Microbiology (2013), 159, 176–190
DOI 10.1099/mic.0.062935-0
Transcriptome changes initiated by carbon
starvation in Aspergillus nidulans
Melinda Szilágyi,1 Márton Miskei,1 Zsolt Karányi,2 Béla Lenkey,1
István Pócsi1 and Tamás Emri1
Correspondence
Tamás Emri
[email protected]
Received 17 August 2012
Revised
19 October 2012
Accepted 9 November 2012
1
Department of Microbial Biotechnology and Cell Biology, University of Debrecen, Egyetem tér 1,
4032 Debrecen, Hungary
2
Department of Medicine, University of Debrecen, Nagyerdei Körút 98, 4032 Debrecen, Hungary
Carbon starvation is a common stress for micro-organisms both in nature and in industry. The
carbon starvation stress response (CSSR) involves the regulation of several important processes
including programmed cell death and reproduction of fungi, secondary metabolite production and
extracellular hydrolase formation. To gain insight into the physiological events of CSSR, DNA
microarray analyses supplemented with real-time RT-PCR (rRT-PCR) experiments on 99 selected
genes were performed. These data demonstrated that carbon starvation induced very complex
changes in the transcriptome. Several genes contributing to protein synthesis were upregulated
together with genes involved in the unfolded protein stress response. The balance between
biosynthesis and degradation moved towards degradation in the case of cell wall, carbohydrate,
lipid and nitrogen metabolism, which was accompanied by the production of several hydrolytic
enzymes and the induction of macroautophagy. These processes provide the cultures with longterm survival by liberating nutrients through degradation of the cell constituents. The induced
synthesis of secondary metabolites, antifungal enzymes and proteins as well as bacterial cell walldegrading enzymes demonstrated that carbon-starving fungi should have marked effects on the
micro-organisms in their surroundings. Due to the increased production of extracellular and
vacuolar enzymes during carbon starvation, the importance of the endoplasmic reticulum
increased considerably.
INTRODUCTION
Carbon starvation can be defined as a certain type of stress,
which commences when the quantity and/or quality of carbon
(energy) sources are no longer sufficient to maintain vegetative
growth. Therefore, starving cells have to set into operation
special stress response programmes to survive. Carbon
starvation occurs frequently both in nature and in industrial
technological processes. In submerged shaken cultures it is
observed after the exponential growth phase when the carbon
source has been depleted. However, in surface cultures, due to
the local depletion of carbon sources, actively growing and
carbon-starved hyphae exist simultaneously.
Abbreviations: CP, cycle number of crossing points; cRNA, complementary RNA; CSSR, carbon starvation stress response; DCM, dry cell
mass; ROS, reactive oxygen species; rRT-PCR, real-time RT-PCR.
The GEO accession number for the microarray data discussed in this
paper is GSE42610.
Two supplementary tables, showing oligonucleotides and annealing
temperatures used in rRT-PCR experiments, and the original microarray
dataset and detailed tables for functional characterization of upregulated
and downregulated genes in carbon-starved Aspergillus nidulans
cultures, are available with the online version of this paper.
176
The carbon starvation stress response (CSSR) is associated
with complex physiological, morphological and ultrastructural changes in fungi (Winderickx et al., 2003). In
filamentous fungi, carbon starvation influences several
important processes. They include development of programmed cell death such as apoptosis and macroautophagy, and the production of secondary metabolites (e.g.
mycotoxins, antibiotics, antimycotics, pigments), hydrolytic enzymes (e.g. proteinases, chitinases, glucanases,
nucleases), and antifungal and antibacterial enzymes/
proteins, as well as the initiation of asexual, sexual and
parasexual cycles (White et al., 2002; Fischer & Kües, 2006;
Keller et al., 2005; Emri et al., 2008; Kim et al., 2011). Some
of these processes have been investigated in detail (Fischer
& Kües, 2006; Keller et al., 2005), but only a few studies
have applied a holistic approach to understand the global
changes caused by carbon starvation. The emerging
regulatory elements that influence the CSSR provide us
with good opportunities to manipulate the behaviour of
fungi during carbon starvation. Increasing the production
of antibiotics and antimycotics is important in the
fermentation industry but can also enhance the efficiency
of biocontrol agents (Reino et al., 2008). Inhibition of
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Carbon starvation stress in A. nidulans
mycotoxin formation is crucial in food and pharmaceutical
fermentations or during post-harvest decay of food and
animal feed (Edlayne et al., 2009). Controlling extracellular
proteinase or autolytic chitinase and glucanase formation
lets us increase the production of these industrially
important hydrolases or, by enhancing autolytic cell wall
degradation, these hydrolases accelerate the release of
certain cell wall-bound metabolites and enzymes (Emri
et al., 2008). Inhibition of self-digesting hydrolase formation helps to maintain pelleted culture morphology and
prevent proteolytic degradation of the product (White
et al., 2002). A deeper understanding of programmed cell
death processes observed during carbon starvation may
also support antifungal drug research in the future.
Inhibition of CSSR can also help us to control fungal
activities in agriculture or in industry.
In our previous research, we demonstrated that carbon
starvation induced complex, well-regulated and energyconsuming physiological and morphological changes in
Penicillium chrysogenum and Aspergillus nidulans (Pócsi
et al., 2003; Emri et al., 2008). We found that the observed
hyphal fragmentation as well as the disintegration of the
pellets were consequences of the production of cell wallhydrolytic enzymes, including ChiB endochitinase and
EngA b-1,3-endoglucanase (Pócsi et al., 2009; Szilágyi et al.,
2010). We also demonstrated that the BrlA transcription
factor, responsible for conidial development (Fischer &
Kües, 2006), was essential for induction of chiB and engA,
and was also important in the formation of extracellular
proteinases during carbon starvation (Szilágyi et al., 2010,
2011). According to our previous data, carbon starvation
induced extracellular hydrolase production, which was
accompanied by accumulation of reactive oxygen species
(ROS), changes in glutathione metabolism, production of
certain antioxidant enzymes, release of ammonia and a
decrease in respiration (Emri et al., 2004). We also
demonstrated that the CSSR of A. nidulans involves
apoptotic cell death in aged cultures (Emri et al., 2005).
To gain deeper insights into the physiological events and
signalling of the CSSR we performed global transcriptome analyses. Carbon starvation-initiated transcriptional
changes were also quantified with real-time RT-PCR (rRTPCR) of 99 selected genes. These data clearly demonstrated
that carbon starvation induced more complex alterations
in the transcriptome than we had considered before,
including the expression of genes encoding important
elements of primary and secondary metabolism and
programmed cell death processes, as well as cell wall and
redox homeostasis. At the level of subcellular organelles,
the importance of the endoplasmic reticulum was emphasized, shedding light on possible future antifungal drug
development strategies.
