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Keywords:

  • carbon allocation;
  • nitrogen (N);
  • phosphorus (P);
  • monoxenic arbuscular mycorrhiza (AM) cultures;
  • metabolic activity;
  • phosphate transporter;
  • GiPT

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    The influence of external nitrogen (N) on carbon (C) allocation and processes related to phosphorus (P) metabolism were studied in monoxenic arbuscular mycorrhiza (AM) cultures of Daucus carota.
  • • 
    Fungal hyphae of Glomus intraradices proliferated from colonized roots growing on solid medium into C-free liquid minimal medium with two different N and P levels. Furthermore, we exposed the colonized roots to high or low N availability and then studied the mycelial development. Roots were provided with 13C-glucose in order to follow the C allocation. The mycelium was analysed for phosphatase activity and transcription levels of two nutrient regulated genes.
  • • 
    High N availability to the monoxenic AM root reduced the C allocation to the AM fungus while N availability to the mycelium was important for the upregulation of the fungal inorganic phosphorus (Pi)-transporter GiPT.
  • • 
    We found that N availability can regulate nutritional processes in arbuscular mycorrhiza. We conclude that negative impacts of N on AM abundance are caused by reduced C allocation from the plant. Upregulation of the fungal Pi-transporter GiPT indicated that increased N availability might induce P limitation in the mycelium.

Introduction

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

The phosphorus (P) nutrition in arbuscular mycorrhiza (AM) is well documented (Smith et al., 2003), but the study of N nutrition in AM has been relatively limited, despite the importance of nitrogen (N) in both the fungal and plant part of the symbiosis. First, AM fungi may mediate plant N uptake (Hodge et al., 2002) and readily assimilate both ammonium and nitrate (NH4+ and NO3) into amino acids, probably through the GS-GOGAT enzyme system (Johansen et al., 1996). Second, N supplied to root-free soil patches may increase hyphal length by 20–50% in AM fungi such as Glomus intraradices (Johansen et al., 1994). Third, high N availability can also reduce AM fungal abundance (Johnson et al., 2003), in particular at high P levels (Bååth & Spokes, 1989), suggesting that these two nutrients interact in their influence on the AM symbiosis.

The adverse effect of high soil P levels on AM formation is for the most part due to imbalanced symbiotic benefit (Mosse, 1973; Menge et al., 1978; Jasper et al., 1979; Abbott et al., 1984). The plant can sufficiently supply itself with enough P from the surrounding environment, yet the plant still provides the fungus with a considerable amount of carbon (C). This fungal C demand can constitute a significant cost to the host plant, as indicated by the reduced growth of mycorrhizal plants at high P levels relative to uncolonized plants (Peng et al., 1993). However, it is not only P that is involved in this process. Nitrogen amendments, for example, which influence P demand in the symbiosis, can have a large effect on fungus–plant P relations (Treseder & Allen, 2002).

We studied the influence of external N availability on C allocation and P metabolism in a monoxenic system with carrot root-organ cultures in symbiosis with the AM fungus G. intraradices. This system has proved suitable for the study of growth strategies of this fungus (Bago et al., 1998, 2000; Maldonado-Mendoza et al., 2001; Fortin et al., 2002). We used a two-compartment Petri dish system to test the hypothesis that responses of the AM fungus to N availability are dependent on the P availability. In a second experiment we tested how increased N availability to the mycorrhizal roots influence the C allocated to AM structures as well as immediate responses in the mycelium to N enrichment of the hyphal environment. We measured fungal growth and C allocation responses to external N supply and also determined how some key P metabolic processes, such as enzyme activity and P transporter gene induction, were influenced by these N amendments.

Materials and Methods

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

Monoxenic AM cultures

Clones of carrot roots (Daucus carota L., line DC1), which originally had been transformed by a root-inducing plasmid using Agrobacterium rhizogenes (Bécard & Fortin, 1988). These were colonized by the AM fungus Glomus intraradices Schenck & Smith (DAOM 197198; Biosystematics Research Center, Ottawa, Canada) in monoxenic cultures on Petri dishes with minimal medium (Bécard & Fortin, 1988) containing 10 g sucrose l−1 as the C source, 35 µm P (as 4.8 mg KH2PO4 l−1) and 0.3% (w : v) Phytagel (Sigma-Aldrich, St Louis, MO, USA) as the gelling agent. The cultures were maintained in the dark at a constant temperature of 24°C. Plugs of solid medium containing carrots roots as well as mycelium and spores of G. intraradices were transferred at the start of each experiment from 3- to 4-month-old cultures to the mycorrhizal root side of the two-compartment Petri dish. When the colonized roots covered the mycorrhizal root side, the plates were subjected to specific treatments depending on the experiment. Roots passing the barrier between the two compartments were removed periodically. Cultures were harvested when the average coverage of the mycelia was c. 50% of the liquid compartment, which was approx. 4–5 week after adding the liquid medium (see later).

