Progress 09/15/02 to 12/31/04
Outputs
Studies have shown mycorrhizae enhance the N nutrition of their hosts although little is known about the mechanisms of uptake of various forms of N, and virtually nothing is known about its movement and transfer from the fungus to the host. Using 15N labeled ammonium and doubly labeled (13C and 15N) arginine in conjunction with mass spectrometric analysis of amino acids in an in vitro culture system we have discovered the operation of a novel metabolic route in which nitrogen is moved by the AM fungus from the soil to its host. We observed that doubly labeled arginine is taken up by the extraradical fungal mycelium and is translocated to the intraradical mycelium, however only 15N and no 13C is transferred to the host as evidenced by the exclusive 15N enrichment in the host protein amino acids. This mechanism of nitrogen movement consists of metabolic processes known to operate in fungi (the assimilation of inorganic nitrogen) together with a new variant of the urea
cycle in which the anabolic and catabolic parts are separated by the long-distance translocation of arginine. The assimilation of N into arginine allows it to be moved in a concentrated (four nitrogens per molecule), non-toxic form; the use of the catabolic arm of the urea cycle allows the transfer of N to the host plant with minimal loss of carbon by the fungus. To further test the model we directly measured fungal mRNA levels for genes encoding key enzymes and transporters using quantitative real-time PCR. Glutamine synthase (GS) was shown to be expressed in both ERM and IRM tissues isolated from the carrot hairy root co-culture system. GS transcripts were more than two fold higher when NH4+ was substituted for NO3- in the ERM compartment of the split plate system. Transcript levels encoding a urease subunit (UAP) and ornithine aminotransferase (OAT) also were significantly induced in IRM tissues after ERM feeding of ammonium, consistent with the activation of the catabolic arm of
the urea cycle to release NH4+ from arginine. These same conditions lead to enhanced expression of an ammonium transporter (AMT) gene in the IRM tissue, consistent with translocation of inorganic nitrogen to the plant host. The expression of the fungal AMT, OAT and UAP genes was down-regulated in IRM tissues as the glucose provided to carrot roots was depleted by growth. Supplemental glucose reversed this trend, suggesting that regulation of fungal nitrogen translocation to the host is dependent on carbon availability. The high flux of N apparently conducted by these mechanisms invites a re-examination of the widely held view that N transfer in the AM symbiosis is of little or no nutritional importance to plants. We have also examined the uptake of other forms of nitrogen containing compounds and have discovered that these fungi are able to degrade soluble proteins and access nitrogen directly as amino acids . Although the fungi in the symbiotic state accepts carbon from the host
plant, it will not share it with the host or any other host plant it may colonize.
Impacts
By understanding the effects of AM fungi on nitrogen uptake, transport and metabolism by host plants, the use of nitrogen fertilizers may be better managed in terms of the forms, levels and timing of application. Such improved management would decrease costs and pollution associated with nitrogen application. The management of AM fungal populations in agriculture will also benefit from an improved understanding of their role in nitrogen cycling since large residues of N remain in our soils due to inefficient recovery by crops. These residues produce NOx and N2O emissions that contribute to stratospheric ozone depletion and global warming . Lastly, our inability to cultivate AM fungi axenically despite prolonged effort has greatly hindered the mass production of inoculum, and prevented the large-scale use of AM fungi in agriculture. We believe that a greater understanding of the regulation of the nitrogen flows in response to the development of this important symbiosis
is a necessary step in this endeavor.
Publications
- Jin, H. Pfeffer, P.E.*,Douds, D.D., Piotrowski, E., Lammers, P.J., Shachar-Hill, Y. 2005 The form of nitrogen stored and transported by the extraradical hyphae of an arbuscular mycorrhizal symbiosis New Phytologist (in press).
- Jun, J., Abubaker, J., Rehrer, C., Pfeffer, P.E., Shachar-Hill, Y., Lammers, P.J. 2002 Expression in an arbuscular mycorrhizal fungus of genes involved in metabolism, transport, the cytoskeleton and the cell cycle. Plant and Soil 244:141-148
- Exploring the Bidirectional Transfer of Carbon and Nitrogen Between Fungus and Host in the Arbuscular Mycorrhizal SymbiosisP.E.Pfeffer, D.D. Douds Jr D.P. Schwartz, M. Govindarajulu, H. Bucking, Y. Shachar-Hill International Conference on Microrrhizas (ICOM) Montreal Canada Aug 10-15, 2003 08/10/2003 #778 p704 Presentation
- The Effect of Root Exudates on Spore Germination, Uptake, metabolism and Gene Expression of the Arbuscular Mycorrhizal Fungus Glomus intraradices H. Bucking, J. Abubaker, M. Govindarajulu, P.E. Pfeffer, P. Lammers, Y. Shachar-Hill International Conference on Microrrhizas (ICOM) Montreal Canada Aug 10-15, 2003. 08/11/2003 #239 p282.
