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Nitric oxide homeostasis is required for light-dependent regulation of conidiation in Aspergillus
Fungal Genetics and Biology 137 (2020) 103337
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Nitric oxide homeostasis is required for light-dependent regulation of conidiation in Aspergillus Ana T. Marcosa,1, María S. Ramosa,2, Thorsten Schinkob,3, Joseph Straussb, David Cánovasa,b,
T ⁎
a
Department of Genetics, Faculty of Biology, University of Seville, Spain Department of Applied Genetics and Cell Biology, BOKU University of Natural Resources and Life Science, University and Research Center – Campus Tulln, Tulln - Donau, Austria
b
ARTICLE INFO
ABSTRACT
Keywords: Aspergillus Nitric oxide Light Conidiation Flavohemoglobin Arginase
Nitric oxide (NO) can be biologically synthesized from nitrite or from arginine. Although NO is involved as a signal in many biological processes in bacteria, plants, and mammals, still little is known about the role of NO in fungi. Here we show that NO levels are regulated by light as an environmental signal in Aspergillus nidulans. The flavohaemoglobin-encoding fhbB gene involved in NO oxidation to nitrate, and the arginine-regulated arginase encoded by agaA, which controls the intracellular concentration of arginine, are both up-regulated by light. The phytochrome fphA is required for the light-dependent induction of fhbB and agaA, while the white-collar gene lreA acts as a repressor when arginine is present in the media. The intracellular arginine pools increase upon induction of both developmental programs (conidiation and sexual development), and the increase is higher under conditions promoting sexual development. The presence of low concentrations of arginine does not affect the light-dependent regulation of conidiation, but high concentrations of arginine overrun the light signal. Deletion of fhbB results in the partial loss of the light regulation of conidiation on arginine and on nitrate media, while deletion of fhbA only affects the light regulation of conidiation on nitrate media. Our working model considers a cross-talk between environmental cues and intracellular signals to regulate fungal reproduction.
1. Introduction Reproduction is an essential part of the biological cycle of fungi and their dispersion in the environment. As a consequence, during asexual development infectious propagules are formed and are disseminated in the air. Among fungi, Aspergillus nidulans has been used as a model organism to study reproduction for decades. A. nidulans displays two modes of reproduction: sexual and asexual (Adams et al., 1998). The asexual program (conidiation) is initiated when aerial hyphae are exposed to oxygen and light. The asexual reproductive structure of A. nidulans is the conidiophore, which forms green-pigmented conidiospores approximately 24 h after induction (Adams et al., 1998). Conidiation is controlled by a central regulatory pathway encompassing BrlA, AbaA and WetA (see reviews by (Adams et al., 1998; Etxebeste et al., 2010; Park and Yu, 2012) and references therein) that is well conserved in Eurotiomycetes (de Vries et al., 2017; Ojeda-López et al., 2018). The sexual program is promoted by signals such as hypoxia and
dark conditions. A. nidulans is homothallic, i.e. each strain harbors both mating type genes, MAT1 and MAT2 (Paoletti et al., 2007). During this program A. nidulans develops spherical fruiting bodies called cleistothecia, which contain meiospores called ascospores (Dyer and O'Gorman, 2012). Light is one of the signals that regulates the balance between asexual and sexual development (Bayram et al., 2010; Mooney and Yager, 1990; Rodriguez-Romero et al., 2010), and it has been shown that light increases the accumulation of brlA mRNA (Mooney and Yager, 1990; Ruger-Herreros et al., 2011). Several photoreceptors have been discovered in A. nidulans (Corrochano, 2019; Rodriguez-Romero et al., 2010): a phytochrome, FphA, for red light detection (Blumenstein et al., 2005; Corrochano, 2019; Purschwitz et al., 2008), a homolog of WC-1, LreA, and a homolog of WC-2, LreB, for blue light detection (Purschwitz et al., 2008), and a cryptochrome, CryA, for blue/UV light detection (Bayram et al., 2008a). The phytochrome forms a large complex with the A. nidulans homologs of WC-1 and WC-2, and the velvet A (VeA)
Corresponding author at: Department of Genetics, Faculty of Biology, University of Seville, Spain. E-mail address: [email protected] (D. Cánovas). 1 Present address: Instituto para el Estudio de la Reproducción Humana (Inebir), Avda de la Cruz Roja 1, 41009 Sevilla, Spain. 2 Present address: Faculty of Psychology, University of Sevilla, Spain. 3 Present address: Novartis Pharma Stein AG, Switzerland. ⁎
https://doi.org/10.1016/j.fgb.2020.103337 Received 8 January 2020; Accepted 15 January 2020 Available online 25 January 2020 1087-1845/ © 2020 Elsevier Inc. All rights reserved.