Mycelium N/P availability study

In this study, fungal hyphae could proliferate from the mycorrhizal root side into liquid medium with two different N and P levels in a full factorial design. One plug of solid medium containing carrot roots, mycelium and spores of G. intraradices was transferred to each new dish containing c. 20 ml of solid minimal nutrient medium on the mycorrhizal root side and inserted into a hole (Maldonado-Mendoza et al., 2001) and sealed with Parafilm ‘M’ (American National Can, Chicago, IL, USA). The second compartment of the Petri dish was at this time still empty. At 70 d, 15 ml of the minimal medium without gelling agent and lacking sucrose was added to the second compartment. Four treatments were applied to the liquid medium containing: (1) normal N and P content (3.2 mm N as KNO3 and 35 µm P as KH2PO4; Control treatment); (2) normal N and high P content (3.2 mm N as KNO3 and 350 µm P as KH2PO4; HighP treatment); (3) high N and normal P content (5.2 mm N as KNO3 and 35 µm P as KH2PO4; HighN treatment); or (4) high N and P content (5.2 mm N as KNO3 and 350 µm P as KH2PO4; HighNP treatment). At 32 d after adding the liquid medium the mycorrhizal root side was supplied with 10 mg of [U-13C]d-glucose (99% (w : w) 13C, Cambridge Isotope Laboratories, Andover, MA, USA) in four replicate dishes. The systems were harvested 1 wk after labelling and were at that time 109 d old.

The mycelium from each disk was collected and transferred to the same type of liquid medium as it came from. Subsamples of mycelium were taken for microscopic assessment of acid- and alkaline-phosphatase-active hyphal length on the same day. Another subsample of mycelium was fixed in 96% ethanol (v : v) for microscopic determination of polyphosphate accumulation. The remaining mycelium was stored at −20°C, freeze-dried and used for determination of 13C-enrichment, lipid extraction and analysis. The liquid medium was collected for determination of pH, total N and total P content, and external phosphatase activity. The solid medium of the mycorrhizal root side was dissolved in 250 ml of 10 mm sodium citrate by mixing for 1 h at low speed on a magnetic stirrer. The roots were stored at −20°C; a subsample of the roots was used for determination of Kjeldahl-N and total-P content. The remaining roots were freeze-dried and used for determination of 13C-enrichment, lipid extraction and analysis.

Root N availability study

In this study, fungal hyphae could proliferate from the mycorrhizal root side with solid medium having two different N levels into liquid medium with two different N levels in a full factorial design. The mycorrhizal root side of a two-compartment Petri plate was filled with c. 20 ml of solid minimal nutrient medium, and covered with a layer of sterilized cellophane. A plug of solid medium containing carrots roots and mycelium and spores of G. intraradices was placed on top of this cellophane. Sterile deionized water was added to the second compartment and the cultures were sealed with Parafilm. After 48 d the cellophane with mycorrhizal root-organ cultures was transferred to new solid medium (SM) containing normal (3.2 mm N as KNO3; HighSM treatment) or reduced (1.6 mm N as KNO3; LowSM treatment) N (Fig. 1). The fungal hyphae were after this transfer allowed to proliferate for 40 d in 15 ml of a liquid minimal medium without gelling agent and lacking sucrose, but with normal N content. One week before harvest, the liquid medium was replaced with 10 ml of new liquid medium (LM), still without gelling agent and lacking sucrose, though containing either normal (3.2 mm N as NH4NO3; HighLM treatment) or reduced (0.1 mm N as NH4NO3; LowLM treatment) N. To obtain these low N contents, the Ca(NO3)2 of the minimal medium was replaced by CaCO3. This resulted in four sets of treatments with four replicates each, namely (1) LowLM/LowSM, (2) LowLM/HighSM, (3) HighLM/LowSM and (4) HighLM/HighSM. The roots were at this time provided with 13C-glucose as described for the previous experiment. The systems were 95 d old at time of harvest.

image

Figure 1. Time-flow diagram showing the experimental treatments in the root nitrogen (N) availability study. The ‘days’ denote ‘days after inoculation in the solid medium’.

