Download Carbohydrate and Amino Acid Metabolism in the

Plant Physiol. (1998) 118: 627–635
Carbohydrate and Amino Acid Metabolism in the Eucalyptus
globulus-Pisolithus tinctorius Ectomycorrhiza during
Glucose Utilization1
Francis Martin*, Vincent Boiffin, and Philip E. Pfeffer
Equipe de Microbiologie Forestière, Institut National de la Recherche Agronomique, Centre de Recherches de
Nancy, F-54280 Champenoux, France (F.M., V.B.); and Plant-Soil Biophysics, United States Department of
Agriculture-Agricultural Research Service, 600 East Mermaid Lane, Wyndmoor, Pennsylvania 19038 (P.E.P.)
The metabolism of [1-13C]glucose in Pisolithus tinctorius cv
Coker & Couch, in uninoculated seedlings of Eucalyptus globulus
bicostata ex Maiden cv Kirkp., and in the E. globulus-P. tinctorius
ectomycorrhiza was studied using nuclear magnetic resonance
spectroscopy. In roots of uninoculated seedlings, the 13C label was
mainly incorporated into sucrose and glutamine. The ratio (13C3 1
13
C2)/13C4 of glutamine was approximately 1.0 during the timecourse experiment, indicating equivalent contributions of phosphoenolpyruvate carboxylase and pyruvate dehydrogenase to the
production of a-ketoglutarate used for synthesis of this amino acid.
In free-living P. tinctorius, most of the 13C label was incorporated
into mannitol, trehalose, glutamine, and alanine, whereas arabitol,
erythritol, and glutamate were weakly labeled. Amino acid biosynthesis was an important sink of assimilated 13C (43%), and anaplerotic CO2 fixation contributed 42% of the C flux entering the Krebs
cycle. In ectomycorrhizae, sucrose accumulation was decreased in
the colonized roots compared with uninoculated control plants,
whereas 13C incorporation into arabitol and erythritol was nearly
4-fold higher in the symbiotic mycelium than in the free-living
fungus. It appears that fungal utilization of glucose in the symbiotic
state is altered and oriented toward the synthesis of short-chain
polyols.
Carbohydrate metabolism in ectomycorrhizae has received considerable attention (for review, see Hampp and
Schaeffer, 1995; Smith and Read, 1997). Using carbohydrates for storage, for increasing biomass, and for conversion into metabolic energy, ectomycorrhizal fungi create
strong assimilate sinks. Photoassimilates move into the
phloem of trees primarily as Suc and reach the ectomycorrhizal tissues in this form (Jakobsen, 1991; Hampp and
Schaeffer, 1995). Suc is the main labeled carbohydrate in
root cells but is not detected in symbiotic fungal tissues,
where mannitol, trehalose, and glycogen are the main labeled carbohydrates (Söderström et al., 1988; Hampp and
Schaeffer, 1995; Smith and Read, 1997). Glc resulting from
Suc catabolism is thought to be the primary source of
1
This work was supported by a travel grant from the Institut
National de la Recherche Agronomique (to F.M.). V.B. was supported by a doctoral scholarship from the Ministère de
l’Enseignement Supérieur et de la Recherche.
* Corresponding author; e-mail [email protected]; fax 33–
383–394069.
carbon for the generation of ATP, reducing power, and
carbon skeletons for biosynthetic pathways in ectomycorrhizae (Hampp and Schaeffer, 1995). The metabolic pathways leading to the synthesis of major fungal carbohydrates such as mannitol and trehalose have been
characterized in several free-living ectomycorrhizal fungi
(Martin et al., 1985, 1988; Ramstedt et al., 1989). These
carbohydrates have also been found in ectomycorrhizae
(Ineichen and Wiemken, 1992), but metabolic routes converting Suc to fungal carbohydrates and other metabolites
in symbiotic tissues have not been characterized.