- The identification and expression of putative homologues of nitrogen metabolic genes in Glomus intraradices M Govindarajulu, J Abubaker, P.E. Pfeffer, JWB Allen, H Bucking, PJ LammersY Shachar-Hill international Conference on Microrrhizas (ICOM) Montreal Canada Aug 10-15, 2003. 0 8/13/2003 #268 p303
- Nitrogen and Carbon metabolism and transfer in the AM symbiosis. Abubaker J, Allen JWB, Bago B, Bucking H, Douds DD Jr, Govindarajulu M, Jin H, Jun J, Lammers PJ, Pfeffer PE, Shachar-Hill Y international Conference on Microrrhizas (ICOM) Montreal Canada Aug 10-15, 2003. 08/14/2003 #275 p310 Presentation
- Pfeffer, P.E., Douds D.D. Jr., Bucking, H., Schwartz, D. P., Shachar-Hill, Y. 2004 The fungus does not transfer carbon to or between roots in an arbuscular mycorrhizal symbiosis. New Phytologist 163:617-627
- Govindarajula, M. Pfeffer, P.E*., Jin H., Abubaker J., Douds, D.D., Allen, W., Bucking, H., Lammers, P.J., Shachar-Hill, Y. 2005 Nitrogen transfer in the arbusular mycorrhizal symbiosis. Nature (in press)
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Progress 09/15/02 to 03/14/03
Outputs
Studies have shown mycorrhizae enhance the N nutrition of their hosts although little is known about the mechanisms of uptake of various forms of N, and virtually nothing is known about its movement and transfer from the fungus to the host. We have cloned 5 genes that are activated in this process. Interestingly, these activated genes, four of which are associated with urea cycle strongly suggest that nitrogen is translocated from fungus to root by the movement of arginine. Labeled ammonium given to the external hyphae produces highly enriched arginine, which can be degraded inside the root-containing hyphae to ammonium and transferred to the host . Athough symbiotic AM fungi can efficiently supply host plants with nitrogen from the soil, lowering the need for excessive applications of fertilizer, it is unclear what forms of nitrogen can be utilized by these fungi. We have examined the uptake of various forms of nitrogen containing compounds and have discovered that
these fungi are able to degrade soluble proteins and access nitrogen directly as amino acids . We have also determined that (1) the fungus will accept carbon from the host plant, but will not share it with the host or any other plant it may colonize, (2) nitrogen and carbon compounds that are transferred from the host plant to the fungus do not come from the host plant cells but from the free space between the plant cells, (3) fungal DNA is synthesized within the host root and exported to the fungal tissue outside the host plant.
Impacts
By understanding the effects of AM fungi on nitrogen uptake, transport and metabolism by host plants, the use of nitrogen fertilizers may be better managed in terms of the forms, levels and timing of application. Such improved management would decrease costs and pollution associated with nitrogen application. The management of AM fungal populations in agriculture will also benefit from an improved understanding of their role in nitrogen cycling since large residues of N remain in our soils due to inefficient recovery by crops. These residues produce NOx and N2O emissions that contribute to stratospheric ozone depletion and global warming . Lastly, our inability to cultivate AM fungi axenically despite prolonged effort has greatly hindered the mass production of inoculum, and prevented the large-scale use of AM fungi in agriculture. We believe that a greater understanding of the regulation of the nitrogen flows in response to the development of this important symbiosis
is a necessary step in this endeavor.
Publications
- The Effect of Root Exudates on Spore Germination, Uptake, metabolism and Gene Expression of the Arbuscular Mycorrhizal Fungus Glomus intraradices H. Bucking, J. Abubaker, M. Govindarajulu, P.E. Pfeffer, P. Lammers, Y. Shachar-Hill international Conference on Microrrhizas (ICOM) Montreal Canada Aug 10-15, 2003. 08/11/2003 #239 p282.
- The identification and expression of putative homologues of nitrogen metabolic genes in Glomus intraradices M Govindarajulu, J Abubaker, P.E. Pfeffer, JWB Allen, H Bucking, PJ LammersY Shachar-Hill international Conference on Microrrhizas (ICOM) Montreal Canada Aug 10-15, 2003. 0 8/13/2003 #268 p303
- Nitrogen and Carbon metabolism and transfer in the AM symbiosis. Abubaker J, Allen JWB, Bago B, Bucking H, Douds DD Jr, Govindarajulu M, Jin H, Jun J, Lammers PJ, Pfeffer PE, Shachar-Hill Y international Conference on Microrrhizas (ICOM) Montreal Canada Aug 10-15, 2003. 08/14/2003 #275 p310 Presentation
- Exploring the Bidirectional Transfer of Carbon and Nitrogen Between Fungus and Host in the Arbuscular Mycorrhizal SymbiosisP.E.Pfeffer, D.D. Douds Jr D.P. Schwartz, M. Govindarajulu, H. Bucking, Y. Shachar-Hill international Conference on Microrrhizas (ICOM) Montreal Canada Aug 10-15, 2003. 08/10/2003 #778 p704 Presentation
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