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The mycelium was collected and sampled as in the previous experiment. The liquid medium was collected for determination of pH, total N and total P content, and measurement of external phosphatase and chitinase activity. Roots were collected from the cellophane and stored at −20°C, freeze-dried and used for determination of 13C-enrichment, lipid extraction and analysis.

Gene induction study

The experimental design was the same as in the root N availability study, except that the mycelia were not labelled with 13C-glucose and were harvested at 110 d instead of at 95 d. At harvest, mycelium from each plate was rapidly collected from the liquid compartment, temporarily placed in sterile water at 4°C for < 5 min, drained, frozen in liquid nitrogen and stored at −80°C until extraction of total RNA. Three split-plate replicates of each treatment were collected.

Lipid quantification

The lipids from ground mycelium and mycorrhizal roots were extracted in a one-phase mixture of citrate buffer, methanol and chloroform (0.8 : 2 : 1, v : v : v, pH 4.0) as described in detail by Olsson et al. (2002b). The lipids were fractionated into neutral lipids, glycolipids and phospholipids on silica columns by eluting with chloroform, acetone and methanol, respectively. The fatty acid residues in neutral lipids and phospholipids were transformed into free fatty acid methyl esters and analysed by gas chromatography using a 50 m HP5 capillary fused silica column (Hewlett Packard, Wilmington, DE, USA) with H2 as carrier gas (Frostegård et al., 1993). The fatty acids were identified from their retention times in relation to that of the internal standard (fatty acid methyl ester 19 : 0).

Determination of 13C-enrichment in solid samples and in fatty acids

Freeze-dried mycelium (approx. 20 µg) or freeze-dried and ball-milled root material (approx. 100 µg) was enclosed in tin capsules: 13C atom-% was determined on an isotope ratio mass spectrometer (20–20 Stable Isotope Analyser; PDZ Europa Scientific Instruments, Crewe, UK) interfaced to a combustion module (ANCA-NT). In order to determine the 13C atom-% in neutral lipid fatty acids and phospholipid fatty acids the fatty acid methyl esters (prepared as described earlier) were analysed on the isotope ratio mass spectrometer, interfaced to a Hewlett Packard gas chromatograph. The gas chromatograph was equipped with a 50 m HP5 capillary column (Hewlett Packard) with He as carrier gas. The 13C-enrichment (excess atom-%13C) was calculated by subtracting the natural abundance of 13C (1.14%). The AM fungal excess 13C in neutral lipid fatty acid (NLFA) 16 : 1ω5 (including both intraradical and extraradical mycelium) was calculated by multiplying the 13C-enrichment in NLFA 16 : 1ω5 with total amount of NLFA 16 : 1ω5-C.

Hyphal polyphosphate accumulation and phosphatase activity

Subsamples of the extraradical mycelium were fixed and polyphosphates were precipitated in 96% ethanol at 5°C for 1 wk. Samples were washed for 10 min in water, and then stained for 5 min in 0.05% Toluidine blue O in 25 mm sodium acetate buffer at pH 4.4 (Ashford et al., 1975). By spreading the mycelium out in the staining solution and shaking this suspension continuously, an improvement in the penetration of the stain was obtained. Mycelia were then washed for 1 min in 1% HCl in order to remove excess staining of compounds other than polyphosphate. The samples were mounted in water on a microscope slide, and a coverglass was sealed immediately with nail varnish. The polyphosphate accumulation was assessed as described by Olsson et al. (2002b). The proportion (%) of hyphal length with polyphosphate was determined for hyphae with cytoplasm.

Subsamples of extraradical mycelium were subjected to histochemical assessment of acid phosphatase and alkaline phosphatase activity. Collected samples were incubated with a fluorogenic phosphatase substrate (enzyme-labelled fluorescence substrate (ELF); Molecular Probes, Leiden, the Netherlands), buffered at either pH 4.8 or 8 (van Aarle et al., 2001). The samples were mounted on microscope slides with the ELF mounting medium. The proportion (%) of acid-phosphatase and alkaline-phosphatase-active hyphal length was determined as described by Olsson et al. (2002b) for hyphae with cytoplasm.

