Evaluation of Drought Tolerance and Trehalose Response in Auricularia heimuer

Abstract

Auricularia heimuer is drought tolerant, but the mechanism underlying its physiological response to drought has not been systematically studied. We selected 13 strains of A. heimuer and simulated drought stress using a complete yeast medium (CYM) containing 20% polyethylene glycol (PEG), while the medium used for the control treatments did not contain PEG. Strains were cultured on a shaker incubator at 25 °C at 120 rpm for 15 days under dark conditions. The contents of soluble sugars (SS) and soluble proteins (SP), the activities of superoxide dismutase (SOD) and catalase (CAT), the content of malondialdehyde (MDA), and the biomass were measured. Between the 20% PEG treatment and the control, as well as among different strains, there were significant differences in all of the physiological indices. The tested strains were classified into the following four categories according to their membership function values: the first category consisted of the highly drought-tolerant strain A; the second consisted of the drought-tolerant strains A127 and C; the third consisted of the moderately drought-tolerant strains A124, A14, A386, A462, A184, A496, A125, and B; and the fourth consisted of the drought-sensitive strains A356 and A508. Transcriptome analysis was performed on C before and after drought stress treatment, and 1762 differentially expressed genes (DEGs) were obtained, including 798 up- and 964 down-regulated genes. Through KEGG enrichment analysis, it was found for the first time that the synthesis pathway for trehalose in A. heimuer is trehalose phosphate synthase–trehalose phosphate phosphatase (TPS-TPP), which is involved in the response of A. heimuer to drought stress. In addition, two key enzyme genes involved in trehalose synthesis, namely trehalose-6-phosphate synthase (AhTPS) and trehalose-6-phosphate phosphatase (AhTPP), were significantly up-regulated after drought stress. The trehalose content significantly increased in 11 strains after drought stress treatment. This study discovered, for the first time, that the synthesis pathway of trehalose is involved in the response of edible fungi to drought stress, thus providing a reference for the genetic improvement of A. heimuer and the selection of drought-tolerant strains, laying a theoretical foundation for the resistance breeding of other edible fungi.

1. Introduction

As China’s second largest edible fungus, Auricularia heimuer holds considerable economic and medicinal value [1,2,3]. In cultivation, it is managed using full-day intermittent misting techniques, creating a “dry–wet alternation” environment [4]. During periods of water deficiency, its fruiting bodies swiftly dehydrate and enter dormancy; however, they resume growth when moisture is sufficient [5]. The ability of A. heimuer to survive drought stress, similar to resurrection plants, contributes to a noteworthy model for studying the adaptations of organisms to dry conditions. The life cycle of the A. heimuer comprises two crucial and closely related stages: the mycelium and the fruiting body [6]. Unlike the visible “shrinking when dry, swelling when wet” phenomenon in its fruiting bodies, the changes in mycelium are not visible to the naked eye [4], and research on the response mechanism of mycelium against drought stress in A. heimuer remains scarce.

Polyethylene glycol (PEG) is a biologically inert polymeric osmotic agent used to mimic the water stress faced by organisms under drought conditions by reducing the water potential of a solution [7]. Apart from PEG, other commonly used osmoregulators include mannitol, and sorbitol. Due to their small molecular weights, however, mannitol and sorbitol can easily penetrate cell walls and plasma membranes, increasing intracellular osmotic pressure and leading to plasma-wall separation [8]. PEG molecules hardly penetrate cell membranes and do not cause physiological damage [9]. As such, the PEG-induced drought stress model is one of the widely used systems in stress biology studies [10]. PEG is widely used to simulate drought stress in organisms such as Arabidopsis thaliana [11], wheat [12], rice [13], and yeast [14].