There is evidence that ectomycorrhizal symbiosis brings
about considerable modification of carbon metabolism in
the host roots and in the mycobiont forming the association
(Martin et al., 1987; Hampp and Schaeffer, 1995). An important question in relation to the physiology of ectomycorrhizal associations concerns the extent to which each
partner contributes to the metabolism of carbohydrates. A
full understanding of the metabolic fate of Glc in ectomycorrhizae requires the characterization of (a) the metabolic
pathways converting Glc to other carbohydrates and metabolites, (b) the carbon compounds accumulated in symbiotic tissues, and (c) the changes induced by the symbiosis
on the partner metabolism. We used NMR spectroscopy in
conjunction with [1-13C]Glc labeling to study carbohydrate
and amino acid metabolism in a eucalypt (Eucalyptus globulus subsp. bicostata) and in Pisolithus tinctorius, growing
separately and in mycorrhizal association. The results demonstrated significant mutual effects on fungal and hostplant metabolism.
MATERIALS AND METHODS
Biological Material and in Vitro Synthesis
of Ectomycorrhizae
Eucalyptus globulus subsp. bicostata ex Maiden Kirkp.
seeds (Kylisa Seeds Co., Weston Creek, Australia) were
sterilized with 20% calcium hypochloride (v/v) for 20 min,
rinsed with four changes of sterile water, and plated on
low-sugar Pachlewski medium (2.7 mm di-ammonium tartrate, 7.3 mm KH2PO4, 2.0 mm MgSO4z7H2O, 5 mm Glc; 2.9
mm thiamine-HCl, and 1 mL of a trace-element stock solution [Kanieltra 6Fe, Hydro Azote Co., Ambès, France];
Hilbert et al., 1991) in 2.0% (w/v) agar. After 7 d, asepti-
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628
Martin et al.
cally germinated seedlings were placed on the edge of
14-d-old fungal mats of the ectomycorrhizal gasteromycete
Pisolithus tinctorius Coker & Couch isolate 441, grown on
low-sugar Pachlewski medium in 2.0% agar, and left for 7 d
in a controlled-environment growth chamber with 16 h of
light (25°C, 150 mmol m22 s21) and 8 h of dark (Hilbert et
al., 1991) for ectomycorrhiza formation. After 7 d, the ectomycorrhizal sheath and Hartig net were differentiated on
the main root and lateral roots of seedlings (Dexheimer et
al., 1994). Petri dishes of free-living mycelium and uninoculated control seedlings were grown under the same
conditions. Fungal colonization of root tissues was measured by the ergosterol assay (Martin et al., 1990). Uninoculated and 7-d-old ectomycorrhizal seedlings, together
with the edges of 21-d-old fungal mats, were then sampled
and preincubated in 5 mL of Pachlewski medium containing 5 mm Glc for 1.5 h prior to [13C]Glc labeling.
Labeling Studies
[1-13C]Glc labeling of uninoculated and ectomycorrhizal
seedlings was carried out with the roots fully submerged in
5 mL of Pachlewski medium containing 5 mm [1-13C]Glc
(99 atom % 13C, Sigma-Aldrich) for 6 to 30 h. Fungal mats
were floated on the surface of the labeled solution. Samples
(approximately 0.1 g dry weight) were taken for natural
abundance NMR analysis immediately before the addition
of the labeled [1-13C]Glc (0-time sample). After this, the
labeled samples (roots and mycelium) were rinsed thoroughly with Glc-free Pachlewski medium to remove any
remaining labeled Glc, blot-dried, frozen in liquid nitrogen,
and lyophilized. Lyophilized samples were extracted using
cold (220°C) methanol:water (70:30, v/v; Martin and Canet, 1986) and processed for NMR analysis of water-soluble
carbon compounds (Martin, 1991). Two labeling experiments were performed for each time course, with very
similar results (65% to 610%); in each case data from one
of the replicates are shown.
NMR Spectroscopy
1
H-decoupled 13C-NMR spectra were recorded at 100.55
MHz using a spectrometer (Unity Plus 400, Varian Instruments, Sugarland, TX) with a superconducting magnet
(Oxford Instruments, Oxford, UK). Spectra were recorded
at 25°C with the following spectrometer conditions: proton
decoupling by WALTZ-16 composite pulse sequence,
25,000-Hz spectral width, quadrature phase detection, 16 K
data storage array, 11.6–ms observation pulse (corresponding to a 45° flip angle), 2.38-s recycle time, and 25,000 free
induction decays. The lock signal was obtained from the
99% (v/v) D2O in which the extract was dissolved. Spectra
were processed with 3.0-Hz exponential line broadening.
