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Article

Transcriptomic Insights and Quantification of Free Amino Acids in Auricularia heimuer Cultivated on Corncob Substrate

by
Xu Sun
1,
Fangjie Yao
1,2,*,
Fanchao Lai
2,
Ming Fang
2,
Lixin Lu
2,
Xiaoxu Ma
2,
Wei Wang
2,
Jingjing Meng
2 and
Kaisheng Shao
1
1
Internationally Cooperative Research Center of China for New Germplasm Breeding of Edible Mushroom, Jilin Agricultural University, Changchun 130118, China
2
Lab of Genetic Breeding of Edible Mushroom, College of Horticulture, Jilin Agricultural University, Changchun 130118, China
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(6), 563; https://doi.org/10.3390/horticulturae11060563
Submission received: 16 April 2025 / Revised: 15 May 2025 / Accepted: 17 May 2025 / Published: 22 May 2025
(This article belongs to the Special Issue Edible Mushrooms: Genetics, Genomics, and Breeding)
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Abstract

:
Substrate type exerts a critical influence on the growth, development, and nutritional quality of Auricularia heimuer. In this study, agricultural waste-derived corncob was used as the treatment group (T1), with sawdust serving as the control (CK), to systematically investigate the variation in free amino acid (FAA) content and transcriptomic expression profiles in fruiting bodies of A. heimuer under the two substrate conditions. Principal component analysis (PCA) revealed a clear separation between CK and T1 samples in terms of FAA composition, indicating that substrate type significantly affects FAA profiles. The corncob substrate notably increased the total FAA content in A. heimuer (2624.57 mg/kg), representing an 11.4% elevation compared to the sawdust group (2355.86 mg/kg), and markedly enhanced the proportion of flavor-associated amino acids (49.2% vs. 42.6%). In particular, the umami amino acid content was 74% higher than in the CK group. Transcriptome analysis identified 20 differentially expressed genes associated with FAA biosynthesis and degradation, including key enzymes involved in umami amino acid metabolism, such as aspartate decarboxylase (ADC), glutamate decarboxylase (GAD), and glutamate N-acetyltransferase (GNA), which were downregulated in T1. This suggests that glutamate and aspartate may have accumulated due to suppressed catabolism. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis further indicated that the differentially expressed genes were significantly enriched in pathways related to branched-chain amino acid metabolism, carbon metabolism, and secondary metabolism. Collectively, these findings demonstrate that corncob substrate significantly alters the accumulation and metabolic profile of FAAs in A. heimuer by modulating the expression of key metabolic enzymes, providing a theoretical foundation for the efficient cultivation of A. heimuer using agricultural waste and for enhancing its flavor quality.

