Biochemical and molecular characterization of NADP‐glutamate dehydrogenase from the ectomycorrhizal fungus Tuber borchii

Abstract

Summary• NADP-glutamate dehydrogenase (NADP-GDH) from Tuber borchii was purified and the corresponding gene was cloned in order to elucidate the physiological role of the enzyme in this ectomycorrhizal fungus.• NADP-GDH was purified using an anion-exchange column followed by affinity chromatography. The complete gene was cloned from a 30-d-old-mycelium cDNA library and characterized.• T. borchii NADP-GDH appears to be physically and kinetically similar to those from other fungi and the deduced amino acid sequence of the gdh gene showed a significant similarity to other fungal NADP-dependent GDHs. Biochemical and Northern blotting analyses carried out with mycelia grown on different nitrogen sources clearly showed that the regulation of T. borchii NADP-GDH in response to different nitrogen sources was markedly different from the responses of the NADPGDHs of other ascomycetes. Northern blotting analyses highlighted that the gdh gene was also expressed in the symbiotic phase.• The biochemical and molecular data suggest that the fungal NADP-GDH contributes to the primary nitrogen metabolism in the ectomycorrhizal tissues.

Gene expression profiling of the nitrogen starvation stress response in the mycorrhizal ascomycete Tuber borchii

“…A number of genes coding for inorganic N transporters and N assimilation enzymes have been isolated from these organisms (Guescini et al, 2003;Jargeat et al, 2000Jargeat et al, , 2003Javelle et al, 2001Javelle et al, , 2003bJuuti et al, 2004;Montanini et al, 2002Montanini et al, , 2003Vallorani et al, 2002). Most of them display the same kind of N status responsiveness previously documented in non-mycorrhizal fungi (Magasanik and Kaiser, 2002;Marzluf, 1997).…”

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

“…In practice, Govindarajulu et al [20] demonstrated N assimilation in the ERM via the GS/GOGAT pathway by measuring mRNA levels for key enzymes in the ERM and IRM tissues with quantitative real-time polymerase chain reactions. They also demonstrated the expression of a putative nicotinamide adenine dinucleotide (NAD)-dependent glutamate dehydrogenase (GDH) gene, which is down-regulated in the ERM tissues supplied with either 3 NO − or NH 4 + in the fungal compartment, consistent with this enzyme playing a catabolic role [21] . Also they found the involvement of the catabolic arm of the urea cycle in transcript levels of genes with a high similarity to known ornithine aminotransferase, urease accessory protein and NH 4 + transporters which are preferentially expressed in the IRM.…”

Functional properties and differential mode of regulation of the nitrate transporter from a plant symbiotic ascomycete

“…This renewed perception of the ecological importance of ectomycorrhizae has revived efforts to elucidate the molecular processes underlying nitrogen assimilation and the related starvation stress responses in ectomycorrhizal fungi [13][14][15]. A number of genes coding for ammonium transporters and nitrogen assimilation enzymes have been isolated from these organisms [16][17][18][19][20][21][22]. In contrast, only one gene coding for an as yet functionally uncharacterized NT has been identified so far in a mycorrhizal fungus, the basidiomycete Hebeloma cylindrosporum [23].…”