Molecular mechanism of delayed development by interfering RNA targeting the phenylalanine ammonia lyase gene (pal1) in Pleurotus ostreatus

Blocking the development of edible mushrooms will affect the production cycle and yield of fruiting bodies. Phenylalanine ammonia lyase (PAL, EC 4.3.1.24.) is an enzyme that catalyzes the deamination of phenylalanine to form trans-cinnamic acid. Previous studies have shown that a decrease in pal1 gene transcription delays fruiting body development in Pleurotus ostreatus. Herein, we used wild type (WT) and RNA interference (RNAi) strains to study the molecular regulation of pal1 by RNA sequencing and Agrobacterium-mediated genetic transformation. Our results showed that interference with the pal1 gene resulted in reductions in the total PAL enzyme activity and the total phenol content, as well as an increase in the intracellular H₂O₂ content. RNA-Seq data demonstrated that the significantly enriched KEGG terms were mainly related to the peroxisome pathway, MAPK signaling pathway-yeast and three other pathways, and the catalase (CAT) gene cat1 is also involved in multiple pathways that were enriched above. Exogenous H₂O₂ significantly enhanced the transcription of the cat1 gene and elevated total CAT enzymatic activity. Moreover, the levels of cat1 gene transcription and the total CAT enzymatic activity in the RNAi-pal1 strains gradually become closer to those in the WT strain through the removal of H₂O₂, which indicated that pal1 regulated the expression of cat1 by affecting the intracellular H₂O₂ content. Finally, the overexpression of the cat1 gene in P. ostreatus caused growth retardation, especially during the process of primordia formation. In conclusion, this study demonstrated that PAL1 affects cat1 gene expression through the signaling molecule H₂O₂ and regulates the development of P. ostreatus. The findings of this study enhance our understanding of the molecular developmental mechanism of edible mushrooms.