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protein, a repressor of light-regulated conidiation and an activator of sexual development (Purschwitz et al., 2008). VeA interacts with additional proteins to regulate sexual development and the synthesis of secondary metabolites (Bayram et al., 2008b; Sarikaya Bayram et al., 2010). In addition, the UV/blue light sensing cryptochrome is involved in the regulation of sexual development in A. nidulans by light (Bayram et al., 2008a). Nitric oxide (NO) is a signaling molecule in living organisms from bacteria to mammals. For example, NO is involved in the response to hypoxic conditions in bacteria (Seth et al., 2012, 2018), or regulates growth, development, photoperiod and flowering in plants (He et al., 2004; Kwon et al., 2012). In mammals it was shown to control a vast number of physiological processes such as smooth muscle tone, platelet aggregation and adhesion, cell growth, apoptosis, neurotransmission, vasoconstriction, the reproductive system and sexual behavior (Ignarro and Freeman, 2017). However, little is known about the role of NO in the biology of fungi. Biologically NO can be synthesized through different pathways. An oxidative pathway is mediated by the nitric oxide synthase and converts L-arginine and O2 into citrulline and NO (Gorren and Mayer, 2007). This pathway has been extensively studied in mammals, where several isoforms are expressed in different tissues (Ignarro and Freeman, 2017). The synthesis of NO from arginine has also been demonstrated in plants, although the responsible gene has not been unequivocally identified yet (Gupta et al., 2011; Prochazkova et al., 2014). The reductive pathway employs nitrite as a substrate, which is reduced to NO in two different pathways either using mitochondrial cytochrome c oxidase or cytosolic nitrate reductase. Both pathways have been demonstrated in plants (Prochazkova et al., 2014; Yamasaki and Sakihama, 2000) and also in fungi (Castello et al., 2006; Marcos et al., 2016). Since NO is a radical, organisms have developed mechanisms to metabolize NO for detoxification purposes (Canovas et al., 2016). One of these enzymes is the flavhohemoglobin, which converts NO to harmless nitrate that can be stored (in plant vacuoles), secreted (in animals) or assimilated (in bacteria, fungi and plants) (Gardner et al., 2000; Liu et al., 2000). A. nidulans contains two flavohaemoglobin genes, fhbA and fhbB. FhbA is located in the cytoplasm and induced when nitrate is present in the medium, while FhbB is mitochondrial and regulated during development (Marcos et al., 2016; Schinko et al., 2010; te Biesebeke et al., 2010; Zhou et al., 2011). Early work by Ninneman and Maier found indications that nitric oxide was involved in the regulation of photoconidiation in Neurospora crassa (Ninnemann and Maier, 1996). Here we present data showing that the genes known to be involved in the regulation of NO levels are regulated by light, and how these genes interact to affect the balance between conidiation and sexual development.
