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Keywords:

  • amino acid;
  • carbon;
  • ectomycorrhiza;
  • nitrogen;
  • organic nutrients

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    While it is accepted that many ectomycorrhizal fungi can assimilate organic substrates and facilitate transfer of their elemental components to plants, the fate of the carbon contained in these materials remains uncertain. Here we investigate the compartmentation of carbon and nitrogen in ectomycorrhizal seedlings of Pinus sylvestris fed with double-labelled (15N and 13C) glycine as their sole N source.
  • • 
    Using isotope ratio mass spectrometry, the quantities of N and C derived from this glycine were determined in sequentially harvested samples of mycorrhizas, roots and shoots.
  • • 
    Whereas considerable quantities of 15N were observed in the mycorrhizal tips, roots and shoots, comparable amounts of 13C were observed only in mycorrhizal tips and roots.
  • • 
    It is clearly important to resolve the role of compound specificity as a factor determining the extent of amino-acid C transfer from roots to shoots. However, from the standpoint of the C budget of the whole plant, wherever heterotrophically acquired C is available as an energy source it will reduce demands on photosynthetically fixed sources of the element.

Introduction

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

It has been known for some time that many ectomycorrhizal fungal symbionts of forest trees have the ability both to assimilate amino acids (Melin, 1925; Abuzinadah & Read, 1988) and to facilitate the transfer of the amino nitrogen contained in these substrates to their autotrophic hosts (Melin & Nilsson, 1953; Abuzinadah & Read, 1989a; Finlay et al., 1992). However, emphasis on the role of amino compounds in the N nutrition of mycorrhizal partners has left us with little knowledge of their possible role as a source of carbon in the symbiosis.

The conventional view is that, in all types of mycorrhizal associations apart from those involving orchids or mycoheterotrophs, the C requirements of the symbiosis are satisfied entirely by photosynthate from the phytobiont (Smith & Read, 1997). Recognition of the ability of the fungi to assimilate amino acids brings with it the need to consider these as sources of C, as well as of N. Some challenge to the conventional view was provided by the observation of Abuzinadah & Read (1989b) that, in mycorrhizal seedlings of birch, a significant proportion of the C requirement of mycorrhizal roots could be met by heterotrophic assimilation of glutamine. As the ‘cost’ of maintaining the symbiosis has been variously estimated as equivalent to between 10 and 30% of photosynthate production, supplementation of the C supply could be of considerable biological importance for the plant. However, little is known about either the extent of occurrence or the pathways of decarboxylation of amino acids in mycorrhizal systems.

Detailed analysis of amino acid uptake systems of fungi has suggested that most, if not all, amino acids are absorbed intact by a general amino acid transport system (Jennings, 1995). In the absence of any evidence for decarboxylation in the uptake process, it is clear that assimilation of these compounds by mycorrhizal fungi provides the potential for supplementation of the C requirements of the fungus and, as a consequence, a reduction in the considerable demand on the resources of the host plant. However, while it may be safe to assume amino acids are assimilated intact by ectomycorrhizal fungi, there is great uncertainty not only about the occurrence of decarboxylation within them, but also about the relative extent of any onward transport of their component C and N atoms to the plant. Here, using the simplest amino acid, glycine, double-labelled with 15N and 13C, as the sole source of N supplied to mycorrhizal and nonmycorrhizal seedlings of Scots pine (Pinus sylvestris), we evaluate compartmentation of the two isotopes into mycorrhizal tips, roots and shoots using isotope ratio mass spectrometric analysis of sequentially harvested plants.

Materials and Methods

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

Seeds of Scots pine (Pinus sylvestris L.) were surface sterilized by rinsing in 30% hydrogen peroxide for 20 min, washed in sterile distilled water, then germinated on water agar. After 4 wk, seedlings were transferred aseptically to 9 cm plastic Petri dishes. The dishes had been prefilled with sterile, acid-washed, horticultural grade perlite moistened with 25 ml modified Melin–Norkrans (MMN) medium which contained glucose as a C source (2.0 g l−1), from which malt extract and mineral N sources were excluded. A ‘v’ notch was cut in the side wall of each dish, enabling the seedlings to be inserted with their roots in contact with the perlite but with their shoots exposed to the atmosphere. The space around the emerging hypocotyls was sealed with sterile anhydrous lanolin to maintain sterility in the root compartment.