Osmotic regulation and antioxidant defense are important strategies for withstanding drought stress [15]. Osmotic regulation involves the accumulation of osmotic adjustment substances such as trehalose, polyols, and glycerol, which help stabilize cell turgor, reduce water loss, lower cellular water potential, and thereby enable the absorption of more water from the substrate [16]. Moreover, drought stress disrupts the cellular redox balance, triggering an increase in the production of reactive oxygen species (ROS), affecting cellular development, and potentially leading to cell death [17]. The antioxidant defense is a network of enzyme systems like SOD, CAT, and glutathione reductase (GR), as well as non-enzyme systems including melanin and vitamin E [18].

Trehalose, a structurally stable non-reducing disaccharide, is abundantly present (up to 20% of dry weight) in dehydrated organisms like fungal spores, yeast cells, and resurrection plants [19]. Trehalose confers to fungi an ability to adapt to stress conditions [20]. It participates in osmotic regulation, maintaining cell turgor, and minimizing water loss [21]. Under drought stress, there is a substantial accumulation of trehalose within the fungal system, which is crucial in stabilizing protein structures and cell membrane integrity [20]. Currently, five trehalose synthesis pathways have been identified [22]. The primary pathway for trehalose synthesis is the TPS-TPP pathway, where trehalose-6-phosphate synthase (TPS) catalyzes the synthesis of trehalose-6-phosphate (T6P) and uridine diphosphate (UDP) from uridine diphosphate glucose (UDPG) and glucose-6-phosphate (G6P). T6P is further catalyzed by trehalose-6-phosphate phosphatase (TPP) to produce trehalose and inorganic phosphate [23]. Under drought stress, the accumulation of trehalose in wheat is correlated with increased TPS activity [24]. Tobacco engineered with the MeTPS1 gene of cassava shows enhanced drought tolerance [25]. The overexpression of TPP increases drought tolerance in rice [26]. Presently, trehalose’s involvement in responses to high-temperature stress has been observed in edible fungi, such as Stropharia rugosoannulata [27], Pleurotus ostreatus [28,29], and Lentinula edodes [30], but research on the role of trehalose in responses to drought stress in edible fungi remains unreported.

We conducted whole-genome sequencing and annotation for A. heimuer [31], assessed its nutritional composition [32], and proposed a scientifically rigorous method for quantitative trait assessment [33]. As a follow-up to our previous studies, the current study subjected 13 strains of A. heimuer to drought stress treatment and assessed their drought responses by measuring various physiological parameters including SS, SP, SOD, CAT, MDA, and biomass. Using these parameters, we assessed the drought tolerance of the strains and selected a drought-tolerant strain for RNA sequencing to uncover the mechanisms underlying the response of mycelium to drought stress. This research provides insights into the potential genetic improvement of A. heimuer and the resistance breeding of other edible fungi.

2. Materials and Methods

2.1. Auricularia heimuer Test Strains

The strains used in this study were sourced from the Auricularia heimuer breeding station of the national edible fungus industry’s technology system. These strains were selected to represent key production regions (northern, central, and southern China). The fungal strains are preserved at the College of Horticulture, Jilin Agricultural University, China (

Table 1. Information on test strains.

Experiment ID	Preservation ID	Strain Type	Source
A	JAUAH590	Cultivated	Jilin Province
B	JAUAH591	Cultivated	Jilin Province
C	JAUAH592	Cultivated	Jilin Province
A14	JAUAH14	Cultivated	Heilongjiang Province
A184	JAUAH184	Cultivated	Shandong Province
A124	JAUAH124	Wild	Heilongjiang Province
A125	JAUAH125	Wild	Heilongjiang Province
A127	JAUAH127	Wild	Jilin Province
A356	JAUAH356	Wild	Yunnan Province
A386	JAUAH386	Wild	North Korea
A462	JAUAH462	Wild	Shandong Province
A496	JAUAH496	Wild	Jilin Province
A508	JAUAH508	Wild	Yunnan Province

).

2.2. Experimental Design

We used a complete yeast medium (CYM) including 1% maltose, 2% glucose, 0.2% peptone, 0.2% yeast extract, 0.05% MgSO₄·7H₂O, and 0.46% KH₂PO₄. The liquid culture conditions were 25 °C, and the culture was shaken at 120 rpm and kept in the dark for 15 days, with three biological replicates.