1. Introduction

Auricularia heimuer is an edible and medicinal jelly fungus, rich in various nutritional components such as amino acids, dietary fiber, and minerals [1,2,3]. Edible fungi generally contain a high proportion of branched-chain amino acids (BCAAs), which are typically found in animal-derived proteins. As a result, edible fungi have become an important source of high-quality protein for vegetarians [4]. Free amino acids (FAAs) are not only precursors for protein synthesis but also play crucial roles in flavor development, cellular signal transduction, and stress responses [5,6,7]. In edible fungi, amino acids are generally classified into four categories based on their taste profiles: umami, sweet, bitter, and tasteless. Among them, the primary umami amino acids are glutamic acid and aspartic acid. The sweet-tasting amino acids include serine, glycine, threonine, and alanine, while the main bitter amino acids are histidine, tyrosine, isoleucine, phenylalanine, and leucine. Umami and sweet amino acids contribute most significantly to the overall flavor profile of edible fungi, whereas the other amino acid categories primarily play auxiliary roles, enhancing and modulating the overall taste [8]. The composition and concentration of amino acids play a crucial role in determining nutritional quality and may also serve as indicators of the organism’s adaptability to different cultivation substrates [9]. However, studies investigating the regulatory mechanisms of FAA metabolism in A. heimuer under varying cultivation conditions remain limited.
Traditionally, sawdust has served as the primary substrate for the cultivation of A. heimuer, demonstrating effectiveness through long-term application. However, with the increasing scarcity and fluctuating cost of sawdust resources, the search for sustainable and efficient alternative substrates has become a key concern in the industry. Corncobs are a major agricultural by-product generated in large quantities during corn processing. In China, their utilization is primarily concentrated in four areas: as industrial raw materials, substrates for edible mushroom cultivation, biofeed, and fuel. Among all crop straw resources, corncobs exhibit the highest utilization efficiency, with a collection and utilization rate reaching up to 97%. Owing to their abundance and rich composition of cellulose, hemicellulose, and lignin, corncobs possess excellent biodegradability, low cost, renewability, and environmental compatibility. In recent years, they have received considerable attention as a sustainable alternative to traditional cultivation substrates such as sawdust in edible fungi production [10,11]. Corncob, a widely available agricultural by-product, features a relatively low carbon-to-nitrogen (C/N) ratio and high organic matter content, making it a promising substrate for A. heimuer cultivation [12,13]. In recent years, corncob has gained attention for its environmental and resource-recycling value [14,15,16], and research on its application in edible mushroom cultivation has gradually expanded [17,18]. The lower C/N ratio of corncob facilitates rapid organic matter decomposition and nitrogen release, potentially providing A. heimuer with a more favorable nutrient supply and thereby enhancing its growth rate and nutrient accumulation [12]. Nevertheless, replacing sawdust with corncob may impact the nutritional composition and metabolic activity of A. heimuer, particularly with respect to amino acid biosynthesis and transport. The underlying molecular mechanisms of these effects remain unclear. In recent years, transcriptomic technologies have emerged as powerful tools for elucidating complex metabolic regulatory networks in non-model organisms. Comparative analysis of transcriptional profiles under different cultivation conditions enables the identification of differentially expressed genes related to free amino acid metabolism in A. heimuer, thus providing molecular insights into how corncob-based substrates influence fungal quality.
This study, incorporating high-throughput transcriptomic sequencing, aims to explore the effects of corncob as a cultivation substrate on the accumulation of FAA in A. heimuer and to investigate the underlying regulatory mechanisms involved in amino acid metabolism. The findings aim to provide valuable theoretical insights and practical guidance for optimizing cultivation substrates, controlling quality, and promoting the efficient utilization of resources in edible fungi production.

2. Materials and Methods

2.1. Cultivation and Sample Collection of A. heimuer

Strain A184 was provided by the College of Horticulture, Jilin Agricultural University (Changchun, China) [19]. The control cultivation substrate (CK) consisted of 78% sawdust, 20% wheat bran, 1% lime, and 1% gypsum. The treatment substrate (T1) was composed of 78% corncob, 20% wheat bran, 1% lime, and 1% gypsum [20,21]. After thorough mixing, the two substrates were each packed into 50 polypropylene cultivation bags (approximately 300 g per bag, with a moisture content of 60%) [22]. The bags were sterilized at 121 °C for 2 h, followed by inoculation with A. heimuer mycelia. The cultures were incubated in the dark at 25 °C until the substrates were fully colonized by mycelia. Fruiting was then induced under controlled conditions: 25 °C, approximately 85% relative humidity, diffused light, and good ventilation. The fruiting bodies were harvested and immediately stored at –80 °C for subsequent RNA extraction. Three biological replicates per treatment were used for RNA-seq analysis.

2.2. Determination of Free Amino Acids

Free amino acids were quantified using high-performance liquid chromatography (HPLC) [22]. Briefly, 1 g of ground sample was weighed into a 10 mL volumetric flask, dissolved, and then brought to volume with 0.02 mol/L hydrochloric acid. The solution was sonicated for 20 min and then centrifuged at 6000 rpm for 5 min. The supernatant was collected for purification. A C18 solid-phase extraction (SPE) column was activated sequentially with 5 mL methanol and 5 mL water. Subsequently, 2.5 mL of the sample extract was loaded onto the column, followed by 1.5 mL of 0.02 mol/L hydrochloric acid. After elution, 100 µL of the purified extract was transferred into a 15 mL centrifuge tube and dried in a vacuum oven at 60 °C for 2 h. The tube was then flushed with nitrogen gas, and exactly 50 µL of derivatization reagent—ethanol/phenyl isothiocyanate/water/triethylamine (7:1:1:1, v/v/v/v)—was added. The mixture was allowed to react at room temperature for 30 min. After derivatization, 0.45 mL of mobile phase A was added, mixed thoroughly, filtered through a 0.45 µm organic membrane, and then injected into the HPLC system for analysis.

