3 Discussion
cAMP is an important second messenger in cells, which plays a role in regulating physiological activities and material metabolism within cells. Studies on some fungi have shown that the intracellular cAMP expression level is rapidly increased through the Ga/Ras pathway, which in turn activates the cPKA catalytic subunit [32,33] (Hatanaka and Shimoda, 2001; Xue et al., 1998). The activation of cAMP leads to the degradation of trehalose in the cell and the synthesis of glucose [34]. cAMP can regulate various functions of fungi, including endogenous and exogenous carbon source utilization, spore formation, spore germination, and phototaxis. In Saccharomyces cerevisiae , several signaling pathways allow cells to sense glucose levels in the environment and initiate a transcriptional response. These pathways include the activation of cAMP/PKA and glucose expression (Snf1 kinase gene/MigI), which controls transcription [35]. Similar to yeast, the germination of the conidia of Aspergillus nidulans andNeurospora crassa is also involved in the activation of the cAMP signaling pathways and trehalose degradation [36]. Related studies have shown that the spore germination and appressorium formation of plant pathogens, particularly M. grisea , are mediated by the cAMP pathway and the cascade reaction of Pmk1 and Mps1 kinases [37,38].
In this study, the content of cAMP in the dormant spores was significantly higher than that in the non-dormant spores of the twoD. flagrans isolates. Li et al. (2016) proved that the cAMP content in chlamydospore was negatively correlated with the germination rate, and the cAMP content in resting spores (black chlamydospore) was higher than that in non-resting spores (yellow chlamydospore) [39]. Although the methods used to determine the strains in the two studies were different, the former using enzyme-labeled colorimetry and the latter using gas chromatography showed similar results. Virdy et al. (1999) reported that the cAMP content of dormant spores of the fungusDictyostelium discoideum is more than 11 times higher than that of newly formed spores [40]. Spores can achieve spontaneous germination in 14–18 days, and cAMP has a typical surge effect. However, the duration is not long, and the high level of cAMP significantly decreases during successful spore germination. These results support the hypothesis that externally activated (e.g., heating) and automatically activated spores germinate through different mechanisms. During heat activation, the transcription of ACG (a gene encoding adenylate-activating enzymes) was strongly correlated with the cAMP content in spore. For young wild-type spores that cannot spontaneously germinate, high cAMP levels are often maintained. Therefore, once spore germination was inhibited, cAMP levels increased. For example, when the activated spores were placed in a hypertonic environment to inhibit their activity, the concentration level of cAMP increases in dormant spores. Barhoom and Sharn (2004) reported that cAMP early activated the germination and expansion of A. nidulansspore [41]. Their results also show that plant surface signals induce spore germination specific to the pathogenic fungusColletotrichum gloeosporioides f. sp. aeschynomene in a cAMP-dependent manner, and that cAMP is necessary for saprophytic germination and appressorium formation.
C. gloeosporioides have two different germination strategies, namely, pathogenic development and saprophytic germination. The growth of this bacterium in liquid soybean extract can induce pathogenic germination, but no appressorium is observed [41]. Appressorium is only produced on the surface of a solid medium containing a soybean extract. The formation of appressorium in M. rosea requires the induction of functional Pmk1 MAP kinase and cAMP. Mutants with the deletion of Pmk1 and various cAMP pathways do not form appressorium, whereas the addition of cAMP can save some mutants and enhance the formation of appressorium [37, 38, 42]. Treatment of the conidia ofC. gloeosporioides with cAMP changed the pattern of germination and appressorium formation. In particular, cAMP induced a large number of conidial enlargement on the bean infusion medium, and enlargement was one of the signs before spore germination. Studies of C. gloeosporioides and other fungal germination and signaling pathways have shown that germination in the early stages of saprophytism involves the activation of the cAMP pathway [41]. In the early stage, pathogenic germination is regulated by the cAMP-independent pathway and not by cAMP, and cAMP can interrupt pathogenic germination. Fillinger et al. (2002) studied trehalose degradation and germination tube development in the conidium of three mutant strains of A. nidulans (chaA△, PKA△, and schA△) germinated on the medium with minimum concentration of glucose to evaluate the role of the cAMP signal during the germination of A. nidulans conidium [13]. The results showed that the degradation rate of trehalose in the conidium of mutant chaA△ and PKA△ was slower than that of wild strains. These results suggest that adenylate cyclase (AC) and PKAc are important determinants of A. nidoris conidium germination. Therefore, cAMP is necessary to transmit information and fully germinate A. nidulans conidium.