2.3. RNA isolation and real time RT-PCR Isolation of RNA and quantification of mRNA was performed as previously described (Ruger-Herreros et al., 2011). The primers employed for real time RT-PCR are detailed in Table S2. Real time RT-PCR experiments were performed in a LightCycler 480 II (Roche) by using the One Step SYBR® PrimeScript™ RT-PCR Kit (Takara Bio Inc.). The fluorescent signal obtained for each gene was normalized to the corresponding fluorescent signal obtained with benA to correct for sampling errors. Expression data are the average of at least three independent experiments. 2.4. Physiological experiments Strains were grown in liquid medium for 18 h at 37 °C and then transferred to solid media. At the indicated time intervals, plugs were cut from the plate and resuspended in Tween 0,1% buffer. Conidia were counted. Data shown are the average of at least three independent experiments. 2.5. Amino acid quantification Amino acids were extracted by adding boiling 75% ethanol to frozen mycelia and then breaking up the cells in a cell homogeneizer with glass beads. Extraction was repeated three times and the supernatants were pooled after spinning down the cell debris (Berger et al., 2008). Samples were derivatized with 6-aminoquinolyl-N-hydrosysuccinimidyl carbamate following the manufacturer’s directions (Waters AccQ tag kit). Amino acids were analyzed by HPLC following the Waters AccQ-Tag Instruction Manual (Millipore Corporation, Milford, Mass) in a chromatographer equipped with a pre-column WAT044380, Waters AccQ-Tag column 3.9 × 150 mm WAT052885, an automatic injector (Waters Alliance 2695), and a scanning fluorescence detector (Waters 474). Five dilutions (7.8 mM, 15.61 mM, 31.25 mM, 62.5 mM, 100 mM) of an equimolar solution of amino acids in HCl (Amino Acid Standard H, Pierce) were used as standards. Integration and processing of data were performed with the Waters Millennium 32 software. 2.6. NO quantification Nitric oxide was quantified in samples by using the NO indicator DAF-FM (Invitrogen) as previously reported (Marcos et al., 2016). Briefly, A. nidulans wild type strain FGSC4 was grown in solid media with the indicated nitrogen source in the dark or under constant light for 18 h as reported (Canovas et al., 2017) and 2.5 µM DAF-FM was added. Fluorescence was detected and quantified in a Synergy HT Multi-mode Microplate Reader (Biotek) with the appropriate filter sets. Data was analyzed with Gen5TM Data Analysis Software.
2. Materials and methods 2.1. Strains, media and culture conditions
3. Results
Strains used in this study are listed in Table S1. Strains were grown in complete or minimal media containing the appropriate supplements (Cove, 1966). 1% glucose was used as carbon source. Ammonium tartrate, sodium nitrate and L-arginine were used as sole nitrogen sources at 5 mM, 10 mM or 3 mM respectively, unless otherwise indicated.
3.1. NO levels are regulated by light Our previous work in A. nidulans showed that nitrate reductase was responsible for part of the NO synthesis (Marcos et al., 2016). Here A. nidulans FGSC A4 (wild type veA+) conidia were inoculated on the surface of solid media containing nitrate as sole N-source and incubated in the dark or under constant light for 18 h. Then, NO was quantified by addition of the NO-sensitive fluorescent dye DAF-FM, and fluorescence was recorded during 60 min. Under these conditions fluorescence was higher in mycelia grown under constant light than in the dark (Fig. 1A), indicating that the levels of NO were higher when A. nidulans grows on nitrate media in the light than in the dark, and it suggests that nitrate-dependent NO levels are either induced by light or repressed in the dark. In order to get further insight into the light-dependent regulation of NO production, the expression of genes known to be involved in NO
2.2. Light induction experiments The experiments involving induction by light were performed as previously described (Ruger-Herreros et al., 2011). Briefly, cultures were grown for 18 h at 37 °C in the dark and then, mycelial mats were exposed to light (11 W/m2) for the indicated times. After the exposure to light mycelia were collected in the dark and immediately frozen in liquid nitrogen. Samples were stored at −80 °C. Control samples were harvested in complete darkness. 2
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Fig. 1. Modulation of NO levels by light. (A) A. nidulans wild type FGSC A4 (veA+) was grown on minimal media containing nitrate as sole nitrogen sources for 18 h in the dark or under constant light, and then 2.5 µM DAF-FM was added to the cultures. The emitted fluorescence was detected by fluorometry. Fluorescence is shown as relative units (r.u.). (B-D) A. nidulans FGSC A4 was grown on minimal media containing nitrite or arginine as sole nitrogen source in the dark. The mycelial mat was illuminated for 5, 30 or 60 min and then immediately frozen in liquid nitrogen. Gene expression was quantified by real time RT-qPCR. Data shown are the average of at least three biological replicates and the standard error of the mean, and were normalized against the expression of the ßtubulin gene (benA) (rel. expression). The nitrate reductase niaD (B) and the flavohaemoglobin fhbA (C) expression was induced by nitrate, but not by light. The flavohaemoglobin fhbB (D) expression was induced by light.