Chemical shifts are quoted relative to the Suc C59 resonance (82.48 d ppm; plant extracts) or the trehalose C1
resonance (94.1 d ppm; fungal and ectomycorrhizal extracts) and expressed in 100 d ppm downfield from tetramethylsilane. The resonances were assigned by comparing
observed chemical shifts with previously published values
for carbohydrates (Dijkema et al., 1985; Martin et al., 1985,
Plant Physiol. Vol. 118, 1998
1988; Shachar-Hill et al., 1995; Fan, 1996) and amino acids
(Martin and Canet, 1986; Fan, 1996). The identification of
each carbohydrate and amino acid component was also
made by peak matching with authentic samples, spiking
with authentic samples, or analyses of heteronuclear
single-quantum coherence spectroscopy spectra.
Acquisition conditions that do not permit complete relaxation of all carbon signals between pulses were used.
However, this had a constant effect on intensities throughout the series of experiments, and the relative intensities
could therefore be measured accurately. To compare the
amounts of 13C incorporated into metabolites in ectomycorrhizal and fungal extracts sampled during the timecourse experiments, the peak intensities of the various 13C
resonances was standardized to the peak intensity of the
natural abundance of mannitol C2,5 within each spectrum
and then to mannitol C2,5 resonance in the natural abundance (T0) spectrum. The 13C enrichment (atom % 13C) of
mannitol C1,6 and C3,4 was calculated by comparison with
natural abundance 13C enrichment (1.1%) of mannitol C2,5
within the same spectrum. Suc 13C enrichment was evaluated by comparing the intensity of the C1 and C19 resonances (62.4 and 93.3 d ppm) with the intensity of the
unlabeled natural abundance resonance of the C59 (82.4 d
ppm). Absolute 13C content in trehalose C1 was determined from the 1H-13C satellite spectra, and 13C labeling in
C6,69 was calculated from the C6,69-to-C1,19 ratio in 13C
spectra.
RESULTS
Glc Assimilation in Free-Living P. tinctorius
Methanolic extracts of P. tinctorius harvested after
growth in a medium containing 5 mm [1-13C]Glc for 29 h
gave rise to the 13C-NMR spectra shown in Figure 1. The
largest resonances observed in the carbohydrate region of
Figure 1A arose from C1,6 of mannitol and C1,19 of trehalose. The C1,6 of mannitol was the most highly labeled
component in the time-course experiment (Fig. 4A), incorporating 31% of total NMR observable 13C at 29 h. It is
possible to deduce the percentage of mannitol C1,6 labeling
by comparing natural abundance mannitol C2,5 with the
C1,6 intensity. Mycelium showed 8.5%, 20%, and 29% 13C
enrichment (i.e. atom % 13C) after 6, 21, and 29 h, respectively. This distribution of label is consistent with the hypothesis that mannitol, the most prominent soluble carbohydrate of free-living P. tinctorius (natural abundance
spectrum not shown), was synthesized by a direct route
from labeled [1-13C]Glc.
Mannitol C3,4 showed a very low 13C enrichment (approximately 2%), indicating that the flux of 13C from Glc
through the pentose phosphate pathway was limited (Martin et al., 1988). Apart from the rapid incorporation of 13C
into mannitol, a number of changes occurred with time,
notably the marked increase in intensity of trehalose C1,19
(Figs. 1A and 4A). From the 1H-13C satellite spectrum of
trehalose (data not shown), it appears that the 13C enrichment of this C1,19 position was 61%, whereas the C6,69
position exhibited a 14% 13C enrichment. The occurrence of
13
C labeling at the C6,69 position of trehalose indicates an
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Carbon Metabolism in Eucalypt Ectomycorrhizae
629
Figure 1. 13C-NMR spectra of intracellular 13C-carbohydrates (A) and free 13C-amino acids (B) in P. tinctorius mycelium
obtained after feeding [1-13C]Glc (99 atom %) for 29 h. G, Glc; T, trehalose; M, mannitol; E, erythritol; A, arabitol. Subscripts
refer to the carbon positions.