At the time of transfer, four inoculum plugs consisting of discs of vegetative mycelium of one of four ectomycorrhizal fungal species were also added to each dish. Two of the fungi were selected on the basis that they were pine-specific (Suillus luteus[L.Fr.] Roussel and Suillus collinitus[Fr.] O. Kuntze); one (Lactarius deterrimus Gröger) was considered to be spruce-specific (at least under field conditions); while the last (Amanita rubescens[Pers:Fr.] SF Gray) was considered to be a generalist. Cultures were derived from sporocarps and were, at most, 2 yr old at the time of the experiment. The two Suillus and A. rubescens sporocarps were collected from a pine plantation at Newborough Warren, Island of Anglesey, north Wales, UK. Lactarius deterrimus was collected in a spruce plantation near Sheffield, England, UK. There were 12 dishes of each fungus combination (only nine with L. deterrimus), as well as of an uninoculated (nonmycorrhizal) control treatment.

All dishes were sealed with parafilm, individually wrapped in aluminium foil to exclude light from the roots, and incubated in a controlled environment growth cabinet with a day–night temperature of 15–10°C. The shoots were exposed to a daylength of 18 h and an irradiance of 120 µmol m2 s−1. Dishes were visually examined periodically during the course of the incubation to evaluate the progress of mycorrhizal formation. After 60 d incubation, extensive mycelial and mycorrhizal development (50–60% of root tips mycorrhizal) were observed in dishes supporting the two pine-specific fungi. Seedlings in dishes inoculated with A. rubescens and L. deterrimus also showed mycorrhizal formation, but to a lesser degree (30–40% of tips mycorrhizal). Neither mycorrhizal formation nor saprotrophic contamination occurred in the uninoculated control treatments.

At this point the first of a two-stage nutrient supplementation programme was initiated. Each dish first received a further 5 ml aliquot of the N-free MMN solution to ensure the seedlings were free of nutrient limitations other than those involving N. In the second stage, applied 5 d later, N was added in the form of double-labelled [99%13C, 10%15N] glycine mixed from nine parts 99%13C-labelled glycine and one part 98%13C + 98%15N labelled glycine (both from Cambridge Isotope Laboratories, Andover, MA, USA). Glycine was added to each dish at a concentration of 4 µmol dissolved in 5 ml sterile distilled water. One harvest (three dishes from each treatment combination) was carried out immediately before the addition of glycine (T0). The remaining dishes were returned to the incubation chamber, from which three further dishes were sequentially harvested after 24 (T1), 48 (T2) and 96 (T3) h. Seedlings were removed from dishes and carefully separated into three compartments: shoots, roots and mycorrhizal tips. The root fraction was examined under a stereo dissection microscope (×50) and all visible fungal material was removed in order to ensure the quantity of fungal material associated with the root fraction was minimized. The three fractions were oven-dried (70°C, 48 h), weighed and ground to a fine powder using a ball mill.

Two sets of stable isotope determinations were carried out. The first set of plants harvested at T0 provided a measure of natural abundance of 15N and 13C in the three compartments. The second set, carried out after the addition of the labelled glycine at T1, T2 and T3, were used to determine enrichment in 15N and 13C as a result of glycine uptake. Total uptake of glycine was estimated from uptake of 15N, assuming that once taken up no 15N was then subsequently lost from the seedling.

Total N concentrations and 15N natural abundances were measured using an elemental analyser (Carlo Erba NA 1500) for Dumas combustion coupled to a Finnigan MAT delta E gas isotope ratio mass spectrometer via an open–split interface. The mass spectrometer was equipped with three Faraday cups for the selective detection of dinitrogen with different isotope composition (14N/14N, 15N/14N, 15N/15N). Total C concentrations and 13C natural abundances were measured separately using an elemental analyser (Fisons 1108) coupled to a Finnigan Mat delta S gas isotope mass spectrometer equipped with six Faraday cups via a Finnigan Mat ConFlo II interface. Dinitrogen and CO2 isotope ratios were measured vs standard gases, which were calibrated with respect to international standard (N2 in air and CO2 in Pee Dee Belemnite (PDB)) by use of the reference substances N1 and N2 for N isotope ratios and NBS19 and ANU sucrose for C isotope ratios provided by the International Atomic Energy Authority, Vienna.