Based on the research of S. cerevisiae , the regulation of adenylate activity depends on the GTP enzymes Ras1 and Ras2. However, several studies have shown that the G-protein 2 subunit Gpa2 regulates yeast growth and pseudofilament development through the cascade amplification of the cAMP/PKA response to glucose [43]. For A. nidus , the alpha subunit of the three heterotrimers of G protein, namely, FadA, GanA, and GanB, has been identified, but its role in regulating AC remains unclear [13]. Some studies have shown that RasA is involved in the regulatory development of A. nidulans[44]. However, Fillinger et al. (2002) showed that RasA did not regulate the activity of AC in A. nidulans , and cAMP levels did not increase when RasA was overexpressed [13]. RasA may regulate the development of A. nidoris by regulating the activated mitogen kinase pathway, which has also been observed in several other fungi. Fillinger (2002) pointed out that the cAMP/PKA signaling pathway is a major but not necessary component for the asexual spore germination of the filamentous fungus A. nidus [13]. For A. nidusspores, more than one cAMP target pathway and another PKA-catalyzed subnodal pathway are required for germination. The polytropic effect of cAMP was also manifested in the withdrawal of spores from continuous dormancy involving cAMP and AC [45]. The cAMP content ofStreptomyces spores is low, but the cAMP level initially increases during spore germination and then decreases during subsequent colony growth [46]. The spores of the AC mutant strain ofStreptomyces Str. coelicolor showed no germination, but when cAMP with a concentration of more than 1 mM was added to the culture medium, spore germination was observed [45]. When this mutant grows on the agar surface, the colony morphology changes [45]. In later research, a receptor for cAMP of Str. coelicolor was discovered, which is a protein homologous to theEscherichia coli cyclic adenylate receptor protein (CRP). The Crp gene knockout mutant exhibited similar defects in spore germination, whereas other physiological effects were also observed on the mutant. The abovementioned research concludes that the cAMP-AC-CRP system plays an important role in controlling the spore germination of Str. coelicolor [47]. The high cAMP content in dormant spores is necessary to initiate germination, and once the spores germinate, the cAMP content decreases. Although the content of cAMP in germinating spores was not determined in this study, A. flagrans dormant chlamydospores contain high levels of cAMP compared with non-dormant spores. The results of this study indicate that cAMP is necessary to initiate the germination of this fungus, and its content may rapidly decrease after germination, with a mechanism similar to that of other fungi.
Protein is one of the three major nutrients in living organisms, and little research has been conducted on its content in fungal spores. In this study, the protein content in the chlamydospores of two D. flagrans strains was determined using the Coomassie brilliant blue method. The results showed a higher soluble protein content in the dormant spores, indicating a difference in protein content between dormant and non-dormant spores. In a study on the spore germination ofBotryodiplodia theobromae [48], dormant spores had two distinct protein bands (A and B). Protein band A decreased with the prolongation of spore germination time, while protein A was not present or detected in the mycelium. The content of protein B in dormant spores is second only to protein A, and the amount of protein B also decreases with the prolongation of germination time. However, some proteins are similar to protein B in the mycelium. The abovementioned two proteins do not exist in fresh spores and hyphae, so such proteins are similar to storage proteins in many higher plant seeds. During the germination of plant seeds, amino acids produced by the degradation of stored proteins in plant seeds are used as synthetic sources of new proteins required for germination. In some non-fungal organism studies, diatom dormant cells are rich in organelle proteins such as membrane proteins, ribosomal proteins, energy-related proteins, pigment proteins, and phosphoproteins. These proteins may be involved in the accumulation of chlorophyll during the formation of dormant cells. Some bacterial spores, such as Bacillus megaterium and B. cereus , contain specific proteins that degrade during germination, and their amino acids are used to synthesize new proteins [49,50]. The results of the study of D. flagrans are also similar to those of the abovementioned studies. However, the types of proteins in the two types of spores have not been studied. Moreover, the protein content, types, and functions in dormant spores remain unknown. Thus, further research is necessary.