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Article

Integration of Physiological and Comparative Transcriptomic Analyses Reveal the Toxicity Mechanism of p-Coumaric Acid on Morchella importuna

by
Qi Yin
1,
Wenchang Zhang
1,
Yingli Cai
2,
Xiaofei Shi
2,3,
Fuqiang Yu
3,
Jianzhuang Guo
4,
Xinhua He
5,6,
Peixin He
1,* and
Wei Liu
2,*
1
School of Food and Biological Engineering, Zhengzhou University of Light Industry, Zhengzhou 450002, China
2
Key Laboratory of Chemistry in Ethnic Medicinal Resources, School of Ethnic Medicine, Yunnan Minzu University, Kunming 650500, China
3
Germplasm Bank of Wild Species & Yunnan Key Laboratory for Fungal Diversity and Green Development, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China
4
Agricultural Industry Development and Service Center of Luanchuan County, Luoyang 471500, China
5
School of Biological Sciences, University of Western Australia, Perth, WA 6009, Australia
6
Department of Land, Air and Water Resources, University of California at Davis, Davis, CA 95616, USA
*
Authors to whom correspondence should be addressed.
Horticulturae 2025, 11(7), 755; https://doi.org/10.3390/horticulturae11070755
Submission received: 16 April 2025 / Revised: 21 June 2025 / Accepted: 23 June 2025 / Published: 1 July 2025
(This article belongs to the Special Issue Morchella: Innovative Mushrooms from Forests to Farms, Then to Our Tables)
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Abstract

p-coumaric acid (p-CA) is one of the main allelochemicals of cultivable Morchella mushrooms. However, its toxicity mechanism has not been elucidated. Therefore, we used physiological and comparative transcriptomic analyses to reveal its toxicity mechanism. The results suggest that the mycelial growth and sclerotial production of M. importuna were promoted under treatment with a low dosage of p-CA (10 μg/mL). The treatment induced moderate reactive oxygen species (ROS) accumulation, with an upregulation of genes associated with antioxidant regulation, energy supply and damage repair. In contrast, oxidative stress induced under treatment with a high dosage of p-CA (50 μg/mL) led to strain ageing. The contents of ROS were significantly increased, along with decreased peroxidase and catalase activity. Moreover, the genes associated with H2O2 synthesis were upregulated, while those responsible for H2O2 decomposition, non-enzymatic antioxidant components and damage repair were downregulated. Meanwhile, the carbohydrate and lipid metabolic pathways, and the signal transduction and cell division pathways, were impaired. Taken together, moderate stress induced under a low concentration of p-CA promotes the mycelial growth and sclerotial metamorphosis of M. importuna. This study provides new insights into the potential mechanisms of continuous cropping obstacles in the cultivation of morel mushrooms, which is of great importance for the practical aspects of mushroom cultivation.

1. Introduction

True morels (Morchella spp., Pezizales, Ascomycota) are valuable edible and medicinal mushrooms known for their delicious taste and prominent bioactivities [1,2,3]. The commercial cultivation of Morchella strains (particularly M. importuna, M. sextelata, and M. eximia) in China started in 2012 and has been expanding ever since. In the 2021–2022 production season, the total cultivated area exceeded 16,467 hectares [4]. However, along with the rapid development of morel cultivation, the inconsistency in cropping has become a bottleneck for the industry. The main problems are stunted growth and development, significant decline in yield and even complete failure, and serious occurrence of fungal diseases, which may be closely related to the reduced spawn activity in continuous cropping soil [5,6,7]. Existing studies have shown that the accumulation of morel-specific allelochemicals in soil, including p-coumaric acid, exhibit a certain extent of toxicity on the growth of morel mycelium, which may induce strain ageing, thereby bringing great “uncertainty” to the production of Morchella mushrooms [7,8,9,10].
Allelopathy refers to the suppression of growth of one plant species by another due to the release of toxic substances (allelochemicals). Allelochemicals are secondary metabolites and/or their degradation products, such as phenolic acids, aldehydes, coumarins, quinones, alkaloids, and terpenoids, among which phenolic acids were extensively investigated [11,12,13,14]. p-Coumaric acid (p-CA) is a phenolic acid compound synthesized in the phenylpropanoid biosynthesis pathway and an important intermediate involved in the metabolism of phenylalanine or tryptophan [9]. It exerts antioxidant effects in biology and can induce the expression of reactive oxygen species (ROS) protective enzymes to help maintain ROS homeostasis [15,16]. During plant growth, p-CA is also secreted into the environment to protect plants from biotic and abiotic stress [17]. p-CA is a common phenolic acid in soil, and low concentrations of p-CA can help plants consolidate their ecological advantages. However, when the concentration of p-CA accumulated in soil is too high, it can inhibit seed germination, root elongation, and the seedling growth of plants. The allelopathy caused by p-CA is a main cause of plant continuous cropping obstacles [18,19,20]. As for the mechanism underlying plant allelopathy, existing studies demonstrate that the allelochemicals can directly affect root growth by damaging cell membranes and/or cell walls. They can intrinsically impact antioxidant enzyme activity, interfere with the metabolism of ROS, and consequently induce excessive ROS (e.g., H2O2) accumulation within cells, leading to oxidative stress and lipid peroxidation, and ultimately root cell death. In bacteria, it was found that p-CA can damage cell membranes, and its binding to bacterial genomic DNA may inhibit cell function, ultimately leading to cell death [21]. Moreover, transcriptomic analyses revealed that differentially expressed genes (DEGs) under stress conditions are involved in antioxidant synthesis, DNA damage repair and metabolism, and stress responses. Furthermore, the expression of genes related to root browning, rotting, morphological deterioration, and plant death is also affected [12,13,14,22].
Our previous study determined p-CA as a morel allelochemical that exhibited inhibitory effect on mycelial growth of M. importuna at concentrations ≥20 μg/mL p-CA [9]. The mycelial growth was promoted at concentrations ≤10 μg/mL, whereas the transition from stimulation to inhibition appeared to occur between 15 and 20 μg/mL, as confirmed by newly conducted dose–response experiments (see Supplementary Attachment S2, Figure S1). Nonetheless, the underlying mechanism of its toxicity is unavailable. In the present study, an integration of physiological and transcriptomic analysis was conducted to reveal the toxicity mechanism of p-CA on cultivable morel species of M. importuna. p-CA is a common secondary metabolite that can be accumulated up to 183.6 μg/mL in the rhizosphere of certain plants. Considering the actual p-CA concentration in soils of morel continuous cropping fields, we chose the concentrations of 10 and 50 μg/mL for the study of its toxicity mechanism [9,23,24]. This study provides novel insights into morel continuous cropping obstacle, which will promote the final resolution of the bottleneck issue that restrains the sustainable development of the morel industry.