2.3. RNA Extraction and cDNA Library Construction

Total RNA was extracted from A. heimuer samples using the RNAprep Pure Plant Kit for Polysaccharide- and Polyphenol-Rich Plants (TIANGEN Biotech Co., Ltd., Shanghai, China), following the manufacturer’s instructions. RNA integrity and purity (including potential DNA contamination) were assessed by agarose gel electrophoresis. RNA concentration and integrity were further evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Polyadenylated mRNA was enriched using Oligo(dT) magnetic beads. The purified mRNA was fragmented and used as a template for first-strand cDNA synthesis using random hexamer primers. Subsequently, cDNA libraries were constructed following the standard NEB protocol [20]. Libraries that passed quality control were subjected to high-throughput sequencing on the Illumina platform.

2.4. Bioinformatics Analysis

To ensure the quality and reliability of the transcriptomic data, raw sequencing reads were subjected to quality control and filtering, with low-quality reads being removed. The filtered clean reads were then aligned to the reference genome using HISAT2 v2.0.5. Novel transcript prediction was performed using StringTie [23]. The reference genome used for alignment was obtained from NCBI (accession: https://www.ncbi.nlm.nih.gov/assembly/GCA_002287115.1, accessed on 19 December 2022) (Auricularia heimuer Dai13782, WGS accession: NEKD01). Gene expression levels were quantified using the FPKM (fragments per kilobase of transcript per million mapped reads) method. Differentially expressed genes (DEGs) between the two groups were identified using thresholds of adjusted p-value (padj) < 0.05 and |log2FoldChange| > 1. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment, and GO (Gene Ontology) functional enrichment analyses of DEGs were conducted using the ClusterProfiler v4.0. A padj value of <0.05 was used as the significance threshold for both KEGG and GO enrichment. The number of DEGs in each comparison group was calculated, and enrichment significance was assessed using the hypergeometric distribution method.

2.5. RT-qPCR Validation

To validate the transcriptomic results, real-time quantitative polymerase chain reaction (RT-qPCR) was conducted. Total RNA was extracted from fruiting body samples of A. heimuer using TRIzol reagent (Phygene Biotechnology, Fuzhou, China), following the manufacturer’s instructions. First-strand cDNA was synthesized using the TransScript® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (Allwegene, Beijing, China), with total RNA from each sample as the template. The APRTase gene was used as the internal reference for normalization [24]. Primers for selected differentially expressed genes (DEGs) were designed using Primer Premier 5.0 software and synthesized by Kumei Biological Company (Changchun, China) (Supplementary Table S1). RT-qPCR reactions were performed using the TransStart® TOP Green qPCR SuperMix (Allwegene, Beijing, China). Relative gene expression levels were calculated using the 2−ΔΔCt method [25]. Reaction components and thermal cycling conditions were applied according to the manufacturer’s protocol.

2.6. Data Statistics and Analysis

Statistical analyses were performed using IBM SPSS Statistics 20. Each measurement was conducted with three biological replicates, and the results are presented as mean ± standard deviation (SD). An independent samples t-test was used to compare the free amino acid contents of A. heimuer cultivated on corncob versus sawdust substrates. Differences were considered statistically significant at p < 0.05. For each replicate, three fruiting bodies were pooled and homogenized to generate one composite sample, resulting in a total of three biological replicates (n = 3)

3. Results

3.1. Determination of Free Amino Acid Content

A total of 17 FAAs were quantified in A. heimuer samples (Figure 1), comprising all 7 essential amino acids and 10 non-essential amino acids. Cystine content was low in both the sawdust and corncob treatment groups (<5.17 mg/kg), while arginine was the most abundant amino acid in both groups, accounting for 27% and 32% of the total free amino acids, respectively. The total FAA content in the sawdust (CK) was 2355.86 mg/kg, whereas the corncob (T1) reached 2624.57 mg/kg. Notably, the T1 group exhibited distinct changes in characteristic amino acid profiles, particularly with a significantly higher content of the essential amino acid phenylalanine compared to the CK group (53.42 ± 2.15 mg/kg vs. 44.69 ± 1.89 mg/kg), representing a 19.5% increase. In addition, the T1 group showed a clear advantage in the accumulation of flavor-related amino acids, which accounted for 49.2% of the total FAA content, significantly higher than the 42.6% observed in the CK group. Among these, the content of umami amino acids in the T1 group was 74% higher than in the CK group, with glutamic acid and aspartic acid levels being 2.2 times and 1.1 times higher, respectively. In contrast, the content of sweet amino acids in the T1 was 16% lower than that in the CK.
To investigate whether the amino acid profiles of A. heimuer cultivated on different substrates exhibited any grouping or associations, principal component analysis (PCA) was performed on the full spectrum of FAAs from the CK and T1 groups (Figure 2). The first two principal components accounted for the majority of variance within the dataset (98.1%), with PC1 explaining 91.9% and PC2 explaining 6.2%. Projection of the samples onto PC1 and PC2 revealed two clearly separated clusters corresponding to the CK and T1 groups, with distinct separation along the PC1 (x-axis), indicating that substrate type had a significant influence on amino acid composition. The proximity of a treatment group to the direction of a variable’s loading vector suggests a strong association. Accordingly, valine, proline, threonine, phenylalanine, isoleucine, alanine, serine, methionine, cystine, and glycine appeared to be more dominant in the CK group, whereas aspartate, tyrosine, leucine, histidine, arginine, and glutamate were more prominent in the T1 group. These results demonstrate that different cultivation substrates significantly impact the FAA profiles of A. heimuer.