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synthesis and metabolism was examined. niaD and the flavohaemoglobin fhbA were both highly expressed in the presence of nitrate, as expected, but light did not change this expression pattern (Fig. 1B-C). In contrast, the expression of fhbB, which was formerly considered to be constitutive (Schinko et al., 2010) but later found to be developmentally regulated (Marcos et al., 2016), appeared to be photoinducible (Fig. 1D). Already after 5 min of illumination, fhbB was detectable and steadily increased over 50 fold until the end of the light incubation period in both nitrogen sources.
(Ruger-Herreros et al., 2011). In this dataset we searched for light-induced genes with possible enzymatic functions in NO biosynthesis. Interestingly, this search revealed only one gene that meets such criteria: the arginase gene agaA was among the light-induced genes, and showed a 22-fold up-regulation under illumination. Arginase catalyzes the conversion of arginine into ornithine in the urea cycle in A. nidulans (Dzikowska et al., 1994), and also regulates arginine levels in plants (Flores et al., 2008). As arginine might serve as substrate for an unknown NO synthase, levels of this amino acid may also influence NO production as it happens in plants. The other genes in the urea cycle, the ornithine transaminase otaA and the zinc-finger transcription factor regulating arginine catabolism arcA were not found to be regulated by light in this transcriptomics dataset (Ruger-Herreros et al., 2011). When we tested the expression of agaA in our illumination conditions and on media containing arginine or nitrate as sole nitrogen sources, we found that agaA was induced in the presence of arginine, as previously reported (Borsuk et al., 2007; Empel et al., 2001), but this level was further increased by exposure to light (Fig. 2B). As already observed for fhbB, also the regulation of agaA by light appeared to be independent of the nitrogen source tested. agaA was also induced 40 fold after 60 min of illumination in the presence of nitrate. However, the accumulation of
3.2. Effect of light and arginine on NO production When arginine was employed as sole nitrogen source in the culture media, the effect of light on NO production was similar to the effect on nitrate media with an increase in fluorescence in the mycelia grown under constant light (Fig. 2A), suggesting that light regulates the production of NO in A. nidulans, regardless whether the nitrogen source was nitrate or arginine. In order to identify genes putatively involved in NO generation during light exposure, we examined transcriptomics data previously obtained from mycelia grown on the surface of complete media in the dark, and subjected to 30 min of illumination
Fig. 2. Light-dependent modulation of NO levels on arginine. (A) A. nidulans wild type FGSC A4 (veA+) was grown in minimal media containing arginine as sole nitrogen sources for 18 h in the dark or under constant light, and then 2.5 µM DAF-FM was added to the cultures. The emitted fluorescence was detected by fluorometry. Fluorescence is shown as relative units (r.u.). (B) Expression of the arginase, agaA, under the same conditions as in Fig. 1. agaA was induced by arginine and by light independently. Gene expression was quantified by real time RTqPCR. Data shown are the average of at least three biological replicates and the standard error of the mean, and were normalized against the expression of the ß-tubulin gene (benA) (rel. expression).