2. Materials and Methods

2.1. Strain, Growth Conditions and Quantitative Assessment of Strain Ageing

M. importuna L16-1 is a high-yielding strain domesticated from wild morels. The strain can be obtained from Peixin He at Zhengzhou University of Light Industry, China, for reasonable request. The taxonomic status of the strain was confirmed through sequence analysis of ITS, EF1-α, RPB1, and RPB2, with sequence accession numbers of PQ524485, PQ559332, PQ559333, and PQ559331, respectively. A mycelial plug from the stock culture was inoculated on one side of a Petri dish (9 cm diameter) of a complete yeast extract medium (CYM) (glucose 20 g/L, yeast extracts 2 g/L, peptone 2 g/L, K2HPO4 1 g/L, MgSO4 0.5 g/L, KH2PO4 0.46 g/L, and agar 20 g/L) and incubated at 22 °C in dark. Mycelial plugs (4 mm diameter) from the respective activated cultures were placed in the centre of CYM plates covered with autoclaved cellophane membranes (aperture: 0.016 μm, thickness: 20 μm) with different concentrations of p-CA (0, 10, 50 μg/mL) (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The plates were incubated in the dark at 22 °C for 2, 4, 6, 8, and 10 d and the mycelial mat was then collected. The fresh biomass was measured and then divided into two portions: one portion was frozen in liquid nitrogen and stored at −80 °C for further analysis, while the other was kept on ice for physiological and biochemical assays. In addition, the mycelial growth was observed every 12 h. When the mycelium under any treatment condition was about to cover the entire CYM plate, the colony diameters of all test plates were recorded. The area of the sclerotia on each plate was also recorded after 10 days of incubation. The enzymatic activities of amylase and xylanase were negatively correlated with the ageing degree of Morchella spp., and thus the ageing degree of the strains was evaluated by measuring the activity of amylase and xylanase. The crude enzyme solutions were prepared from the culture of compost containing different concentrations of p-CA, and the activity assay of amylase and xylanase was conducted using dinitrosalicylic acid method [25].

2.2. Physiological and Biochemical Assays

The activity of antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), and the content of O2•−, H2O2 and malondialdehyde (MDA) of the fresh mycelia were determined. The SOD activity was measured using the nitro blue tetrazolium (NBT) method and the POD activity was determined by the method of the guaiacol oxidation [26]. The CAT activity was assessed using the ammonium molybdate method [27]. The contents of O2•− and H2O2 were determined using the method of hydroxylamine hydrochloride reaction and titanium sulphate [28,29]. The MDA content was quantified using the thiobarbituric acid method [7].

2.3. RNA Extraction, Library Construction, and Sequencing

Total RNA of the 48 h culture of p-CA treatments under concentration of 0, 10, and 50 μg/mL, respectively, was extracted using the Trizol® reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. The integrity of the RNA was assessed via agarose gel electrophoresis, while the RNA concentration was determined using the Qubit 2.0 RNA Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). The RNA quality was further evaluated with the Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). Six libraries were then constructed using the Hieff NGS™ MaxUp Dual-mode mRNA Library Prep Kit for Illumina® (Yeasen Biotechnology, Shanghai, China), in accordance with the manufacturer’s protocol. These libraries were sequenced on the Hiseq 4000 platform (Illumina, San Diego, CA, USA) by Sangon Biotech (Shanghai, China), with sequencing reads of 2 × 150 bp. All reads have been deposited in the Sequence Read Archive (SRA) at NCBI (https://www.ncbi.nlm.nih.gov/sra, accessed on 26 November 2024) under the accession number SRR31512980-SRR31512982.

2.4. Data Processing and Bioinformatic Analysis

Data processing was conducted using Trimmomatic [30] to remove poly-N sequences, adapters, and low-quality reads, resulting in clean reads. The clean reads were then aligned to the M. importuna genome (assembly ASM344463v2, https://www.ncbi.nlm.nih.gov/assembly/GCF_003444635.1, accessed on 26 November 2024) [31] using HISAT2 software (v2.1.0) [32]. For each transcript, the read count is standardized as the number of fragments per kb of base per million mapped reads (FPKM). Differential expression analysis was performed using the DEGseq R package under R v4.4.0, with significant changes defined by a Q-value (adjusted p-value for multiple hypothesis testing) < 0.05 and |log2(Fold Change)| ≥ 1 [33]. Functional annotation of DEGs was carried out using the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Eukaryotic Orthologous Groups (KOG) databases.