3.2. Transcriptome Analysis

3.2.1. Quality Assessment of Sequencing Data

The RNA Integrity Numbers (RINs) of all samples, as determined by the Agilent 2100 Bioanalyzer, were greater than 6 (ranging from 6.10 to 9.40), indicating that the RNA was intact and free from degradation, thus making it suitable for library construction and sequencing (Supplementary Table S2). After filtering the raw data, checking for sequencing error rates, and examining the GC content distribution, high-quality clean reads were obtained for subsequent analyses (Supplementary Table S3). The results showed that in all samples, the percentage of bases with a Phred quality score greater than 30 (>Q30) exceeded 93%, indicating that high-quality clean reads were generated for further analysis. Correlation analysis was performed among the three biological replicates of each sample from the CK and T1 groups to assess the reproducibility of the experiment. The strong correlations observed between replicates demonstrate a high degree of experimental reproducibility (Figure 3). These findings indicate that the RNA-Seq dataset generated in this study is reliable.

3.2.2. RT-qPCR Validation

In this study, six differentially expressed genes (DEGs) were randomly selected for RT-qPCR analysis to validate the A. heimuer transcriptome. The RT-qPCR results were consistent with the gene expression data obtained from the transcriptome analysis (Figure 4 and Supplementary Figure S1), confirming the reliability of the RNA-Seq dataset.

3.2.3. Differential Gene Expression

A total of 12,568 differentially expressed genes (DEGs) were identified between the CK and T1 samples. Based on the filtering criteria of |log2(FoldChange)| ≥ 1 and padj ≤ 0.05, 856 genes were determined to be significantly differentially expressed, including 264 upregulated genes and 592 downregulated genes (Figure 5).
Based on the filtering criteria of |log2(FoldChange)| ≥ 1 and padj ≤ 0.05, a total of 20 DEGs encoding enzymes involved in free amino acid biosynthesis and degradation were identified (Figure 6). Among them, 10 genes were associated with flavor-related amino acids. Five genes were related to umami amino acid metabolism, including g10120, encoding aspartate decarboxylase (ADC, EC 4.1.1.12), which was downregulated; g12280, encoding glutamate decarboxylase (GAD, EC 4.1.1.15); g1846 and g407, both encoding glutamate dehydrogenase (GDH, EC 1.4.1.2); and g9767, encoding glutamate N-acetyltransferase (GNA, EC 2.3.1.35). Except for g1846, all other genes exhibited downregulated expression in T1 compared to CK. Five genes were associated with sweet amino acid metabolism, including g12252 and g11518, encoding serine/threonine-protein kinases (PK, EC 2.7.11.-); g5534, encoding serine carboxypeptidase (SCPs, EC 3.4.16.1); g1013, encoding threonine dehydratase (TDH, EC 4.3.1.19); and g1912, encoding L-threonine 3-dehydrogenase (ThrDH, EC 1.1.1.103). All of these genes were downregulated in the T1 group compared to CK, except for g1013 (TDH), which was upregulated. Overall, the downregulation of key enzymes involved in glutamate and aspartate degradation suggests that these amino acids may be retained within cells under T1 rather than being further catabolized. Similarly, the general downregulation of pathways related to serine degradation and regulation implies a potential accumulation of serine. Notably, the threonine metabolic pathway exhibited bifurcated regulation, with upregulation of TDH and downregulation of ThrDH, indicating differential control of threonine catabolism.