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the agaA transcript was higher when both arginine and light signals were applied at the same time, suggesting that independent and additive inducing mechanisms are regulating the expression of the arginase. Whether the arginase is involved in the regulation of the arginine levels available for NO synthesis that are concomitantly increased upon light exposure is still obscure.
media. Plates were further incubated under conditions promoting sexual development in the dark or conidiation under illumination at the indicated times, and the intracellular amino acids were quantified. The arginine pool increased 4 h after the fungal mycelia were transferred to solid media to induce either of the developmental programs (3–4 fold), and decreased continuously afterwards to reach the basal levels found during vegetative growth (Fig. 4 and Table S3). The pool of ornithine also increased slightly after induction of development (1.4 fold after 4 h), and steadily decreased during the remaining incubation period (10–48 h). The intracellular pool of these two amino acids were basically the same after placing pre-grown mycelia onto solid media in the dark promoting sexual development. These data suggest that arginine pools are responding to the change of the cellular environment from liquid to solid medium rather than to light conditions.
3.3. Role of the photoreceptors in light- and N-dependent regulation of agaA and fhbB expression The next question was whether the photoreceptors were responsible for the light regulation of agaA and fhbB. For that, single deletion mutants of lreA, lreB and fphA and the triple deletion mutant were used. A cryA mutant was also analyzed. In these experiments strains were grown on the surface of minimal media containing nitrate or arginine as sole nitrogen source, and under continuous light or dark conditions. Deletion of the blue photoreceptor lreA led to the derepression of agaA and fhbB in the dark, and increased expression under illumination (12–25 fold compared to the wild type), when cells were grown in the presence of arginine (Fig. 3). Interestingly, no differences between the wild type and the ΔlreA mutant were found on nitrate. Hence, LreA seems to be acting as a repressor of agaA and fhbB expression, but this effect is dependent on the presence of arginine. It could be reasoned that LreA may play a general role in arginine utilization. Indeed, while the wild type strain grew faster on arginine than on nitrate, the ΔlreA deletion strain grew at the same rate on nitrate and on arginine (Fig. S1). Combination of arginine and nitrate did not increase the growth rate above the growth rate on nitrate in the ΔlreA mutant, as occurred in the wild type strain. This suggests a general role of the lreA photoreceptor in the metabolism and physiology of arginine. LreB does not contain a photoreceptor LOV domain but it also forms part of a large photoreceptor complex in A. nidulans (Purschwitz et al., 2008). At difference to the deletion of lreA, the deletion of lreB did not affect the regulation of agaA and fhbB by light. In the phytochrome ΔfphA mutant strain both agaA and fhbB were not induced by light (Fig. 3), suggesting that FphA acts as a light-dependent positive regulator of agaA and fhbB. Differently from LreA, the effects observed in the ΔfphA strain were independent of the nitrogen source. When the three photoreceptors (lreA, lreB, fphA) were deleted, the light-induced overexpression observed in the ΔlreA mutant was lost indicating that FphA and LreA have opposite functions in the same pathway. Deletion of the cryptochrome cryA did not have any significant effects on the expression of either gene. Taken altogether, the photoreceptor complex is required for the correct light-dependent regulation of agaA and fhbB transcription, where FphA seems to function as an activator, and LreA is an arginine-specific repressor.