We selected the cultivated strain B and the wild strains A462 and A496 from the test strains for preliminary experiments. Simulated drought stress was applied using 10%, 20%, and 30% PEG 6000, with a control of 0% PEG 6000, and the biomass under different concentrations was compared to determine the appropriate concentration of PEG 6000 for inducing drought stress in A. heimuer.

Subsequently, the 13 fungal test strains were subjected to drought stress treatment using a CYM containing 20% PEG 6000 or to a control treatment, namely a CYM without PEG 6000. The mycelium of the 20% PEG 6000-treated group and the control group was sampled to determine the physiological indices. The drought-tolerant strain C was selected for transcriptome sequencing to explore the drought tolerance mechanism of A. heimuer during the mycelial stage.

2.3. Physiological Index Determination

The soluble sugar (SS) content was determined using the anthrone–sulfuric acid method [34]. The soluble protein (SP) content was assessed using the Coomassie brilliant blue method [35]. The SOD activity was measured using the photochemical reduction of nitro blue tetrazolium [36]. The CAT activity was determined using ammonium molybdate [37]. The malondialdehyde (MDA) content was determined using the thiobarbituric acid method [38]. The biomass determination was slightly modified based on the method of Scheid [39]. Briefly, the mycelium was filtered through a four-layer gauze, washed three times with distilled water, and dried to a constant weight in an 80 °C electric convection oven.

2.4. Evaluation of Drought Tolerance in A. heimuer

To evaluate differences in drought tolerance among A. heimuer strains, the drought tolerance coefficient (DTC) was calculated for all traits in this study according to the equations of Chen et al. [40], as follows:

$$\mathrm{D}\mathrm{T}\mathrm{C}={\displaystyle \frac{\mathrm{P}\mathrm{E}\mathrm{G}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}}{\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}}}$$

(1)

Drought tolerance was assessed using the membership function value, which quantifies an object’s degree of membership in a fuzzy set by mapping each element to a value between 0 and 1. In the evaluation of drought tolerance for crops like wheat, membership function values are often used to quantitatively express the crops’ tolerance to drought conditions. We linked the physiological indicators, including SS, SP, SOD, CAT, MDA, and biomass, with the fuzzy set of drought levels. The modified membership function value was calculated according to the equations of Chen et al. [40], as follows:

$$\mathrm{U}\mathrm{i}\mathrm{j}={\displaystyle \frac{(\mathrm{D}\mathrm{T}\mathrm{C}\mathrm{i}\mathrm{j}-\mathrm{D}\mathrm{T}\mathrm{C}\mathrm{j}\mathrm{m}\mathrm{i}\mathrm{n})}{(\mathrm{D}\mathrm{T}\mathrm{C}\mathrm{j}\mathrm{m}\mathrm{a}\mathrm{x}-\mathrm{D}\mathrm{T}\mathrm{C}\mathrm{j}\mathrm{m}\mathrm{i}\mathrm{n})}}$$

(2)

$$\mathrm{D}\mathrm{i}={\displaystyle \frac{1}{\mathrm{n}}}{\sum }_{\mathrm{j}=1}^{\mathrm{n}}\mathrm{U}\mathrm{i}\mathrm{j}$$

(3)

where Uij is the membership function value of the trait (j) for the strain (i) for drought tolerance; DTCjmax is the maximum value of the drought tolerance coefficient for the trait (j); and DTCjmin is the minimum value. The average membership value of all traits is denoted as the comprehensive value Di.

Drought tolerance is classified into five categories based on the mean value ($\overline{\mathrm{D}}$) and standard deviation (SD) of the comprehensive value. When Di ≥ $\overline{\mathrm{D}}$ + 1.64 SD, it is categorized as highly tolerant (HDT); when $\overline{\mathrm{D}}$ + 1 SD ≤ Di < $\overline{\mathrm{D}}$ + 1.64 SD, it is considered tolerant (DT); when $\overline{\mathrm{D}}$ − 1 SD ≤ Di < 1 + SD, it is moderately tolerant (MDT); when $\overline{\mathrm{D}}$ − 1.64 SD ≤ Di < $\overline{\mathrm{D}}$ − 1 SD, it is classified as sensitive (S); and when Di < $\overline{\mathrm{D}}$ − 1.64 SD, it is regarded as highly sensitive (HS) [40].

