Analysis of primary metabolites of Morchella fruit bodies and mycelium based on widely targeted metabolomics

As a medicinal and edible homologous fungi, Morchella is rich in multiple metabolites. The metabolite is a kind of essential substance with active components. In this study, Morchella fruit bodies and mycelium were selected to identify their metabolite components. The primary metabolites of the two experimental groups were analyzed using a method of widely targeted metabolome based on UPLC-ESI-MS/MS. A total of 354 different metabolites were characterized, including 188 upregulated ones and 166 downregulated ones in the fruit bodies. Further, the main 20 metabolic pathways of the metabolites were analyzed. The first 9 ones are tyrosine metabolites, thyroid hormone biosynthetic pathway, phenylalanine metabolites, linoleic metabolites synthetic pathway, glycerophosphate metabolic pathway, choline in tumors, methyl butyl metabolites, arginine synthetic pathway, arginine and proline metabolites. This study provides theoretical basis for the analysis of metabolic pathway of Morchella fruit bodies and mycelium that serve for further research of their medicinal mechanism and effective components.


Introduction
Morchella belongs to Eumycota, Aseomycotina, Discomycetes, Pezizales, and Morchellaceae, which has attracted great interest due to its high nutritional value and medicinal benefits (Kanwal and Reddy 2014). The fruit bodies and mycelium of Morchella are rich in protein, fat, carbohydrates, crude fiber, riboflavin, niacin, folic acid, vitamins and other components (Enkhjargal et al. 2011). Morchella is sweet in taste and is characterized by its antioxidant properties (Harpreet et al. 2011). Besides, it also shows extremely high medicinal value (Böckerman and Maliranta 2013). Modern medical research has shown that Morchella has various biology activities such as lowering blood lipids, regulating body immunity, anti-fatigue, protecting liver, anti-virus, inhibiting tumors, and reducing the side effects caused by radiotherapy and chemotherapy (Hai-Bin 2019). Nevertheless, a thorough and dynamic evaluation of the metabolites in Morchella fruit bodies and mycelium has not been done before. Metabolomics is a rapidly emerging discipline in Bionomics as an important part of systems biology (Widiarsih et al. 2021), using high-throughput chemical analysis technology to perform qualitative and quantitative analyses of small molecule metabolites in biological samples (Lin et al. 2011). Based on the quantitative analysis of metabolites, metabolomics can be used for the analysis of metabolic pathways or metabolic networks, the basic research of metabolism of the macroscopic phenotypic phenomena of different organisms, and the metabolites of different diseases, drugs and other physical, chemical or pathogenic organisms (Zhou et al. 2020). Wang et al. (Wang et al. 2018) identified the nutrients in black sesame seeds and related metabolites that play a role in traditional Chinese medicine based on extensively targeted metabolomics technology. Ho et al. (Ho et al. 2018) identified 6 compounds with antibacterial activity from black walnut with metabolomics technology. For the past few years, ultra-performance liquid chromatography tandem electrospray Ionization mass spectrometry (UPLC-ESI-MS/MS)-based, widely targeted metabolome has become an effective tool to deeply research secondary metabolites due to its advantages of high throughput, fast separation, high sensitivity, and wide coverage (Wang et al. 2019). Up to now, researchers have reported that the types of bioactive compounds in fungi are often different. As to Morchella, the fruit bodies are the woody stage of mature mushrooms while the mycelium is formed by liquid deep fermentation, so the components and functions of them are different. There will be different metabolites in the fruit bodies and mycelium, and they will have different functions. If the fruit bodies is rich in N-methylephedrine, it will have a strong anti-allergy and antitussive effect. If the mycelium is rich in anti-4-hydroxy-Lproline, the main use is flavor enhancer and nutritional fortifier. However, there is a lack of research to analyze the metabolic components of the fruit bodies and mycelium of Morchella with the technology of widely targeted metabolome. In this study, we selected Morchella fruit bodies and mycelium as research materials, and detect the types of metabolites of them with UPLC-ESI-MS/MS technology. By means of comparing and analyzing their differences, we characterized the metabolic pathway in Morchella fruit bodies and mycelium. Our study may help to understand the biological processes and mechanisms of the fruit bodies and mycelium of Morchella more intuitively and effectively (Nadia et al. 2015) and provide a reference for their sufficient utilization in the future.
Morchella mycelium: The Morchella strain was obtained from the Academy of Biological Science and Technology, Shenyang Agricultural University (Shenyang, China). Morchella mycelia were cultured on potato-glucose-agar medium in flasks at 28 °C for 7 days in a rotary shaker at 120 rpm. The suspension was centrifuged and lyophilized to obtain the best fermented Morchella mycelium.