METHODS AND METHODS
Strain and culture conditions. The A. nidulans FGSC26 strain (biA;
veA1) was maintained on minimal nitrate medium (Barratt et al.,
http://mic.sgmjournals.org
1965) supplemented with 25 mg biotin l21. Cultures were incubated
at 37 uC for 7 days, and only freshly made conidia were used for the
experiments. Minimal nitrate medium (100 ml) supplemented with
5 g yeast extract l21 was inoculated with 56107 conidia and
incubated for 20 h at 37 uC and 3.7 Hz shaking frequency. Mycelia
from these exponential growth phase cultures were separated and
washed with the same prewarmed medium used for further
cultivation. The washed mycelia (10 g wet weight) were transferred
into 100 ml glucose-free minimal nitrate medium (carbon-starved
cultures) or minimal nitrate medium containing 10 g glucose l21
(growing cultures). The starting dry cell masses were approximately
4 g l21. Cultures were cultivated at 37 uC and 3.7 Hz shaking
frequency and samples were taken at 4 and 24 h.
Analytical procedures. The extracellular b-1,3-glucanase, b-gluco-
sidase, proteinase and chitinase activities were measured using
laminarin, p-nitrophenyl-b-D-glucose, azocasein and CM-ChitinRBV substrates, respectively, as described previously (Szilágyi et al.,
2010). Intracellular peroxide and superoxide levels were characterized
by the formation of 29,79-dichlorofluorescein (DCF) and ethidium
from 29,79-dichlorofluorescin diacetate and dihydroethidium, respectively (Emri et al., 1999). To measure sterigmatocystin formation,
20 mg lyophilized mycelium was extracted using 70 % (v/v) acetone,
and the sterigmatocystin content of the solutions was detected on
silica gels according to Klich et al. (2001). The formation of
conidiophores and vacuoles was followed by light microscopy.
rRT-PCR assays. To quantify the expression of selected genes, total
RNA was isolated from lyophilized 4 h mycelia originating from four
parallel experiments following the procedure of Chomczynski (1993).
rRT-PCR experiments were carried out as described previously (Pócsi
et al., 2009) with the primers and annealing temperatures presented in
Table S1 available with the online version of this paper. Primers were
designed based on the locus sequences of A. nidulans FGSC A4
obtained from The Broad Institute’s homepage (www.broadinstitute.
org). Relative transcript levels were quantified with DCP5
(CPtarget2CPreference) rRT-PCR cycle number of crossing point
differences, where the CP values represent the cycle numbers of
crossing points recorded for the tested target gene and for the
reference housekeeping gene (eEF-3; AN6700), respectively.
Microarray analysis. The DNA chip was produced on Agilent 60-
mer oligonucleotide high-density arrays 4644 K with 42 034
available features (Kromat). Oligonucleotides were designed with
the eArray software from Agilent (design number 024712). Total RNA
was isolated from mycelia after 4 h carbon starvation and from
growing mycelia (for microarray and rRT-PCR analysis, total RNA
was isolated from separate experiments). RNA samples from three
parallel experiments were pooled in 1 : 1 : 1 ratios. Hybridization
targets were prepared according to Agilent’s Two-Colour MicroarrayBased Gene Expression Analysis protocol (Version 5.7) using Cy3 and
Cy5 cyanine dyes. Purification of the labelled and amplified
complementary RNA (cRNA) was conducted using RNeasy Mini
spin columns (Qiagen). The quality of labelled cRNA was evaluated
on the Agilent Bioanalyser 2100 and quantified using an ND-1000
NanoDrop spectrophotometer. Fragmented cRNA samples (825 ng
each) were applied to the individual arrays. The slides were placed in
an Agilent hybridization oven and hybridized at 65 uC and 10 r.p.m.
for 17 h. Slides were scanned with an Agilent MicroArray Scanner and
intensities were extracted using Agilent’s Feature Extraction software
(version 9.1). The log2 ratios of the pre-normalized data were
normalized by local regression (LOESS) intensity-dependent blockby-block normalization (log2R) using the SAS for Windows software
(SAS Software Release 9.2, SAS Institute).
In order to identify proteins potentially secreted during carbon
starvation, genes encoding putative saccharide, lipid, protein,
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177
M. Szilágyi and others
bacterial, fungal and plant cell wall-degrading (hydrolytic) enzymes as
well as antifungal proteins were tested for signal peptide sequences by
the SignalP program of Bendtsen et al. (2004).
RESULTS AND DISCUSSION
Transcriptional changes caused by carbon starvation in A.
nidulans, which is a well-known filamentous fungus model
organism, were studied. For microarray experiments,
samples were taken at 4 h incubation time after transferring mycelia into either glucose-containing (growing
cultures) or glucose-free (carbon-starved cultures) minimal
media. These cultures showed characteristic physiological
differences. In carbon-starved cultures, elevated extracellular hydrolase production, increased intracellular superoxide and peroxide contents, and intensive vacuolization
were observed (Table 1). It is worth mentioning that
significant changes in the dry cell mass (DCM), hyphal
fragmentation, conidiophore formation, sterigmatocystin
production (2 mg DCM g21) as well as an increase in pH
(up to 8.5) were detected only after 24 h incubation under
carbon starvation. Genes characteristically upregulated or
downregulated by carbon starvation (the normalized log2 R
value was ¡2 or .2, respectively) were selected and
grouped. Among the stress-responsive genes, those where
the intensity of processed signals generated by Agilent’s
Feature Extraction software were too low (,1000 units) for
both labelled cRNA pools were omitted from further
analysis. Following these criteria, 1676 genes, 816 downregulated during carbon starvation and 860 upregulated
during carbon starvation, were identified. Among them, 99
genes with well-characterized or at least putative functions
from all the major gene function categories were selected
for rRT-PCR experiments. The microarray (log2 R values)
and rRT-PCR (DDCP values) data showed a strong
correlation with a correlation coefficient of 0.78 (Fig. 1).
During the proteome analysis of A. nidulans cultures,
Kim et al. (2011) identified 10 proteins characteristically
upregulated and five proteins characteristically downregulated in carbon-starved cultures. In our experiments,
among these proteins/genes, only four, aldA, AN8223,
AN7298 and AN4102 (Table S2), behaved similarly to
those observed by Kim et al. (2011). This significant
difference may be explained by the fact that Kim et al.
(2011) studied older (14 h) cultures than us (4 h).
DNA microarray and rRT-PCR gene expression data
suggested a remarkably complex, multilevel and sophisticated response to carbon starvation stress (Fig. 2, Tables 2
and S2.
