Yuansen Hu

Quantitative Proteomic Analysis of Wheat Seeds during Artificial Ageing and Priming Using the Isobaric Tandem Mass Tag Labeling

Wheat (Triticum aestivum L.) is an important crop worldwide. The physiological deterioration of seeds during storage and seed priming is closely associated with germination, and thus contributes to plant growth and subsequent grain yields. In this study, wheat seeds during different stages of artificial ageing (45°C; 50% relative humidity; 98%, 50%, 20%, and 1% Germination rates) and priming (hydro-priming treatment) were subjected to proteomics analysis through a proteomic approach based on the isobaric tandem mass tag labeling. A total of 162 differentially expressed proteins (DEPs) mainly involved in metabolism, energy supply, and defense/stress responses, were identified during artificial ageing and thus validated previous physiological and biochemical studies. These DEPs indicated that the inability to protect against ageing leads to the incremental decomposition of the stored substance, impairment of metabolism and energy supply, and ultimately resulted in seed deterioration. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the up-regulated proteins involved in seed ageing were mainly enriched in ribosome, whereas the down-regulated proteins were mainly accumulated in energy supply (starch and sucrose metabolism) and stress defense (ascorbate and aldarate metabolism). Proteins, including hemoglobin 1, oleosin, agglutinin, and non-specific lipid-transfer proteins, were first identified in aged seeds and might be regarded as new markers of seed deterioration. Of the identified proteins, 531 DEPs were recognized during seed priming compared with unprimed seeds. In contrast to the up-regulated DEPs in seed ageing, several up-regulated DEPs in priming were involved in energy supply (tricarboxylic acid cycle, glycolysis, and fatty acid oxidation), anabolism (amino acids, and fatty acid synthesis), and cell growth/division. KEGG and protein-protein interaction analysis indicated that the up-regulated proteins in seed priming were mainly enriched in amino acid synthesis, stress defense (plant-pathogen interactions, and ascorbate and aldarate metabolism), and energy supply (oxidative phosphorylation and carbon metabolism). Therefore, DEPs associated with seed ageing and priming can be used to characterize seed vigor and optimize germination enhancement treatments. This work reveals new proteomic insights into protein changes that occur during seed deterioration and priming.

Expression of feruloyl esterase A from Aspergillus terreus and its application in biomass degradation

Feruloyl esterases (FAEs) are key enzymes involved in the complete biodegradation of lignocelluloses, which could hydrolyze the ester bonds between hemicellulose and lignin. The coding sequence of a feruloyl esterase A (AtFaeA) was cloned from Aspergillus terreus and the recombinant AtFaeA was constitutively expressed in Pichia pastoris. The SDS-PAGE analysis of purified AtFaeA showed two protein bands owing to the different extent of glycosylation, and the recombinant AtFaeA had an optimum temperature of 50°C and an optimum pH of 5.0. The substrate utilization and primary sequence identity of AtFaeA demonstrated that it is a type-A feruloyl esterase. The hydrolysis of corn stalk and corncob by xylanase from Aspergillus niger could be significantly improved in concert with recombinant AfFaeA.

Insight into the global regulation of laeA in Aspergillus flavus based on proteomic profiling

In Aspergillus flavus, laeA affects cell morphology and contributes to the production of secondary metabolites (SMs) production including aflatoxin, cyclopiazonic acid, and aflatrem. Here, we investigated the function of this transcription factor by performing proteomics analysis of the wild-type (WT) and ΔlaeA mutant growing on corn. Notably, our proteomics profile confirmed the functions of extracellular hydrolases, conidial hydrophobin, and response to oxidative stress during the induction of aflatoxin biosynthesis regulated by laeA. Unexpectedly, deletion of laeA resulted in the significant upregulation of the NAD+-dependent histone deacetylase sirA involved in silencing SM clusters via chromatin remodeling. Accompanying the chromatin modification, enzymes participating in SM, including aflatoxin and cyclopiazonic acid biosynthesis, were drastically decreased. Another unexpected finding was that enzymes in the recently identified ustiloxin B biosynthesis pathway might be regulated by laeA. These data provided novel insights into the complex regulation of laeA and suggested a potential link between laeA deletion, NAD+-dependent histone deacetylation, and SM production in A. flavus.

Expression of a wheat β-1,3-glucanase in Pichia pastoris and its inhibitory effect on fungi commonly associated with wheat kernel

β-1,3-glucanases, the plant PR-2 family of pathogenesis-related (PR) proteins, can be constitutively expressed and induced in wheat crop to enhance its anti-fungal pathogen defense. This study aimed to investigate the inhibitory effect of wheat β-1,3-glucanase on fungi most commonly associated with wheat kernel. A β-1,3-glucanase from wheat was successfully expressed in Pichia pastoris X-33 and its biochemical and antifungal properties were characterized herein. The molecular weight of recombinant β-1,3-glucanase is approximately 33 kDa. β-1,3-glucanase displays optimal activity at pH 6.5, remaining relatively high at pH 5.5-8.0. The optimal reaction temperature of β-1,3-glucanase is 50 °C, retaining approximately 84.0% residual activity after heat-treated at 50 °C for 1 h. The steady-state kinetic parameters of β-1,3-glucanase against laminarin was determined and the Km and Vmax were 1.32 ± 0.20 mg/ml and 96.4 ± 4.4 U mg-1 protein, respectively. The inhibitory effect of purified β-1,3-glucanase against the seven fungi commonly associated with wheat kernel was assessed in vitro. β-1,3-glucanase exerted differential inhibitory effects on hyphal growth of Fusarium graminearum, Alternaria sp., A. glaucus, A. flavus, A. niger, and Penicillium sp. Spore formation and mycelial morphology of Alternaria sp., A. flavus, and A. niger were significantly affected by β-1,3-glucanase (1U). The present results would help elucidate the mechanism underlying the inhibition of wheat β-1,3-glucanases on pathogens.