Sample preparation
Thaw the sample on ice. Take 50 mg of the sample and homogenize it with 1000 µL of ice-cold methanol/water (70%, V/V). Add cold steel balls to the mixture and homogenize at 30 Hz for 3 min. Whirl the mixture for 1 min, and then centrifuge it with 12,000 rpm at 4 ℃ for 10 min. The collected supernatant will be used for LC-MS/MS analysis (Xian et al. 2008).
LIT and triple quadrupole (QQQ) scans were acquired on a triple quadrupole-linear ion trap mass spectrometer (QTRAP), QTRAP ® LC-MS/MS System, equipped with an ESI Turbo Ion-Spray interface, operating in positive and negative ion mode and controlled by Analyst 1.6.3 software (Sciex). The ESI source operation parameters were as follows: source temperature 500 °C; ion spray voltage (IS) 5500 V (positive), − 4500 V (negative); ion source gas I (GSI), gas II (GSII), curtain gas (CUR) were set at 55, 60, and 25.0 psi, respectively; the collision gas (CAD) was high. Instrument tuning and mass calibration were performed with 10 and 100 μmol·L −1 polypropylene glycol solutions in QQQ and LIT modes, respectively. A specific set of MRM transitions were monitored for each period according to the metabolites eluted within this period (Oh et al. 2011).

Quality control analysis
Quality control samples (QC) were prepared by mixing sample extracts and were used to monitor the repeatability of analytical samples under the same processing method. In the process of instrumental analysis, a quality control 1 3 Page 3 of 9 98 sample was inserted into every 10 test analysis samples to monitor the repeatability of the analysis process (Bürger et al. 2012).

Data analysis
Qualitative analysis of metabolites was based on MVDB V2.0 database of Maiwei Biotechnology Co., Ltd. and metabolite information in public databases. The primary and secondary mass spectrometry analyses were based on the existing MassBank, KNAPSACK, HMDB and MET-LIN mass spectrometry databases (Isoherranen et al. 2009). A triple quadrupole mass spectrometry multiple reaction monitoring mode (MRM) was used for quantitative analysis of metabolites. After obtaining the metabolite data of different samples, the Analyst 1.6.3 software was used to perform mass spectrometry qualitative and quantitative analyses, including baseline filtering, peak identification, integration, retention time correction, peak alignment, and mass spectrometry fragment attribution analysis (Bessa et al. 2013). The data were normalized and annotated based on the obtained retention time, massto-severity ratio and peak intensity. At the same time, most substances were further confirmed compared to standard products. A reliable mathematical model was established by multi-dimensional statistical analysis to analyze the metabolites (Muazu et al. 2021). First, principal component analysis (PCA) of unsupervised pattern recognition was used to analyze the detected metabolites to get a preliminary understanding of the overall metabolite differences between samples in each group and the degree of variability between samples of each group. Then, the partial least squares-discriminant analysis (PLS-DA) with supervised pattern recognition was used to distinguish the overall differences in metabolites between groups to find different metabolites (Cozzolino et al. 2014). The orthogonal partial least squares discriminant analysis (OPLS-DA) was used to remove irrelevant differences to screen the difference variables to find the differences between the samples in each group, and the variable weight value (Variable importance in projection, VIP) greater than 1 was considered to be a difference variable (Boccard and Rutledge 2013). Evaluation of PLA-DA and OPLS-DA predictive model parameters were R 2 X, R 2 Y and Q 2 . The closer the 3 indicators were to 1, the more stable and reliable the model was. Use multi-dimensional statistics VIP value (VIP > 1), single-dimensional statistics (p < 0.05) and multiple of difference (fold change) to screen different metabolites. The multiple of difference was transformed by Log 2, and select VIP > 1, p < 0.05, Log 2 FC ≥ 2 or metabolites with Log 2 FC ≤ 0.5 as differential metabolites.

Overall analysis of metabolic components
Based on Maiwei (Wuhan) self-built metabolite database and related mass spectrometry database, the multi-peak map of MRM metabolites was detected, the characteristic ions of each substance were screened through the triple quadrupole rod, and the signal intensity of the characteristic ions was obtained in the detector (CPS). Use Multi-Quant software to open the sample offline mass spectrometry file and analyze the main metabolites qualitatively and quantitatively. In Morchella fruit bodies and mycelium, 34 types of 610 metabolites were identified (Table 1).

