Regulation of the nitrogen transfer pathway in the arbuscular mycorrhizal symbiosis: gene characterization and the coordination of expression with nitrogen flux

Abstract

The arbuscular mycorrhiza (AM) brings together the roots of over 80% of land plant species and fungi of the phylum Glomeromycota and greatly benefits plants through improved uptake of mineral nutrients. AM fungi can take up both nitrate and ammonium from the soil and transfer nitrogen (N) to host roots in nutritionally substantial quantities. The current model of N handling in the AM symbiosis includes the synthesis of arginine in the extraradical mycelium and the transfer of arginine to the intraradical mycelium, where it is broken down to release N for transfer to the host plant. To understand the mechanisms and regulation of N transfer from the fungus to the plant, 11 fungal genes putatively involved in the pathway were identified from Glomus intraradices, and for six of them the full-length coding sequence was functionally characterized by yeast complementation. Two glutamine synthetase isoforms were found to have different substrate affinities and expression patterns, suggesting different roles in N assimilation. The spatial and temporal expression of plant and fungal N metabolism genes were followed after nitrate was added to the extraradical mycelium under N-limited growth conditions using hairy root cultures. In parallel experiments with (15)N, the levels and labeling of free amino acids were measured to follow transport and metabolism. The gene expression pattern and profiling of metabolites involved in the N pathway support the idea that the rapid uptake, translocation, and transfer of N by the fungus successively trigger metabolic gene expression responses in the extraradical mycelium, intraradical mycelium, and host plant.

Figure 1.

Working model of N transport and metabolism in the symbiosis between plant roots and arbuscular mycorrhizal fungi. N moves (black arrows) from the soil into the fungal ERM, through a series of metabolic conversion reactions into Arg, which is transported into the IRM within the root (host) and there is broken down; N is transferred to and assimilated by the host as ammonia. Red circles refer to the sites of action of the genes identified and analyzed in this study. Blue arrows indicate mechanisms hypothesized to regulate gene expression by N metabolites involved in the pathway.

Figure 2.

Heterologous expression, purification, and enzymatic kinetics of the two GS isoforms identified, GiGS1 and GiGS2. A, Western blot of the expressed GiGS1 and GiGS2 in yeast. Whole cell protein extracts from the yeast transformed with GiGS1 and GiGS2 were fractionated on a denaturing 12% polyacrylamide gel, transferred to polyvinylidene fluoride membranes, and detected with Anti-His(C-term)-HRP antibody. The yeast knockout mutants were independently transformed with the empty pYES2.1 vector or the vector fused with GiGS1 or GiGS2. Lanes a and c, The expression of GiGS on the medium without Gal; lanes b and d, the expression of GiGS on the medium with Gal; lane e, the expression of the empty vector on the medium with Gal (as a negative control). B, The purification stages of GiGS1 and GiGS2. Purified proteins were subjected to SDS-PAGE (12% NuPAGE Novex Bis-Tris Gel) and silver stained. Low-M_r markers are in lane f. Crude yeast extracts before purification were loaded in lane e, and successive elution fractions were loaded in lanes a to d. Each lane contained 1 μg of protein. C, Enzymatic kinetics for GiGS1 and GiGS2 detected by synthetase assay using the purified proteins. Biosynthetic GS activity assays were carried out by varying the concentration of Glu in the reaction mixture. Plots of 1/V versus [S] are shown. Velocity data, expressed as proportions of V_max, were based on the three biological replicates; absolute V_max values were 3.68 and 4.01 μm min⁻¹ mg⁻¹ for GiGS1 and GiGS2, respectively. Lineweaver-Burk plots of the data were obtained with GiGS1 (K_m = 1.87 mm, r² = 0.99) and GiGS2 (K_m = 3.80 mm, r² = 0.999).

Figure 3.

Functional complementation of S. cerevisiae knockout mutants using the genes identified from G. intraradices. M, The knockout mutant expressing the empty vector; M:GiXXX, the knockout mutant expressing the GiXXX gene; WT, the wild type. A, GiGS1 and GiGS2. Left, Growth of colonies on rich medium plus 2% Gln; right, medium without Gln. B, GiASS. Left, Minimal medium plus all essential amino acids including 0.01% l-Arg; right, minimal medium with all essential amino acids excluding l-Arg. In A and B, 5 μL of cells was used for spreading on the plate. The optical density at 600 nm values for the cells are 1, 0.1, 0.01, 0.001, and 0.0001 from the left to the right columns. C, GiCAR1. Left, Minimal medium with all essential amino acids including 0.01% l-Arg; right, medium with 0.01% l-Arg as the sole N source. D, GiOAT1. Left, Medium with all essential amino acids; right, medium with only 0.01% Orn and Leu as N sources. E, GiODC. Left, Medium with 0.001% Leu, His, Lys, and putrescine as N sources; right, medium with 0.001% Leu, His, and Lys as N sources.

Figure 4.

The expression of primary N metabolic and transport genes in the arbuscular mycorrhizal symbiosis after the addition of 4 mm KNO₃ to the fungal ERM. Bars are as follows: 2 h (white bars), 4 h (black bars), 8 h (dotted bars), 24 h (gray bars), and 72 h (hatched bars). Gene expression was measured by quantitative real-time PCR. A and B, ERM (A) and IRM (B) with fungal ribosomal protein S4 gene expression as the reference. C, Carrot GS expression in the colonized root tissue, with the expression of elongation factor 1-α from carrot used as the reference.

Figure 5.

N movement from the ERM to the IRM in mycorrhizal carrot roots based on the timing of labeling free amino acids in the mycorrhizal roots after the addition of 4 mm K¹⁵NO₃ to the fungal ERM. A, The percentage of total free amino acids represented by Arg (including ¹⁵N-labeled and unlabeled molecules; white bars) and the percentage of total free amino acids represented by only ¹⁵N-labeled Arg (black bars) in the colonized roots (plant plus IRM tissue) after 4 mm K¹⁵NO₃ was applied to the ERM. B, The percentage labeling in the free amino acids in the mycorrhizal roots at 4 h (white bars), 8 h (black bars), 24 h (gray bars), and 72 h (hatched bars) after adding 4 mm K¹⁵NO₃ to the ERM compartment. GABA, γ-Aminobutyrate.