External phosphatase and chitinase activity

In order to measure acid phosphatase and alkaline phosphatase activity of the liquid medium, a modified procedure based on that of Tabatabai & Bremner (1969) was used. Phosphatase activities were determined spectrophotometrically using p-nitrophenyl phosphate as substrate (Sigma Chemical Co., St Louis, MO, USA) as described in detail by Olsson et al. (2002b). Chitinase activity was analysed according to a similar procedure, using p-nitrophenyl N-acetyl-β-d-glucosaminide (Sigma Chemical Co.) as substrate. Chitinase activity was estimated in unbuffered liquid samples. One unit of enzyme activity was defined as the amount of enzyme activity that had liberated 1 µmol of p-nitrophenol in 1 h.

Phosphorus and nitrogen content in medium and roots

Total P and N concentrations in roots were determined after Kjeldahl combustion. Ammonia and orthophosphate were analysed with a flow injection analysis system for root samples of the N/P availability study. Kjeldahl-N and total-P were calculated as mg g−1 dry weight of root. The liquid samples of the N/P study were analysed with a flow injection analysis system for nitrate and orthophosphate, whereas those of the root availability study were analysed for ammonium, nitrate and orthophosphate. Total-N and total-P were calculated as mg l−1 liquid.

Reverse transcriptase polymerase chain reaction (RT-PCR)

A semiquantitative RT-PCR was carried out to measure the transcript levels of three G. intraradices genes in the fungal hyphae; the phosphate transporter GiPT (AF395112), the N-regulated gene Gi-1(AJ315337) and a β-tubulin (BE603903). Following Burleigh (2001), total RNA was isolated from the −80°C frozen fungal hyphae using an RNeasy Plant Mini Kit (Qiagen, Courtaboeuf, France), DNAse-treated (Stratagene, La Jolla, CA, USA) and reverse-transcribed using Superscript reverse transcriptase (Gibco BRL, Paisley, UK) following the manufacturer's instructions. The primers 5′-GCTTCCGCCTTTACTCTTTC (GiPT), 5′-TAGAGCCCAGTATCTGAGG (Gi-1) and 5′-CCACCTTCTTCATATTCACC (β-tubulin) were used to prime the RT reactions. The RT treatments were first standardized by polymerase chain reaction (PCR) based on their β-tubulin content using the 5′ primer 5′-TTTCCGTTCTCTCTCAGTTC and the β-tubulin 3′ primer described above at a cycle number that corresponded to the linear phase of the PCR reaction (predetermined empirically, see Burleigh, 2001). Quantitative PCR of GiPT and Gi-1 was then carried out with the standardized volumes using the 5′ primers 5′-CGTTCCTGGTTATTGGGTTAC (GiPT) and 5′-ATAAGGAAAGCGAGATGTCTG (Gi-1) and their corresponding 3′-primers. The following cycle conditions were used: 94°C for 5 min followed by 30 cycles at 94°C for 30 s, 54°C for 30 s and 72°C for 30 s. As with the β-tubulin PCR, the cycle number chosen to PCR GiPT and Gi-1 corresponded to the linear phases of their reactions. Products were gel electrophoresed and band intensity calculated using a Geldoc 2000 imager with Quantity One, v. 4 software (Bio-Rad, Marnes-la-Coquette, France). The approximate product size for all PCR products was 500 bp, as predicted by their respective cDNA sequences. Hyphae collected from three split-plates were subjected to independent RNA isolations. Hence, each estimate of gene expression was based on the average of three replicates.

Statistics

Averages and standard errors were calculated. Data were analysed for differences between treatments with a two-way anova.

Results

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

Mycelium N/P availability study

This study was designed for testing if mycelium N availability influenced the mycelium irrespective of P availability. Liquid medium N content was c. 1 mm for the control treatment and 2 mm for the highN treatment at the time of harvest (Table 1) compared with N concentrations of 3.2 mm N and 5.2 mm N, respectively, at the beginning. All P in the liquid medium was depleted at the end of the experiment in the highN treatment. Phosphorus concentration of the medium was, at harvest, still under influence of the original P application (Table 1). Root nutrient concentration was influenced by the nutrient applications to the liquid medium. Root P was nearly twice as large in the highP treatment compared with the control treatment and N concentration was slightly increased by the highN treatment.