PCA results
PCA results can generally reflect the differences of the metabolite between the two groups of samples. Through the principal component analysis (PCA) of the samples, the degree of variability between the groups of Morchella fruit bodies and mycelium samples and between the samples within the group could be discriminated. The results contained three principal components (PCs), PC1 (99.79%), PC2 (0.15%) and PC3 (0.03%). The two groups of samples showed a clear separation trend on the graph (Fig. 1).

OPLS-DA results
Although the PCA analysis method can effectively extract the main information, it is not sensitive to less correlated variables. The PLS-DA can maximize the distinction between groups, which is conducive to finding different metabolites (Peng et al. 2018). Orthogonal Partial Least Square Discriminant Analysis (OPLS-DA) combined orthogonal signal correction (OSC) and PLS-DA methods can screen difference variables by removing irrelevant differences. The OPLS-DA model (Suppl. Fig. 1) of 610 metabolites set of data analysis showed that the sample distribution of Morchella fruit bodies was on the right side of the confidence interval, while mycelium samples were on the left side. The difference effect between the two samples was often obvious. Two principal components were analyzed by OPLS-DA, of which the contribution rate were 98.6% and 0.623%. R 2 X = 0.992, R 2 Y = 1, Q 2 = 1 (Fig. 2), showing that the grouping model had a strong interpretation and prediction ability, and the clustering results were reliable. The effect was better than the PCA model. Perform permutation verification to OPLS-DA (n = 200, that is, conduct 200 permutation experiments).

Identification of differential metabolites
In the obtained multivariate analysis of the variable importance in project (VIP) of the OPLS-DA model, VIP value represents the difference between the groups of the corresponding metabolites in the classification of each group of samples in the model. The metabolites with differences between the two types of samples were initially screened, and then select the metabolites with VIP ≥ 1. (The intensity of the impact is considered to be significant for metabolites with VIP ≥ 1). Morchella fruit bodies and mycelium were then screened to obtain a difference metabolite 32 class 354 species. Overall, the different metabolic components (354 kinds) of Morchella fruit bodies and mycelium accounted for 58.03% of total metabolic components (610 kinds), indicating the metabolites of Morchella fruit bodies and mycelium were significantly different. Among them, there were five categories of organic acids and derivatives, amino acids and their metabolites, nucleotides and their metabolites, benzene and their derivatives, carbohydrates and their metabolites, accounting for 20.0%, 16.8%, 10.0%, 10.0%, and 6.9%, respectively.

Analysis of main differences in metabolic components
Comparing the difference fold change of the quantitative information of the metabolic components in the fruit bodies and mycelium of Morchella and processing it by log 2, the top 20 differentially expressed metabolic components are shown in Fig. 3. The relative content of Trans-4-hydroxy-L-proline, N-α-acetyl-L-asparagine, 3-iodo-L-tyrosine, L-phenylalanyl-L-proline, N-acetyl-L-alanine, N-amidino-L-aspart-ic acid, 3-ketone-sphingosine, N-phenylacetyl glycine, 3-methoxy tyramine, and Ellagic acid was significantly high in mycelium. The relative content of N-methyle-phedrine, 2-Furoylglycine, herniarin, Caffeic acid, indolelactic acid, 3,5-dimethoxy-4-hydroxycinnamicacid, 3-hydroxy-DL-kynurenine, Xanthosine, Melatonin, lysoPC(14:0) was significantly high in the fruit bodies. N-Methylephedrine is one of the main components of Chinese herbal medicine Ephedra, similar to ephedrine hydrochloride, with an effect of expanding the bronchus (Wei et al. 2009;Holzgrabe et al. 2015), it is suitable for the treatment of influenza, bronchial wheezing, allergic reactions and other pathogens (Zhang et al. 2013). Some metabolic components are enriched in fruit bodies, some are enriched in mycelium, mainly because of the difference between their condition of culture. The mycelium was cultivated mainly in deep fermentation, and the medium was mainly artificially prepared with carbon source, nitrogen source, inorganic salt, growth factor. Fruit bodies were mainly cultured in the soil, which is the main source of nutrient matrix components.

Differential metabolites' volcanic map analysis
To observe the changes of metabolites more easily, we normalize the metabolites with significant differences and draw a volcanic map, in which differential metabolites (188 upregulated and 166 downregulated) were detected (Fig. 4), representing 53.11% and 46.89%.

Pathway analysis of differential metabolites
The results of pathway enrichment analysis of differential metabolites through the KEGG (Kyoto Encyclopedia of Genes and Genomes) database (Zhang et al. 2017) (Fig. 5) showed that the identified 354 significantly different metabolites were mainly distributed in 20 metabolic pathways. The first nine pathways with the largest number of differential metabolites were tyrosine metabolism, thyroid hormone synthesis, phenylalanine metabolites synthesis, Linoleic acid metabolism and synthesis, glycerophospholipid metabolism, choline metabolism in cancer, butanoate metabolism, arginine biosynthesis, arginine and proline metabolism.