2.5. Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) Detection of Differentially Expressed Genes

The RT-qPCR was utilized to confirm the accuracy of the RNA-seq results. Total RNA was extracted using Trizol® reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesized via reverse transcription using the MightyScript Plus First Strand cDNA Synthesis Master Mix (gDNA digester) (Sangon Biotech, Shanghai, China), according to the manufacturer’s protocols. RT-qPCR was then performed to analyze gene expression. The RT-qPCR protocol included an initial denaturation at 95 °C for 5 min, followed by 42 cycles of 95 °C for 10 s and 60 °C for 30 s, with fluorescence measurement taken at the end of each cycle. After the completion of the 42 cycles, a melting curve was generated by gradually heating the amplicons from 65 °C to 95 °C to verify the production of a single specific product. To validate the RNA-seq findings, 9 genes related to antioxidant enzymes were selected for RT-qPCR validation. Expression levels for each gene were evaluated using three technical replicates. The housekeeping gene ACT1 was employed to normalize the Ct values, and relative expression levels were determined using the 2−ΔΔCT method [34]. The genes and primers used in RT-qPCR analysis were listed in Supplementary Table S1.

2.6. Statistical Analysis

Data were presented as means ± standard error (S.E.) (n = 3). Statistical analysis was performed using one-way ANOVA, followed by Duncan’s multiple range test. Probability values (p) < 0.05 and <0.01 were considered statistically significant and highly significant, respectively. All analyses were conducted using IBM SPSS Statistics 22. Graphs were generated using Origin 2022 (OriginLab Corporation, Northampton, MA, USA) and processed with Adobe Photoshop 2025 (v26.0.0).

3. Results

3.1. Physiological and Biochemical Responses to p-CA Treatment

The general trend in the allelopathic effect of p-CA on the mycelial growth of the tested morel strain is similar to hormesis. Mycelial growth (biomass) and sclerotia production (number of sclerotia unit area) were promoted under the treatment with a low concentration of p-CA (10 μg/mL) (Figure 1A–C). However, ageing in the tested morel strain was induced by a high concentration of p-CA (50 μg/mL), manifested as mycelial growth inhibition, decline in amylase and xylanase activity (Figure 1D,E), pigment aggravation, and a decreased production of sclerotium (Figure 1B, Supplementary Figures S2 and S3). The results were similar to those of total phenolic acid extracts from both continuous cropping soils and control ones [8]. Moreover, the trends in antioxidant enzyme activities under different p-CA concentrations were different. For instance, stronger SOD activities were induced under a high concentration of p-CA (50 μg/mL) (Figure 1F), where the activities of POD (Figure 1G) and CAT (Figure 1H) were lowest, implying a distinctive role of the three antioxidant enzymes responding to the stress of the morel allelochemical. The content of O2•− increased in a dose-dependent manner during the first 6 d of culture and then decreased along with the extended culture time (Figure 1I). However, the contents of H2O2 (Figure 1J) and MDA (Figure 1K) increased in a dose-dependent manner with increasing p-CA concentrations. The results suggest a greater ROS production response to the high-concentration-p-CA (50 μg/mL) treatment and the inducement of antioxidant enzymes such as SOD to scavenge ROS. Oxidative stress may occur when the balance between ROS production and antioxidant defence is disrupted.

3.2. Overall Analysis of mRNA Sequencing

To elucidate the molecular mechanisms underlying p-CA stress, RNA sequencing (RNA-seq) was performed on 48 h old cultures of M. importuna L16-1 cultivated with the addition of 0 (CK), 10 (CA10), and 50 μg/mL p-CA (CA50). A total of six cDNA libraries were constructed. The raw read counts were 102.55 million for CK, 100.23 million for CA10, and 97.39 million for CA50, respectively. Following the removal of poly-N sequences, adapters, and low-quality reads, 97.95% to 98.40% of the clean reads were successfully mapped to the M. importuna genome for further analysis. The percentage of Q30 bases (bases with a quality score of 30 or higher) exceeded 95% in all samples (Supplementary Table S2). The Pearson correlation analysis results of sequencing data (Supplementary Figure S4) indicated good reproducibility.

3.3. Identification of DEGs

DEGs were identified by applying |log2 (Fold Change)| ≥ 1 and p < 0.05 as thresholds for fragments per kilobase of exon model per million mapped reads (FPKM) values. Compared to the control, a total of 3739 DEGs exhibited significantly differential expression among the three groups under p-CA stress. The DEGs between CA10 and CK were designated as D10, while DEGs between CA50 and CK were designated as D50. There were 1250 DEGs in D10, of which 1026 genes were upregulated and 224 genes were downregulated. Further, 2959 DEGs existed in D50, of which 420 were upregulated and 2539 were downregulated (Figure 2A, Supplementary Table S3). A large number of downregulated DEGs were enriched in D50 (compared to D10), which indicated that high concentrations of p-CA exert greater pressure on hyphae and limit the normal expression of more genes. Meanwhile, D10 enriched more upregulated genes, which indicated that low concentrations of p-CA stimulate gene expression.