2.5. Total RNA Extraction and Transcriptome Sequencing

A drought-tolerant strain C was selected for RNA sequencing to explore the drought tolerance mechanism of the mycelium of A. heimuer, based on the evaluation of drought tolerance and agronomic traits (unpublished data). Strain C was inoculated into a liquid CYM with different treatments (0 and 20% PEG) and cultured at 25 °C and 120 rpm in the dark for 15 days. The mycelium was filtered through a four-layer gauze and washed three times with distilled water. The mycelium was frozen in liquid nitrogen and stored at −80 °C.

Total RNA was isolated using Trizol Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), after which the concentration, quality, and integrity were determined using a NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA). Subsequently, we selected total RNA with a quantity of ≥1 µg and used the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs Inc.; Ipswich, MA, USA) to construct the RNA sequencing library. The sequencing library was then sequenced on the NovaSeq 6000 platform (Illumina, San Diego, CA, USA).

2.6. Real-Time Quantitative PCR Analysis

Real-time quantitative PCR (RT-qPCR) was employed to validate the transcriptome results using nine randomly selected DEGs. Total RNA was extracted from mycelium of A. heimuer using the RNAiso Plus (Takara, Shiga, Japan). cDNA synthesis was conducted using the TransScript All-in-One First-Strand cDNA Synthesis Supermix for the qPCR kit (Transgene Biotech, Beijing, China). Primer design was conducted using the NCBI Primer-BLAST tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/, (accessed on 23 November 2023)). The primers used are listed in Table S1. RT-PCR was performed using the PerfectStart Green qPCR SuperMix kit (Transgene Biotech, Beijing, China) on the ABI StepOne Plus Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). The thermal cycling profile included an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. We employed 18S rRNA as the internal reference gene. Gene expression was analyzed using the 2^−ΔΔCt method.

2.7. DEG Identification and Enrichment Analysis

We used fastp version 0.22.0 (https://github.com/OpenGene/fastp, (accessed on 23 April 2023)) software to filter and clean sequencing data for further analysis. The filtered reads were mapped to the reference genome of Auricularia heimuer using HISAT2 version 2.1.0 (https://daehwankimlab.github.io/hisat2/, (accessed on 23 April 2023)). In this study, we used the reference genome of Auricularia heimuer (Dai 13782), which is available through the National Center for Biotechnology Information (NCBI) with the accession number GCA_002287115.1 [3]. We used HTSeq version 0.9.1 (https://htseq.readthedocs.io/en/latest/, (accessed on 23 April 2023)) statistics to compare the read count values on each gene as the original expression of the gene and then used the fragments per kilobase of exon per million mapped reads (FPKM) to standardize the expression.

The difference in the expression of genes was analyzed using DESeq version 1.38.3 (https://bioconductor.org/packages/release/bioc/html/DESeq2.html, (accessed on 23 April 2023)), with screening conditions as follows: expression difference multiple |log₂FoldChange| > 1; significant p-value < 0.05. ClusterProfiler version 4.6.0 (https://www.bioconductor.org/packages/release/bioc/html/clusterProfiler.html, (accessed on 23 April 2023)) software was used for KEGG pathway enrichment analysis, with a p-value < 0.05 indicating significant enrichment.

2.8. Measurement of Trehalose Content

The trehalose content was assessed using the trehalose content assay kit (enzymatic method—Visible Colorimetric) from Grace Biotechnology Co., Ltd., Suzhou, China, following the kit’s provided instructions for reaction systems and procedures.

