Combined Proteomic and Metabolomic Analyses Reveal the Comprehensive Regulation of Stropharia rugosoannulata Mycelia Exposed to Cadmium Stress.

The potential of Stropharia rugosoannulata as a microbial remediation material for cadmium (Cd)-contaminated soil lies in its capacity to absorb and accumulate Cd in its mycelia. This study utilized the TMT and LC-MS techniques to conduct integrated proteomic and metabolomic analyses with the aim of investigating the mycelial response mechanisms of S. rugosoannulata under low- and high-Cd stresses. The results revealed that mycelia employed a proactive defense mechanism to maintain their physiological functions, leading to reduced sensitivity to low-Cd stress. The ability of mycelia to withstand high levels of Cd stress was influenced primarily by the comprehensive regulation of six metabolic pathways, which led to a harmonious balance between nitrogen and carbohydrate metabolism and to reductions in oxidative stress and growth inhibition caused by Cd. The results provide valuable insights into the molecular mechanisms involved in the response of S. rugosoannulata mycelia to Cd stress.

Composition and contents of fatty acids and amino acids in the mycelia of Lentinula edodes.

With the global shortages of animal protein foods, mycoprotein as a low-cost alternative source of protein by its high-protein and low-fat content has become a development trend. Lentinula edodes (L. edodes) is a healthy food with high protein and low fiber. This work evaluated the nutritional value of L. edodes mycelia, and determined the composition and contents of fatty acids and amino acids. Eleven saturated fatty acids (SFAs) and 12 unsaturated fatty acids (UFAs) were detected in the mycelia of L. edodes. The UFA content accounted for 75.7% and 73.1% of the total fatty acid content in the mycelia of strains 18 and 18N44, respectively. Linoleic acid was the major polyunsaturated fatty acid (PUFA) in the mycelia, accounting for 91.0% and 86.3% of the UFAs, respectively. The mycelia of the two strains contained 17 types of amino acids, and the essential amino acids were sufficient (357.92 ± 0.42 and 398.38 ± 4.52 mg/g pro, respectively), both close to the WHO/FAO reference protein pattern value. The most abundant essential amino acid was Lys, and the limiting amino acids were Met + Cys and Ile, respectively. The SRC values in the mycelia of the two strains were 68.07 and 54.86, and the EAAI values were 67.70 and 74.42, respectively, both being close to those of ovalbumin. It is concluded that L. edodes mycelia are rich in easily absorbed high-quality proteins and PUFAs, and can be used as a source for meat analog required by vegetarians. This study provides a scientific basis for the further utilization of mycelial resources.

Integrated physiologic and proteomic analysis of Stropharia rugosoannulata mycelia in response to Cd stress.

Soil Cd pollution seriously threatens environment and human health. Due to its ability to absorb and accumulate Cd in mycelia, Stropharia rugosoannulata could be a potential candidate for bioremediation of Cd-contaminated soils; however, the response mechanism of mycelia to Cd stress is still unclear. In this study, the physiologic and proteomic differences of S. rugosoannulata mycelia under 0.2 mg/L (low) and 2 mg/L (high) Cd stress were investigated. The results showed that Cd accumulation and mycelial growth inhibition exhibited a concentration-depended trend. Analysis of antioxidant system indicated that SOD, GR, GSH, GSSG and ASA played key roles in resisting the toxic effects of Cd. Via proteome analysis, 24 and 267 differentially expressed proteins (DEPs) were observed under low and high Cd stress, respectively. GO and KEGG analysis found that the mycelial growth inhibition might due to the down-regulation of some DEPs involved in "valine, leucine and isoleucine biosynthesis" and "tyrosine metabolism"; the certain tolerance to high Cd stress might attribute to the regulation of DEPs referred to energy metabolism and antioxidant system-related pathways, maintaining cellular energy homeostasis and removing ROS. These results provide a theoretical basis for further elucidation of response mechanisms in S. rugosoannulata to Cd stress.

Comparative Proteomics Study on the Postharvest Senescence of Volvariella volvacea.