3.4. GO and KEGG Analysis of DEGs

The functional annotation of DEGs using GO analysis suggested that the top five categories significantly enriched in D10 were cytoplasmic part (GO: 0044444), intracellular organelle part (GO: 0044446), organelle part (GO: 0044422), cellular biosynthetic process (GO: 0044249), and organonitrogen compound metabolic process (GO: 1901564) (Figure 2B). The five most enriched categories in D50 were response to stimulus (GO: 0050896), biological regulation (GO: 0065007), regulation of cellular process (GO: 0050794), regulation of biological process (GO: 0050789), and catalytic activity (GO: 0003824) (Figure 2C). In particular, the response to stimulus was the most significantly enriched among the downregulated genes in D50, suggesting that the biological process of responding to external stimuli may be markedly suppressed.
The differential metabolic pathways involved in p-CA stress defences were analyzed by KEGG analysis (Supplementary Tables S4–S7). The KEGG pathways with p < 0.05 for DEGs in D10 and D50 are shown in Figure 2D–G. Overall, the upregulated pathways in D10 are mostly related to the metabolic pathways required for antioxidant regulation, material transportation, energy supply and damage repair (Figure 2D, Supplementary Table S4), while the downregulated pathways are related to specific substance metabolism and signal transduction (Figure 2E, Supplementary Table S5). The upregulated pathway in D50 is closely related to energy supply and specific substance synthesis (Figure 2F, Supplementary Table S6), while the downregulated group enriches a large number of pathways related to signalling and regulation, metabolism and repair (Figure 2G, Supplementary Table S7).

3.5. DEGs Associated with Oxidative Stress

A total of 29 genes associated with H2O2 metabolism exhibited differential expression in both D10 and D50 (Figure 3A, Supplementary Table S8). Genes involved in H2O2 synthesis, such as SOD encoding genes, were upregulated. Conversely, genes involved in H2O2 decomposition, including encoding genes of CAT and POD, were mainly downregulated in D50. However, most of the genes involved in H2O2 decomposition in D10 were upregulated in expression. Notably, although the expression of most antioxidant enzymes decreased in D10, GPX showed no significant downregulation, whereas in D50, GPX was slightly upregulated, indicating a possible compensatory response to higher oxidative stress in this state.
Three genes related to vitamin digestion, absorption, and metabolism were identified. Among them, LAJ45_00740 was upregulated in D10, while LAJ45_07924 and LAJ45_05546 were downregulated in D50. Moreover, two genes associated with trehalose metabolism were downregulated in D50, with no significant expression changes observed in D10 (Figure 3A, Supplementary Table S8). In addition, a large number of genes in the MAPK signalling pathway related to oxidative stress regulation were upregulated in D10 and downregulated in D50 (Figure 3A, Supplementary Table S9).

3.6. DEGs Related to Ageing

A total of 34 DEGs related to nucleic acid and protein repair were identified as differentially expressed under D10 and D50 stress (Figure 3B, Supplementary Table S10). Most of these genes were upregulated in D10, with six genes significantly upregulated. In D50, only 3 genes significantly upregulated, while the remaining 31 genes were downregulated. Therefore, the repair ability of D50 was greatly weakened. These DEGs were related to pathways such as nucleotide excision repair (ko03420), base excision repair (ko03430), and mismatch repair (ko03430). These pathways are crucial for regulation of the repair of nucleic acids and proteins. The downregulated genes within these pathways may result in cell dysfunction and contribute to morel strain ageing.

3.7. DEGs Related to Metabolism

Metabolic pathways were significantly impacted in response to p-CA treatment. In a total of 60, 35 DEGs related to carbohydrate and lipid metabolism pathways were identified at D10 and D50, respectively. Carbohydrate catabolism serves as a major energy source for biological activities and is essential for maintaining normal physiological processes. At D10 and D50, 11/3 and 21/29 genes related to carbohydrate synthesis and metabolism were upregulated/downregulated, respectively (Figure 3C, Supplementary Table S11). The upregulated genes at D10 were mainly associated with pathways such as starch and sucrose metabolism (ko00500), pentose phosphate pathway (ko00030), glycolysis/gluconeogenesis (ko00010), and N-glycan biosynthesis (ko00510). On the other hand, the DEGs related to these pathways were generally downregulated at D50.
Lipid transport and metabolism are crucial for the maintenance of cell membrane stability and are also integral to signal transduction. A total of 3/1 DEGs were upregulated/downregulated at D10 (Figure 3D, Supplementary Table S12). The upregulated genes were associated with pathways including steroid biosynthesis (ko00100), inositol phosphate metabolism (ko00562), and glycerophospholipid metabolism (ko00564), which contribute positively to cell membrane stability. On the other hand, 37 DEGs were downregulated at D50 (Figure 3D, Supplementary Table S12). The downregulated DEGs were linked to pathways such as glycerophospholipid metabolism (ko00564), inositol phosphate metabolism (ko00562), and sphingolipid metabolism (ko00600). The downregulated DEGs may impact the structure, function, and stability of the cell membrane, resulting in reduced signal transduction capability and a diminished ability of stress resistance.

3.8. RT-qPCR Validation of Candidate Genes

Nine genes related to antioxidant enzymes were selected for RT-qPCR validation and their relative expression abundance in different samples was calculated (Figure 4). Following p-CA treatment, the expression levels of SOD2 and SOD4 genes were upregulated in a concentration-dependent manner, whereas SOD1 expression showed an increasing trend with concentration that was not statistically significant. CAT3 expression was progressively downregulated with increasing p-CA concentrations. In addition, under the D10 treatment, the expression of SOD3 and SOD5 was inhibited, while CAT1 and CAT2 expressions were induced. POD expression is significantly decreased under D10 treatment, while no statistically significant difference between D50 treatment and CK (p > 0.05). Conversely, the D50 treatment exhibited an opposite regulatory trend. The results showed that the expression patterns of DEGs in RT-qPCR were similar to RNA-seq data, demonstrating the reliability of RNA-seq results.