3.2.4. KEGG Pathway Enrichment Analysis

A total of 66 significantly enriched metabolic pathways were identified through KEGG-based pathway analysis, primarily involving primary and secondary metabolism (Supplementary Figure S2). Differentially expressed amino acid-related genes were mainly enriched in the following pathways: biosynthesis of secondary metabolites (adl01110); protein processing in the endoplasmic reticulum (adl04141); alanine, aspartate, and glutamate metabolism (adl00250); arginine biosynthesis (adl00220); biosynthesis of amino acids (adl01230); and carbon metabolism (adl01200) (Figure 7). In the pathway of protein processing in the endoplasmic reticulum (adl04141), the gene g7450, encoding the translocation protein SEC62, was upregulated, suggesting that T1 may enhance the activity of specific metabolic pathways—particularly those dependent on ER-associated processing and protein modification. In the context of carbon metabolism, amino acids function not only as metabolic intermediates but also participate in a wide range of biological processes, including energy metabolism, protein synthesis, signal transduction, and regulation.

3.2.5. GO Enrichment Analysis

Gene Ontology (GO) enrichment analysis of the T1 vs. CK comparison revealed 340 enriched biological activities, with most significantly different terms falling under the categories of biological process (BP) and molecular function (MF). A total of 12 GO terms showed significant enrichment, including 7 MF terms: hydrolase activity, hydrolyzing O-glycosyl compounds, hydrolase activity, acting on glycosyl bonds, cofactor binding, flavin adenine dinucleotide binding, threonine-type endopeptidase activity, threonine-type peptidase activity, and iron ion binding. These functional shifts may be closely related to differences in nutrient acquisition and metabolic regulation of A. heimuer under different substrate conditions. In addition, four significantly enriched cellular component (CC) terms—proteasome core complex, proteasome complex, endopeptidase complex, and peptidase complex—were all associated with the proteasome, suggesting distinct protein degradation and quality control mechanisms depending on the cultivation substrate. One significantly enriched BP term was the carbohydrate metabolic process, which indicates that A. heimuer may have adjusted its carbohydrate utilization pathways when grown on a corncob-based substrate, likely in response to the higher cellulose and lower lignin content of corncob compared to sawdust (Figure 8).