Discussion
Morchella is an important kind of fungi that exhibits various bioactivity due to its variety of metabolites. However, its metabolites and main pathways have not been previously investigated. The utilization rate of Morchella is extremely inadequate. Thus, it becomes a necessity of this study aimed to provide a theoretical basis for the utilization of Morchella. In this study, the primary metabolites of Morchella were comprehensively analyzed. The analysis of the experimental data showed that 354 different compounds were reported for the first time. The first 10 richest compounds in the fruit bodies and mycelium of Morchella were different and exhibited diverse efficacy. Comparison groups showed that the anti-4-hydroxy-Lproline was higher in mycelium, which was mainly used as flavor agent, nutritional intensifier, flavor material and biochemical reagents in the present study. Aromatic amino acids, including phenylalanine, tyrosine and tryptophan, were important essential amino acids in organisms for their important biological functions. 3-Iodide tyrosine and N-acetyl-L-alanine were richer in mycelium which could be widely used in fields of medicine, food and feed . N-Methyl ephedrine rich in Morchella fruit bodies was one of the main Chinese medicinal herbs, that exhibited the effects of dilating bronchial lines and a stronger antiallergy and antitussive. They were suitable for the treatment of influenza, bronchial wheezing, allergic reaction and other bacteria (Abreu et al. 2021). 7-Methoxycoumarin, which could be used as an organic synthesis intermediate, was also a drug with the effect of flat asthma, expectorant, cough suppression (Han et al. 2021).The basic function of melatonin was to participate in the antioxidant system and prevent cells from causing oxidative damage, which was the strongest endogenous free radical scavenger ever found. In this respect, it outperforms all known substances in vivo (Cruz et al. 2014). Recent research proves that melatonin was the commander in chief of endocrine and it controlled the activity of various endocrine glands in the body, thus to indirectly control the function of our whole body (Cezary et al.  2021). The above analysis results revealed the differential metabolites between Morchella fruit bodies and mycelium that serving for the development and utilization of Morchella resources. The enrichment analysis of different metabolites showed that different metabolites mainly participated in the synthesis of tyrosine, thyroid hormones, phenylalanine metabolic product, linoleic acid metabolites, glycerol phospholipid metabolites, choline metabolic pathways, butyric acid, arginine methyl metabolites synthesis pathway as well as arginine and proline biosynthesis of metabolites. They were the main pathways of Morchella metabolism and directly affect the accumulation of the main substances in Morchella (Dobson et al. 2012). The phenylalanine and tyrosine metabolic pathways mainly determined phenolic difference metabolites. The changes of these material contents were related to the accumulation of phenylalanine and tyrosine, and then indirectly affected the umami flavor of Morchella. Thyroid hormone signaling pathway was associated with improving the efficacy of myocardial ischemia and provided a theoretical basis for its clinical application (Zeng et al. 2019). Therefore, it was speculated that the active components in Morchella may achieve the purpose of improving myocardial ischemia through the key acts in the above signaling pathway, providing a basis for the drug research of Morchella to improve myocardial ischemia, and new ideas and methods for the research and development of traditional Chinese medicine. The biosynthesis pathways of arginine and proline metabolites could increase the content of anti-4-hydroxyl-L-proline and L-amphetamine-L-proline in the mycelium significantly (He et al. 2019).
The metabolites and main pathways are different in mycelium and fruit bodies, mainly because of the difference between their development in liquid fermentation and solid cultivation. Morchella mycelia were cultured on potato-glucose-agar medium in flasks at 28 °C for 7 days in a rotary shaker at 120 rpm. Fruit bodies were mainly cultured in the soil. These findings have improved to our understanding of the molecular mechanisms accounting for the biological activities of Morchella fruit bodies and mycelium. It provides a basis for the drug research of Morchella to provide novel insight into further applications in drug industry.

Conclusions
In this paper, the primary metabolites in fruit bodies and mycelium of Morchella based on extensive target metabolomics were analyzed. 354 significantly different metabolites were identified, and the main 20 metabolic pathways of the metabolites were analyzed. The relationship between the detected various compounds and the pharmacological activity of Morchella may become an orientation of scientific research for the development and utilization of Morchella in the future. Besides, metabolic group information can provide references for the separation and purification identification of many active components in Morchella. Associated metabolic pathways also provide a basis for subsequent studies of the function of key genes in the metabolic pathways and the biosynthesis of the major metabolites.