BackgroundPhenylalanine ammonia-lyase (PAL, EC 4.3.1.24) is the first key enzyme in the phenylpropanoid pathway. The pal gene has been widely studied in plants and participates in plant growth, development and defense systems. However, in Pleurotus ostreatus, the biological functions of pal during organismal development and exposure to abiotic stress have not been reported.ResultsIn this study, we cloned and characterized the pal1 (2232 bp) and pal2 (2244 bp) genes from the basidiomycete P. ostreatus CCMSSC 00389. The pal1 and pal2 genes are interrupted by 6 and 10 introns, respectively, and encode proteins of 743 and 747 amino acids, respectively. Furthermore, prokaryotic expression experiments showed that PAL enzymes catalyzed the conversion of L-phenylalanine to trans-cinnamic acid. The function of pal1 and pal2 was determined by constructing overexpression (OE) and RNA interference (RNAi) strains. The results showed that the two pal genes had similar expression patterns during different developmental stages. The expression of pal genes was higher in the reproductive growth stage than in the vegetative growth stage. And the interference of pal1 and pal2 delayed the formation of primordia. The results of heat stress assays showed that the RNAi-pal1 strains had enhanced mycelial tolerance to high temperature, while the RNAi-pal2 strains had enhanced mycelial resistance to H2O2.ConclusionsThese results indicate that two pal genes may play a similar role in the development of P. ostreatus fruiting bodies, but may alleviate stress through different regulatory pathways under heat stress.
Pleurotus ostreatus is widely cultivated in China. However, its cultivation is strongly affected by seasonal temperature changes, especially the high temperatures of summer. Nitric oxide (NO) was previously reported to alleviate oxidative damage to mycelia by regulating trehalose. In this study, we found that NO alleviated oxidative damage to P. ostreatus mycelia by inhibiting the protein and gene expression of aconitase (ACO), and additional studies found that the overexpression and interference of aco could affect the content of citric acid (CA). Furthermore, the addition of exogenous CA can induce alternative oxidase (aox) gene expression under heat stress, reduce the content of H 2 O 2 in mycelium, and consequently protect the mycelia under heat stress. An additional analysis focused on the function of the aox gene in the heat stress response of mycelia. The results show that the colony diameter of the aox overexpression (OE-aox) strains was significantly larger than that of the wild-type (WT) strain under heat stress (32°C). In addition, the mycelia of OEaox strains showed significantly enhanced tolerance to H 2 O 2 . In conclusion, this study demonstrates that NO can affect CA accumulation by regulating aco gene and ACO protein expression and that CA can induce aox gene expression and thereby be a response to heat stress. IMPORTANCE Heat stress is one of the abiotic stresses that affect the growth and development of edible fungi. Our previous study found that exogenous NO had a protective effect on mycelia under heat stress. However, its regulatory mechanism had not been elucidated. In this study, we found that NO altered the respiratory pathway of mycelia under heat stress by regulating aco. The results have enhanced our understanding of NO signaling pathways in P. ostreatus.
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