Changes in cell wall homeostasis
Carbon starvation-induced autolysis involves the degradation of cell wall biopolymers (Emri et al., 2008), resulting
in declining DCM and progressive fragmentation of
hyphae (Emri et al., 2004). Previously, we demonstrated
that ChiB chitinase and EngA b-1,3-endoglucanase were
178
essential in this decomposition process (Pócsi et al., 2009;
Szilágyi et al., 2010). Deletion of one or both genes
significantly inhibited the DCM decline and hyphal
fragmentation (Pócsi et al., 2009; Szilágyi et al., 2010). In
the present study, we gained a more complex view of the
changes taking place in cell wall homeostasis (Fig. 3). We
detected the induction of several genes encoding hydrolytic
enzymes potentially involved in chitin (chiB, Pócsi et al.,
2009; nagA, Kim et al., 2002), b-1,3-glucan (engA, Szilágyi
et al., 2010; AN0779, AN4825 and AN0245) and a-1,3
glucan (mutA, Wei et al., 2001) degradation. Repression of
genes encoding synthases (chsB, chsF, fksA and agsB; de
Groot et al., 2009) and enzymes involved in cell wall
polysaccharide biosynthesis, including transglycosylases
(gelA, gelE and AN10779; de Groot et al., 2009) and
hydrolases (eglC, Choi et al., 2005; chiA, Yamazaki et al.,
2008) was also observed (Tables 2 and S2). Induction of the
gene AN9380 (Table 2), encoding a chitin deacetylase
(Wang et al., 2010), suggested that this alternative chitin
degradation pathway (chitin/chitooligomersAchitosanA
glucosamine) might also operate in carbon-starved cultures, as has been suggested by Alfonso et al. (1995).
Induction of several genes encoding cell wall-hydrolysing
enzymes (including chitinases, a- and b-glucanases and a1,6-mannannases) has also been detected in carbon-starved
cultures of Aspergillus niger (Nitsche et al., 2012).
Upregulation of the putative b-glucosidase-encoding genes
bglM (Table 2) and AN4102 (Table S2) might also be
related to the degradation of the fungal cell wall, and was in
good accordance with the observed elevated b-glucosidase
activity found previously under carbon starvation (Szilágyi
et al., 2010). The AN4102 gene product was also recorded
in a proteome analysis performed on carbon-starved
cultures by Kim et al. (2011). Induction of the putative
glucosamine-6-phosphate isomerase and N-acetylglucosamine-6-phosphate deacetylase, as well as the activation of
several potential high-affinity carbohydrate transporters,
including mstA and hxtA (Tables 2 and S2), were also
observable. Upregulation of high-affinity glucose transporters has also been detected in carbon-starved A. niger
cultures (Nitsche et al., 2012). In recent experiments, we
found that a DchiB DengA double mutant grew significantly
slower in surface cultures than its control strain on culture
media containing weak carbon sources (Szilágyi et al.,
2012). All these data suggest that fungi not only split their
own cell wall polysaccharides but also utilize them, as has
also been suggested by Kim et al. (2011) in their proteome
study.
Programmed cell death during carbon starvation
Carbon starvation is accompanied by a continuous
decrease in the viability of the culture. Although necrotic
cell death is possible during any kind of stress, programmed cell death processes have also been observed
during carbon starvation in several fungi (Emri et al., 2005;
Cebollero & Gonzalez, 2006; Robson, 2006; Pollack et al.,
2009; Sharon et al., 2009). In the case of A. nidulans, the
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Microbiology 159
Carbon starvation stress in A. nidulans
Table 1. Characteristic properties of the studied cultures as compared by DNA chip-based transcriptome analyses
Mycelia from exponential phase A. nidulans FGSC26 cultures were washed and transferred into either glucose-containing minimal medium
(growing cultures) or glucose-free minimal medium (carbon-starved cultures). Mean±SD values calculated from five independent experiments are
presented. DCF, 29,79-dichlorofluorescein; Et, ethidium.
Property
Growing culture
21
Glucose content (g l )
b-1,3-Glucanase activity (U ml21)
b-Glucosidase activity (U ml21)
Proteinase activity (U ml21)
Chitinase activity (U ml21)
Superoxide content [pmol Et (mg DCM21)]
Peroxide content [pmol DCF (mg DCM21)]
Changes in the DCM (g l21)
pH
Vacuolization
Conidiogenesis
Sterigmatocystin production
Hyphal fragmentation
9±0.5
2±0.3
0.24±0.03
0.2±0.03
0.2±0.03
0.03±0.01
0.5±0.1
1±0.1
6.6±0.1
No
No
No
No
Carbon-starved culture
0*
9±1*
0.9±0.08*
1.3±0.2*
0.88±0.3*
0.06±0.01*
0.8±0.1*
0±0.1*
6.7±0.1
Yes
No
No
No
*Significant differences between the growing and starved cultures according to Student’s t test (P,0.05).
appearance of apoptotic markers (membrane inversion,
DNA fragmentation) was detected in late autolytic cultures,
and macroautophagy has also been observed in both
carbon-starved and rapamycin-treated cultures by Emri
et al. (2005) and Kim et al. (2011), respectively. In their
proteome study, Kim et al. (2011) found the upregulation
of Pho8, a marker protein of autophagy. Although in our
experiments pho8 was not induced (Table S2), upregulation of five other genes potentially involved in autophagy
was observed (Tables 2 and S2). Among these genes, the
induction of genes AN2876, AN5174 and tipA was verified
by rRT-PCR (Table 2). Gene AN2876 is an orthologue
8
6
y = 0.7813x + 0.1342
R2 = 0.6072
rRT-PCR data (ΔΔCP)
4
2
0
–2
–4
–6
–8
–10
–8
–6
–4
–2
0
Microarray data [log2(R)]
2
4
6
Fig. 1. Correlation between DNA-microarray and rRT-PCR data
from the transcriptional analyses of 99 selected genes.
http://mic.sgmjournals.org
of Saccharomyces cerevisiae atg22, encoding a vacuolar,
autophagy-related amino acid permease (Yang et al., 2006).
The genes AN5174 and AN7428 are orthologues of atg5
and atg7, respectively, and both are involved in the
formation of autophagosomes in S. cerevisiae (Pollack
et al., 2009). TipA is the orthologue of baker’s yeast Tip41
(Fitzgibbon et al., 2005), which induces autophagy by
inhibiting TOR signalling. Induction of Atg7 and Atg26
together with other autophagy-related genes was also
observed in carbon-starved A. niger cultures (Nitsche
et al., 2012). These data together with the intensive
vacuolization observed in 4 h carbon-starved cultures
demonstrated that macroautophagy was an important part
of the early response to carbon starvation in A. nidulans.
Little information is available on the regulation of apoptosis
in A. nidulans. According to the microarray data (Table S2),
neither aifA (Savoldi et al., 2008; Dinamarco et al., 2010),
nor casA (Cheng et al., 2003) and prpA (Semighini et al.,
2006), were induced. However, repression of ndeA and ndiA,
which are both putative anti-apoptotic NADH-dehydrogenases (Dinamarco et al., 2010), was observed in the
microarray experiment (Table S2). Our previous physiological experiments demonstrated that the ratio of apoptotic
cells (protoplasts) was low immediately after glucose
depletion, and apoptosis became important only in old
(168 h) cultures, when there was an accumulation of ROS
(Emri et al., 2004, 2005). All these data indicated that
macroautophagy was responsible for most of the (programmed) cell death observed under carbon starvation. We
assume that macroautophagy and apoptosis had different
functions: macroautophagy was responsible for the reutilization of intracellular compounds, while apoptosis eliminated cells that were not functioning properly well before
they would become burdensome for the culture.
