SWATH label-free proteomics analyses revealed the roles of oxidative stress and antioxidant defensing system in sclerotia formation of Polyporus umbellatus.

Understanding the initiation and maturing mechanisms is important for rational manipulating sclerotia differentiation and growth from hypha of Polyporus umbellatus. Proteomes in P. umbellatus sclerotia and hyphae at initial, developmental and mature phases were studied. 1391 proteins were identified by nano-liquid chromatograph-mass spectrometry (LC-MS) in Data Dependant Acquisition mode, and 1234 proteins were quantified successfully by Sequential Window Acquisition of all THeoretical fragment ion spectra-MS (SWATH-MS) technology. There were 347 differentially expressed proteins (DEPs) in sclerotia at initial phase compared with those in hypha, and the DEP profiles were dynamically changing with sclerotia growth. Oxidative stress (OS) in sclerotia at initial phase was indicated by the repressed proteins of respiratory chain, tricarboxylic acid cycle and the activation of glycolysis/gluconeogenesis pathways were determined based on DEPs. The impact of glycolysis/gluconeogenesis on sclerotium induction was further verified by glycerol addition assays, in which 5% glycerol significantly increased sclerotial differentiation rate and biomass. It can be speculated that OS played essential roles in triggering sclerotia differentiation from hypha of P. umbellatus, whereas antioxidant activity associated with glycolysis is critical for sclerotia growth. These findings reveal a mechanism for sclerotial differentiation in P. umbellatus, which may also be applicable for other fungi.

Oxalic acid and sclerotial differentiation of Polyporus umbellatus.

The present investigation aimed to uncover the effects of exogenous oxalic acid during the sclerotial formation of Polyporus umbellatus, with an emphasis on determining the content of the endogenic oxalic acid in the fungus. To this end, the oxalic acid content of the vegetative mycelia, sclerotia, culture mediums and sclerotial exudate were measured using High Performance Liquid Chromatography (HPLC). Furthermore, the lipid peroxidation was estimated by detecting thiobarbituric bituric acid reactive substances (TBARS). The results showed that the exogenous oxalic acid caused a delay in sclerotial differentiation (of up to 9 or more days), suppressed the sclerotial biomass and decreased the lipid peroxidation significantly in a concentration-dependent manner. Oxalic acid was found at very low levels in the mycelia and the maltose medium, whereas it was found at high levels in the mycelia and sucrose medium. After sclerotial differentiation, oxalic acid accumulated at high levels in both the sclerotia and the sclerotial exudate. Oxalic acid was therefore found to inhibit P. umbellatus sclerotial formation.

Sclerotial formation of Polyporus umbellatus by low temperature treatment under artificial conditions.

BACKGROUND: Polyporus umbellatus sclerotia have been used as a diuretic agent in China for over two thousand years. A shortage of the natural P. umbellatus has prompted researchers to induce sclerotial formation in the laboratory. METHODOLOGY/PRINCIPAL FINDING: P. umbellatus cultivation in a sawdust-based substrate was investigated to evaluate the effect of low temperature conditions on sclerotial formation. A phenol-sulfuric acid method was employed to determine the polysaccharide content of wild P. umbellatus sclerotia and mycelia and sclerotia grown in low-temperature treatments. In addition, reactive oxygen species (ROS) content, expressed as the fluorescence intensity of mycelia during sclerotial differentiation was determined. Analysis of ROS generation and sclerotial formation in mycelia after treatment with the antioxidants such as diphenyleneiodonium chloride (DPI), apocynin (Apo), or vitamin C were studied. Furthermore, macroscopic and microscopic characteristics of sclerotial differentiation were observed. Sclerotia were not induced by continuous cultivation at 25°C. The polysaccharide content of the artificial sclerotia is 78% of that of wild sclerotia. In the low-temperature treatment group, the fluorescent intensity of ROS was higher than that of the room temperature (25°C) group which did not induce sclerotial formation all through the cultivation. The antioxidants DPI and Apo reduced ROS levels and did not induce sclerotial formation. Although the concentration-dependent effects of vitamin C (5-15 mg mL(-1)) also reduced ROS generation and inhibited sclerotial formation, using a low concentration of vitamin C (1 mg mL(-1)) successfully induced sclerotial differentiation and increased ROS production. CONCLUSIONS/SIGNIFICANCE: Exposure to low temperatures induced P. umbellatus sclerotial morphogenesis during cultivation. Low temperature treatment enhanced ROS in mycelia, which may be important in triggering sclerotial differentiation in P. umbellatus. Moreover, the application of antioxidants impaired ROS generation and inhibited sclerotial formation. Our findings may help to provide new insights into the biological mechanisms underlying sclerotial morphogenesis in P. umbellatus.

Involvement of Ca(2+) channel signalling in sclerotial formation of Polyporus umbellatus.

Growth and morphogenesis transformation in Polyporus umbellatus were examined in the presence of various pharmacological compounds, to investigate signal transduction pathways that influence the development of sclerotia. Both the calcium channel blocker nifedipine and the calcium ionophor A23187 reduced sclerotial production in P. umbellatus; four classes of Ca(2+) signal agent-including calcium chelators, calcium channel blockers, calcium ionophors and calmodulin inhibitors-were further studied. Among them, EGTA and BAPTA, as calcium chelators, exhibited a complete inhibitory effect on sclerotial formation, among the levels tested. Calcium channel blockers and calcium ionophors at the concentrations used in this study could not eliminate sclerotia formation completely, but did greatly reduce sclerotial production. Notoginsenoside in dosages >250 microg/ml produced a significant negative effect on mycelial growth, and it prevented sclerotial formation entirely at a dosage of 500 microg/ml; no other drug influenced vegetative growth at all. The calcium ionophor A23187 did not decrease sclerotial mean weight at low doses (20 nM); at higher doses (200 nM), however, sclerotial development was significantly reduced, albeit not completely halted. The CaM inhibitors (W-7 and chlorpromazine) could each completely stop sclerotial formation. Using Fluo-3/AM as the indicator of cytosolic free calcium, the Ca(2+) content in the cytoplasm was found to have decreased significantly when hyphae were treated with different drugs, and there was no active Ca(2+) signal in the sclerotial mycelium. In general, the results suggest that Ca(2+) signal transduction may play an important role in sclerotial formation in P. umbellatus.

First synthesis of (+/-)-basidifferquinone C, an inducer for fruiting-body formation in Polyporus arcularius.

Basidifferquinones, isolated from Streptomyces sp., are potent inducers of fruiting-body formation in the basidiomycete, Polyporus arcularius. The first synthesis of (+/-)-basidifferquinone C was accomplished by starting from 3,5-dihydroxy-2-naphthoic acid.