4. Discussion

The composition of the cultivation substrate plays a crucial role in regulating the nutritional quality and metabolic processes of edible fungi [26,27,28]. Among various nutritional indicators, amino acid composition is a key determinant of food quality. FAAs serve not only as functional bioactive compounds but also as major contributors to the taste profile of edible fungi [29,30,31]. In this study, the use of agricultural waste-derived corncob as a cultivation substrate significantly enhanced the accumulation of FAAs in A. heimuer. Compared to the traditional sawdust substrate, the total FAA content in the corncob group increased by 11.4%, highlighting its potential to improve both the nutritional and functional properties of A. heimuer. Of particular interest was the marked increase in flavor-related amino acids, which primarily include umami and sweet amino acids—key contributors to the sensory qualities of edible fungi [32,33]. In this study, flavor amino acids accounted for 49.2% of total FAAs in the corncob group, notably higher than the 42.6% observed in the sawdust group, indicating that corncob-based substrates may help improve the flavor profile of A. heimuer. This improvement may be related to the carbon-to-nitrogen (C/N) ratio of the substrate. Compared with sawdust, corncob contains higher nitrogen content and thus exhibits a lower C/N ratio [20], which is consistent with previous studies suggesting that a lower C/N ratio favors amino acid accumulation [34,35]. This study demonstrated that both sawdust and corncob treatments were rich in arginine, with a significantly higher concentration observed in the corncob group. Arginine is a nitrogen-rich amino acid and a well-known precursor of nitric oxide (NO) [36] The enhanced nitrogen availability in the corncob substrate may have promoted the synthesis and accumulation of arginine, reflecting a more active nitrogen metabolism in A. heimuer grown on corncob. As a result, the relatively high nitrogen availability in corncob may have shifted amino acid metabolism toward nitrogen-rich amino acids. In contrast, sweet-tasting amino acids generally contain lower nitrogen levels [37], which may explain their reduced accumulation in the corncob group compared with the sawdust group. This could be attributed to substrate-driven differences in carbon–nitrogen balance and nitrogen-allocation strategies. These findings suggest that substrate composition not only affects the total content of FAAs but also shapes their qualitative profiles through metabolic regulation. In contrast, the cysteine content in both corncob and sawdust treatments remained below 5.17 mg/kg. Cysteine is a key component of glutathione (GSH) [38], playing a crucial role in the antioxidant defense system of A. heimuer and other fungi [39,40]. Therefore, a portion of cysteine may have been rapidly converted into glutathione rather than accumulating in its free form.
Free amino acids, primarily derived from protein metabolism, are widely involved in key physiological processes such as energy metabolism, gluconeogenesis, fatty acid synthesis, antioxidant stress response, and immune regulation [41,42,43,44,45]. Among the EAAs, phenylalanine content was significantly higher in the T1 compared to the CK. As a key precursor in lignin biosynthesis, phenylalanine is involved in multiple steps of the lignin synthesis pathway [46]. Therefore, its consumption is closely associated with the lignification process. In substrates with high lignin content, such as sawdust, phenylalanine may be preferentially utilized via the phenylalanine ammonia-lyase (PAL; EC 4.3.1.24) pathway to promote the synthesis of lignin-derived secondary metabolites, thereby reducing its accumulation in the fungal tissue.
Leucine, isoleucine, and valine are branched-chain amino acids (BCAAs) that serve as essential amino acids for protein synthesis and are known to support immune function and protein anabolism [47]. Interestingly, although the BCAA metabolic pathway was significantly upregulated in the corncob group, the concentrations of these free amino acids were significantly lower than those in the sawdust group. Further analysis revealed that most upregulated genes were associated with BCAA degradation, rather than biosynthesis. These included genes encoding branched-chain amino acid aminotransferase (EC 2.6.1.42), 2-oxoisovalerate dehydrogenase E1 component subunit alpha (EC 1.2.4.4), 3-methylcrotonyl-CoA carboxylase beta subunit (EC 6.4.1.4), and aldehyde dehydrogenase (EC 1.2.1.3), indicating that BCAAs were likely catabolized more actively under corncob-based cultivation conditions. This degradation process produces short-chain acyl-CoA intermediates (e.g., propionyl-CoA), which can feed into the TCA cycle and support energy metabolism [48,49]. A similar mechanism involving the GABA shunt has been observed in Arabidopsis thaliana during ROS homeostasis and phosphorus deprivation stress, where BCAA catabolism is linked to energy and redox balance. This shift toward BCAA degradation may be attributed to the relatively lower C/N ratio and carbon-limited environment of the corncob substrate. Under such conditions, A. heimuer may mobilize amino acid catabolism to fulfill its energy demands. Comparable trends have also been reported in Vicia faba seedlings and soil microbial communities, where amino acid catabolism is enhanced in response to carbon and nitrogen availability [50,51]. In addition, the expression of the key biosynthetic gene hydroxymethylglutaryl-CoA synthase (EC 2.3.3.10) was significantly downregulated in the corncob group, suggesting a reduction in BCAA biosynthetic activity. Collectively, these findings indicate that although the BCAA metabolic pathway was transcriptionally activated, the predominant flux was directed toward degradation rather than accumulation. This also highlights the complexity of amino acid metabolism, which is regulated by multiple factors, including gene expression, substrate availability, and the organism’s energy status. It is worth noting that the gene g7857, encoding gluconolactonase (EC 3.1.1.17), was upregulated in the corncob group. This enzyme is a key component of the oxidative phase of the pentose phosphate pathway (PPP), which has been identified as a critical target of reactive oxygen species and plays a central role in NADPH production Enhanced expression of gluconolactonase may accelerate NADPH generation, thereby supporting the antioxidant defense system and promoting the biosynthesis of aromatic amino acids [52,53]. This would ultimately contribute to enhanced cellular activity and overall metabolic efficiency. Consistent with this, the levels of phenylalanine and tyrosine, both aromatic amino acids, were higher in the corncob group compared to the sawdust group. This trend agreed with transcriptome expression data, further validating the role of carbon-metabolism regulation in promoting amino acid biosynthesis pathways under corncob-based cultivation.