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179
M. Szilágyi and others
100
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80
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40
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Although the production of cell wall-hydrolysing enzymes
may also trigger the cell death process in old carbonstarved cultures (Szilágyi et al., 2012), their major function
might be to release nutrients from the cell wall of dead cells
and not to initiate cell death (Emri et al., 2008). Autolysis
can be defined as a physiological process of self-digestion,
in which macroautophagy is followed by (autolytic) cell
wall degradation. It also means that living cells can protect
themselves against the deleterious effect of endogenous
hydrolytic enzymes (Szilágyi et al., 2012).
In our experiments, the transcriptionally regulated mpkA
and rlmA, which are both key members of the cell
wall integrity pathway (Fujioka et al., 2007), were not
upregulated. Repression of genes involved in the biosynthesis of cell wall polysaccharides (Table 2, Fig. 3) suggests
that the cell wall integrity pathway was not upregulated at
the protein level either. Besides intensified cell wall
biosynthesis, the production of melanin may be an
alternative defensive process against hydrolytic enzymes.
In the case of Wangiella dermatidis, melanin-producing
cells are more resistant to cell wall hydrolases than mutants
defective in melanin synthesis (Dixon et al., 1991).
Moreover, melanin itself inhibits glucanase and chitinase
activities as well as the enzymic lysis of cells in A. nidulans
(Kuo & Alexander, 1967). Our microarray data indicated
the upregulation of four genes potentially involved in
melanin formation (ivoA, ivoB, AN0230 and AN2091;
Table S2). Among them, the induction of ivoA and the gene
AN0230 was confirmed by rRT-PCR (Table 2). Both ivoA
and ivoB are involved in the Tyr-based melanin biosynthesis (Birse & Clutterbuck, 1990), while AN0230 (Nahlik,
2007) together with AN2091 encodes putative tyrosinases
and is potentially also involved in this process. Although
melanin formation was not detected at the 4 h incubation
180
Fig. 2. Functional characterization of downregulated (white) and upregulated (grey)
genes in carbon-starved cultures of A. nidulans. Note that 572 upregulated and 591
downregulated genes with uncharacterized or
unknown functions are not presented.
time, the formation of this pigment was visible in older
cultures.
Changes in carbon and nitrogen metabolism
In general, genes connected to the utilization of glucose
from glycolysis to oxidative phosphorylation were downregulated (Table S2). These genes included mstE, gpdA,
pfkZ, gsdA, citA, aoxA and AN10585 (putative cytochrome
c oxidase) (Table 2). In previous experiments, decreased
specific glucose-6-phosphate dehydrogenase activities as
well as decreased cytochrome c-dependent and cyanideresistant respiration were detected during carbon starvation (Emri et al., 2004). In the case of trehalose
metabolism, the repression of biosynthesis (tpsA) and the
induction of degradation (treA) were observed (Table 2).
These changes could be connected to the utilization of
trehalose as a potentially abundant carbon source in soil
(Jorge et al., 1997) rather than to the stress-related
functions of this disaccharide, since the treA gene encodes
an extracellular acid trehalase (Fillinger et al., 2001). The
production of enzymes responsible for the degradation of
plant cell wall polysaccharides is also possible according to
the microarray data. However, the number of induced and
repressed genes potentially involved in the utilization of
plant materials was similar (Table S2). It is worth
mentioning that cultivation of A. nidulans on plant
materials resulted in the induction of several hydrolytic
enzymes, suggesting the importance of induction in their
regulation in addition to derepression (Schneider et al.,
2010).
In both lipid and nitrogen metabolism, catabolic processes
became dominant over the anabolic pathways (Fig. 2, Table
S2). Among them, downregulation of synthases (AN4923, a
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Carbon starvation stress in A. nidulans
Table 2. rRT-PCR-based transcriptional analyses of selected genes
All rRT-PCR data presented here represent mean±SD values calculated from four parallel experiments. Asterisks indicate significant (P¡0.05,
calculated by Student’s t test) alterations in mRNA levels between the growing and carbon-starved cultures. Microarray and rRT-PCR data were
gained from separate experiments.
Gene ID
(Putative) product
Microarray
data
[log2(R)]
rRT-PCR data
ReferenceD
Growing
Starving
culture (DCP) culture (DCP)
Carbohydrate metabolism, excluding metabolism of cell wall polysaccharides
AN8041
Glyceraldehyde-3-phosphate dehydrogenase
3.05
(GpdA)
AN5144
Putative 6-phosphofructo-2-kinase (PfkZ)
2.55
AN2981
Putative glucose-6-phosphate 12.37
dehydrogenase (GsdA)
AN8275
Citrate synthase (CitA)
3.54
AN2099
Alternative oxidase (AoxA)
2.17
AN10585
Putative cytochrome c oxidase polypeptide
2.57
AN5523
Trehalose-6-phosphate synthase subunit 1
2.37
(TpsA)
AN9340
Acid trehalase (TreA)
22.14
AN7396
AN6620
AN2017
AN5860
AN8737
AN6923
AN5104
AN8347
AN9168
AN3357
AN6669
Lipid metabolism
AN8242
AN6464
AN4923
AN9408
Nitrogen metabolism
AN1006
AN1007
AN1899
AN8559
AN5558
AN7962
AN3393
AN8445
AN6438
AN2572
http://mic.sgmjournals.org
Punt et al. (1988)
23.2±0.4
21.4±0.3*
5.3±0.8
0.7±0.2
10±1*
0.7±0.2*
Flipphi et al. (2009)
Flipphi et al. (2009)
5.1±0.5
7±0.6
23.9±0.4
3±0.3
6.2±0.5*
8.2±0.6*
22.8±0.1*
4.4±0.5*
Park et al. (1997)
Suzuki et al. (2012)
3.9±0.5
2.1±0.3*
Fillinger et al. (2001)
d’Enfert & Fontaine
(1997)
Putative b-glucosidase (BglM)
Glycosyl hydrolases family 16 domaincontaining protein
Putative a-glucosidase (AgdA)
27.47
24.84
12.6±1
4±0.5
8.1±0.7*
3.7±0.3
23.00
5.4±0.4
5.9±0.6
Low-affinity glucose transporter (MstE)
Putative major facilitator superfamily
(MFS) monosaccharide transporter
(MstA)
High-affinity hexose transporter (HxtA)
Putative MFS monosaccharide transporter
Putative hexose transporter
Putative sugar transporter
Putative MFS monosaccharide transporter
Putative MFS monosaccharide transporter
4.26
24.04
3±0.2
6.6±0.7
9.4±0.8*
3.5±0.4*
22.77
22.39
24.77
21.85
4.78
21.9
5.6±0.4
5.3±0.6
6±0.7
6.6±0.8
13.1±0.9
14.1±1
20.9±0.2*
1±0.2*
3.9±0.4*
2.8±0.3*
15±1*
14±1.2
Putative lipase
Putative esterase
Putative hydroxymethylglutaryl-CoA
synthase
Fatty acid synthase (FasB)
23.13
25.35
3.08
7.1±0.8
12.3±1
0.6±0.1
2.4±0.4*
6.5±0.8*
4.7±0.7*
3.23
0.4±0.2
1.5±0.3*
Brown et al. (1996)
Nitrate reductase (NiaD)
Nitrite reductase (NiiA)
4-Hydroxyphenylpyruvate dioxygenase
(HpdA)
Putative 2-oxoisovalerate dehydrogenase
Alkaline protease (PrtA)
4.21
4.33
28.58
6.1±0.7
23±0.3
12±0.9
11±0.9*
3.5±0.4*
6.5±0.6*
Johnstone et al. (1990)
Johnstone et al. (1990)
da Silva Ferreira et al.