4. Discussion

4.1. Sclerotial Metamorphosis and Ageing

Our study suggests that p-CA stress induced ROS production in hyphal cells of M. importuna under a low dose of p-CA (10 μg /mL), while oxidative stress occurred in response to high concentration of p-CA (50 μg /mL) (Figure 1, Supplementary Figure S2). It is noteworthy that the actual content of p-CA in morel continuous cropping soils is usually around 40 μg/mL, while the concentration of 50 μg/mL p-CA used in this study is slightly higher than this level [9]. Therefore, the experimental conditions may not fully simulate the soil environment, but they still contribute to elucidating the response patterns of morel mycelium under extreme stress conditions. This provides a valuable reference for understanding the relationship between allelochemicals and continuous cropping obstacles.
ROS (H2O2, ·OH−, O2•−, and so on) are metabolic byproducts that act as signalling molecules under normal physiological conditions. Nonetheless, excessive ROS production will disrupt redox homeostasis and cause cell damage and oxidative stress. The dynamic equilibrium of ROS is regulated by an antioxidant system that includes both enzymatic (e.g., SOD, POD and CAT) and non-enzymatic components (e.g., vitamins and trehalose) [35,36,37]. The fluctuation phenomenon of O2•− content (Figure 1I) suggests spatial heterogeneity in O2•− production and removal. NADPH oxidase on plasma membranes can overproduce O2•− under stress conditions, forming localized high concentration areas, while the compartmentalized distribution of SOD enzyme in cells (such as mitochondria and peroxisomes) may not respond promptly to such transient signals [35]. In addition, O2•− can be easily converted to H2O2 under non-enzymatic conditions, which further dissociates the correlation between O2•− content and SOD activity. Therefore, the production of superoxide radicals in CA50 does not fully correspond to SOD activity [35,37]. Under CA50 stress, the production of H2O2 in cells continued to increase with time (Figure 1J). On the one hand, the mitochondrial electron transport chain is damaged by stress, leading to increased electron leakage (as described in detail below) and the generation of a large amount of O2•−. Catalyzed by superoxide dismutase (SOD) (Figure 1F), O2•− is rapidly converted to H2O2. Moreover, metabolic activities in peroxisomes, such as fatty acid β-oxidation, can directly produce H2O2, and this process does not rely on the O2•− conversion pathway [35]. On the other hand, the H2O2 scavenging system showed significant dysfunction—the activities of POD (Figure 1G) and CAT (Figure 1H) were consistently lower than the control levels, limiting the ability to clear H2O2. Ultimately, it leads to sustained and significant accumulation of H2O2 in cells. Unfortunately, due to experimental limitations, the activity of other antioxidant enzymes (such as GPX) and non-enzymatic antioxidant components were not detected, which may be one of the important reasons for the incomplete match between O2•− and H2O2 levels and SOD/POD/CAT activity [35,37]. Interestingly, transcriptomic data revealed a different expression pattern of GPX genes under different treatment concentrations. In D10, the slight but not significant downregulation of GPX suggests that the antioxidant defence may still rely primarily on major enzymes such as SOD and CAT, without requiring activation of the GPX pathway. In contrast, under D50, GPX expression genes were upregulated, which may reflect a compensatory mechanism to alleviate H2O2 accumulation caused by the downregulation of CAT and POD. GPX uses reduced glutathione as a cofactor to reduce H2O2 into water and may serve as an alternative detoxification pathway when the primary clearing enzymes are damaged [38].
Fungal sclerotial metamorphosis is believed to be induced by the rapid and transient production of significant amounts of ROS (oxidative burst). The sclerotium is an important energy storage structure in morel mushroom, and its formation is regulated by environmental stressors such as hypoxia, nutrient deprivation and ROS stress [10,39,40,41]. H2O2 can induce sclerotial formation with concentration-dependent efficiency. Low concentrations of H2O2 (5 mM) can significantly promote sclerotial formation, whereas higher concentrations (>10 mM) inhibit sclerotial formation [10,40,41,42]. Our physiological measurements are consistent with these findings. Moreover, our study revealed that treatment with a low concentration of p-CA(D10) upregulated the expression of genes associated with the MAPK signalling pathway and lipid metabolism, whereas these gene expression levels downregulated under high-concentration p-CA treatment (D50) (Figure 3A). The MAPK signalling pathway can mediate oxidative stress responses and promote sclerotial formation, while lipid metabolism is essential for energy storage in sclerotia [39,40]. Therefore, we speculate that low concentrations of p-CA promote sclerotial formation by regulating ROS metabolism, MAPK signalling, and lipid accumulation. In contrast, oxidative stress induced by high p-CA concentrations may disrupt these regulatory mechanisms, thereby inhibiting extensive sclerotium accumulation.
Ageing is a time-related process of progressive decline in the ability to withstand stress, damage, and disease [7,43]. As ascomycetes fungi, strain ageing and spawn ageing in Morchella spp. are more prominent than most basidiomycetes mushrooms. Ageing may be the main reason underlying the “uncertainty” of morel farming. Oxidative stress may accelerate the ageing process of morel cells [28,44]. Oxidative stress is an important factor contributing to the decreased efficiency of DNA repair pathways such as nucleotide excision repair and base excision repair. Reactive oxygen species (ROS) can promote cellular ageing by causing oxidative damage to DNA and repair-related enzymes [45,46]. In addition, prolonged oxidative stress may impair the function of ubiquitin-mediated proteolysis, leading to disrupted protein degradation and cellular homeostasis [47]. These changes may be closely associated with the widespread downregulation of related metabolic pathway genes in D50. Fungal ageing is closely related to mitochondrial dysfunction, and oxidative damage can affect the stability of mitochondrial DNA (mtDNA) and weaken respiratory chain function [48,49,50]. In D10, a large number of genes related to repair are upregulated, which actively maintain the stability of cell structure and function. However, in D50, most genes related to damage repair are downregulated, which greatly reduced the self-repair ability of cells. When ROS accumulation exceeds the cell’s regulation and repair capacity, the damage may be irreversible, and thus the final response appears to be the induction of a programmed cell death [50,51,52]. Notably, following D50 treatment, the expression of a large number of genes involved in nucleocytoplasmic transport (ko03013), RNA degradation (ko03018), ribosome biogenesis in eukaryotes (ko03008), and Cell cycle—yeast (ko04111) pathways was significantly downregulated (Figure 2G). This suggests that D50 treatment may inhibit the essential cellular functions and proliferation ability of cells, leading to inhibited cell division, reduced growth rate, and potentially fatal cell cycle arrest [53,54]. A similar phenomenon was observed in M. importuna, where low ROS stress promoted the formation of sclerotia, while high ROS stress led to cell ageing [40,41].