2.9. Data Processing and Analysis

Each measurement was conducted with three biological replicates, and the results are presented as the mean ± standard deviation. We applied a square root transformation to the data and conducted a one-way analysis of variance, followed by Duncan’s multiple range test, to determine a suitable PEG concentration for inducing drought stress. Physiological indicators and trehalose content were compared before and after drought stress treatment using the paired t-test. A p value of < 0.05 was considered significant.

All data processing was conducted using Microsoft Excel 2019. Variance analysis was performed using SPSS 16.0 software, and paired sample testing was conducted using the R version 4.3.1 (R Core Team (2023). https://www.R-project.org/, (accessed on 16 February 2024)).

3. Results

3.1. Study on Drought Tolerance of A. heimuer

Biomass was measured after treatment with PEG concentrations of 0, 10, 20, and 30% to determine the optimal PEG concentration for simulating drought stress in A. heimuer. As illustrated in Figure 1, the biomass of strains B and A496 following the 10% PEG treatment exceeded that of the 0% PEG treatment; however, the biomass of all three fungal strains decreased with the 20% PEG treatment, compared to both 0 and 10% treatments. Mycelium growth was severely inhibited after treatment with 30% PEG. Therefore, this study proceeded with the simulated drought stress experiment using the 20% PEG treatment.

3.2. Physiological Response of A. heimuer to Drought Stress

Drought stress caused significant changes in six physiological indicators (Figure 2, Table S2). Following the drought stress treatment, the soluble sugar content decreased for strains B, C, and A508 to 0.57, 0.66, and 0.43 times that of the control, respectively. Conversely, the soluble sugar content increased for the other 10 strains, with strains A462 and A127 exhibiting the highest increments, at 1.93 and 1.90 times that of the control, respectively. Notably, strain A14 showed a significant decline in soluble protein content to half that of the control, while nine other strains had an increase, with strain C exhibiting the most substantial rise, of 4.10 times that of the control.

SOD activity decreased for strains C and A462, to 0.12 and 0.63 times that of the control, respectively, while eight other strains experienced an increase, with strain A showing the most significant increase at 13.83 times that of the control. The CAT activity increased for 10 strains, with strains B and A125 exhibiting the highest increments at 4.36 and 3.65 times higher than that of the control, respectively. Following drought stress, the malondialdehyde content significantly increased across all the tested strains, ranging from 2.47 to 8.60 times that of the control, while the biomass of 10 strains declined by 0.05 to 0.74 times that of the control.

In summary, the general physiological responses of A. heimuer under drought stress are as follows: decreased biomass, an overall increase in SS, SP, SOD, and CAT, and a significant rise in MDA content.

3.3. Identification and Evaluation of Drought Tolerance in A. heimuer

Based on the membership values, 13 strains of A. heimuer were assessed for drought tolerance and classified into the following four categories (

Table 2. Evaluation of drought tolerance in A. heimuer.

Strain	D	Rank	Drought Tolerance
A	0.565	1	HDT
A127	0.515	2	DT
C	0.508	3	DT
A124	0.470	4	MDT
A14	0.465	5	MDT
A386	0.451	6	MDT
A462	0.441	7	MDT
A184	0.430	8	MDT
A496	0.426	9	MDT
A125	0.397	10	MDT
B	0.391	11	MDT
A356	0.385	12	S
A508	0.357	13	S

Note: HDT: highly drought tolerant; DT: drought tolerant; MDT: moderately drought tolerant; S: sensitive.

): the first comprises highly drought-tolerant strain A; the second includes drought-tolerant strains A127 and C; the third encompasses moderately drought-tolerant strains, including A124, A14, A386, A462, A184, A496, A125, and B; and the fourth comprises drought-sensitive strains A356 and A508.

3.4. High-Throughput RNA Sequencing of Strains Under Drought Stress

High-throughput RNA sequencing was performed on drought-tolerant strain C, which was subjected to 0 and 20% PEG treatments. Based on the expression levels of samples, Pearson correlation coefficients were used to depict the correlation of gene expression among these samples. The control 3 sample exhibited significant deviations from the other ones under the same treatment; hence, it was excluded from subsequent analysis. The correlation coefficients between these samples under the same treatment were all greater than 0.9, indicating a robust correlation among these samples (Figure 3a). Cluster map analysis revealed significant differences in gene expression before and after the drought stress treatment of strain C (Figure 3b).

