The research team, led by investigators studying the commercial cultivation of Morchella, formulated two culture media using quartz particles as an inorganic matrix to mimic soil conditions. All nutrients were identical between the two media except for the nitrogen source: one contained nitrate-nitrogen (NN) and the other ammonium-nitrogen (AN) as the sole nitrogen source. Nitrogen concentrations were set to approximate previously measured soil nitrogen levels in large-scale morel cultivation sites across China. A dikaryotic M. importuna strain widely cultivated on a commercial scale in China was inoculated into both media and incubated at 15 °C, reflecting typical soil temperatures during field cultivation. Samples were collected at five time points over 25 days post-inoculation, and mycelia grown for 15 days were selected for subsequent multi-omics and biochemical experiments.

Growth monitoring using the chloroform-fumigation method revealed that biomass carbon in the nitrate treatment peaked at 10 days post-inoculation, while the ammonium treatment peaked at 15 days. The peak biomass in nitrate medium was significantly lower than in ammonium medium, suggesting that nitrate imposes a higher energetic cost on the fungus. Stereomicroscopic observation showed no significant phenotypic differences between the two treatments when biomass was at its peak, indicating that the divergent effects of the two nitrogen forms operate at the molecular level rather than producing immediately visible morphological changes.

Transcriptomic profiling generated nearly 48 million reads across ten samples, with an average sequence alignment efficiency of approximately 85.5%. Of the 11,965 genes expressed, 2,073, or 17.3%, were identified as differentially expressed genes between the two treatments. On nitrate, 843 genes were upregulated while 1,230 were downregulated. As expected, genes encoding the nitrate transporter, nitrate reductase and nitrite reductase, all involved in nitrate uptake and reduction, were strongly upregulated in nitrate medium with fold changes exceeding 8. In contrast, NADP-glutamate dehydrogenase, a key enzyme in the alternative nitrogen assimilation pathway, was downregulated on nitrate. Glutamate synthase, or GOGAT, was marginally upregulated, suggesting a potential role in nitrogen assimilation when nitrate is the primary source.

Fungi assimilate nitrogen through two primary pathways. The first is the glutamine synthetase/glutamate synthase cycle, in which glutamine synthetase incorporates ammonium into glutamate to form glutamine, and GOGAT then transfers the amido group of glutamine to 2-oxoglutarate, yielding two glutamates. The second pathway involves NADP-dependent glutamate dehydrogenase, which directly catalyses the conversion of ammonium and 2-oxoglutarate into glutamate. The study found that nitrate inhibited both the gene expression and enzymatic activity of NADP-glutamate dehydrogenase while simultaneously increasing glutamate levels through the GS/GOGAT cycle. This confirmed that when nitrate is the sole nitrogen source, the GS/GOGAT pathway plays the dominant role in glutamate synthesis in morel mycelia, whereas ammonium preferentially activates the NADP-glutamate dehydrogenase pathway.

Proteomic analysis using tandem mass tag labelling and high-performance liquid chromatography coupled with tandem mass spectrometry identified 4,392 proteins, of which 270, or 6%, were differentially expressed between treatments. Among these, 174 proteins were upregulated in the nitrate treatment while 96 were downregulated. Consistent with the transcriptomic results, the nitrate transporter and nitrite reductase proteins were upregulated after nitrate treatment. KEGG enrichment analysis of the differentially expressed proteins identified five regulated pathways: nitrogen metabolism, tyrosine metabolism, thiamine metabolism, starch and sucrose metabolism, and the phosphatidylinositol signalling pathway. Gene set enrichment analysis further revealed that protein sets related to tyrosine metabolism and N-glycan biosynthesis were upregulated by nitrate, while protein sets for ribosome biogenesis and RNA transport were downregulated.