Table 1.  Mycelium nitrogen (N)/phosphorus (P) availability study
 Liquid medium nutrient conc.Root nutrient contentDry mass13C-enrichment (%)
N (mm)P (µm)N (mg g−1 d. wt)P (mg g−1 d. wt)Roots (mg)Mycelium (mg)AM fungal hyphaeNLFA 16 : 1ω5PLFA 16 : 1ω5
  1. Roots collected from the solid medium were analysed for their nutrient content and dry mass, while mycelium was collected from the liquid medium compartment and analysed for dry mass and total 13C-enrichment as well as 13C-enrichment in neutral lipid (NLFA) and phospholipid (PLFA) fatty acids extracted from the same mycelia. Results were analysed with a two-way anova (means ± SE; ns, no significant differences; nd, not detectable). AM, arbuscular mycorrhiza.

Control0.91 ± 0.34 45 ± 3519 ± 1.70.9 ± 0.148 ± 5.8 1.8 ± 0.552.4 ± 0.442.1 ± 0.285.3 ± 0.73
High P 1.3 ± 0.55150 ± 5816 ± 1.11.6 ± 0.255 ± 2.9 1.1 ± 0.387.9 ± 1.18.0 ± 2.2 11 ± 1.6
High N 2.2 ± 0.60nd21 ± 0.71.1 ± 0.154 ± 1.8 1.2 ± 0.363.5 ± 0.942.2 ± 0.354.6 ± 0.73
High P and N 2.4 ± 0.36 74 ± 3221 ± 1.51.9 ± 0.251 ± 5.90.78 ± 0.315.2 ± 1.44.8 ± 0.876.6 ± 1.3
anova         
(P-value)         
N< 0.05ns< 0.05nsnsnsnsns< 0.05
Pns< 0.05ns< 0.001nsns< 0.01< 0.01< 0.01
N × Pnsnsnsnsnsnsnsnsns

The 13C-enrichment in total C of AM fungal hyphae growing in liquid medium was increased by high P availability (Table 1), while N availability did not influence the 13C-enrichment in hyphal carbon. The 13C-enrichment in the AM fungal signature NLFA 16 : 1ω5 showed a pattern very similar to the 13C-enrichment in total hyphal carbon. This was also true for 13C-enrichment in phospholipid fatty acid (PLFA) 16 : 1ω5, but the 13C-enrichment, however, was higher than in total hyphal C and in NLFA 16 : 1ω5. 13C-enrichment in PLFA 16 : 1ω5 was significantly reduced by high N availability (Table 1).

By using the 13C-enrichment in NLFA 16 : 1ω5 from both extraradical mycelium in the liquid compartment and intraradical mycelium in the colonized roots from the solid compartment, we calculated the C flow to this AM fungal signature in the total mycelium. There was no significant effect of N or P treatment on the C-flow to the mycelium (Fig. 2a).

image

Figure 2. Mycelium nitrogen (N)/phosphorus (P) availability study. (a) Total carbon flow from the root to the mycorrhizal mycelium (intraradical and extraradical). Carbon flow was measured as µg 13C incorporated into the neutral lipid fatty acid (NLFA) 16 : 1ω5 (mean values, ± SE, n = 4, no significant treatment effects on carbon flow as revealed by the two-way anova). (b) Acid (ACP) and alkaline (ALP) phosphatase active proportion of extraradical mycelium (mean values, ± SE, n = 4, significant effect of P treatment on ALP active hyphae: P < 0.05). Filling of bars are as indicated in panel (a).

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Microscopic investigations of the mycelium in the liquid medium showed that less than half of the length of hyphae contained an intact cytoplasm, except for the highP treatment, where 60% contained an intact cytoplasm (data not shown). The proportion of hyphal length with ALP activity was larger than that with ACP activity for all treatments (Fig. 2b). We could observe that higher P content of the medium increased the ALP activity compared with that at lower P (Fig. 2b). At higher N content there was a reduced ALP activity compared with the lower N, but an increased ACP activity. No significant interaction between N and P treatment was found.

Root N availability study

In this study, we tested if root N availability influenced mycelium growth by applying N treatments either to the root available solid medium or to the mycelium available liquid medium. The liquid medium with high N availability was much depleted for most of the new N at harvest and the same was true for the lowLM treatment, but differences between the two treatments remained at the time of harvest (Table 2). A high N availability to the solid root medium reduced the amount of the extraradical mycelium in the liquid medium and intraradical mycelium compared with the lowSM treatment. This was shown by both the PLFA (data not shown) and NLFA 16 : 1ω5 content and for the extraradical mycelium also by the fungal dry weight (Table 2). The reduced N availability of the liquid medium did not influence the amount of either extraradical or intraradical mycelium (Table 2). The lowLM treatment in the liquid medium increased the 13C-enrichment in hyphal carbon, while the N treatment to the solid medium did not.