Volvariella volvacea is difficult to store after harvest, which restricts the production and circulation of V. volvacea fruiting bodies. Low-temperature storage is the traditional storage method used for most edible fungi. However, V. volvacea undergoes autolysis at low temperatures. When fruiting bodies are stored at 15 °C (suitable temperature), V. volvacea achieves the best fresh-keeping effect. However, the molecular mechanism underlying the postharvest senescence of V. volvacea remains unclear. Based on this information, we stored V. volvacea fruiting bodies at 15 °C after harvest and then analyzed the texture and phenotype combined with the results of previous physiological research. Four time points (0, 24, 60, and 96 h) were selected for the comparative proteomics study of V. volvacea during storage at 15 °C. A variety of proteins showed differential expressions in postharvest V. volvacea at 15 °C. Further comparison of the gene ontology (GO) enrichment analysis and KEGG pathways performed at different sampling points revealed proteins that were significantly enriched at several time points. At the same time, we also analyzed differentially expressed proteins (DEPs) related to the RNA transport, fatty acid biosynthesis and metabolism, and amino acid biosynthesis and metabolism pathways, and discussed the molecular functions of the PAB1, RPG1, ACC1, ADH3, ADH2, ALD5, and SDH2 proteins in postharvest V. volvacea senescence. Our results showed that many biological processes of the postharvest senescence of V. volvacea changed. Most importantly, we found that most RNA transport-related proteins were down-regulated, which may lead to a decrease in related gene regulation. Our results also showed that the expression of other important proteins, such as the fatty acid metabolism related proteins increased; and changes in fatty acid composition affected the cell membrane, which may accelerate the ripening and perception of V. volvacea fruiting bodies. Therefore, our research provides a reference for further studies on the aging mechanism of V. volvacea.

Strategies to Study Dark Growth Deficient or Slower Mutants in Chlamydomonas reinhardtii.

Photosynthesis is the most important chemical reaction on the earth, and about 60% of the CO2 is fixed by algae through photosynthesis. Photosynthetic organisms including algae experience half of the entire life in the dark due to diel cycles, and dark metabolism is critical and necessary for photosynthetic organisms to restart photosynthesis when receiving light again. Briefly, dark metabolism provides necessary materials and energy for restoring photosynthesis, reoxidizes NADH to form NAD+, rationally stores photosynthates, and maintains correct redox balance. Chlamydomonas reinhardtii grows under both autotrophic and heterotrophic conditions, making it an ideal organism to study photosynthesis, dark metabolism, and light dark transitions as well. In addition, it provides a good model to identify key molecular components and elucidate the molecular regulatory mechanisms of heterotrophic, which provides new clues to understand how photosynthetic organisms restart photosynthesis from the dark. Chlamydomonas mutants with dark growth deficiency or slower growth phenotypes are likely caused by the inefficient uptake and transport of acetate, the damaged proteins of mitochondrial electron transport chain, the malfunctioned mitochondrion, the redox state alteration in the dark or failed communication between mitochondrion and other organelles, the imbalanced redox or the disrupted distribution of the photosynthetic products. Here we summarize the methods and strategies for analyzing these mutants in Chlamydomonas reinhardtii.

The Response of Volvariella volvacea to Low-Temperature Stress Based on Metabonomics.

Low temperature can lead to the autolysis of Volvariella volvacea (V. volvacea), hindering its growth and preservation and severely reducing its yield and quality. This autolysis of V. volvacea at low temperature has been reported, but a metabolomics-based investigation of the underlying mechanisms of the V. volvacea response to low temperature has not been reported. Therefore, this study aimed to explore the changes, levels and expression patterns of V. volvacea metabolites at low temperature. To understand the metabolic differences within V. volvacea, two strains with different levels of low-temperature tolerance were treated in an ice bath at 0°C for 2, 4, 8, and 10 h, while the blank control group was treated for 0 h. Metabonomics analysis was adopted to study the changes in V. volvacea in response to low temperature and the differences between the two different strains. Metabolic curves were analyzed at different time points by high-performance liquid chromatography-mass spectrometry (HPLC-MS). A total of 216 differential metabolites were identified and enriched in 39 metabolic pathways, mainly involving amino acid metabolism, carbohydrate metabolism, the TCA cycle, energy metabolism, etc. In this paper, we report the metabonomic analysis of V. volvacea in response to low temperature and compare the differences in metabolite expression between the low-temperature-resistant strain VH3 and the low-temperature-sensitive strain V23. Finally, the putative low-temperature resistance mechanism of VH3 is revealed at the metabolic level. This study provides a theoretical basis for revealing the regulatory mechanism of low-temperature resistance in V. volvacea and for future molecular breeding efforts.