Transcriptome sequencing yielded 38,785,758 to 51,027,092 raw reads. After filtering the raw data using fastp (0.22.0), 37,001,576 to 48,367,260 clean reads were obtained, with a Q30 score ranging from 94.22 to 94.79%, indicating a high sequencing quality. HISAT2 (v2.1.0) was employed to construct an index of the reference genome, and the paired-end clean reads were aligned to the reference genome using HISAT2 with default parameters. The gene alignment rate ranged from 88.67 to 89.89%, with 9204 to 10,183 expressed genes identified (

Table 3. RNA sequencing and assembly results.

Sample	Raw Read Number	Clean Read Number	Q30 Score	Gene Map Rate (%)	Expressed Gene
Control 1	38,785,758	37,001,576	94.22	88.14	9683
Control 2	46,536,944	44,469,130	94.79	90.89	9204
20% PEG1	49,262,512	46,986,024	94.57	91.15	9797
20% PEG2	46,805,826	44,663,836	94.45	91.65	10,054
20% PEG3	51,027,092	48,367,260	94.78	91.04	10,183

).

3.5. KEGG Enrichment Analysis and Quantitative Real-Time PCR Verification

A total of 1762 DEGs were identified after drought stress treatment, comprising 798 up- and 964 down-regulated genes (Figure 4a). A KEGG enrichment analysis was conducted to further elucidate the metabolic pathways involved in DEGs. This analysis identified significant enrichment in 10 metabolic pathways (p ≤ 0.05, Figure 4b), including DNA replication, homologous recombination, base excision repair, mismatch repair, nicotinate and nicotinamide metabolism, starch and sucrose metabolism, other glycan degradation, methane metabolism, cell cycle, and meiosis.

Nine DEGs were randomly selected for the validation of the sequencing results using RT-qPCR. The RT-qPCR outcomes were largely consistent with the transcriptome sequencing results, thus confirming the reliability of the sequencing (Figure 4c).

3.6. Involvement of Trehalose in the Drought Stress Response of A. heimuer

The synthesis pathway of TPS-TPP involves two enzymatic reactions (Figure 5a): trehalose-6-phosphate synthase (TPS) catalyzes the synthesis of trehalose-6-phosphate (T6P) from uridine diphosphate glucose (UDPG) and glucose-6-phosphate (G6P), which is then catalyzed by trehalose-6-phosphate phosphatase (TPP) to produce trehalose. KEGG enrichment analysis revealed a significant up-regulation of both key enzymes, TPS and TPP, involved in trehalose synthesis in the starch and sucrose metabolism pathway (

Table 4. Genes in the trehalose biosynthesis pathway.

Gene	Fold Change (20% PEG/Control)	Pval	Regulation
trehalose 6-phosphate synthase (TPS)	2.23	0.019	Up
trehalose 6-phosphate phosphatase (TPP)	2.12	0.025	Up
trehalase (TREH)	/	/	/
trehalose phosphorylase (TreP)	2.37	0.010	Up

Note: The presence of ‘/’ indicates that the gene lacks annotation in the RNA sequencing results.

), while the genes encoding trehalase (TREH) and trehalose phosphorylase (TreP) involved in trehalose degradation were not significantly enriched, according to the KEGG enrichment analysis. Therefore, A. heimuer possibly accumulates trehalose in response to drought stress.

To further validate the involvement of trehalose in the response of A. heimuer to drought stress, the trehalose content of the tested strains was measured before and after drought stress treatment. The results (Figure 5b) showed that, after drought stress treatment, the trehalose content significantly increased in 11 strains tested, while strain A127 exhibited a decrease in trehalose content, overall indicating the involvement of trehalose in the drought stress response of A. heimuer. However, the correlation between trehalose content in mycelium and drought tolerance was not significant.