5. Conclusions

In this study, corncob—a renewable agricultural waste—was evaluated as an alternative substrate to sawdust for the cultivation of A. heimuer, with a focus on its effects on free amino acid accumulation and related metabolic pathways. The results demonstrated that corncob substrates significantly enhanced the total free amino acid content in fruiting bodies, particularly flavor-related amino acids, thereby improving both the nutritional and sensory qualities of A. heimuer. This enhancement was closely associated with the lower carbon-to-nitrogen (C/N) ratio of the corncob substrate, which promoted amino acid biosynthesis and altered the transcriptional regulation of key metabolic pathways.
Transcriptomic analysis further revealed that while several amino acid metabolic pathways were upregulated under corncob treatment, the predominant direction of metabolic flux differed among amino acid types. Notably, genes related to branched-chain amino acid (BCAA) degradation were activated, suggesting increased catabolic activity to meet the energy demands under lower carbon availability. In contrast, aromatic amino acids such as phenylalanine and tyrosine showed both transcriptional and metabolic-level increases, possibly supported by enhanced NADPH generation via the pentose phosphate pathway.
Collectively, these findings not only provide new insights into the metabolic responses of A. heimuer to substrate composition but also highlight the potential of corncob as a sustainable and value-added cultivation material. Future research integrating metabolomics approaches could further uncover the coordinated regulation of key metabolites and genes involved in carbon and nitrogen metabolic pathways, providing new insights for the precision enhancement of edible mushroom quality.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/horticulturae11060563/s1, Figure S1: KEGG pathway enrichment analysis of DEGs between T1 (corncob) and CK (sawdust) treatments in A. heimuer; Figure S2: Melting curve analysis of qPCR products for one reference gene and six target genes. Table S1: Primers and sequence for RT-qPCR; Table S2: RNA Integrity Number of A. heimuer samples; Table S3: Sequencing results of transcriptome; Table S4: Abbreviations of enzymes name.

Author Contributions

Conceptualization, F.Y.; methodology, F.Y. and X.S.; software, M.F. and J.M.; formal analysis, X.S. and X.M.; writing—original draft preparation, X.S.; writing—review and editing, X.S. and F.L.; supervision, L.L., W.W. and K.S.; project administration, F.Y.; funding acquisition, F.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Jilin Provincial Key Scientific and Technological Project for Targeted Innovation—Development of Bionic Auricularia heimuer Cultivars.

Data Availability Statement

Data is contained within the article or Supplementary Materials.