Differential proteomics study of postharvest Volvariella volvacea during storage at 4 °C.

The postharvest storage of Volvariella volvacea is an important factor limiting the industry development. Low-temperature storage is the traditional storage method used for most edible fungi, but V. volvacea undergoes autolysis at low temperature. To understand the molecular mechanism underlying the low-temperature autolysis of V. volvacea after harvesting, fruiting bodies of V. volvacea strain V23 were stored at 4 °C. Based on our previous study, in which the changes of morphological and physiological indexes during storage for 0, 6, 12, 24, 30, 36, 48 and 60 h were measured; four time points, namely, 0, 12, 24 and 60 h, were selected for this differential proteomics study. The proteomic changes in the postharvest storage samples were studied by isobaric tags for relative and absolute quantification-coupled two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS). A total of 2,063 proteins were identified, and 192 differentially expressed proteins (DEPs), including 24 up-regulated proteins and 168 down-regulated proteins, were detected after 12 h of storage. After 24 h of storage, 234 DEPs, including 48 up-regulated and 186 down-regulated proteins, were observed, and after 60 h, 415 DEPs, including 65 up-regulated proteins and 350 down-regulated proteins, were observed. An in-depth data analysis showed that the DEPs participated in various cellular processes, particularly metabolic processes. In this study, we combined Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses, and the results focused on oxidative phosphorylation and ubiquitin mediated proteolysis pathways. In addition, sdh2, uba1 and ubc1 was confirmed by quantitative real-time polymerase chain reaction, and the results showed that the expression of these genes were consistent with their protein level. Based on the literature and our results, it is speculated that the identified DEPs, such as ATP1, SDH2, COR1, UBA1, COX4, UBC1 and SKP1 play a key role in the low-temperature autolysis of V. volvacea.

SUMOylation Stabilizes the Transcription Factor DREB2A to Improve Plant Thermotolerance.

Heat stress (HS) has serious effects on plant development, resulting in heavy agricultural losses. A critical transcription factor network is involved in plant adaptation to high temperature. DEHYDRATION RESPONSIVE ELEMENT-BINDING PROTEIN2A (DREB2A) is a key transcription factor that functions in plant thermotolerance. The DREB2A protein is unstable under normal temperature and is degraded by the 26S proteasome; however, the mechanism by which DREB2A protein stability dramatically increases in response to HS remains poorly understood. In this study, we found that the DREB2A protein of Arabidopsis (Arabidopsis thaliana) is stabilized under high temperature by the posttranslational modification SUMOylation. Biochemical data indicated that DREB2A is SUMOylated at K163, a conserved residue adjacent to the negative regulatory domain during HS. SUMOylation of DREB2A suppresses its interaction with BPM2, a ubiquitin ligase component, consequently increasing DREB2A protein stability under high temperature. In addition, analysis of plant heat tolerance and marker gene expression indicated that DREB2A SUMOylation is essential for its function in the HS response. Collectively, our data reveal a role for SUMOylation in the maintenance of DREB2A stability under high temperature, thus improving our understanding of the regulatory mechanisms underlying HS response in plant cells.

Low Temperature (15 °C) Reduces Bacterial Diversity and Prolongs the Preservation Time of Volvariella volvacea.

Straw mushroom (Volvariella volvacea) is the most commonly cultivated edible fungus in the world, but the challenges associated with the preservation have limited its marketability. Microbiology, especially bacteria, play a key role in the deterioration of food, this study aimed to reveal the succession of the bacterial community on the surfaces of V. volvacea fruit bodies under different temperature conditions. We amplified 16S rRNA genes of V4 regions, obtained the bacterial species information by using high-throughput sequencing technology, and analyzed the effects of environmental temperature and preservation time on bacterial communities. The relative abundances of Firmicutes, Bacilli, and Bacillales increased significantly when straw mushrooms began to rot. Furthermore, the relative abundances of Paenibacillus, Lysinibacillus and Solibacillus, which belong to Bacillales, increased with the decay of straw mushroom. The Shannon and Simpson indices of V. volvacea stored at 30 °C were significantly higher than those of V. volvacea stored at 15 °C, which indicates that a high temperature contributes to the improvement in the species diversity. According to the linear discriminant analysis (LDA) effect size (LEfSe) results, the number of biomarkers in the 30 °C group (32, 42.11%) was significantly higher than that in the 15 °C group (17, 22.37%), indicating that a high temperature has a clustering effect on some bacterial communities. A Spearman correlation analysis showed that Pseudomonas, Stenotrophomonas and Solibacillus promoted the decay of straw mushroom. In conclusion, a high temperature increases the bacterial diversity on the straw mushroom surfaces and has a clustering effect on the bacterial communities. The bacterial community consisting of Firmicutes, Bacilli, Bacillales, Paenibacillus, Lysinibacillus, Pseudomonas, Stenotrophomonas and Solibacillus could promote the decay of straw mushroom, so new preservation materials research can focus on inhibiting anaerobic and decay-causing bacteria to prolong preservation time.