Table 2.  Root nitrogen (N) availability study
 RootsLiquid mediumMyceliumIn roots13C-enrichment in mycelium
 Dry mass (mg)N content (mm)Dry mass (mg)NLFA 16 : 1ω5 (nmol)NLFA 16 : 1ω5 (nmol mg−1)(%) AM fungal hyphaeNLFA 16 : 1ω5PLFA 16 : 1ω5
  1. Low or high N treatment implemented in the liquid medium (LM) containing mycelium or solid medium (SM) containing mycorrhizal roots. Mycelium collected form liquid medium was analysed for dry mass and fatty acid content and both total mycelium and fatty acid were analysed for 13C-enrichment. Neutral lipid fatty acid (NLFA) 16 : 1ω5 was also analysed in roots collected from the solid compartment. Results were analysed with a two-way anova (means ± SE; ns, no significant differences). AM, arbuscular mycorrhiza; PLFA, phospholipid fatty acid.

LowLM/LowSM47 ± 2.2 0.11 ± 0.043 1.4 ± 0.49 940 ± 360120 ± 192.3 ± 0.41 1.9 ± 0.334.20 ± 0.62
LowLM/HighSM36 ± 8.40.092 ± 0.0290.68 ± 0.13 390 ± 72 50 ± 52.9 ± 0.62 2.4 ± 0.314.39 ± 0.72
HighLM/LowSM43 ± 7.3 0.46 ± 0.043 1.9 ± 0.621300 ± 530120 ± 191.2 ± 0.270.87 ± 0.102.69 ± 0.44
HighLM/HighSM37 ± 5.1 0.37 ± 0.0570.54 ± 0.18 370 ± 94 42 ± 111.6 ± 0.56 1.0 ± 0.162.72 ± 0.44
anova
(P-value)
SMnsns< 0.01< 0.01< 0.001nsnsns
LMns< 0.001nsnsns< 0.05< 0.001< 0.05
SM × LMnsnsnsnsnsnsnsns

The high N availability in the solid medium reduced the total C-flow to the AM fungal mycelium as indicated by excess 13C in NLFA 16 : 1ω5, while the N treatments in the liquid medium had no significant effect on the total C-flow (Fig. 3a).

image

Figure 3. Root nitrogen (N) availability study. Low or high N treatment implemented in the liquid medium (LM) containing mycelium or solid medium (SM) containing mycorrhizal roots. (a) Total carbon flow from the root to the mycorrhizal mycelium (intraradical and extraradical). Carbon flow was measured as µg 13C incorporated into the neutral lipid fatty acid (NLFA) 16 : 1ω5 (mean values, ± SE, n = 4, significant effect of solid medium N treatment: P < 0.001). (b) Acid (ACP) and alkaline (ALP) phosphatase active proportion of extraradical mycelium (mean values, ± SE, n = 4, no significant treatment effects). Filling of bars are as indicated in panel (a).

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More than half of the hyphae in the liquid medium contained an intact cytoplasm, and this proportion was slightly higher with a reduced N availability in the liquid medium (data not shown). The extraradical mycelium of all treatments contained low amount of poly P, but it was not related to the nutrient availability to the extraradical mycelium (data not shown). No treatment-related differences in ALP activity were observed.

A low amount of acid phosphatase was released into the growth medium of the extraradical mycelium, but the activity in neither of the studies was related to the nutrient availability (data not shown). No external alkaline phosphatase activity or chitinase activity were detected in the liquid medium.

Gene induction study

The semiquantitative RT-PCR experiment was carried out to measure transcript levels of the phosphate transporter GiPT (AF395112), the N-regulated gene Gi-1 (AJ315337) and a β-tubulin (BE603903) in the fungal hyphae. The results showed that the P transporter GiPT was downregulated in the extraradical mycelium under conditions of N limitation (Fig. 4A). However, when the mycorrhizal roots had access to high N availability in the solid medium GiPT transcript levels increased (P < 0.05, Fisher's LSD). Also, when the extraradical mycelium had access to higher N availability, no such downregulation was observed. The expression of Gi-1 was more variable, but it appeared that Gi-1 was downregulated in the extraradical mycelium when N was available, in particular when both the roots and the extraradical mycelium had access to higher N availability. A representative gel is shown in Fig. 4c.

image

Figure 4. Gene expression of GiPT and Gi-1 in extraradical mycelium exposed to low or high nitrogen (N) treatment implemented in the liquid medium (LM) containing mycelium or solid medium (SM) containing mycorrhizal roots, as measured by transcripts levels estimated by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). (A) GiPT transcription levels relative to β-tubulin (mean values ± SE, n = 3, significant effect of liquid medium N treatment: P < 0.01; and a significant interaction: P < 0.05). (B) Gi-1 transcription levels relative to β-tubulin (mean values ± SE, n = 3, no significant treatment effects). (C) One representative gel of the three used for estimating expression levels.