4. Discussion

4.1. Physiological Responses to Abiotic Stress

The accumulation of osmotic regulatory substances, such as soluble sugars, is a fundamental stress adaption strategy in yeast [41]. This study found that, among the 13 tested strains, 10 showed an increase in both osmotic regulatory substances (Figure 2a), indicating the significant role of these substances in the response of A. heimuer mycelium to drought stress. Soluble sugars also serve as a source of energy for cells. The decrease in soluble sugar content in strains B, C, and A508 (Figure 2a) may be attributed to their participation as substrates in other metabolic processes, thereby enhancing the drought tolerance of Auricularia heimuer mycelium. Aspergillus creber also exhibited the same phenomenon [42].

SOD and CAT are important reactive oxygen species scavengers [43]. Under abiotic stress, SOD and CAT activity increases in edible fungi such as S. rugosoannulata [44], Volvariella volvacea [45], and A. heimuer [46]. It was found that the SOD activity of strains C and A462 decreased (Figure 2c), possibly due to drought-induced damage to SOD. Similar phenomena also existed in the research conducted by Ma [46].

Malondialdehyde is often used as an indicator to assess the degree of membrane damage cause by oxidative stress [47]. We found that, after drought stress treatment, the MDA content of the tested fungal strains significantly increased (Figure 2e), indicating that the drought stress induced oxidative damage in the fungal strains. Under air humidity stress, the MDA content of A. heimuer showed a similar trend [46]. Similarly, the biomass of the tested strains showed a decreasing trend (Figure 2f), which is consistent with previous research findings.

4.2. Drought Tolerance Assessment

Drought tolerance is a complex trait influenced by both environmental and genetic factors, thus necessitating the evaluation of various indicators, namely SS, SP, SOD, CAT, MDA, and biomass, in A. heimuer [40]. Membership values can overcome the limitations of using single indicators to assess drought response [48]. In this study, for the first time, membership values were employed to comprehensively assess the drought tolerance of 13 strains of A. heimuer in the mycelial stage, identifying one highly drought-tolerant and two moderately drought-tolerant strains (Table 2). Subsequent evaluations of drought tolerance in A. heimuer in the fruiting body stage can be conducted, and integrating data from both stages can provide a holistic assessment of drought tolerance in A. heimuer strains.

4.3. Study on the Drought Tolerance of A. heimuer

Auricularia heimuer demonstrates rare drought tolerance, but research into the mechanisms behind this trait is currently limited. Trehalose participates in turgor maintenance and the protection of macromolecular structures against the effect of drought stress [20]. This study has identified that the trehalose TPS-TPP synthesis pathway is implicated in the mycelial response to drought stress in A. heimuer. Ma [49] observed a significant increase in the activity of two key enzymes in the pentose phosphate pathway (PPP) during drought stress, glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH), suggesting that the PPP may be involved in the drought stress response of the fruiting bodies of A. heimuer. However, the current study did not find the PPP involved in the drought stress response during the mycelial stage of A. heimuer, and the genes encoding the two key enzymes did not show differential expression. This might be attributed to the fact that the mycelium and the fruiting body represent two distinct phases in the life cycle of A. heimuer, during which the gene expression levels of key enzymes in the PPP may vary. Further transcriptome sequencing of the fruiting body stage of A. heimuer could elucidate the role of the PPP in its response to drought stress.

Additionally, future research endeavors may also incorporate metabolomic methodologies to elucidate the pivotal metabolites underlying the drought stress response in A. heimuer. Such an approach will facilitate a comprehensive dissection of the mycelial stress adaptation mechanisms, enhancing our understanding of the complex physiological adjustments enacted by this fungus in response to drought stress.