isotopic scrambling between C1,19 and C6,69 positions,
suggesting that Glc carbon used to form trehalose was
cycled through the metabolically active mannitol pool or
the pentose phosphate cycle enzyme transaldolase (Martin
et al., 1988; Pfeffer and Shachar-Hill, 1996). However, the
13
C6,69-to-13C1,19 ratio of trehalose was approximately
0.18, suggesting low randomization of the 13C label. After
29 h of labeling, mannitol, trehalose, erythritol, arabitol,
and Glc accounted for 54%, 26%, 9%, 6%, and 5% of the
total carbohydrate 13C, respectively.
Figure 1B shows an expanded 13C-NMR spectrum of the
amino acid region after 29 h of labeling. The most intense
peaks of this spectrum had chemical shifts that correspond
to C2, C3, and C4 of Gln and C3 of Ala. Glu positions were
weakly labeled. In Gln and Glu, C4 exhibited a greater 13C
content than the C2 or C3 positions of these amino acids
(Fig. 1B). The ratio (13C3 1 13C2)/13C4 of Gln was approximately 1.0 during the time-course experiment. The proportion of the label entering the free amino acid pools
represented 22% (6-h time sample) to 43% (29-h time sample) of the 13C observed by NMR. After 29 h, Gln (59%)
accounted for the largest incorporation in the amino acid
pool, followed by Ala (30%) and Glu (11%). Multiple 13C
resonances of Gln C3 at 27 ppm showed that the synthesis
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630
Martin et al.
of multilabeled [13C3-13C4]Gln occurred. This indicates cycling of amino acid precursors through the Krebs cycle
(Malloy et al., 1988).
Glc Assimilation in Roots of Uninoculated E. globulus
Suc is the main soluble carbon metabolite in uninoculated eucalypt roots, as shown by the presence of natural
abundance 13C resonances of this disaccharide (Fig. 2A).
Plant Physiol. Vol. 118, 1998
After addition of [1-13C]Glc, the majority of labeling was
incorporated into the glucosyl C1 and fructosyl C19 moieties of Suc. These positions showed about 15% 13C enrichment after 29 h. The labeling of the C1 of Fru (the precursor
of the C19 of Suc) and C1 of the Glc used to synthesize Suc
were identical when the contribution of free Glc a C1 was
subtracted. This suggests a rapid labeling of the Fru pool
from absorbed Glc. There was a weak scrambling between
the C1,19 of Glc into the C6,69 positions of Suc.
Figure 2. 13C-NMR spectra of intracellular 13C-carbohydrates (A) and free 13C-amino acids (B) in noncolonized E. globulus
roots obtained after feeding [1-13C]Glc (99 atom %) for 28 h. G, Glc; S, Suc; F, Fru; T, trehalose; M, mannitol; E, erythritol;
A, arabitol.
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Carbon Metabolism in Eucalypt Ectomycorrhizae
Gln was the only free amino acid detected in uninoculated eucalypt roots (Figs. 2B and 4B). The ratio (13C3 1
13
C2)/13C4 of Gln was approximately 1.0 during the timecourse experiment, indicating equivalent contributions of
PEP carboxylase and pyruvate kinase/pyruvate dehydrogenase to the production of Krebs cycle intermediates.
Glc Assimilation in Ectomycorrhizae
Based on the ergosterol assay (Martin et al., 1990), the
analyzed ectomycorrhizae contained approximately 25% to
30% fresh weight of mycelium (data not shown). As shown
in Figure 3, ectomycorrhiza formation had a dramatic effect
on Glc metabolism in the mycobiont and the host roots.