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Discussion

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

This study shows that increased N availability to the root had significant impact on the C allocation to the AM fungal mycelium, while the direct effects of N availability on AM fungal growth are limited. The gene expression responses were related to the N availability of both the mycelium and the root, showing that the mycelium does respond to the N availability in the external environment.

The study confirms the negative effects of increased N on AM fungal root colonization found earlier (Bååth & Spokes, 1989) and spore abundance in field vegetation (Johnson et al., 2003). Both AM colonization and sporulation are parameters indicating the C allocation to AM fungi and therefore one may say also that these studies indicate that high N availability may reduce plants allocation to the AM fungi. It was very clear that it is mainly an increased N availability to the roots that reduced the C allocation to the AM fungal mycelium. Application of N to the mycelium in the liquid medium had much less of an effect compared with when applied directly to the root. Where both P and N are concerned it is clear that moderate levels of fertilization can stimulate AM fungal abundance and the responses to nutrients can thus be assumed to be rather complex (Olsson et al., 1997; Treseder & Allen, 2002). Treseder & Allen, 2002 showed that both P and N can enhance AM fungal growth when these nutrients are limiting, while at fertile sites AM fungal growth are reduced at further nutrient application. In ectomycorrhizal plants N availability changes the C allocation by reducing the amount of C allocated to storage. This increases C allocation to consumption-related N assimilation and shoot growth (Wingler et al., 1994; Wallenda et al., 1996), and may reduce extramatrical ectomycorrhizal mycelium growth (Arnebrant, 1994). Wallander (1995) gave an alternative explanation to the reduced ectomycorrhizal colonization at high N availability. His argument was that high N availability consumed much of the carbon allocated to mycorrhizal fungi and that the fungi could not regulate the allocation to N uptake. Our results show that in the AM fungal symbiosis, it is mainly the N availability to the root that reduces the C availability to the fungus. The results are thus in accordance with the effects seen for P availability that P had a negative influence only when available to the plant host (Sanders, 1975). In a meta-analysis of field experiments it was found that N fertilization decreased mycorrhizal abundance by 15% on average although effects varied between experiments (Treseder, 2004). This supports that the negative effects seen at the laboratory scale also have significance for the interpretation of field effects. At the ecosystem-level N fertilization to forest soil reduce soil respiration (Nohrstedt et al., 1989). The general explanation has been that the N-limited vegetation consumes more of the newly fixed C and less is allocated to rhizosphere microorganisms. This explanation corresponds well with the finding that newly fixed C drives large part of the soil respiration (Högberg et al., 2001). It means that any changes in allocation pattern within plants will rapidly affect the root colonizing fungi since they to a large extent depend on recent assimilates.

Studies of direct responses of AM fungal mycelium to different substrates are few (Olsson et al., 2002a). A recent study showed that an AM fungus does change its growth pattern in relation to N availability (Bago et al., 2004). When applied to solid medium in compartments of monoxenic cultures only reached by the fungus nitrate increased both hyphal growth and sporulation, while the opposite effect was seen for ammonium. It has been shown that AM fungal mycelium proliferates in the presence of organic matter, dry yeast or albumin, in contrast to simple C sources such as starch or cellulose (St John et al., 1983a, 1983b; Joner & Jakobsen, 1995; Larsen & Jakobsen, 1996; Ravnskov et al., 1999). The response of the extraradical AM fungal mycelium to P additions has been unclear (Olsson et al., 2002a, 2002b) and difficult to study because there seems to be a negative feedback mechanism between C availability for AM fungi and plant P status (Peng et al., 1993). Despite their central role in P nutrition of the symbiosis, the AM fungi do not seem to actively forage for P (Li et al., 1991; Olsson & Wilhelmsson, 2000). Carbon allocation to extraradical hyphae in response to P addition was estimated as 13C-enrichment in the NLFA 16 : 1ω5 in an in vitro system with transformed carrot roots after applying 13C-labelled glucose. The 13C-enrichment in the NLFA 16 : 1ω5 was negatively related to P concentration in roots (Olsson et al., 2002b).