(2006)
23.88
24.58
9.9±0.8
4.9±0.5
9.4±1
0.6±0.2*
Metalloproteinase (PepJ)
Putative deuterolysin metalloprotease
family protein (PepI)
Putative aminopeptidase
Putative dipeptidyl-peptidase
Putative dipeptidyl-peptidase
22.47
24.22
4±0.4
11.9±1.2
1±0.2*
7.4±0.7*
25.33
23.93
24.96
5.7±0.7
3±0.3
12.1±1.1
1±0.2*
21.5±0.1*
9.8±0.9*
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Nakamura et al.
(2006)
Forment et al. (2006)
Wei et al. (2004)
Wei et al. (2004)
Peña-Montes et al.
(2008)
Emri et al. (2009)
Katz et al. (2008)
Schneider et al. (2010)
Schneider et al. (2010)
181
M. Szilágyi and others
Table 2. cont.
Gene ID
(Putative) product
Microarray
data
[log2(R)]
rRT-PCR data
ReferenceD
Starving
Growing
culture (DCP) culture (DCP)
AN8498
AN2237
AN2092
AN4282
AN1723
AN11897
AN11062
AN4809
Cell wall metabolism
AN4367
AN2523
AN8710
AN8241
AN4871
AN9390
AN9380
AN1502
AN1418
Putative proteinase
Putative carboxypeptidase
Putative prolyl aminopeptidase (PapA)
Putative aminopeptidase
Putative T1 RNase
Putative T2 RNase
Putative liver perchloric acid-soluble
protein (L-PSP) endoribonuclease Brt1
Putative glutaminase A (GtaA)
21.87
20.66
24.48
25.05
27.48
24.11
27.21
2.7±0.3
3.6±0.4
15.5±1.3
11.3±0.9
7.8±0.8
3.3±0.4
5.9±0.6
1±0.2*
2.6±0.4*
14.5±1.2
10.2±1
2.7±0.2*
2±0.1*
0.9±0.2*
23.66
6.4±0.6
1.9±0.3*
7±0,6
4.7±0.6
2.7±0.6
4.4±0.5
4±0.6
12.3±1
3.1±0.4
4.1±0.4
3.4±0.4
11±1.2*
7.6±0.7*
3.7±0.7
8.5±1*
0.2±0.1*
11.5±0.9
1.6±0.3*
1±0.2*
1±0.2*
7.5±1
4.5±0.6*
4.7±0,6
7.8±0,8*
21.8±0.3
3.8±0.5
12±1
1.7±0.3
9±1.3
4.8±0.6
2.5±0.3
6.3±0.7
2.9±0.4
20.4±0.2*
5.9±0.7*
14±1.1*
5.4±0.6*
2.1±0.4*
0.8±0.2*
1.1±0.2*
20.6±0.2*
6.7±0.7*
de Groot et al. (2009)
de Groot et al. (2009)
de Groot et al. (2009)
Choi et al. (2005)
Szilágyi et al. (2010)
de Groot et al. (2009)
de Groot et al. (2009)
de Groot et al. (2009)
de Groot et al. (2009)
9.8±1
21.2±0.2
7±0.7
4.3±0.5
15.3±1.2
3.6±0.3*
4.4±0.4*
1.4±0.2*
3.9±0.4
16.1±1.3
Wei et al. (2001)
de Groot et al. (2009)
Stringer et al. (1991)
de Groot et al. (2009)
Bussink & Osmani
(1999)
Fujioka et al. (2007)
Chitin synthase (ChsF)
3.09
Chitin synthase (ChsB)
20.92
Putative chitin biosynthesis protein
22.32
Chitinase (ChiA)
4.94
Chitinase (ChiB)
23.47
Putative chitinase (ChiC)
24.14
Chitin deacetylase
28.14
24.69
N-Acetyl-b-D-glucosaminidase (NagA)
Putative glucosamine-6-phosphate
24.39
isomerase
AN1428
Putative N-acetylglucosamine-6-phosphate
26.10
deacetylase
21.30
AN3729
Catalytic subunit of 1,3-b-glucan synthase
complex (FksA)
2.82
AN7657
Putative 1,3-b-transglucosylase (GelA)
3.74
AN7511
Putative 1,3-b-transglucosylase (GelE)
3.45
AN10779
Putative 1,6-b-transglycosidase
2.27
AN7950
1,3-b-Glucanase (EglC)
AN0472
Endo-1,3-b-glucanase (EngA)
22.75
25.62
AN0245
Putative 1,3/1,4-b-glucanase
21.34
AN0779
Putative exo-1,3-b-glucanase
24.44
AN4825
Putative exo-1,3-b-glucanase
AN3307
Putative catalytic subunit of the 1,3-a4.35
glucan synthase complex (AgsB)
23.66
AN7349
1,3-a-Glucanase (MutA)
AN7539
Putative hydrophobin
2.48
AN8803
Hydrophobin (RodA)
22.74
AN0940
Putative hydrophobin
24.31
AN5666
Mitogen-activated protein (MAP) kinase
20.01
(MpkA)
AN2984
Transcription factor (RlmA)
1.90
Glutathione metabolism, thioredoxin metabolism, catalases, superoxide dismutases
AN3150
Putative glutamate–cysteine ligase
1.40
22.97
AN5658
Putative c-glutamyltranspeptidase
23.04
AN10444
Putative c-glutamyltranspeptidase
AN5652
Putative hydantoinase/5-oxoprolinase
23.44
AN8218
AN0241
Putative thioredoxin reductase (TrxB)
Superoxide dismutase (SodA)
AN5577
Putative manganese superoxide dismutase
(SodB)
182
1.3±0.3
1.8±0.3
3.6±0.4
5.4±0.5
5.6±0.6
2.5±0.4
3.9±0.3
3.2±0.4*
2.8±0.2*
4.6±0.6*
22.79
0.58
3±0.4
0.1±0.1
2.5±0.3
2.2±0.3*
2.50
4±0.3
1.7±0.2*
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Schneider et al. (2010)
Basten et al. (2005)
Koibuchi et al. (2000)
Horiuchi (2009)
Borgia et al. (1996)
Yamazaki et al. (2008)
Erdei et al. (2008)
de Groot et al. (2009)
Alfonso et al. (1995)
Kim et al. (2002)
Kelly et al. (1996)
Hortschansky et al.
