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

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.

Figure 1. Scheme for Polyporus umbellatus proteomics.

Red circle: sclerotia (S); black circle: hypha (H). I: initial; D: developmental; M: mature.

Figure 2. GO annotations of all quantified proteins.

Figure 3. PCA score plots of proteome data in sclerotia and hyphae.

PCA plots compared between sclerotia and hyphae were shown in (A) (IS and IH), (B) (DS and DH) and (C) (MS and MH), and among sclerotial proteomes at the three time points (D).

Figure 4. Venn diagram and the relative ratio of peak area of differentially expressed proteins in P. umbellatus sclerotia and hyphae.

(A) Venn diagram of differentially expressed proteins between sclerotia and hyphae at initial, developmental and mature phases. (B) Venn diagram of differentially expressed proteins in sclerotia at initial, developmental and mature phases. (C) relative ratio of peak area of representative differentially expressed proteins between sclerotia and hyphae at initial, developmental and mature phases. (D) relative ratio of peak area of representative differentially expressed proteins in sclerotia at initial, developmental and mature phases.

Figure 5. Cellular component of GO analyses on differentially expressed proteins between sclerotia and hyphae at initial phase.

Figure 6. Protein-protein interactions network of differentially expressed proteins in P. umbellatus sclerotia and hyphae at initial phase.

Figure 7. Glycerol induced and facilitated P. umbellatus sclerotia formation.

(A) Sclerotia formed on fructose complete medium (control). (B to F) Sclerotia formed on fructose complete media containing 1% (B), 3% (C), 5% (D), 6% (E) and 7% (F) glycerol, respectively. The biomass of sclerotia was significantly increased at 5% glycerol compared with that on control medium (p < 0.05), and it was decreased at 6% and 7% glycerol. The colony diameter of mycelia (green arrow) was not affected by glycerol.

Figure 8. Proteins involved in ROS generation and elimination as a response to hypoxia in P. umbellatus.

This schematic diagram was composed of electron transfer chain, TCA cycle and glycolysis. The expression of SDH subunits of complex II and IDH in TCA cycle was decreased in P. umbellatus sclerotia at initial phase leading to the increased ratio of NAD(P)+/NAD(P)H and ROS accumulation. NAD(P)H required for GSH to eliminate ROS could be accumulated with elevated alcohol dehydrogenase and aldehyde dehydrogenase following glycolysis/gluconeogenesis activation.