Phosphorus availability may stimulate C allocation to the AM fungal mycelium on short-term, but in the long-term mainly negative effects can be seen in C allocation to the AM fungus (Olsson et al., 2002b). No such initial stimulatory effects could be seen in response to increased N availability and N also inhibited C allocation in cases when 13C-enrichment was stimulated by P availability. This indicates that C allocation to the AM symbiosis is not an important strategy to gain N uptake. Again, we could show that P starvation may enhance the total C allocation to the colonizing AM fungus, while both high P and N conditions can reduce the AM allocation in plants. We found increased C-enrichment at high P and this is partly contradictory to earlier findings. Recent studies showed that this contradiction may arise from the timing of harvests (P. A. Olsson et al., unpublished). Initially we found increased C allocation towards the P-enriched medium, but when cultures aged higher P status reduced C allocation to the AM fungus. This shows the complexity in C allocation and also that the total C allocation to AM fungi is a critical parameter when estimating the outcome of the symbiosis for the fungus. Therefore the signature lipid method is particularly good since it is the only method that can differentiate fungal C-enrichment in roots from that of the root itself (Olsson et al., 2005).

Phosphorus-regulated gene expression in AM fungi includes phosphatases (Kaffman et al., 1994), P transporters (Versaw, 1995) and proteins related to polyphosphate metabolism (Ogawa et al., 2000). One such gene is the high affinity P transporter GiPT from G. intraradices (Maldonado-Mendoza et al., 2001), which is expressed in the extraradical mycelium in response to P demand (Harrison & van Buuren, 1995). Here, we showed that GiPT expression was also influenced by N availability, whereby high N availability resulted in higher levels of GiPT expression. Such results are in agreement with our current understanding of the ‘dilutive effect’ that can occur when the sudden availability of one primary limiting nutrient leads to growth and thus to an increased demand of other limiting nutrients (Timmer & Leyden, 1980; Clark & Zeto, 2000). In this case, it appeared that N availability increased fungal growth and the demand for P, which subsequently induced the GiPT P transporter gene. Our results strengthen evidence that GiPT has a relatively complex transcriptional regulation, since Maldonado-Mendoza et al. (2001) found that this gene, while upregulated by P demand, is actually turned off when no P is available, perhaps as a means to conserve uptake efforts. Thus, the regulation of GiPT by N nutrition does not come as a complete surprise.

However, one surprise was that GiPT's upregulation by high N availability to the extraradical mycelium occurred when N was applied to not only to the extraradical mycelium-side, but also the mycorrhizal root side of the split plate. Hence, N or another signal must have crossed over from the root-side of the split-plate to influence gene expression in the extraradical mycelium. Such results suggest that the AM fungal mycelium possess the ability to communicate effects that occur near or at the mycorrhizal roots to the extraradical mycelia. While communication in this direction was also indicated in an earlier study involving secondary compounds, whereby poly P was detected in extraradical mycelia growing in a P-free environment (Olsson et al., 2002b), the results shown here demonstrate that effects at/near the mycorrhizal root can also influence gene expression in the external hyphae.

Gi-1 was identified by Ruiz-Lozano et al. (2002) as an AM fungal gene expressed in colonized lettuce roots and upregulated in intraradical mycelium following increased N availability. Here we show that this gene is also expressed in the extraradical mycelium of G. intraradices; however, we found no statistically significant corresponding positive trend. Rather our results suggested that, at least in our system, Gi-1 is either not influenced by N or this gene is actually slightly down-regulated in external hyphae by N additions. Hence, Gi-1 may have a function not directly related to the N metabolism, but rather with some other process associated with fungal N nutrition, although this varied between our system and that of Ruiz-Lozano et al. (2002). Breuninger et al. (2004) studied an N-related AM fungal gene with known function, the glutamine synthetase gene. Surprisingly, they found no regulation of transcription levels by N supply.

In summary, our results support the hypothesis that a high-nutrient regime's negative impact on AM fungi results from less C allocation and that the mechanism behind this is an increased C immobilization in the plant. We conclude that plant N availability may reduce C-flow to the AM fungus in the same way as P availability may do and that localized nutrient availability may stimulate the AM fungus near the enriched patch just as was earlier found in whole-plant systems (Gavito & Olsson, 2003).

Acknowledgements

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

This work was supported by The Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (Formas).

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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