(2007)
Oberegger et al.
(2000)
Microbiology 159
Carbon starvation stress in A. nidulans
Table 2. cont.
Gene ID
(Putative) product
rRT-PCR data
Microarray
data
[log2(R)]
ReferenceD
Starving
Growing
culture (DCP) culture (DCP)
AN1131
AN8637
AN9339
AN5918
Putative
Catalase
Catalase
Catalase
copper/zinc superoxide dismutase
A (CatA)
B (CatB)
C (CatC)
24.57
4.86
2.48
23.93
3.8±0.5
9.3±1
3±0.4
5.5±0.7
0.3±0.1*
15.6±1.3*
6±0.5*
4.5±0.6
Genes involved in autophagy
AN5174
Putative autophagy protein (Atg5)
24.58
14±0.8
12.6±0.7*
AN2876
23.91
6.4±0.5
2.2±0.2*
Putative autophagy protein (Atg22)
22.07
4.8±0.5
2.9±0.2*
Putative target of rapamycin (TOR)
signalling pathway protein (TipA)
Genes involved in folding and post-translational modification of proteins as well as intracellular trafficking
AN7436
Putative disulfide isomerase (PdiA)
22.52
5.1±0.5
1.6±0.3*
AN5814
AN9397
Transcription factor (HacA)
22.28
2.6±0.2
1.2±0.1*
AN0787
Putative a-1,2- mannosidase (Mns1B)
24.31
2.5±0.3
0.2±0.1*
23.25
4.5±0.5
3.1±0.4*
22.47
2.1±0.2
0.8±0.1*
24.20
8.2±0.8
2.9±0.4*
AN2738
Putative coat protein complex-II (COPII)coated vesicle membrane protein (Erv46)
AN1117
Putative COPII-coated vesicle protein
(SurF4/Erv29)
Genes involved in secondary metabolism and inter-specific interactions
AN10576
Non-ribosomal peptide synthetase (IvoA)
AN2091
AN0230
AN9129
AN7820
AN6470
AN5046
AN11510
Other
AN1414
AN0973
AN2265
AN5457
Putative tyrosine decarboxylase
Putative tyrosinase
Putative non-ribosomal peptide synthetase
C6 transcription factor (AflR)
N,O-Diacetylmuramidase
Anizin
Putative atesin-3
24.08
24.07
23.91
22.62
27.16
24.96
24.95
3.9±0.3
5.6±0.7
11.3±1.1
6±0.7
9.1±1
7.5±0.8
12.8±1.2
3.4±0.4
20.4±0.1*
5.4±0.6*
0.3±0.1*
2.2±0.4*
2.5±0.3*
5.6±0.5*
Transcription factor (XprG)
Transcription factor (BrlA)
Putative serine/threonine protein kinase
NADPH oxidase (NoxA)
22.36
24.28
22.20
21.00
2.2±0.3
8±1
5.5±0.5
0.4±0.2
0.8±0.2*
2.2±0.3*
20.1±0.2*
0.7±0.2
AN5712
Putative metacaspase (CasA)
20.48
21±0.2
20.8±0.2
Navarro et al. (1996)
Kawasaki et al. (1997)
Kawasaki and Aguirre
(2001)
Kiel and van der Klei
(2009)
Kiel and van der Klei
(2009)
Fitzgibbon et al.
(2005)
Colabardini et al.
(2010)
Saloheimo et al.
(2003)
Eades and Hintz
(2000)
Birse & Clutterbuck
(1990)
Nahlik (2007)
von Döhren (2009)
Yu et al. (1996)
Bauer et al. (2006)
Eigentler et al. (2012)
Katz et al. (2006)
Adams et al. (1988)
Lara-Ortı́z et al.
(2003)
Cheng et al. (2003)
DWhere a reference is not given, annotation is based on the Aspergillus Genome Database (www.aspergillusgenome.org) and the Aspergillus
Comparative Database (www.broadinstitute.org).
putative hydroxymethylglutaryl-CoA synthase and fasB) as
well as upregulation of hpdA, which is involved in the
degradation of Tyr, was also confirmed by rRT-PCR (Table
2). Our data, similarly to the observations of Kim et al.
(2011), supported the view that cells metabolized their
amino acids and nucleotides during carbon starvation,
which is in good accordance with the observed ammonia
http://mic.sgmjournals.org
production (Emri et al., 2004) and alkalification of culture
media (Szilágyi et al., 2011). It is worth mentioning that
alcR, encoding an ethanol regulon regulatory protein, was
also induced by carbon starvation together with the aldA
aldehyde dehydrogenase gene (Table S2), which is a target
gene of AlcR (Pateman et al., 1983). Upregulation of
AldA was also observed in the proteome analysis of
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183
M. Szilágyi and others
Degradation of monomers AN1428, AN1418 ( )
chiB, nagA ( )
AN9380 ( )
Hydrolysis
Uptake of monomers
hxtA, mstA, AN5104 ( )
AN8347, AN9168 ( )
mstE ( )
engA, AN0779 ( ),
AN4825, AN0245 ( )
mutA ( )
Proteinase production
prtA, pepJ ( )
brlA ( )
MELANIN
ivoA ( )
AN0230 ( )
CELL WALL POLYSACCHARIDES
Chitin
α- and β-Glucans
Conidiogenesis
rodA ( )
chsB ( )
chsF ( )
chiA ( )
Biosynthesis
Tyr
fksA, gelE, gelA, eglC ( )
AN10779, agsB ( )
Monomers
mpkA, rlmA (0)
CWI pathway-dependent and -independent signalling
Fig. 3. Proposed changes in the cell wall metabolism and related processes in A. nidulans during carbon starvation based on
rRT-PCR experiments.
carbon-starved cultures (Kim et al., 2011). Furthermore,
the induction of alcR was also detected in the presence of
ethanol and acetate (Pateman et al., 1983) or hypoxia
(Terabayashi et al., 2012), while oxidative stress caused by
menadione downregulated this protein (Pusztahelyi et al.,
2011). These findings demonstrated the importance of
AlcR in the coordination of several stress responses. Since
AldA is necessary for both ethanol and Thr utilization
(Pateman et al., 1983), its induction during carbon
starvation might also be related to the utilization of
acetaldehyde, which is formed during the degradation of
Thr to Gly, and not to the degradation of ethanol.
The downregulation of niaD and niiA, encoding nitrate
reductase and nitrite reductase, respectively, was also notable
(Table 2), since their promoters are often used to construct
overproducer mutants. Our data call attention to the fact
that the behaviour of these overproducer mutants can be
very different in surface cultures, where growing and starving
cells are present simultaneously, or in submerged cultures,
where only growing or starving cells are likely to occur.