The downregulation of ribosome biogenesis and RNA transport pathways is particularly significant. A total of 37 proteins associated with ribosome biogenesis and 28 proteins involved in RNA transport were suppressed by nitrate treatment. These included 15 translation initiation factors, five ribonucleases, three nuclear GTP-binding proteins, three H/ACA ribonucleoproteins and nine U3 small nucleolar RNA-associated proteins. This suppression of the core translational machinery suggests that morel mycelia experience a state analogous to nitrogen starvation when nitrate serves as the sole nitrogen source. This finding explains the phenotypic observation that maximum biomass in nitrate medium was lower than in ammonium medium, as the energetic cost of nitrate uptake and reduction, combined with translational suppression, creates a physiological burden that limits vegetative growth.

Metabolomic analysis identified 296 metabolites in negative ion mode and 824 in positive ion mode. Screening revealed that L-glutamate was enriched while L-aspartate was found at lower concentrations in response to nitrate, confirming the upregulation of GOGAT at the metabolic level. Notably, gamma-glutamylcysteine, the precursor of reduced glutathione, was the most upregulated metabolite after nitrate treatment, with a fold change of 73.0. Reduced glutathione and its precursor function as major antioxidants that prevent cell damage caused by oxidative stressors. The accumulation of these metabolites, combined with the finding that malondialdehyde, hydrogen peroxide and superoxide anion levels were all significantly higher in nitrate-treated mycelia, demonstrates that nitrate induces genuine oxidative stress in morel cells.

The study also revealed that nitrate activated pathways associated with sexual morphogenesis. Both proteomic and metabolomic data showed that tyrosine metabolism was the most enriched pathway after nitrate treatment. Melanin synthesis substrates, including L-tyrosine and L-dopa, were upregulated, along with three tyrosinase proteins and overall tyrosinase activity, which increased more than fourfold. Melanin deposition stabilises the fungal cell wall and protects cells from extreme environmental conditions and pathogen infections, facilitating reproductive development. Additionally, nitrate upregulated metabolites associated with amino sugar metabolism, such as fructose, D-glucosamine 6-phosphate and uridine 5′-diphosphoglucuronic acid, which act as precursors for chitin and other fungal cell wall polysaccharides. The upregulation of N-glycan biosynthesis proteins, including six oligosaccharyl transferases and four mannan polymerases, further indicated that nitrate enhanced glycosylation to supply cell-wall formation.

The integrated analysis revealed minimal concordance between the transcriptome and proteome data, with a Pearson’s correlation coefficient of -0.134. This discrepancy may be attributed to temporal delays in translation, protein synthesis and post-translational modifications, as well as metabolite compensation mechanisms. However, both proteomic and metabolomic data independently identified tyrosine metabolism as the most enriched pathway, underscoring its pivotal role in regulating the response of morel mycelia to different nitrogen forms. The findings collectively demonstrate that nitrate and ammonium shape different cellular homeostasis and physiological states in Morchella importuna by activating distinct nitrogen assimilation pathways. Nitrate induces a nitrogen starvation-like state that triggers oxidative stress, which in turn activates glutathione metabolism, tyrosine metabolism and N-glycan synthesis, pathways associated with increased antioxidant levels, melanin deposition and cell wall formation that are implicated in sexual morphogenesis and fruiting body formation in macrofungi. These insights provide a scientific foundation for promoting morel productivity through the optimised modulation of nitrogen nutrition in cultivation substrates.

Subject of Research: Multi-omics reveals nitrate-induced oxidative stress and morphogenesis pathways in Morchella importuna

Article Title: Multi-omics reveals nitrate-induced oxidative stress and morphogenesis pathways in Morchella importuna

Article References: Yu, Y., Liu, T., Li, J., Wu, X., Zhang, S., Wang, Y., Tang, J., Peng, W., Martin, F. M., & Tan, H. (2026). Multi-omics reveals nitrate-induced oxidative stress and morphogenesis pathways in Morchella importuna. IMA Fungus, 17, Article e159999. https://doi.org/10.3897/imafungus.17.159999

Image Credits: AI Generated

DOI: 10.3897/imafungus.17.159999

Keywords: Multi-omics, reveals, nitrate-induced, oxidative, stress, morphogenesis, pathways, Morchella, importuna, scientific research