The 15N and 13C enrichments were calculated from concentration data. Accumulation rates and quantities of N and C derived from the labelled glycine were calculated using the following procedures (see also Gebauer, 2000):

  • ΔXa = [(XT − XC) × Xconc × f]/100t(Eqn 1)
  • ΔXb = [(XT − XC) × Xconc × f × B]/100t(Eqn 2)

Xa, amount of N or C taken up from glycine per unit compartment biomass and time (mmol N or C g dw−1 h−1); ΔXb, amount of N or C taken up from glycine per compartment and time (mmol N or C h−1); XT, 15N or 13C abundance after labelling treatment (at%15N or 13C); XC, 15N or 13C abundance of respective unlabelled control (at%15N or 13C); Xconc, total N or C concentration after the labelling experiment (mmol N or C g dw−1, i.e. N[%]/1.4 or C[%]/1.2); f, enrichment factor of tracer (100/15N or 13C enrichment): 15N enrichment was 10%, 13C enrichment of glycine was 99%; t, time after tracer application (h); B, biomass of respective compartment (g dw).)

Data analysis

The experiment consisted of five treatment levels: four fungal isolates, and the nonmycorrhizal seedlings. Data are usually expressed as mean values ± standard error derived from three replicate seedlings harvested within each level at each sampling occasion. The accumulation of 13C and 15N within each fraction at each sampling occasion was compared for the effect of treatment using a one-way anova. The effects of fungal isolates on the rates of N accumulation into roots and mycorrhizal tips over 96 h were examined using a one-way anova. The effect of treatment on the total amount of glycine taken up, as estimated from N uptake, was compared by one-way anova. Pooling the data after the final harvest for seedlings within each treatment allowed a comparison of potential treatment effects on seedling shoot, root and mycorrhizal tip biomass. This was also analysed using a one-way anova within each seedling fraction. Where a significant overall effect was indicated by an anova, differences between means were then sought using Tukey's honest significance difference (HSD) test with a family error of 5%. Differences within treatments in rates of N accumulation into roots and mycorrhizal tips over 96 h were compared using a two-sample t-test. Post-labelling deviations in seedling shoot 13C were analysed using a one-sample t-test to determine if they represent significant shifts from those measured at T0. Relationships in the form of Pearson's product moment correlation coefficients were sought between the quantities of 15N and 13C taken up from the labelled glycine into the different fractions of seedlings. All statistical analyses were done using minitab statistical software ver. 12.21 (Minitab Inc., PA, USA).

Decarboxylation of glycine was estimated by calculating C : N ratios for 13C and 15N taken up into each compartment using the mean values of the three replicate seedlings within each treatment.

Results

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

Nitrogen and C, derived from glycine, were both readily detectable in mycorrhizal tips and roots of mycorrhizal and nonmycorrhizal seedlings (Figs 1b,c, 2b,c) but in contrast with N (Fig. 1a) there was no pattern of C accumulation in shoots in either category of plant (Fig. 2a). With the exception of S. collinitus- and A. rubescens-inoculated seedlings at 48 h, postlabelling deviations in 13C contents of shoots were not significantly different from zero. Small, nonsignificant differences in the 13C atom percentage of postlabelling samples from T0 values give either positive (A. rubescens) or negative (L. deterrimus) mean values.

image

Figure 1. Accumulation of nitrogen after 24 (black bars), 48 (grey bars) and 96 h (open bars), acquired from double-labelled [15N and 13C] glycine in different compartments of pine (Pinus sylvestris) seedlings inoculated with four species of ectomycorrhizal fungi: (a) shoots; (b) roots; (c) mycorrhizal tips (n = 3, mean ± 1 SE). Bars sharing the same letter are not significantly different at P = 0.05. (S. col, Suillus collinitus; S. lut, Suillus luteus; A. rub, Amanita rubescens; L. det, Lactarius deterrimus; Non.myco, nonmycorrhizal seedlings; nd, not determined.)

