Spermidine, a kind of polycation and one important member in the polyamine family, is essential for survival in many kinds of organisms and participates in the regulation of cell growth and metabolism. To explore the mechanism by which spermidine regulates ganoderic acids (GAs) biosynthesis in Ganoderma lucidum , the effects of spermidine on GAs and reactive oxygen species (ROS) contents were examined. Our data suggested that spermidine promoted the production of mitochondrial ROS and positively regulated GAs biosynthesis. Further research revealed that spermidine promoted the translation of mitochondrial complexes I and II and subsequently influenced their activity. With the reduction in eukaryotic translation initiation factor 5A (eIF5A) hypusination by over 50% in spermidine synthase gene ( spds ) knockdown strains, the activity of mitochondrial complexes I and II was reduced by nearly 60% and 80%, respectively, and the protein content was reduced by over 50%, suggesting that the effect of spermidine in mitochondrial complexes I and II was mediated through its influence on eIF5A hypusination. Furthermore, after knocking down eIF5A , the deoxyhypusine synthase gene ( dhs ) and the deoxyhypusine hydroxylase gene ( dohh ), mitochondrial ROS level was reduced by nearly 50%, and GAs content was reduced by over 40%, suggesting that eIF5A hypusination contributed to mitochondrial ROS production and GAs biosynthesis. In summary, spermidine maintains mitochondrial ROS homeostasis by regulating the translation and subsequent activity of complexes I and II via eIF5A hypusination and promotes GAs biosynthesis via mitochondrial ROS signaling. The present findings provide new insight into spermidine-mediated biosynthesis of secondary metabolites. IMPORTANCE: Spermidine is necessary for organism survival and is involved in the regulation of various biological processes. However, the specific mechanisms underlying the various physiological functions of spermidine are poorly understood, especially in microorganisms. In this study, we found that spermidine hypusinates eIF5A to promote the production of mitochondrial ROS and subsequently regulate secondary metabolism in microorganisms. Our study provides a better understanding of the mechanism by which spermidine regulates mitochondrial function and provides new insight into spermidine-mediated biosynthesis of secondary metabolites.

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Fungal AreA is a key nitrogen metabolism transcription factor in nitrogen metabolism repression (NMR). Studies have shown that there are different ways to regulate AreA activity in yeast and filamentous ascomycetes, but in Basidiomycota, how AreA is regulated is unknown. Here, a gene from Ganoderma lucidum with similarity to nmrA of filamentous ascomycetes was identified. The NmrA interacted with the C-terminal of AreA according to yeast two-hybrid assay. In order to determine the effect of NmrA on the AreA, 2 nmrA silenced strains of G. lucidum, with silencing efficiencies of 76% and 78%, were constructed using an RNA interference method. Silencing nmrA resulted in a decreased content of AreA. The content of AreA in nmrAi-3 and nmrAi-48 decreased by approximately 68% and 60%, respectively, compared with that in the WT in the ammonium condition. Under the nitrate culture condition, silencing nmrA resulted in a 40% decrease compared with the WT. Silencing nmrA also reduced the stability of the AreA protein. When the mycelia were treated with cycloheximide for 6 h, the AreA protein was almost undetectable in the nmrA silenced strains, while there was still approximately 80% of the AreA protein in the WT strains. In addition, under the nitrate culture, the content of AreA protein in the nuclei of the WT strains was significantly increased compared with that under the ammonium condition. However, when nmrA was silenced, the content of the AreA protein in the nuclei did not change compared with the WT. Compared with the WT, the expression of the glutamine synthetase gene in nmrAi-3 and nmrAi-48 strains increased by approximately 94% and 88%, respectively, under the ammonium condition, while the expression level of the nitrate reductase gene in nmrAi-3 and nmrAi-48 strains increased by approximately 100% and 93%, respectively, under the nitrate condition. Finally, silencing nmrA inhibited mycelial growth and increased ganoderic acid biosynthesis. Our findings are the first to reveal that a gene from G. lucidum with similarity to the nmrA of filamentous ascomycetes contributes to regulating AreA, which provides new insight into how AreA is regulated in Basidiomycota.

Hydrogen sulfide (H2S) is considered to be a novel gaseous signalling molecule with multiple physiological functions. Recently, the identification of sulfhydrated proteins has become a new hotspot in the analysis of the underlying mechanism of H2S. Our preliminary study has shown that H2S negatively regulates the heat-induced accumulation of ganoderic acids (GAs)，a major secondary metabolite in Ganoderma lucidum. However, a comprehensive understanding of its mechanism is lacking. In this study, sulfhydrated proteins in G. lucidum were quantified by quantitative proteomic mass spectrometry (MS), and the role of H2S in maintaining redox homeostasis under heat stress (HS) was determined. A redox-regulated protein, thioredoxin 1 (Trx1), was selected as a potential target of H2S. Further research revealed that the activity of Trx1 was provoked by sulfhydration at Cys31 and Cys34, contributing to the negative regulation of H2S to ROS accumulation and GAs biosynthesis under HS in G. lucidum. Our results provide a novel target for investigating the molecular mechanism of H2S physiological function. Moreover, new evidence is provided regarding the interaction mechanism between the H2S and ROS signalling pathways.