4.4. Trehalose Synthesis Pathway

To date, five pathways for trehalose synthesis have been identified: TPS-TPP, TreS, TreY-TreZ, TreP, and TreT [22]. Among eukaryotes, the TPS-TPP pathway is conserved [50]. For the first time, the current study discovered the TPS-TPP pathway of trehalose synthesis in A. heimuer, where two key enzymes, the TPS and TPP genes, were significantly up-regulated after drought stress (Figure 5a). Additionally, one TreP gene was found to be up-regulated, although it was not enriched in the trehalose synthesis pathway, suggesting the possible existence of a TreP synthesis pathway in A. heimuer. The TreP synthesis pathway has been identified in macromycetes such as Polyporus frondosus [51], Flammulina velutipes [52], and Pleurotus pulmonarius [53]. Subsequent studies can further verify the presence of the TreP synthesis pathway in A. heimuer.

4.5. Trehalose Involvement in Stress Response

Trehalose acts as a protective agent for proteins and membranes, exerting intracellular protective effects on organisms under various stress conditions [54]. It plays a crucial role in the response of V. volvacea to low-temperature stress, and cold-resistant strains exhibit stronger trehalose accumulation capabilities [55]. Trehalose can also alleviate damage caused by high-temperature stress in P. ostreatus [28]. In this study, the trehalose content significantly increased in 11 tested strains of A. heimuer after drought stress (Figure 5b), indicating the involvement of trehalose in the response to drought stress. In fungi, trehalose can serve as an energy storage carbohydrate, providing the necessary energy for stress responses [56]. However, the cause of the decrease in trehalose content in strain A127 (Figure 5b) is not clear in the current study.

4.6. Potential Mechanisms of Drought Stress Response in A. heimuer

Transcriptome provides valuable information about the stress response mechanisms of edible fungi [57]. The complex trait of drought tolerance is regulated by a network formed by the interactions of multiple metabolic pathways. The discovery of trehalose and its synthetic pathway in this study reveals that it is just one of the many factors involved in the response of A. heimuer to drought stress. Furthermore, we also discovered some metabolic pathways that potentially participate in the drought stress response of this species. Metabolic pathways, including DNA replication, homologous recombination, base excision repair, and mismatch repair (Figure 4b), are activated possibly to counteract DNA damage induced by environmental stress, and similar phenomena exist in lichens [15]. The B6 vitamers are reported to modulate the activities of enzymes involved in ROS scavenging under abiotic stress conditions [58]. We discovered a similar phenomenon in A. heimuer: under drought stress, genes associated with vitamin B6 synthesis are up-regulated. Riboflavin metabolism-related genes were also identified in our study, a finding that is similar to that observed for A. bisporus [59]. Although we have made initial studies to understand the complex metabolic network’s underlying response to drought stress in A. heimuer, numerous mechanisms remain to be unraveled. Consequently, future research endeavors should delve deeper into the interplay and regulatory mechanisms among these metabolic pathways using comparative transcriptomics, gene overexpression, and gene silencing techniques. Such approaches will enable a more comprehensive understanding of the drought adaption mechanisms in A. heimuer.

5. Conclusions

This study used a CYM containing 20% PEG 6000 to subject 13 strains of Auricularia heimuer to drought stress and explore their physiological response mechanisms. Physiological analysis shows that A. heimuer responds to drought stress by increasing SS, SP, SOD, CAT, and MDA, while reducing biomass; also, there are differences in drought tolerance among strains of A. heimuer. Based on the six indicators mentioned above, the thirteen strains were classified into four categories using membership functions: one highly drought-tolerant strain, two drought-tolerant strains, eight moderately drought-tolerant strains, and two drought-sensitive strains. The transcriptome analysis of the drought-tolerant strain C revealed, for the first time, that the biosynthesis pathway of trehalose in A. heimuer is the TPS-TPP pathway, which is involved in drought stress response. Two key enzyme genes, AhTPS and AhTPP, were significantly up-regulated after drought stress, and the trehalose content in A. heimuer increased significantly. This indicates that the accumulation of trehalose is one of the mechanisms by which A. heimuer responds to drought stress. This study lays a foundation for research on A. heimuer resistance and provides valuable insights for its genetic improvement.