3.6. Combined effects of arginine or nitrate, and light to regulate conidiation A direct relationship between arginine auxotrophic mutants and reduced sporulation in fungi was postulated in A. nidulans (SerlupiCrescenzi et al., 1983) and in Coniothyrium minitans (Gong et al., 2007). In view of the above results we investigated the combined effects of arginine or nitrate, and light on conidiation. In both nitrogen sources there were a significant reduction in the conidiation levels in the dark, consistent with the veA+ genotype of the strains. Serlupi-Crescenzi et al. observed that increasing the arginine concentration in the media up to 3 mM increased conidiation of an argB12 auxotrophic mutant. These authors also suggested using a fluffy mutant that arginine could be required for the formation of aerial hyphae (Serlupi-Crescenzi et al., 1983). One possibility is that there is a fine tuning of arginine concentrations to regulate conidiation. Therefore, we tested the effect of higher concentrations of arginine on conidiation. 10 mM arginine inhibited conidiation and resulted in a nearly fluffy aconidial phenotype (Fig. S3). Notably formation of cleistothecia did not appear to be inhibited by 10 mM arginine. Deletion of fhbA resulted in lower conidiation levels in both nitrogen sources in the light (Fig. 5A-B). Deletion of the flavohemoglobin B (fhbB) provoked a partial loss of the light regulation of conidiation, showing an increase in conidiation levels in the dark both on nitrate and on arginine. The double mutant fhbA fhbB behaved similar to the single fhbA deletion mutant and showed lower conidiation levels in the light than the wild type. Two additional interesting patterns were observed. First, on both nitrogen sources there was a loss of light regulation of conidiation when fhbB was deleted either in the single or in the double mutant with increase levels of conidiation in the dark. Second, on nitrate an increase in conidiation levels was observed in the dark, when either one or both flavohemoglobins were deleted. Furthermore, deletion of both flavohemoglobins had an additive effect on the increase in the conidiation levels on nitrate in the dark, which it is probably a reflection of the combined effects of the deletion of the nitrate-regulated fhbA and the light-regulated fhbB. Taken altogether, flavohemoglobins are required for the correct light regulation of conidiation.
3.4. Role of the flavohemoglobins in the light regulation of the NO levels Since the flavohemoglobins are involved in the detoxification of NO (Gardner et al., 2000; Liu et al., 2000), the next question was to study whether they play any role in the regulation of the NO levels by light. For this, first we obtained the deletion mutants in a veA+ background and then, the NO levels were quantified in the dark and in the light (Fig. S2). A general loss of light regulation of the NO levels by light was observed in all the flavohemoglobin mutants, which seems to be slightly more pronounced in the case of the double mutant. Furthermore, it was observed that the NO levels were not increased in any of the mutants compared to the wild-type levels. Although it might seem surprising, these data are in agreement with previous data obtained in our lab (Marcos et al., 2016).
4. Discussion NO is one of the earliest signals accumulating immediately after the induction of development in fungi (Marcos et al., 2016). In this work, we report that NO production is regulated by light, an environmental signal involved in the regulation of different biological processes, such as development and reproduction, secondary metabolism and circadian rhythms (Bayram and Braus, 2012; Bayram et al., 2010; Corrochano, 2019; Corrochano and Avalos, 2010; Rodriguez-Romero et al., 2010). There are several pathways operating in nature for the synthesis of NO, involving either nitrite or arginine as a precursor (Canovas et al., 2016). A nitrate route has been identified in fungi (Marcos et al., 2016), but not the arginine one (Canovas et al., 2016; Pengkit et al., 2016; Samalova et al., 2013).
3.5. Developmental control of the intracellular pool of arginine The expression data of agaA suggest that the intracellular pool of arginine is regulated by light. For that, the wild-type strain was grown in liquid minimal media for 18 h and then transferred to solid minimal 4
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Fig. 3. Light-dependent inducion of agaA and fhbB is regulated by LreA and FphA, but not LreB or CryA. Mycelia of the wild type, ΔlreA, ΔlreB, ΔfphA, ΔlreAΔlreBΔfphA or ΔcryA strains were grown in minimal media containing nitrate or arginine as sole nitrogen source, and then exposed to white light for 60 min (L), or kept in the dark (D) prior to RNA extraction. The expression of the agaA and fhbB genes was quantified by real time RT-qPCR. Data were normalized against the expression of the ß-tubulin gene (rel. expression). Expression of agaA and fhbB was deregulated in the ΔlreA strain only in arginine but not in nitrate. Light induction of both genes was dependent on fphA.