GC⁻MS-Based Nontargeted and Targeted Metabolic Profiling Identifies Changes in the Lentinula edodes Mycelial Metabolome under High-Temperature Stress.

To clarify the physiological mechanism of the Lentinula edodes (L. edodes) response to high-temperature stress, two strains of L. edodes with different tolerances were tested at different durations of high temperature, and the results showed that there were significant changes in their phenotypes and physiology. To further explore the response mechanism, we established a targeted GC-MS-based metabolomics workflow comprising a standardized experimental setup for growth, treatment and sampling of L. edodes mycelia, and subsequent GC-MS analysis followed by data processing and evaluation of quality control (QC) measures using tailored statistical and bioinformatic tools. This study identified changes in the L. edodes mycelial metabolome following different time treatments at high temperature based on nontargeted metabolites with GC-MS and further adopted targeted metabolomics to verify the results of the analysis. After multiple statistical analyses were carried out using SIMCA software, 74 and 108 differential metabolites were obtained, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the metabolic pathways with significant changes included those related to the following: amino acid metabolism, the glycolysis pathway, the tricarboxylic acid (TCA) cycle, and sugar metabolism. Most amino acids and carbohydrates enriched in these metabolic pathways were upregulated in strain 18, downregulated in strain 18N44, or the synthesis in strain 18 was higher than that in strain 18N44. This result was consistent with the physiological phenotypic characteristics of the two strains under high-temperature stress and revealed the reason why strain 18N44 was more heat-sensitive. At the same time, under high temperature, the decrease of intermediate products in glycolysis and the TCA cycle resulted in carbon starvation and insufficient energy metabolism, thus inhibiting the growth of L. edodes. In addition, the results also showed that the metabolites produced by different L. edodes strains under high-temperature stress were basically the same. However, different strains had species specificity, so the changes in the content of metabolites involved in the response to high-temperature stress were different. This provides a theoretical basis for further understanding the mechanism of the L. edodes response to high temperature and can be used to establish an evaluation system of high-temperature-resistant strains and lay the foundation for molecular breeding of new L. edodes strains resistant to high temperature.

Reference Gene Selection for Quantitative Real-Time PCR of Mycelia from Lentinula edodes under High-Temperature Stress.

Housekeeping genes are important for measuring the transcription expression of functional genes; 10 traditional reference genes, TUB, TUA, GADPH, EF1, 18S, GTP, ACT, UBI, UBC, and H2A, were tested for their adequacy in Lentinula edodes (L. edodes). Using specific primers, mRNA levels of these candidate housekeeping genes were evaluated in mycelia of L. edodes, which were treated with high-temperature stress at 37°C for 0, 4, 8, 12, 18, and 24 hours. After treatment, expression stability of candidate genes was evaluated using three statistical software programs: geNorm, NormFinder, and BestKeeper. According to geNorm, TUB had the lowest M values in L. edodes strains 18 and 18N44. Using NormFinder, the best candidate reference gene in strain 18 was TUB (0.030), and the best candidate reference gene in strain 18N44 was UBI (0.047). In BestKeeper analysis, the standard deviation (SD) values of UBC, TUA, H2A, EF1, ACT, 18S, and GTP in strain 18 and those of GADPH and GTP in strain 18N44 were greater than 1; thus, these genes were disqualified as reference genes. Taken together, only UBI and TUB were found to be desirable reference genes by BestKeeper software. Based on the results of three software analyses, TUB was the most stable gene under all conditions and was verified as an appropriate reference gene for quantitative real-time polymerase chain reaction in L. edodes mycelia under high-temperature stress.