After a 27-h incubation in [1-13C]Glc, resonances detected
in the carbohydrate region (Fig. 3A) predominantly arose
from C1,19 of trehalose, C1,6 of mannitol, C1,5 of arabitol,
and C1,4 of erythritol. The 13C natural abundance resonances of Suc were detected, but 13C incorporation into the
glucosyl C1 and fructosyl C19 moieties of the disaccharide
was very low (about 1% above the natural abundance). The
C1,19 of trehalose was labeled to about 75% from the 13C
spectrum (comparison of the C5,59 position at 73.1 d ppm
and the C1,19 position at 94.1 d ppm; Fig. 3A) and approximately 81% from the 1H-13C satellite spectrum (data not
shown). Trehalose C6,69 was more intense than the natural
abundance resonances of this carbohydrate at 73.4, 73.1,
71.9, and 70.5, indicating the occurrence of an isotopic
scrambling (i.e. cycling) between the C1,19 and C6,69 positions. The 13C6,69-to-13C1,19 ratio of trehalose was approximately 0.11, implying low randomization of the 13C label,
as observed in the free-living mycelium (Fig. 1A). The
amount of newly incorporated 13C into trehalose was
nearly 2-fold higher in symbiotic tissues than in free-living
mycelium. The C1,6 of mannitol was also highly labeled
over the time-course experiment (Fig. 4C), reaching 21%
13
C enrichment after 27 h.
Apart from the rapid synthesis of [13C]trehalose and
13
[ C]mannitol, a striking feature of [13C]Glc assimilation in
ectomycorrhizae was the rapid and intense accumulation
of [13C]erythritol and [13C]arabitol (Figs. 3A and 4C). The
distribution of 13C label is consistent with the hypothesis
that these polyols were synthesized by a direct route from
[1-13C]Glc. These sugar alcohols, which barely accumulated in free-living P. tinctorius (Fig. 1A), represented a
large part of the labeling in the soluble ectomycorrhizal
carbon compounds (Fig. 4C). After 27 h, trehalose, mannitol, erythritol, and arabitol accounted for 31%, 25%, 26%,
and 18% of NMR-observable 13C incorporated in fungal
carbohydrates, respectively, whereas root Suc was barely
detectable. The amount of newly incorporated 13C in total
polyols (i.e. mannitol, erythritol, and arabitol) of symbiotic
tissues was about 1.6-fold higher than in free-living mycelium. The increased synthesis of erythritol plus arabitol in
the symbiotic mycelium was even higher: 4.4-fold higher
than in free-living mycelium.
As found in free-living P. tinctorius, the most intense
peaks observed in the amino acid region (Fig. 3B) had
chemical shifts corresponding to C2, C3, and C4 of Gln and
631
C3 of Ala. Glu positions were weakly labeled. In Gln, C4
exhibited a greater 13C content than the C2 and C3 positions of these amino acids (Fig. 3B). The ratio (13C3 1
13
C2)/13C4 of Gln was approximately 1.1, indicating that
the 13C flux through anaplerotic carboxylases was still high
in symbiotic tissues and was not affected by ectomycorrhiza development. As observed in the free-living mycelium, multiplet 13C resonances of Gln C3 revealed the
presence of Gln isotopomers. The proportion of the label
entering the free amino acid pools represented 19% (6-h
time sample) to 25% (27-h time sample) of the 13C observed
by NMR. After 27 h, Gln (72%) accounted for the largest
incorporation, followed by Ala (15%) and Glu (13%). The
proportion of 13C incorporated into Ala was thus 50%
lower in symbiotic mycelium compared with the freeliving mycelium.
DISCUSSION
13
C Metabolism in Free-Living P. tinctorius
In ectomycorrhizal ascomycetes (Martin et al., 1985,
1988), basidiomycetes (Martin et al., 1984; Söderström et
al., 1988; Ramstedt et al., 1989; Ineichen and Wiemken,
1992; Hampp and Schaeffer, 1995), and other fungi (Lewis
and Smith, 1967; Dijkema et al., 1985), trehalose and various polyols (e.g. mannitol and arabitol) have been reported
to be present during active growth. These carbohydrates
form endogenous storage pools that are continuously metabolized and contribute to the osmotic stabilization of the
hyphae. Mannitol contains the highest proportion of carbon from assimilated Glc in Cenococcum geophilum and
Sphaerosporella brunnea (Martin et al., 1985, 1988), whereas
trehalose is prominent in Piloderma croceum (Ramstedt et
al., 1989) and Laccaria bicolor (Martin, 1991). This indicates
that mannitol and trehalose are important components of
carbohydrate conversion and biosynthesis.
