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- Materials and Methods
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.
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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).