4.2. Metabolic Adjustment in Response to p-CA Stress

Metabolism is an essential determinant of cellular and organismal health and lifespan [55]. Our study found that most DEGs involved in carbohydrate and lipid metabolism pathways were upregulated in the treatment of 10 μg/mL p-CA (D10) (Figure 3C,D), which was consistent with the increased metabolic activity and requirement for carbon sources to support mycelial growth and lipid-rich sclerotial metamorphosis in morel. Similar results have been reported on the interspecific interactions between white-rot fungi [56] and host–pathogen interactions between plants and fungi, especially in nutrient competition and the secretion of defence-related compound of against the pathogen [57,58]. However, the expression levels of pathways related to carbohydrate metabolism under 50 μg/mL p-CA were generally downregulated in hyphal cells of M. importuna (Figure 3C, Supplementary Table S11). The downregulation of associated genes may reduce the rates of glucose assimilation and ATP synthesis [59]. Nevertheless, it is worth noting that several genes involved in glycolysis/gluconeogenesis and the pentose phosphate pathway were upregulated, which may reflect a compensatory response to oxidative stress. The pentose phosphate pathway, in particular, generates NADPH, which neutralizes oxidative stress and promotes damage repair [38]. Moreover, pentoses may also serve as precursors for nucleotides synthesis. Both components play vital roles in stress resistance [60,61,62]. The mixed expression pattern of stress-related genes suggests that the p-CA-induced oxidative stress at this concentration may partially exceed the organism’s self-regulatory capacity. Consequently, the energy metabolic pathways in morel hyphal cells are partially inhibited, leading to a decreased rate of material transformation and hampering the organism’s ability to respond to toxic stress.
Furthermore, p-CA stress affects the metabolism of sugars and lipids and damages the structure, function, and stability of cell membranes. N-glycans are primarily involved in N-glycosylation, playing a crucial role in the regulation of protein activity and stability as well as in signal transduction within fungal cells [63,64]. Steroid and inositol phosphate are involved in cell signal transduction. Meanwhile, steroid, glycerophospholipid, and sphingolipid are the main membrane lipids in fungi, participating in transmembrane signal transduction and the maintenance of membrane stability. We observed a significant downregulation of lipid metabolism–related genes in D50. Abnormal lipid metabolism can directly impair membrane structure and function, for example, by enhancing lipid peroxidation and compromising membrane integrity. The increase in MDA content (Figure 1K) suggested a state of lipid peroxidation, which may further accelerate cellular ageing [65]. The disruption of the metabolic pathways of these molecules (Figure 3D, Supplementary Table S12) may compromise membrane integrity and disrupt signalling pathways, hence ultimately affecting intracellular and intercellular interactions, cell growth, and signal transduction [66,67].

4.3. Mechanisms Underlying the Toxicity of p-Coumaric Acid on M. importuna

The mechanisms underlying the toxicity of p-CA on M. importuna involved oxidative stress and the related regulation of signal transduction, as well as metabolic pathways (Figure 5). Our study indicates that moderate ROS levels (e.g., induced by treatment with 10 μg/mL p-CA) may induce fungal sclerotial metamorphosis, manifested as increased mycelial biomass, sclerotial formation, and metabolism of carbohydrate and lipid in cultivated M. importuna. However, high concentrations of p-CA (50 μg/mL) induce excessive ROS accumulation, disrupting the balance between ROS generation and elimination, ultimately leading to oxidative stress. Mitochondria are the primary site for ROS generation. Persistent ROS accumulation within mitochondria leads to oxidative damage to mtDNA and impairs respiratory chain complexes, potentially resulting in mitochondrial dysfunction. The damage to the respiratory chain causes excessive electron leakage, further increasing ROS production while reducing ATP synthesis, thereby creating a vicious cycle. Due to the decreased repair ability of mtDNA and proteins in cells, oxidative stress conditions progressively worsen, ultimately triggering strain ageing [50,51,52]. In addition, oxidative stress can cause lipid peroxidation, damage cell membranes, and create osmotic stress. Moreover, stress induced by high p-CA concentrations leads to the downregulation of most genes involved in carbohydrate and lipid metabolism, which results in the disruption of metabolic equilibrium and insufficient energy supply. Meanwhile, a disruption of the signalling pathways (e.g., N-glycan, phosphatidylinositol, and MAPK signalling pathways) may further disrupt cell growth and metabolic regulation, exacerbating developmental disorders. Ultimately, the interaction of oxidative stress, metabolic disorder, and nutritional deficiency induced by a high dose of p-CA may lead to an inhibition of cell division and growth and the development of tested morel strains, manifesting as inhibited mycelial growth, reduced biomass accumulation, and accelerated ageing.