Insoluble glycogen was not detectable in the extracts, but
likely contributed to the carbohydrate pools (Martin et al.,
1985, 1988). The extensive labeling of mannitol (31% of total
NMR-observable 13C at 29 h) and trehalose (17%) in P.
tinctorius is consistent with this scheme. Free-living mycelium of P. tinctorius strain Lelly/Marx 298 showed high
contents of trehalose and arabitol (Ineichen and Wiemken,
1992), whereas [13C]arabitol was barely detectable (6% at
29 h) in the strain used in the present study. The fact that
trehalose showed only a weak isotopic scrambling between
the C1 and C6 positions in hexose pools in free-living P.
tinctorius and symbiotic fungal cells (Figs. 1A and 3A) is in
marked contrast to observations in free-living C. geophilum
and S. brunnea. In these ectomycorrhizal ascomycetes, cycling through the mannitol cycle is high and leads to intense isotopic scrambling (Martin et al., 1985, 1988).
Free amino acids also represent an important sink of
absorbed and assimilated carbon in P. tinctorius (43% of
total 13C at 29 h). This value was similar to the proportion
of 13C entering the free amino acids of other ectomycorrhizal fungi (France and Reid, 1983; Martin and Canet, 1986;
Martin et al., 1988). Under the conditions of nitrogen (5.4
mm) levels in the growth medium, a large proportion of the
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Martin et al.
Plant Physiol. Vol. 118, 1998
Figure 3. 13C-NMR spectra of intracellular 13C-carbohydrates (A) and free 13C-amino acids (B) in P. tinctorius-E. globulus
ectomycorrhizae obtained after feeding [1-13C]Glc (99 atom %) for 27 h. G, Glc; S, Suc; F, Fru; T, trehalose; M, mannitol;
E, erythritol; A, arabitol.
carbon is therefore shifted toward production of amino
acids. Labeled Gln was already abundant after 6 h of
feeding (Fig. 4A) and its 13C content increased rapidly to
25% of the soluble 13C. In [1-13C]Glc-fed C. geophilum (Martin and Canet, 1986) and P. croceum (Ramstedt et al., 1989),
Gln was also rapidly synthesized. Because Gln has a strong
signal and its labeling pattern reflects the isotopic distribution of a-ketoglutarate, it could be used to track the label
through Krebs cycle intermediates (Martin, 1991; Pfeffer
and Shachar-Hill, 1996).
The intramolecular 13C-labeling pattern of Glu/Gln in P.
tinctorius is in agreement with the operation of the Krebs
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Carbon Metabolism in Eucalypt Ectomycorrhizae
633
the Krebs cycle (Malloy et al., 1988). The 13C isotopic distribution in Gln was used to estimate the contribution of
the anaplerotic C flux to malate via pyruvate carboxylase,
as previously demonstrated by Martin and Canet (1986).
During the time-course experiment, the ratio (13C3 1
13
C2)/13C4 of Gln indicated that the contributions of pyruvate carboxylase and pyruvate dehydrogenase to the production of Krebs cycle intermediates (Martin and Canet,
1986; Martin, 1991) were similar. Anaplerotic CO2 fixation
is therefore an important component of Glc metabolism in
free-living mycelium. It is likely that this anaplerotic role is
particularly significant under conditions of amino acid accumulation to replenish intermediates of the Krebs cycle
that are drawn off for biosynthesis during active growth.
13
C intramolecular enrichment of Glu and Gln in other
ectomycorrhizal fungi (Martin and Canet, 1986; Martin et
al., 1988; Ramstedt et al., 1989) also suggested high activity
of anaplerotic carboxylases during rapid Glc utilization.
