## 4 Discussion
Notable proteins such as endoplasmic reticulum proteins, small secretory molecules, membrane proteins, and enzymes involved in carbohydrate and cellulose degradation contribute to nutrient accumulation and root morphology. These proteins actively participate in nutrient accumulation and the construction of mycorrhizae. In the metabolomics investigation, the KEGG annotation results showed that differential metabolic pathways during the mycelium vs. primordium period were primarily dominated by amino acid metabolic pathways and the biosynthesis of sugar and lipid metabolism precursors. These pathways in amino-acid-based metabolism supply precursor substances for complex macromolecules and organic compounds, emphasizing the substantial energy demand crucial for early developmental stages. Metabolic pathways enriched in mycelium vs. fruiting body, with a \(p\)-value < 0.05, included folate biosynthesis and porphyrin metabolism, both of which are involved in DNA synthesis and repair and have signaling roles. In the primordium vs. fruiting body phase, the key metabolic pathways with a \(p\)-value < 0.05 were 12-, 14-, and 16-member macrolides biosynthesis; this phase enhances fungal immunity as an endogenous antimicrobial agent, reducing the dependence of fungi on plant hosts. The remaining pathways included arginine biosynthesis. Notably, during the late development of the fruiting body, there was an enhancement in signal transduction and hormone analog synthesis pathways, likely involved in interactive processes with the host plant, while there was a decline in energy metabolism activity. We performed a combined analysis of the metabolome and transcriptome to uncover specific substances and unique processes influencing the ectomycorrhizal development of _T. ganhajun_ throughout its life cycle. Co-analysis results suggest that functions such as amino acid metabolism and vitamin biosynthesis may constitute key factors influencing the morphological construction of _T. ganhajun_ during the transition from the mycelium to the primordium. The folate synthesis pathway plays a pivotal role in the synthesis of B vitamins and serves as a precursor for purines and pyrimidines, participating in both the ascorbate pathway and the methylation process [43]. Folic acid's fundamental physiological functions are achieved through its reduced product, tetrahydrofolate (THF), and methylated derivatives of THF [44]. These derivatives primarily act as methyl donors for intracellular methylation reactions and contribute to de novo deoxyribonucleic acid synthesis [45]. In organisms, folic acid undergoes reduction by ascorbic acid, NADPH, and dihydrofolate reductase, resulting in THF production [46]. Subsequently, methylenetetrahydrofolate reductase (MTHFR) generates N-methyltransferasehydrofolate, with 5-methyltetrahydrofolate (5-MTHF) being the most prevalent active form. This active form is transported to peripheral tissues for cellular metabolism [47], or it may undergo methylation to produce homocysteine by methionine synthetic reductase (MTRR) [48], ultimately lowering homocysteine levels [49]. In the biosynthetic pathway, nearly all plants, along with the majority of fungi and bacteria, synthesize folic acid [50,51]. This water-soluble B vitamin is crucial for the synthesis of thymine nucleotides and purines, as well as for methionine metabolism in fungi [50]. It serves as a vital pathway for the synthesis of micronutrients. A comprehensive analysis of both transcriptomic and metabolomic KEGG enrichment revealed a weak presence of the folate biosynthesis pathway in the GM vs. GPM phase. However, this pathway was significantly enriched in the GPM vs. GSM phase. This suggests that adequate access to this vitamin might be constrained under laboratory culture conditions. Nonetheless, the nutrient could potentially be acquired from the plant host or through self-synthesis in a field environment.