Abstract
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.
Highlights
Key inducers of sclerotia formation upon stimulation of external factors, such as starvation, temperature variation and ionizing radiation
The integrative assessment of P. umbellatus proteomes provides molecular bases for unveiling the sclerotia differentiation mechanism, which may be applicable for other fungi
The pooled and tryptic digested protein samples were analyzed by tandem LC-Mass spectrometry (MS) in data dependent acquisiation (DDA) model, and the acquired data were processed by ParagonTM (AB Sciex) (Fig. 1)
Summary
Key inducers of sclerotia formation upon stimulation of external factors, such as starvation, temperature variation and ionizing radiation. Antioxidants (diphenyleneiodonium, DPI) eliminating ROS could suppress sclerotia formation and caused biomass reduction via inhibiting reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and superoxide dismutase (SOD) to impair H2O2 generation[10]. Another experiment indicated that low temperature shift enhanced H2O2 generation in hypha cell wall or around the organelle membranes, and induced P. umbellatus sclerotial formation[11]. Among the various MS technologies, label-free quantitative proteomics based on Sequential Window Acquisition of all THeoretical fragment ion spectra (SWATH)-MS provides good reproducibility, accuracy and precision in quantification of proteins, and is suitable for detecting negligible protein differentiation (less than two folds)[15,16] These approaches facilitated the proteomic analyses of filamentous fungi in the past decade[13]. The integrative assessment of P. umbellatus proteomes provides molecular bases for unveiling the sclerotia differentiation mechanism, which may be applicable for other fungi
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