Ala synthesis was a significant fate for the Glc carbon in
P. tinctorius. The high 13C labeling of the C3 of Ala is in
agreement with the synthesis of this amino acid via pyruvate kinase and Ala aminotransferase.
13
Figure 4. Time dependence of the 13C content of metabolites identified in P. tinctorius mycelium (A), uninoculated E. globulus roots
(B), and P. tinctorius-E. globulus ectomycorrhizae (C) incubated in 5
mM [1-13C]Glc for the indicated times. The 13C contents are from the
peak heights of the carbohydrate and amino acid resonances in the
NMR spectra. E, Trehalose; F, mannitol; f, arabitol; M, erythritol; ‚,
Suc; Œ, Gln; X, Glu; L, Ala. 13C content in fungal and ectomycorrhizal spectra were standardized to the natural abundance mannitol
C2,5. Average values are from duplicate experiments (65% to 610%).
cycle. a-Ketoglutarate, used to synthesize Glu and Gln,
would therefore arise from sequential action of citrate synthase, aconitase, and isocitrate dehydrogenase. There is
evidence of about 32% multiple labeling of Gln, as indicated by 13C-13C spin-spin coupling (e.g. resonance at 27.1
ppm of Gln C3; Fig. 1B) showing that a-ketoglutarate used
to form amino acids does cycle to a minor extent through
C Metabolism in Uninoculated Eucalypt Roots
The majority of labeling from [1-13C]Glc was incorporated into C1 of the glucosyl and fructosyl moieties of Suc
in the uncolonized roots. Gln was the only amino acid
detected and it represented an important sink of absorbed
and assimilated carbon (17% at 20 h). As shown by the
(13C3 1 13C2)/13C4 ratio of Gln, PEP carboxylase and
pyruvate dehydrogenase contributed equally to the production of Krebs cycle intermediates. It is now well documented in plant cells that both malate and pyruvate act as
the point of entry for glycolytic carbon into the Krebs cycle
and that malate is favored during rapid respiration, such
that a significant fraction of glycolytic products enters the
Krebs cycle via the combined action of PEP carboxylase
and malate dehydrogenase (Wiskich and Dry, 1985). Edwards et al. (1998) showed that PEP carboxylase contributed 62% of the malate synthesized in respiring maize root
tips. The flux through PEP carboxylase is comparable in
magnitude in eucalypt roots. This high PEP carboxylase
anaplerotic activity likely sustains the synthesis of Gln.
13
C Metabolism in Ectomycorrhizae
The utilization patterns of the Glc source by seedlings
and mycelium was dramatically influenced by mycorrhizal
colonization, with a greater allocation of carbon to shortchain polyols, arabitol, and erythritol and to trehalose in
the mycelium and a suppression of Suc synthesis in the
roots. The labeling of Suc by the host cells was suppressed
in the mycorrhizal roots despite the significant level of
[1-13C]Glc supplied (5 mm). This finding does not seem to
be a result of the preferential interception of labeled Glc by
the hyphal network ensheathing the roots, because previous studies using 35S-labeled Met and Cys have shown that
synthesis of proteins is taking place at a high rate from the
exogenous precursors in the root cells of ectomycorrhizal
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634
Martin et al.
eucalypt seedlings (Hilbert et al., 1991; Burgess et al., 1995).
Compared with uninoculated roots, the level of Suc was
reduced by 50% in spruce roots colonized by either Amanita
muscaria or C. geophilum (Schaeffer et al., 1995). The mycobiont, which lacks sucrolytic enzymes (Schaeffer et al.,
1995), could possibly induce Suc breakdown to meet its
carbohydrate supply. This could be achieved by inducing a
higher acid-dependent Suc breakdown (Schaeffer et al.,
1997). It is clear that the carbohydrate metabolism of host
roots is regulated by the presence of a fungal partner that
is able to induce strong additional carbon sinks (ShacharHill et al., 1995; Schaeffer et al., 1997).
Conversely, E. globulus-P. tinctorius ectomycorrhizae
contain a high amount of fungus-specific carbohydrates.