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image

Figure 2. Accumulation of carbon after 24 (black bars), 48 (grey bars) and 96 h (open bars), acquired from double-labelled [15N and 13C] glycine in different compartments of pine (Pinus sylvestris) seedlings inoculated with four species of ectomycorrhizal fungi: (a) shoots; (b) roots; (c) mycorrhizal tips (n = 3, mean ± 1 SE). Bars sharing the same letter are not significantly different at P = 0.05. (S. col, Suillus collinitus; S. lut, Suillus luteus; A. rub, Amanita rubescens; L. det, Lactarius deterrimus; Non.myco, nonmycorrhizal seedlings; nd, not determined.)

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There were no treatment effects on the accumulation of C and N in the roots of seedlings (Figs 1b, 2b). However, there were differences in accumulation into mycorrhizal tips, with the two Suillus species generally having higher amounts of N and C than the two other species.

In mycorrhizal seedlings there were strong correlations between the quantities of 15N and 13C absorbed over time into mycorrhizal tips and roots (Table 1). These correlations, and the ratios between the 13C and 15N taken up (Fig. 3), suggest that most of the glycine is taken up intact by both mycorrhizal and nonmycorrhizal plants. As a result of the absence of glycine-derived C transfer from roots to shoots, correlations between 15N and 13C transfer to this compartment were not significant.

Table 1.  Correlation coefficients between quantities of 13C and 15N acquired from double-labelled [15N and 13C] glycine in different compartments of mycorrhizal pine (Pinus sylvestris) seedlings
CompartmentSample (h)nrP
Shoot24 8−0.271  0.517
4812−0.560  0.059
9612−0.108  0.739
Root24 8 0.929  0.001
4811 0.907< 0.001
9612 0.820  0.001
Mycorrhizal tips24 9 0.828  0.006
4812 0.964< 0.001
9612 0.979< 0.001
image

Figure 3. Ratios of 13C to 15N derived from double-labelled [15N and 13C] glycine after 24 (black bars), 48 (grey bars) and 96 h (open bars) in different compartments of pine (Pinus sylvestris) seedlings inoculated with four species of ectomycorrhizal fungi: (a) roots; and (b) mycorrhizal tips. Dotted line at 2 represents the C : N ratio of glycine. (S. col, Suillus collinitus; S. lut, Suillus luteus; A. rub, Amanita rubescens; L. det, Lactarius deterrimus; Non.myco, nonmycorrhizal seedlings; nd, not determined.)

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There was some indication that root tips colonized by the two Suillus species accumulated N and C at faster rates (Fig. 4) and to higher concentrations (Figs 1c, 2c) over 96 h than did those colonized by A. rubescens. In addition, the total amount of glycine, as estimated from N uptake, was generally higher in seedlings colonized by Suillus species (Fig. 5). Only L. deterrimus showed significantly higher rates of N accumulation by mycorrhizal tips than by roots (Fig. 4).

image

Figure 4. Accumulation rate of 15N acquired from double-labelled [15N and 13C] glycine into roots (black bars) and mycorrhizal tips (open bars) of pine (Pinus sylvestris) seedlings inoculated with four species of ectomycorrhizal fungi (n = 3, mean ± 1 SE). Asterisk indicates significant difference (P = 0.05) between uptake rates into root and mycorrhizal compartments of seedlings colonized by L. deterrimus. (S. col, Suillus collinitus; S. lut, Suillus luteus; A. rub, Amanita rubescens; L. det, Lactarius deterrimus.)

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image

Figure 5. Total amounts of glycine taken up in 96 h by nonmycorrhizal and mycorrhizal pine (Pinus sylvestris) seedlings (n = 3) inoculated with four species of ectomycorrhizal fungi. (S. col, Suillus collinitus; S. lut, Suillus luteus; A. rub, Amanita rubescens; L. det, Lactarius deterrimus; Non.myco, nonmycorrhizal seedlings.) Bars sharing the same letter are not significantly different at P = 0.05.

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Overall, there was no effect of treatment on seedling shoot biomass, but nonmycorrhizal seedlings had significantly greater root biomass than seedlings inoculated with S. collinitus, S. luteus and A. rubescens (data not shown). Mycorrhizal development, in terms of mycorrhizal tip biomass, followed the series S. luteus > S. collinitus > L. deterrimus > A. rubescens, but only S. luteus and A. rubescens were significantly different (data not shown).