Acknowledgments

The authors thank the reviewers for their valuable suggestions.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Free amino acid contents in A. heimuer cultivated on sawdust (CK) and corncob (T1) substrates. Statistical significance was assessed using the t-test. ns: not significant, p > 0.05; **** p ≤ 0.0001.
Figure 1. Free amino acid contents in A. heimuer cultivated on sawdust (CK) and corncob (T1) substrates. Statistical significance was assessed using the t-test. ns: not significant, p > 0.05; **** p ≤ 0.0001.
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Figure 2. Principal component analysis (PCA) of free amino acid profiles in A. heimuer cultivated on sawdust (CK, red) and corncob (T1, blue) substrates. Each point represents a biological replicate. Red arrows indicate the direction of correlation between specific amino acids and the principal components.
Figure 2. Principal component analysis (PCA) of free amino acid profiles in A. heimuer cultivated on sawdust (CK, red) and corncob (T1, blue) substrates. Each point represents a biological replicate. Red arrows indicate the direction of correlation between specific amino acids and the principal components.
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Figure 3. Heatmap of sample correlation analysis based on transcriptomic data from A. heimuer cultivated on sawdust (CK) and corncob (T1) substrates. Color intensity indicates the degree of Pearson correlation between biological replicates.
Figure 3. Heatmap of sample correlation analysis based on transcriptomic data from A. heimuer cultivated on sawdust (CK) and corncob (T1) substrates. Color intensity indicates the degree of Pearson correlation between biological replicates.
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Figure 4. Validation of the RNA-Seq of 6 selected DEGs in the A. heimuer transcriptome by RT-qPCR. APRTase was chosen as the reference gene. The gray column represents the log2 ratio (T1/CK) of DEGs, as calculated by the RT-qPCR analysis. The black column represents the log2 ratio (T1/CK) of DEGs, as calculated by the RNA-Seq analysis.
Figure 4. Validation of the RNA-Seq of 6 selected DEGs in the A. heimuer transcriptome by RT-qPCR. APRTase was chosen as the reference gene. The gray column represents the log2 ratio (T1/CK) of DEGs, as calculated by the RT-qPCR analysis. The black column represents the log2 ratio (T1/CK) of DEGs, as calculated by the RNA-Seq analysis.
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Figure 5. Number of DEGs identified between T1 (corncob) and CK (sawdust) treatments in A. heimuer. The gray column indicates the total number of DEGs, the light blue column represents downregulated genes, and the dark blue column represents upregulated genes.
Figure 5. Number of DEGs identified between T1 (corncob) and CK (sawdust) treatments in A. heimuer. The gray column indicates the total number of DEGs, the light blue column represents downregulated genes, and the dark blue column represents upregulated genes.
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Figure 6. The putative encoded amino acid-related enzymes, DEGs, from the transcriptome of T1 vs. CK. The strip color from blue to red represents the extent of significantly downregulated and upregulated expression in the T1 A. heimuer transcriptome compared to that in the CK transcriptome. The numbers represent the ratio of log2(T1/CK). Abbreviations of enzymes name are given in Supplementary Table S4.
Figure 6. The putative encoded amino acid-related enzymes, DEGs, from the transcriptome of T1 vs. CK. The strip color from blue to red represents the extent of significantly downregulated and upregulated expression in the T1 A. heimuer transcriptome compared to that in the CK transcriptome. The numbers represent the ratio of log2(T1/CK). Abbreviations of enzymes name are given in Supplementary Table S4.
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Figure 7. KEGG pathway enrichment dot plot. Dot size represents the number of associated genes, while dot color indicates the statistical significance, −log10 (p-value).
Figure 7. KEGG pathway enrichment dot plot. Dot size represents the number of associated genes, while dot color indicates the statistical significance, −log10 (p-value).
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Figure 8. GO enrichment DAG plot: (a) BP, (b) CC, and (c) MF. Arrows indicate hierarchical relationships between upper and lower levels; elliptical shapes indicate GO terms with enrichment levels not in the top 10; and the box represents the GO terms with enrichment levels in the top 10. Colors indicate the degree of enrichment of differentially expressed genes in GO terms, with darker colors indicating more significant enrichment. Red represents the most significant enrichment, followed by yellow, and colorless indicates insignificant enrichment. The first row in the box represents the term number of GO, the second row represents the functional description of the term, the third row represents the p-value, and the last row of numbers represents the number of differentially expressed genes enriched in the term in the study divided by the total number of differentially expressed genes in the term.
Figure 8. GO enrichment DAG plot: (a) BP, (b) CC, and (c) MF. Arrows indicate hierarchical relationships between upper and lower levels; elliptical shapes indicate GO terms with enrichment levels not in the top 10; and the box represents the GO terms with enrichment levels in the top 10. Colors indicate the degree of enrichment of differentially expressed genes in GO terms, with darker colors indicating more significant enrichment. Red represents the most significant enrichment, followed by yellow, and colorless indicates insignificant enrichment. The first row in the box represents the term number of GO, the second row represents the functional description of the term, the third row represents the p-value, and the last row of numbers represents the number of differentially expressed genes enriched in the term in the study divided by the total number of differentially expressed genes in the term.
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MDPI and ACS Style

Sun, X.; Yao, F.; Lai, F.; Fang, M.; Lu, L.; Ma, X.; Wang, W.; Meng, J.; Shao, K. Transcriptomic Insights and Quantification of Free Amino Acids in Auricularia heimuer Cultivated on Corncob Substrate. Horticulturae 2025, 11, 563. https://doi.org/10.3390/horticulturae11060563

AMA Style

Sun X, Yao F, Lai F, Fang M, Lu L, Ma X, Wang W, Meng J, Shao K. Transcriptomic Insights and Quantification of Free Amino Acids in Auricularia heimuer Cultivated on Corncob Substrate. Horticulturae. 2025; 11(6):563. https://doi.org/10.3390/horticulturae11060563

Chicago/Turabian Style

Sun, Xu, Fangjie Yao, Fanchao Lai, Ming Fang, Lixin Lu, Xiaoxu Ma, Wei Wang, Jingjing Meng, and Kaisheng Shao. 2025. "Transcriptomic Insights and Quantification of Free Amino Acids in Auricularia heimuer Cultivated on Corncob Substrate" Horticulturae 11, no. 6: 563. https://doi.org/10.3390/horticulturae11060563

APA Style

Sun, X., Yao, F., Lai, F., Fang, M., Lu, L., Ma, X., Wang, W., Meng, J., & Shao, K. (2025). Transcriptomic Insights and Quantification of Free Amino Acids in Auricularia heimuer Cultivated on Corncob Substrate. Horticulturae, 11(6), 563. https://doi.org/10.3390/horticulturae11060563

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