The disaccharides trehalose and mannitol are the prominently labeled carbon compounds, and it appears that fungal metabolism dominates the assimilation of exogenous
carbohydrates into symbiotic tissues. 13C incorporation
into arabitol and erythritol was nearly 4-fold higher than in
the free-living mycelium. Arabitol and erythritol accumulated 6% of the total 13C in free-living hyphae, whereas
these polyols incorporated 25% of 13C detected in symbiotic tissues. Arabitol accumulation in P. tinctorius during
spruce ectomycorrhiza formation has also been reported
(Ineichen and Wiemken, 1992).
The initial steps in the ectomycorrhizal interaction include the swelling of hyphal tips and the formation of
fan-like structures on the root surface (Jacobs et al., 1989;
Kottke et al., 1997). The hyphal tip then produces the force
to break the root surface and penetrates between epidermal
cells to initiate the Hartig net (Gea et al., 1994). The internal
turgor pressure is believed to be generated by an influx of
water caused by the osmotic gradient produced in the
fungal cell (Smith and Read, 1997). It is tempting to speculate that the accumulation of the osmolytes arabitol and
erythritol, together with the up-regulation of the synthesis
of cell wall hydrophobins that takes place in P. tinctorius
during eucalypt mycorrhiza development (Tagu et al.,
1996), provides a simple mechanism for plant infection.
Arabitol and erythritol may be the compatible solutes responsible for generating the hydrostatic pressure. The rice
blast fungus (Magnaporthe grisea) simultaneously accumulates a high amount of glycerol and hydrophobins during
the formation of its appressorium (De Jong et al., 1997).
Dark CO2 fixation by fungal and root carboxylases contributes substantially to fulfilling the demands for carbon
compounds in ectomycorrhizae (France and Reid, 1983;
Martin et al., 1988; Hampp and Schaeffer, 1995). In freeliving mycelium of C. geophilum (Martin and Canet, 1986),
S. brunnea (Martin et al., 1988), and P. tinctorius (present
study), a large part of the a-ketoglutarate for Glu and Gln
biosynthesis is also provided by anaplerotic CO2 fixation.
There was a large accumulation of Gln, which displayed a
(13C3 1 13C2)/13C4 ratio in agreement with a high anaplerotic carboxylase activity (Fig. 3B), in symbiotic tissues. The
spectrum (Fig. 3B) does not directly show whether the
amino acids labeled in mycorrhizal roots are of plant or
fungal origin. However, the amino acid labeling (e.g. intramolecular labeling of Gln) in symbiotic tissues was similar to those observed in the free-living fungus, suggesting
Plant Physiol. Vol. 118, 1998
that most of the labeling took place in the fungus. The high
labeling in Gln is in agreement with the known high activity of the Gln synthetase/Glu synthase cycle in eucalypt
ectomycorrhizae (Turnbull et al., 1995), and is likely related
to the high NH41 concentration used in the growth medium. However, the proportion of 13C allocated to Gln and
Ala was lower by 30% and 50%, respectively, in symbiotic
tissues than in the free-living mycelium.
In conclusion, the assimilation of [13C]Glc in free-living
P. tinctorius and E. globulus-P. tinctorius ectomycorrhizae
resulted in the production of a large amount of labeled
polyols, trehalose, Gln, and Ala, whereas E. globulus roots
mainly accumulated Suc and Gln. Ectomycorrhiza development induces striking alterations in the carbohydrate
pools, including enhanced synthesis of arabitol and erythritol. Whether this accumulation of polyols is linked to the
aggregation of hyphae to form the ectomycorrhizal sheath
and/or penetration of root surface by the hyphal tips will
await further biochemical and molecular analyses.
ACKNOWLEDGMENTS
We would like to thank Dr. Yair Shachar-Hill for reading the
manuscript and for helpful discussions and Janine Brouillette for
her technical assistance in obtaining the NMR spectra.
Received April 7, 1998; accepted July 21, 1998.
Copyright Clearance Center: 0032–0889/98/118/0627/09.
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