Discussion

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

The results support the observations of Carrodus (1966) and Näsholm et al. (1998) that mycorrhizal systems have the ability to absorb amino compounds intact. This is also in accordance with earlier work on fungi grown in pure culture, which showed that most species absorb amino acids without decarboxylation (Jennings, 1995). However, most of these studies have considered amino acid incorporation only from the perspective of its role in the N economy of fungi and plants.

Abuzinadah & Read (1989b) showed that C uptake associated with amino acid assimilation could supply up to 8% of the total C in Betula seedlings. In their study, autoradiographic analyses of the shoots of Betula plants fed with 14C-labelled protein revealed some translocation of C to leaves of mycorrhizal plants, particularly if they were deeply shaded. Likewise, Finlay et al. (1996) observed some transfer of C to shoots after feeding mycorrhizal and nonmycorrhizal birch plants with the 14C-labelled amino acid alanine. The amount of light for C gain from photosynthesis may be a major factor in determining C transfer from fungus to host. In this context, it has recently been demonstrated that for the chlorophyllous orchid Cephalanthera damasonium the amount of C gained heterotrophically from associated root fungi was considerably greater when the plants were in deep forest shade (Gebauer & Meyer, 2003). Shading may also play a role in plant-to-plant transfer of C, as Simard et al. (1997) found a positive influence of shading on the net transfer of C from Betula papyrifera to Pseudotsuga menziesii.

It is clear that in the present study there was negligible transfer of glycine-derived 13C from roots to shoots in either mycorrhizal or nonmycorrhizal plants. The lack of glycine-derived 13C in the shoots may be accounted for by the fact that the processes of assimilation and hence distribution of amino acids are compound-specific (Lipson et al., 1999; Näsholm & Persson, 2001). It is known that glycine is metabolized in the roots of plants, so any N subsequently passed on to the shoot would be attached to a new C skeleton (Näsholm & Persson, 2001). In contrast, there is evidence that alanine is transferred from roots to shoots in both mycorrhizal (Abuzinadah & Read, 1989b) and nonmycorrhizal (Finlay et al., 1996) plants. It will clearly be important to resolve the role of compound specificity as a factor determining the extent of amino acid C transfer from roots to shoots, as this process is likely to be fundamental to the success of achlorophyllous (mycoheterotrophic) plants that are associated with ectomycorrhizal fungal symbionts.

In the present study there was little or no effect of either mycorrhization or fungal species on the accumulation of C and N into seedling roots or shoots. However, the two pine-specific fungi, S. collinitus and S. luteus, appeared to have greater rates of uptake than the generalist mycorrhizal fungus A. rubescens. Suillus species form tuberculate mycorrhizas that support considerable development of mycelium close to the root surface (Agerer, 1986–98). The higher uptake rates may therefore be partly a consequence of the greater proportion of fungal biomass in Suillus mycorrhizas compared with the other two species. The functioning of the L. deterrimus mycorrhizas, in terms of rates of uptake, appeared to be superior to A. rubescens. This is surprising given the reported specificity of L. deterrimus with Picea species (Hansen & Knudsen, 1992).

As we saw transport to – but no accumulation of C in –roots it can be assumed that respiratory loss of the element occurred from the root tissue. Rapid loss of C from amino acids taken up by mycorrhizal and nonmycorrhizal systems has been reported previously (Chalot et al., 1994a, 1994b). Chalot & Brun (1998) suggested that C taken up from organic molecules could provide a labile C source for root respiration.

From the standpoint of the C budget of the whole plant, the pattern of distribution of C once within the plant may be of relatively small significance. Wherever the amino acid C is metabolized, this heterotrophically acquired source will be available as an energy source to reduce demands on photosynthetically fixed sources of the element. As amino compounds such as glycine are now known to be present in quantitatively significant amounts in acidic organic soils of the kinds supporting ectomycorrhizal plants (Abuarghub & Read, 1988; Nemeth et al., 1988; Kielland, 1994, 1995; Jones & Kielland, 2002) their potential to supplement the C budgets of mycorrhizal plants is worthy of further examination.

Acknowledgements

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

The authors would like to thank Petra Dietrich for technical assistance in isotope ratio mass spectrometry.

References

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