hodges M,
Bismuth E,
Samson I,
Teller S,
Gadal P
(1995)
Purification and characterization of a fully active recombinant tobacco cytosolic NADP-dependent isocitrate dehydrogenase in Escherichia coli: evidence for a role for the N-terminal region in enzyme activity.
Arch Biochem Biophys
323:
164-168
[Medline]
Gálvez S,
Hodges M,
Decottignies P,
Bismuth E,
Lancien M,
Sangwan RS,
Dubois F,
Le Maréchal P,
Crétin C,
Gadal P
(1996)
Identification of a tobacco cDNA encoding a cytosolic NADP-isocitrate dehydrogenase.
Plant Mol Biol
30:
307-320
[CrossRef][Web of Science][Medline]
Hampp R,
Schaeffer C
(1995)
Mycorrhiza-carbohydrate and energy metabolism.
In
E Varma,
B Hock,
eds, Mycorrhiza Structure, Function, Molecular Biology and Biotechnology.
Springer-Verlag, Berlin, pp 267-296
Hanning I,
Heldt HW
(1993)
On the function of mitochondrial metabolism during photosynthesis in spinach (Spinacea oleracea L.) leaves. Partitioning between respiration and export of redox equivalents and precursors for nitrate assimilation products.
Plant Physiol
107:
905-913
Hayakawa T,
Nakamura T,
Hattori F,
Mae T,
Ojima K,
Yamaya T
(1994)
Cellular localization of NADH-dependent glutamate synthase protein in vascular bundles of unexpanded leaf blades and young grains of rice plants.
Planta
193:
455-460
Hilbert JL,
Costa G,
Martin F
(1991)
Ectomycorrhizin synthesis and polypeptide changes during the early stage of eucalypt mycorrhiza development.
Plant Physiol
97:
977-984
Kamachi K,
Yamaya T,
Hayakawa T,
Mae T,
Ojima K
(1992)
Vascular bundle-specific localization of cytosolic glutamine synthetase in rice leaves.
Plant Physiol
98:
1481-1486
Laurent P (1995) Contribution à l'étude des
protéines régulées par la symbiose chez
l'ectomycorhize d'Eucalyptus-Pisolithus: caractérisation de mannoprotéines pariétales chez le
basidiomycète Pisolithus tinctorius. PhD thesis.
Université Henri Poincaré, Nancy, France
Martin F,
Botton B
(1993)
Nitrogen metabolism of ectomycorrhizal fungi and ectomycorrhiza.
Adv Plant Pathol
9:
83-102
Martin F,
Delaruelle C,
Hilbert JL
(1990)
An improved ergosterol assay to estimate fungal biomass in ectomycorrhizas.
Mycol Res
94:
1059-1064
Martin F,
Ramstedt M,
Söderhäll K
(1987)
Carbon and nitrogen metabolism in ectomycorrhizal fungi and ectomycorrhizas.
Biochimie
69:
569-581
[Medline]
Martin F,
Ramstedt M,
Söderhäll K,
Canet D
(1988)
Carbohydrate and amino acid metabolism in the ectomycorrhizal ascomycete Sphaerosporella brunnea during glucose utilization. A 13C NMR study.
Plant Physiol
86:
935-940
Martin F,
Stewart GR,
Genetet I,
Le Tacon F
(1986)
Assimilation of [15N]NH4+ by beech (Fagus sylvatica L.) ectomycorrhiza.
New Phytol
102:
85-94
[CrossRef]
Martin F,
Tagu D,
Lapeyrie F
(1997)
Altered gene expression during ectomycorrhiza development.
In
P Lemke,
G Caroll,
eds, The Mycota, Vol VI: Plant Relationships.
Springer-Verlag, Berlin, pp 223-242
Nehls U, Martin F (1995) Changes in root gene expression in
ectomycorrhiza. In V Stocchi, P Bonfante, M Nuti, eds,
Biotechnology of Ectomycorrhizae: Molecular Approaches. Plenum Press,
New York, pp 125-138
Peterson RL,
Bonfante P
(1994)
Comparative structure of vesicular-arbuscular mycorrhizas and ectomycorrhizas.
In
AD Robson,
LK Abbot,
N Malajczuk,
eds, Management of Mycorrhizas in Agriculture, Horticulture and Forestry.
Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 79-88
Rieger A,
Güttenberger M,
Hampp R
(1992)
Soluble carbohydrates in mycorrhized and non-mycorrhized fine roots of spruce seedlings.
Z Naturforsch
47:
201-204
Ruiz-Avila L,
Ludevid MD,
Puigdomenech P
(1991)
Differential expression of a hydroxyproline-rich cell-wall protein gene in embryonic tissues of Zea mays L.
Planta
184:
130-136
Sambrook J,
Fritsch EF,
Maniatis T
(1989)
Molecular Cloning: A Laboratory Manual.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Sankaran B,
Chavan AJ,
Haley BE
(1996)
Identification of adenine binding domain peptides of the NADP active site within porcine heart NADP-dependent isocitrate dehydrogenase.
Biochemistry
35:
13501-13510
[Medline]
Schaeffer C,
Johann P,
Nehls U,
Hampp R
(1996)
Evidence for up-regulation of the host and a down-regulation of the fungal phosphofructokinase activity in ectomycorrhizas of Norway spruce and fly agaric.
New Phytol
134:
697-702
Scheible WR,
Gonzàlez-Fontes A,
Lauerer M,
Müller-Röber B,
Caboche M,
Stitt M
(1997)
Nitrate acts as a signal to induce organic acid metabolism and repress starch metabolism.
Plant Cell
9:
783-798
[Abstract]
Smith SE,
Read DJ
(1997)
Mycorrhizal Symbiosis.
Academic Press, London
Stepien V,
Martin F
(1992)
Purification, characterization and localisation of the bark storage proteins of poplar.
Plant Physiol Biochem
30:
399-407
Swofford DL
(1993)
PAUP: Phylogenetic Analysis Using Parsimony, version 3.1.1.
Illinois Natural History Survey, Champaign, IL
Tagu D,
Python M,
Crétin C,
Martin F
(1993)
Cloning symbiosis-related cDNAs from eucalypt ectomycorrhiza by PCR-assisted differential screening.
New Phytol
125:
339-343
[CrossRef]
Turnbull MH,
Goodall R,
Stewart GR
(1995)
The impact of mycorrhizal colonization upon nitrogen utilization and metabolism in seedlings of Eucalyptus grandis Hill ex Maiden and Eucalyptus maculata Hook.
Plant Cell Environ
18:
1386-1394
[CrossRef]
Wallenda T,
Schaeffer C,
Einig W,
Wingler A,
Hampp R,
Seith B,
George E,
Marschner H
(1996)
Effects of varied soil nitrogen supply on Norway spruce (Picea abies L. Karst.). II. Carbon metabolism in needles and mycorrhizal roots.
Plant Soil
186:
361-369
) 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.
View larger version (11K):
[in a new window]
Figure 4.
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.
View larger version (112K):
[in a new window]
Figure 5.
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. phage digested by the restriction enzymes HindIII and EcoRI
were used as a DNA ladder.
-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
Top
Abstract
Introduction
Methods
Results
Discussion
References
; 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).
; 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.
; 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).
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.
FOOTNOTES
2
Present address: Philipps Universität
Marburg, Fachbereich Biologie, Karl-von-Frisch Strasse, 35032 Marburg,
Germany.
*
Corresponding author; e-mail fmartin{at}nancy.inra.fr; fax
33-383-39-40-69.
ABBREVIATIONS
ACKNOWLEDGMENTS
-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.
LITERATURE CITED
Top
Abstract
Introduction
Methods
Results
Discussion
References
-tubulin: cDNA cloning and increased level of transcripts in ectomycorrhizal root system.
Plant Mol Biol
31:
905-910
[Medline]
Copyright Clearance Center: 0032-0889/98/117/0939/10
© 1998 American Society of Plant Physiologists
This article has been cited by other articles:
|
|
 |
|
  M. F. Suarez, C. Avila, F. Gallardo, F. R. Canton, A. Garcia-Gutierrez, M. G. Claros, and F. M. Canovas Molecular and enzymatic analysis of ammonium assimilation in woody plants J. Exp. Bot., April 15, 2002; 53(370): 891 - 904. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Hodges Enzyme redundancy and the importance of 2-oxoglutarate in plant ammonium assimilation J. Exp. Bot., April 15, 2002; 53(370): 905 - 916. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ASPB Publications | PLANT PHYSIOLOGY® | THE PLANT CELL | |
|---|---|---|---|