Similarly to several other fungi (White et al., 2002), the
carbon starvation and autolysis of A. nidulans can be
characterized by the production of hydrolytic enzymes,
including lipases (Garcı́a-Lepe et al., 1997), nucleases
(Reyes et al., 1990; Kim et al., 2011) and proteinases
(Katz et al., 2008; Szilágyi et al., 2011). Our microarray data
also demonstrated the upregulation of several genes
encoding hydrolytic enzymes (Table S2). Among them,
the upregulation of AN8242, encoding a putative lipase,
184
and AN6464, encoding a signal peptide-containing putative esterase, as well as the induction of three Rnase-coding
genes (AN1723, AN11897 and AN11062), was validated by
rRT-PCR. The induction of eight signal peptide-containing
proteinase genes (prtA, pepJ, pepI, AN8445, AN6438,
AN2572, AN8498 and AN2237) was also confirmed by
rRT-PCR (Table 2). Besides PrtA and PepJ, secretion of
proteinases AN8445, AN6438 and AN2237 has also been
detected on beech leaf litter, demonstrating that these
enzymes, in accordance with their signal peptide sequence,
are extracellular proteinases (Schneider et al., 2010). It is
worth mentioning that the accumulation of protein
AN2237 has also been described during carbon starvation
by Kim et al. (2011), and the induction of several genes
encoding extracellular proteinases has also been detected in
carbon-starved cultures of A. niger (Nitsche et al., 2012).
The production of the huge number of proteinases
explained why the DprtA DpepJ double mutant had no
significant phenotype in our previous experiments (Szilágyi
et al., 2011). The bulk production of proteinases observed
during carbon starvation (Szilágyi et al., 2011) was in good
accordance with the upregulation of brlA and xprG,
encoding transcription factors, and hxkC, which encodes
a non-catalytic hexokinase (Tables 2 and S2). The
involvement of BrlA, XprG and HxkC in the regulation
of proteinase production was demonstrated by Szilágyi et al.
(2011), Katz et al. (2006) and Bernardo et al. (2007),
respectively. These proteinases can be important in the
utilization of proteins released by dead cells, which can be
physiologically significant in ageing cultures (data not
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Microbiology 159
Carbon starvation stress in A. nidulans
shown). Moreover, proteinases may also be important
in the degradation of enzymes and antifungal proteins
produced by competitive or parasitic species (Elad, 2000;
Hegedüs et al., 2011) and in autolytic cell wall degradation (Szilágyi et al., 2011). Obviously, these proteinases
themselves may have antifungal/antibacterial effects as well
(De Marco & Felix, 2002).
Production of secondary metabolites and other
compounds with roles in intraspecific interactions
Although the FGSC 26 strain harbours a veA1 mutation,
which decreases its secondary metabolite production
(Stinnett et al., 2007), certain genes involved in polyketide
and non-ribosomal peptide synthesis were induced (Tables
2 and S2). The induction of aflR, encoding the aflatoxin
regulatory protein AflR (Table 2), was in good accordance
with the sterigmatocystin production observed in 24 h
cultures. In addition to secondary metabolites, the
upregulation of genes encoding antibacterial (AN6470;
Bauer et al., 2006) or antifungal (e.g. ChiB and EngA;
Szilágyi et al., 2012) enzymes as well as low-molecular-mass
antifungal proteins (such as anisin1, Eigentler et al., 2012;
AN11510) was observed (Table 2). Some of these proteins
and metabolites can be multifunctional, e.g. in addition to
its antifungal effect, anisin1 also enhances the sporulation
of the producer strain (Eigentler et al., 2012), and ChiB and
EngA also play a key role in autolytic cell wall degradation
(Pócsi et al., 2009; Szilágyi et al., 2010). The production of
these compounds, enzymes and other proteins clearly
demonstrates that fungi have evolved multilevel strategies
to control the biological activity of any competing
organisms occupying the same habitats. The significance
of this capability becomes clear when the available carbon
sources become limited.
Redox balance
Previous physiological experiments demonstrated that
carbon-starved A. nidulans cultures lost the majority of
their glutathione pool, which was accompanied by the
induction of specific c-glutamyltranspeptidase activities
(Emri et al., 2004). rRT-PCR and DNA microarray data
also supported the induction of the glutathione-degrading
c-glutamyltranspeptidases, because genes AN10444 and
AN5658, both encoding putative c-glutamyltranspeptidase
domains, were upregulated in this study (Table 2). The
degradation of glutathione might be significant, because
glutathione is utilized as an energy source, supports
intracellular amino acid transport and protects cells against
ROS (Pócsi et al., 2004). The accumulation of ROS is a
typical event in carbon-starved cultures (Jakubowski et al.,
2000; Sámi et al., 2001; Emri et al., 2004). Interestingly,
while specific superoxide dismutase activities increased
continuously under carbon starvation, the specific catalase
and glutathione peroxidase activities decreased after a
temporary induction (Sámi et al., 2001; Emri et al., 2004;
Molnár et al., 2004, 2006). Similar changes were observed
http://mic.sgmjournals.org
in rRT-PCR experiments (Table 2), because catA, catB and
sodA were all repressed, while sodB and another gene
(AN1131) encoding a putative superoxide dismutase were
induced. The accumulation of ROS was accompanied by
the induction of several genes involved in DNA repair,
including sldI and mreA (Fig. 2, Table S2).
ROS have pleiotropic effects on cells. On one hand, they
can cause serious oxidative damage, resulting in mutations
or even necrotic cell death (Fridovich, 1998), but on the
other hand the accumulation of ROS regulates several
important physiological processes, including apoptosis
(Madeo et al., 2004) and secondary metabolite production
(Jayashree & Subramanyam, 2000) as well as sexual and
asexual differentiation (Hansberg et al., 1993; Lara-Ortı́z
et al., 2003; Emri et al., 2004). Therefore, a delicately
controlled accumulation of ROS can be beneficial for
carbon-starved cultures. The induction of c-glutamyltranspeptidases, the depletion of the glutathione pool and the
repression of certain antioxidant enzymes can be part of
this strategy. This view is supported by several observations, including that NoxA, a superoxide-generating
NADPH oxidase, is necessary for efficient chleistotechium
formation in A. nidulans (Lara-Ortı́z et al., 2003), and
that RgsA, a regulator of GanB/SfaD/GpgA heterotrimeric
G protein signalling (Han et al., 2004), is necessary for
catB repression in carbon-starved cultures (Molnár et al.,
2004).