4.4. Structure–Activity Relationships of Hydroxycinnamic Acid Derivatives: Implications for Antifungal Activity and Toxicology

This study reveals the toxicity and potential mechanisms of p-CA on morel strains, providing preliminary evidence for understanding the biological functions of hydroxycinnamic acids. The antifungal activity and cytotoxicity of hydroxycinnamic acid derivatives are highly dependent on their structural characteristics, particularly the number and position of hydroxyl and methoxy groups on the aromatic ring [68,69]. Compounds bearing an ortho dihydroxyl (catechol) group, such as caffeic acid and rosmarinic acid, exhibit superior bioactivity, probably because of their enhanced metal chelating capacity and ability to induce intracellular oxidative stress. In contrast, mono-hydroxylated p-CA generally shows lower activity [68]. The introduction of a methoxy group (such as ferulic acid) can enhance the lipophilicity of molecules, which may not only increase their cell membrane permeability but also affect their redox potential and target selectivity [69,70]. In addition, conjugation between the aromatic ring and the side chain carboxyl group favours electron delocalization, which can strengthen their interaction with DNA or enzymes targets and thereby induce oxidative stress responses [70,71].
Although ferulic acid may have stronger fungal growth inhibition than p-CA due to its methoxy group, our previous study [9] showed that the concentration of ferulic acid (about 1.34 μg/mL) in the soil of morel continuous cropping was significantly lower than that of p-CA (about 40.22 μg/mL). Therefore, under natural soil conditions, the inhibitory effect of ferulic acid on morel development is weaker than that of p-CA, which supports the selection of p-CA as the focus of further toxicological research. These structural features collectively influence the antifungal activity and cytotoxicity of hydroxycinnamic acid compounds. Notably, the structure–activity relationship of p-CA itself remains unclear. In the future, the relationship between its toxicity and substituent position, hydrophilicity/hydrophobicity, and electronic properties should be systematically explored. This will provide a theoretical basis for the further recognition of the mechanisms underlying its toxicity on morel mushrooms.

5. Conclusions

Our previous study determined p-CA as the main morel allelochemical. In this study, the toxicity mechanism of p-CA on M. importuna was revealed by the integration of physiological and comparative transcriptomic analyses. It was found that moderate ROS accumulation was reduced under treatment with low concentrations of p-CA (10 μg/mL), which promotes morel mycelial growth and sclerotial metamorphosis. However, excess ROS induced by a high dosage of p-CA (50 μg/mL) could cause oxidative stress and thus induce morel strain ageing. Meanwhile, treatments under high p-CA concentrations disrupted metabolic pathways, particularly carbohydrate and lipid metabolism, and impaired signal transduction and gene expression pathways. Overall, the mechanisms underlying the toxicity of p-CA on M. importuna may involve oxidative stress, the regulation of signal transduction, cell division, and corresponding metabolic and repair pathways. The detailed molecular pathways involved need further studies. Our results provide valuable insights into the mechanism of allelochemical toxicity on morels and will be helpful for the remediation and control of obstacles in continuous morel cropping.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/horticulturae11070755/s1, Figure S1: Physiological characteristics of M. importuna L16-1 cultured with under concentration of 0, 5, 10, 15, 20, 25, 30, 40 and 50 μg/mL p-CA for 2, 4, 6, 8, and 10 d, respectively. (A) Mycelial biomass. (B) Sclerotia area. (C) Colony diameter. Significant differences between treatments were indicated by different letters (Duncan’s multiple range test, p < 0.05). Figure S2: Colony morphology of M. importuna L16-1 cultured on CYM plates supplemented with p-CA under concentration of 0, 10, and 50 μg/mL at 22 °C for 2–10 d, respectively. Figure S3: Sclerotia morphology of M. importuna L16-1 cultured on CYM plates supplemented with p-CA under concentrations of 0, 10 and 50 μg/mL at 22 °C for 10 d, respectively. Figure S4: Pearson correlation analysis of sequencing data. The Pearson correlation coefficients were presented in each matrix. Table S1: Gene-specific primers for RT-qPCR; Table S2: Transcriptome sequencing quality data summary; Table S3: Whole transcriptome gene expression information; Table S4: The top 15 upregulated KEGG pathways of differentially expressed genes in D10; Table S5: The top 15 downregulated KEGG pathways of differentially expressed genes in D10; Table S6: The top 15 upregulated KEGG pathways of differentially expressed genes in D50; Table S7: The top 15 downregulated KEGG pathways of differentially expressed genes in D50; Table S8: List of H2O2 metabolism related genes; Table S9: List of oxidative stress signal pathway related genes; Table S10: List of ageing related genes; Table S11: List of energy metabolism related genes; Table S12: List of lipid transport and metabolism related genes. Attachment S1: Detailed Methods of Section 2.2 and Assay of Amylase and Xylanase Activity. Attachment S2: Dose–Response Experiment of p-Coumaric Acid on Morchella importuna.

Author Contributions

All authors contributed to the study conception and design. P.H. and W.L. designed the study. W.L. and F.Y. obtained the research funding. Material preparation, data collection and analysis were performed by Q.Y., W.Z., Y.C., X.S., and J.G. The manuscript was written by Q.Y. and P.H., and edited by X.H. All authors commented on previous versions of the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Yunnan Key Project of Science and Technology (202402AE090030).

