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.