Protein synthesis
Surprisingly, several genes with important functions in
protein synthesis from transcription to protein secretion
were upregulated (Tables 2 and S2). Many of them,
including pdiA and mns1B as well as AN2738 and AN1117
(encoding putative COPII-coated vesicle membrane proteins), were related to endoplasmic reticulum functions. The
upregulation of pdiA (Colabardini et al., 2010) and hacA
(Table 2), an orthologue of the S. cerevisiae hac1 bZIP-type
transcription factor (Mori et al., 1996; Saloheimo et al.,
2003), suggested the activation of the unfolded-protein
stress response under carbon starvation. The induction of
these genes can be explained by the increased need for
formation of extracellular proteins (see above) in carbonstarved cultures. It is worth mentioning that the unfoldedprotein stress response proved to be important in the
virulence of Aspergillus fumigatus (Richie et al., 2009). These
observations suggest that processes linked to the endoplasmic reticulum could be good targets in future antifungal
strategies. The upregulation of several genes involved in
protein synthesis demonstrated that the stress response
induced by carbon starvation was an active process
dependent on de novo protein synthesis. The limited
availability of energy sources, the elevated intracellular
ROS concentrations and the bulk secretion of various
proteins recorded under carbon shortage may explain why
very different sets of genes were necessary to maintain
protein synthesis in growing and in starving cultures.
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M. Szilágyi and others
Released intracellular compounds
Hydrolysed cell wall
compounds
prtA, pepI, pepJ, bglM, gtaA, chiB, nagA, treA ( )
AN9380, AN6464, AN8445, AN6438, AN2572 ( )
AN8498, AN1723, AN11897, AN6470, AN5046, AN11510 ( )
Secreted proteins
brlA, gprX ( )
Protection against other species
hxtA, mstA, AN5104,
AN8347, AN9168 ( )
Cell wall
Intracellular compounds
hacA ( )
Protein synthesis
and secretion
mns1B, pdiA ( )
erv46, erv29 ( )
Glucose (high concentration)
aflR, AN9129 ( )
Synthesis of secondary
metabolites
ivoA, ivoB, AN0230 ( )
Melanin synthesis
brlA ( )
mstE ( )
Cell wall biosynthesis
chsB, chsF, chiA, fksA, gelE ( )
gelA, eglC, AN10779, agsB ( )
brlA ( )
Conidiogenesis
rodA ( )
Utilizable metabolites
AN1428, AN1418 ( )
Protein, nucleic acid
and lipid degradation
AN8242, AN11062 ( )
Biosynthesis of saccharides Glycolysis,PPS
tpsA ( )
gpdA, pfkZ, gsdA ( )
Fatty acid biosynthesis
fasB ( )
citA ( )
Synthesis of amino acids
β-Oxidation
and nucleotides
Citrate cycle
Triglyceride, phospholipid
and steroid biosynthesis
AN4923 ( )
ER and Golgi
aoxA, AN10585 ( )
Degradation of amino acids
and nucleotides
Vacuole
Respiration
sodB ( ) ROS
hpdA ( )
atg22 ( )
Mitochondria
ROS
Living cells
Dead cells
Nucleus
Autophagosome
formation
atg5, atg7 ( )
tipA ( )
Repair
sldI, mreA ( )
niiA, niaD ( )
Ammonia (alkalification)
sodA, catA, catB ( )
AN5658, AN10444 ( )
AN1131 ( )
Nitrate
Fig. 4. Proposed changes in the metabolism of A. nidulans during carbon starvation based on DNA microarray-based and rRTPCR-based transcriptome analyses. Upregulated and downregulated pathways are indicated by lines and dashed lines,
respectively. The names or ID numbers of representative loci are presented. The expression of the underlined genes was not
validated by rRT-PCR.
Development of conidiophores
The induction of brlA, encoding a zinc-finger transcription
factor, is a typical event in carbon-starved cultures
(Skromne et al., 1995; Emri et al., 2008; Table 2). BrlA is
responsible for the initiation of conidiophore development
and conidiogenesis in surface cultures, and induces the
abaA and, via the AbaA transcription factor, the wetA
conidiation regulatory genes (Adams et al., 1998).
Transcription factors BrlA and AbaA with WetA orchestrate all genes responsible for conidiophore and conidia
formation (Adams et al., 1998). In surface cultures, abaA is
induced only 5 h after the induction of brlA (Adams et al.,
1998), which explains why the induction of abaA and wetA
was not observed in our cultures. However, in older
(24 h) cultures, conidiophore development was observed,
although these conidiophores were underdeveloped, similarly to those observed by Skromne et al. (1995), and
186
peeled off easily from the pellets (data not shown). In
contrast to abaA, the induction of several BrlA-dependent
genes was observed, including rodA, which is responsible for
the rodlet surface of conidia (Girardin et al., 1999), ivoA
and ivoB, which play roles in melanin production and
conidiophore pigmentation (Birse & Clutterbuck, 1990),
and several genes encoding extracellular hydrolases (chiB,
engA, prtA and pepJ) (Tables 2 and S2). Upregulation of
genes involved in asexual development (including brlA,
abaA, wetA and several hydrophobin genes) was also
observable during the carbon starvation of A. niger
(Nitsche et al., 2012). According to these data, BrlA is
a multifunctional transcription factor that regulates a
wide spectrum of physiological processes during carbon
starvation, and the initiation of conidiogenesis is only one,
and probably not the main, function of this regulatory
protein.
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Microbiology 159
Carbon starvation stress in A. nidulans
Barratt, R. W., Johnson, G. B. & Ogata, W. N. (1965). Wild-type and
Conclusions
From an ecological point of view, carbon-starved cultures
of fungi need to find appropriate answers to two questions:
(1) ‘how and where should they obtain utilizable
nutrients?’ and (2) ‘how should they use them to give an
adequate response to carbon starvation stress?’ According
to our results summarized in Fig. 4, induced macroautophagy, together with autolytic cell wall degradation and the utilization of organic compounds
liberated from dead cells, can maintain the viability of A.
nidulans cultures for a long time. The production of an
array of biologically active compounds, e.g. various
antifungal proteins, antifungal/antibacterial enzymes, secondary metabolites and mycotoxins, may also help fungi to
survive the lack of metabolizable carbon sources in their
habitats. In addition to the maintenance and protection of
cell viability, conidia formation and, at least in surface
cultures, supporting uninterrupted radial growth (Szilágyi
et al., 2012), can ensure the long-term existence of the
strain. The observed remarkable complexity of the CSSR
suggests the existence of a sophisticated regulatory
network, which delicately controls conidiophore and
conidia formation and influences melanin synthesis,
autolytic cell wall degradation and extracellular proteinase
production. According to our observations, the zinc-finger
transcription factor BrlA could be a key regulator of this
stress response network. It is worth emphasizing that
signalling not regulated transcriptionally and therefore not
studied in these experiments can also be essential in the
regulation of the CSSR. The bulk production of extracellular proteins (Table S2) and the induction of endoplasmic reticulum-specific genes including the HacA
transcription factor (Tables 2 and S2) suggest that the
endoplasmic reticulum plays a central role in the survival
of carbon-starved cultures. Therefore, endoplasmic reticulum functions may be promising future targets in
antifungal drug research. These data also draw our
attention to the importance of the subapical secretion of
extracellular proteins in carbon-starved cultures reported
more recently by Read (2011).
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ACKNOWLEDGEMENTS
This project was supported financially by the European Union and
the European Social Fund co-financed TAMOP 4.2.1/B-09/1/KONV2010-0007 and TAMOP-4.2.2/B-10/1-2010-0024 projects.
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