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Physiological characteristics of M. importuna L16-1 cultured with 0 μg/mL (CK), 10 μg/mL (CA10), and 50 μg/mL p-CA (CA50) for 2, 4, 6, 8, and 10 d, respectively. (A) Mycelial biomass. (B) Sclerotia area. (C) Colony diameter. (D) Amylase activity. (E) Xylanase activity. (F) Superoxide dismutase (SOD) activity. (G) Peroxidase (POD) activity. (H) Catalase (CAT) activity. (I) O2•− content. (J) H2O2 content. (K) Malondialdehyde (MDA) content. Significant differences between treatments were indicated by different letters (Duncan’s multiple range test, p < 0.05).
Figure 1. Physiological characteristics of M. importuna L16-1 cultured with 0 μg/mL (CK), 10 μg/mL (CA10), and 50 μg/mL p-CA (CA50) for 2, 4, 6, 8, and 10 d, respectively. (A) Mycelial biomass. (B) Sclerotia area. (C) Colony diameter. (D) Amylase activity. (E) Xylanase activity. (F) Superoxide dismutase (SOD) activity. (G) Peroxidase (POD) activity. (H) Catalase (CAT) activity. (I) O2•− content. (J) H2O2 content. (K) Malondialdehyde (MDA) content. Significant differences between treatments were indicated by different letters (Duncan’s multiple range test, p < 0.05).
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Figure 2. GO annotation and KEGG enrichment pathway analysis of DEGs in D10 and D50. (A) Number of DEGs identified in D10 and D50. (B,C) The scatter displays the top 20 GO annotation categories that achieved the highest enrichment level. (D–G) Scatter plots of significantly upregulated and downregulated KEGG pathways in D10 and D50, respectively. The vertical axis indicated the functional annotation information, and the horizontal axis represented the enrichment factor (−lg p value). The colour of the points represented the p-value, with smaller p-values corresponding to colours closer to red. The size of each point reflected the number of DEGs associated with each function.
Figure 2. GO annotation and KEGG enrichment pathway analysis of DEGs in D10 and D50. (A) Number of DEGs identified in D10 and D50. (B,C) The scatter displays the top 20 GO annotation categories that achieved the highest enrichment level. (D–G) Scatter plots of significantly upregulated and downregulated KEGG pathways in D10 and D50, respectively. The vertical axis indicated the functional annotation information, and the horizontal axis represented the enrichment factor (−lg p value). The colour of the points represented the p-value, with smaller p-values corresponding to colours closer to red. The size of each point reflected the number of DEGs associated with each function.
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Figure 3. Heatmap representation of gene expression related to H2O2 metabolism (A), ageing (B) and transport and metabolism of carbohydrate (C), and lipid (D) in D10 and D50 response to p-CA stress in M. importuna.
Figure 3. Heatmap representation of gene expression related to H2O2 metabolism (A), ageing (B) and transport and metabolism of carbohydrate (C), and lipid (D) in D10 and D50 response to p-CA stress in M. importuna.
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Figure 4. RT-qPCR analysis of nine antioxidant enzyme genes in M. importuna. CK, CA10, and CA50 represent the treatment under concentration of p-CA of 0, 10, and 50 μg/mL, respectively. Significant differences between treatments are indicated by different letters (Duncan’s multiple range test, p < 0.05).
Figure 4. RT-qPCR analysis of nine antioxidant enzyme genes in M. importuna. CK, CA10, and CA50 represent the treatment under concentration of p-CA of 0, 10, and 50 μg/mL, respectively. Significant differences between treatments are indicated by different letters (Duncan’s multiple range test, p < 0.05).
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Figure 5. A conceptual diagram underlying the toxicity of p-CA on M. importuna. The red and green circle represents harmful and beneficial effects, respectively. “+” represents promotion, and “−” represents inhibition.
Figure 5. A conceptual diagram underlying the toxicity of p-CA on M. importuna. The red and green circle represents harmful and beneficial effects, respectively. “+” represents promotion, and “−” represents inhibition.
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MDPI and ACS Style

Yin, Q.; Zhang, W.; Cai, Y.; Shi, X.; Yu, F.; Guo, J.; He, X.; He, P.; Liu, W. Integration of Physiological and Comparative Transcriptomic Analyses Reveal the Toxicity Mechanism of p-Coumaric Acid on Morchella importuna. Horticulturae 2025, 11, 755. https://doi.org/10.3390/horticulturae11070755

AMA Style

Yin Q, Zhang W, Cai Y, Shi X, Yu F, Guo J, He X, He P, Liu W. Integration of Physiological and Comparative Transcriptomic Analyses Reveal the Toxicity Mechanism of p-Coumaric Acid on Morchella importuna. Horticulturae. 2025; 11(7):755. https://doi.org/10.3390/horticulturae11070755

Chicago/Turabian Style

Yin, Qi, Wenchang Zhang, Yingli Cai, Xiaofei Shi, Fuqiang Yu, Jianzhuang Guo, Xinhua He, Peixin He, and Wei Liu. 2025. "Integration of Physiological and Comparative Transcriptomic Analyses Reveal the Toxicity Mechanism of p-Coumaric Acid on Morchella importuna" Horticulturae 11, no. 7: 755. https://doi.org/10.3390/horticulturae11070755

APA Style

Yin, Q., Zhang, W., Cai, Y., Shi, X., Yu, F., Guo, J., He, X., He, P., & Liu, W. (2025). Integration of Physiological and Comparative Transcriptomic Analyses Reveal the Toxicity Mechanism of p-Coumaric Acid on Morchella importuna. Horticulturae, 11(7), 755. https://doi.org/10.3390/horticulturae11070755

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