Arginine bi-directional translocation and breakdown into ornithine along the arbuscular mycorrhizal mycelium

Abstract

Bi-directional translocation and degradation of Arginine (Arg) along the arbuscular mycorrhizal (AM) fungal mycelium were testified through (15)N and/or (13)C isotopic labeling. In vitro mycorrhizas of Glomus intraradices and Ri T-DNA-transformed carrot roots were grown in dual compartment Petri dishes. [(15)N- and/or(13)C]Arg was supplied to either the fungal compartment or the mycorrhizal compartment or separate dishes containing the uncolonized roots. The levels and labeling of free amino acids (AAs) in the mycorrhizal roots and in the extraradical mycelia(ERM) were measured by gas chromatography/mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC). The ERM of AM fungi exposed in either NH(4) (+) or urea as sole external nitrogen source had much higher (15)N enrichment of Arg, compared with those in nitrate or exogenous Arg; however, glycerol supplied as an external carbon source to the ERM had no significant effect on the level of Arg in the ERM. Meanwhile, Arg biosynthesized in the ERM could be translocated intact to the mycorrhizal roots and thereby the level of Arg in the mycorrhizal roots increased to about 20% after culture of ERM in 4 mmol/L NH(4) (+) for 6 weeks. Also Arg was found to be bi-directionally transported along the AM fungal mycelium through [U-(13)C]Arg labeling either in the mycorrhizal compartment or in the fungal compartment. Once Arg was translocated to the potential N-limited sites, it would be further degraded into ornithine (Orn) and urea since either [U-(13)C] or [U-(15)N/U-(13)C]Orn was apparently shown up in the mycorrhizal root tissues when [U-(13)C] or [U-(15)N/U-(13)C]Arg was labeled in the fungal compartment, respectively. Evidently Orn formation indicated the ongoing activities of Arg translocation and degradation through the urea cycle in AM fungal mycelium.

Chromatographic Analysis of Nitrogen Utilization and Transport in Arbuscular Mycorrhizal Fungal Symbiosis

“…This result suggests that Arg is either taken up or synthesized by the ERM and transported intact to the IRM; the conclusion is also consistent with the outcome of other [U-13 C/U-15 N] Arg labeling experiments. [30] This finding indicates that the AM fungi take up N and incorporate it into Arg, and this organic form of N is then transported to IRM (Fig.1).…”

“…Chromatographic separations are used both for sample preparation and core analytical separation and measurements. To check nitrogen utilization and amino acids metabolism in AM fungal symbiotic system, free amino acids from cultured tissues can be extracted with a mixture of methanol/chloroform/water, then separated on a cation exchange column (DOWEX 50 *4-200, hydrogen form) and recovered after freeze-drying [28][29][30] .…”

“…To follow the uptake, assimilation and transfer of nitrogen in the arbuscular mycorrhizal symbiosis, we added isotopically labeled substrates to in vitro arbuscular mycorrhizal cultures of carrot (Daucus carota L.) roots colonized by G. intraradices. [28][29][30] When grown in divided Petri plates, this model mycorrhiza excludes other microorganisms and prevents diffusion of nonvolatile solutes between the compartments. This model system shows normal life cycle and development of fungal morphology.…”

“…1), GC-MS [30] analysis of Arg isotopomer found either [U- 13 C] or [U-15 N/U-13 C] ornithine(Orn) in the mycorrhizal root tissues. This result demonstrates that once Arg is translocated to the potential N-limited sites in the mycelium of AM fungi, it is degraded into Orn and urea.…”

“…Analysis of 15 N-labeled Glu and Gln isotopomers using GC-MS has demonstrated that, following Arg degradation, Orn is recycled to Glu and Gln, which serve as C donors. Small amounts of 15 N label have been found in Glu and Gln (in spite of their low levels) after [guanido-15 N2] Arg translocation from the ERM to the mycorrhizal roots, whereas other amino acids have displayed only negligible 15 N labeling [30] . Thus, the Orn cycle seems to be an efficient path for Arg biosynthesis, nitrogen transfer to the host, and carbon recycling in AM symbiosis.…”