Enzymatic evidence for the key role of arginine in nitrogen translocation by arbuscular mycorrhizal fungi

Abstract

Key enzymes of the urea cycle and (15)N-labeling patterns of arginine (Arg) were measured to elucidate the involvement of Arg in nitrogen translocation by arbuscular mycorrhizal (AM) fungi. Mycorrhiza was established between transformed carrot (Daucus carota) roots and Glomus intraradices in two-compartment petri dishes and three ammonium levels were supplied to the compartment containing the extraradical mycelium (ERM), but no roots. Time courses of specific enzyme activity were obtained for glutamine synthetase, argininosuccinate synthetase, arginase, and urease in the ERM and AM roots. (15)NH(4)(+) was used to follow the dynamics of nitrogen incorporation into and turnover of Arg. Both the absence of external nitrogen and the presence of L-norvaline, an inhibitor of Arg synthesis, prevented the synthesis of Arg in the ERM and resulted in decreased activity of arginase and urease in the AM root. The catabolic activity of the urea cycle in the roots therefore depends on Arg translocation from the ERM. (15)N labeling of Arg in the ERM was very fast and analysis of its time course and isotopomer pattern allowed estimation of the translocation rate of Arg along the mycelium as 0.13 microg Arg mg(-1) fresh weight h(-1). The results highlight the synchronization of the spatially separated reactions involved in the anabolic and catabolic arms of the urea cycle. This synchronization is a prerequisite for Arg to be a key component in nitrogen translocation in the AM mycelium.

Figure 1.

Specific enzyme activity in the ERM of G. intraradices and AM roots of carrot grown in an in vitro compartmented system after exposing the ERM to (NH₄)₂SO₄ at nil (white symbols), 0.1 (gray symbol), or 5 mm (black symbols). Enzyme activity was normalized against protein concentration of the sample. Symbols with vertical bars represent means and sd (n = 3).

Figure 2.

Specific enzyme activity in the ERM of G. intraradices and AM roots of carrot grown in an in vitro compartmented system after exposing the ERM to 5 mm (NH₄)₂SO₄ (black symbols), 5 mm (NH₄)₂SO₄ plus 20 mm l-norvaline (an inhibitor of Arg synthesis; white symbols), and 5 mm (NH₄)₂SO₄ plus 500 μm PPDA (an inhibitor of urease activity; gray symbols). Enzyme activity was normalized against protein concentration of the sample. Symbols with vertical bars represent means and sd (n = 3).

Figure 3.

Time course of Arg concentration in the ERM of G. intraradices after exposure to 5 mm (NH₄)₂SO₄ in an in vitro compartmented root culture system.

Figure 4.

Time course of ¹⁵N abundance in AM roots of carrot, in the ERM of G. intraradices, and in Arg from the ERM. The ERM was exposed to 5 mm (NH₄)₂SO₄ with 82% ¹⁵N at 0 h. Solid lines indicate the stationary levels attained.

Figure 5.

Isotopomer distribution (¹⁵N₀ [m/z 175], ¹⁵N₁ [m/z 176], ¹⁵N₂ [m/z 177], ¹⁵N₃ [m/z 178], and ¹⁵N₄ [m/z 179]) of Arg in the ERM after 12 h (gray). The distribution is compared with that of unlabeled Arg (black) and the binomial distribution probability calculated on the basis of 82% ¹⁵N (white).

Figure 6.

MS/MS analysis of ¹⁵N₂-Arg (m/z 177) in the ERM after 4 h. Principal fragmentations are indicated on the formula inserted; the m/z values indicated correspond to fragments observed from nonlabeled Arg (m/z 175).

Figure 7.

Time course of depletion of unlabeled Arg from the ERM of G. intraradices grown in an in vitro compartmented system. The ERM was exposed to 5 mm (NH₄)₂SO₄ with 82% ¹⁵N at 0 h. The proportions of unlabeled Arg were log transformed.

Figure 8.

Schematic outline of the results obtained. Ammonium is taken up by the ERM and assimilated via GS into Gln. ASS is the key enzyme for the biosynthesis of Arg. The presence of ammonium in the ERM inhibits the activity of arginase and urease. Arg concentration in the cytoplasm is maintained by compartmentation into the vacuole, where Arg can be transported toward the IRM. The transfer of nitrogen from the ERM into the IRM was estimated as 3 nmol nitrogen g⁻¹ fresh weight h⁻¹. The IRM is deficient in nitrogen, which favors the activity of arginase and urease leading to the production of ammonium, which is transferred to the root where it is integrated into Gln through the action of GS.