Here we have found that the nitrate reductase gene (niaD) and the flavohemoglobin A (fhbA), which are involved in the nitrate route, are not regulated by light. On the other hand, we provide evidences for a role of light in arginine metabolism and an interaction between arginine and light to control the conidiation levels. Therefore, it could happen that arginine is playing a pivotal role in the regulation of the balance between asexual and sexual reproducion. An early work by Serlupi-Crescenzi and coworkers showed that the arginine-auxotrophic mutant in the arginine pathway argB12 was aconidial and sexual sterile (Serlupi-Crescenzi et al., 1983). In green algae, plants and mammals it was found that the concentration of arginine affects NO production (Flores et al., 2008; Foresi et al., 2010; Lavie et al., 2003). However, deletion of some of the genes in the arginine biosynthetic pathway did not result in any loss of NO synthesis in Magnaporthe (Zhang et al., 2015). So the question of whether arginine is a substrate for the
synthesis of NO still remains open. How are the arginine levels regulated? The intracellular concentrations of arginine can be regulated in several ways, one of which is through the activity of the arginase (Flores et al., 2008; Hallemeesch et al., 2002). Our data are in agreement with these previous reports (Borsuk et al., 2007), showing a clear induction of agaA on arginine. Frequently, genes in the same pathway are coordinately regulated (Canovas and Andrianopoulos, 2006; Lorenz and Fink, 2001). Remarkably, other genes in the urea cycle were not regulated by light or development in microarray experiments (Canovas et al., 2014; Ruger-Herreros et al., 2011). agaA was previously reported to be regulated specifically by arginine to the point that it contains an arginine aptamer in the 5′ UTR region, which stabilizes the mRNA (Borsuk et al., 1999; Borsuk et al., 2007). agaA is positively regulated by the Zn2Cys6 transcripition factor ArcA in response to arginine (Empel et al., 2001), and negatively regulated by the GATA factors AreA and AreB
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The regulation of the NO levels by light and development is further supported by the expression profile of genes and the pattern of arginine accumulation during both developmental programs. agaA and fhbB contain both light and developmental response elements in their promoter sequences. Both genes contain one AbaA-binding site (Andrianopoulos and Timberlake, 1994). AbaA is an ATTS transcriptional factor located downstream of BrlA in the central regulatory pathway of conidiation. It specifically regulates conidiation genes (Andrianopoulos and Timberlake, 1994). Genes regulated by light can be classified according to the temporal expression into Early Light Response Genes (ELRGs) and Late Light Response Genes (LLRGs). The expression of ELRGs peaks between 15 and 45 min after onset of light, whereas LLRGs peak later between 45 and 90 min (Chen et al., 2009). Early Light Response Elements (ELREs) and Late Light Response Elements (LLREs) have been identified in N. crassa in the promoters of the corresponding genes (Chen et al., 2009). 4 ELREs and 2 LLREs were found in the promoter of agaA and 1 ELRE in the promoter of fhbB, in addition to a number of other GATA and GATC sequences. The whitecollar homologe LreA is a GATA factor which seems to repress the expression of agaA and fhbB in the presence of arginine, but not nitrate. This arginine-specific and interesting phenomenon is further supported by the lack of growth rate increase in the ΔlreA mutant in arginine compared to nitrate. This suggests that LreA is playing some general role in arginine utilization. Protein binding to the promoter of agaA was shown by gel retardation assays using cell extracts but the proteins responsible have not been identified yet (Borsuk et al., 2007). LreA arises as a possible candidate for binding the agaA promoter. The regulation of NO synthesis by light seems to be a general mechanism conserved from fungi to humans: in the green alga Ostreococcus tauri and in the zygomycete P. blakesleeanus the production of NO increases in light (Foresi et al., 2010; Maier et al., 2001). In mammals, NO has a role in the synchronization of the circadian rhythms. The circadian deadenylase nocturnin stabilizes iNOS mRNA in mice (Niu et al., 2011). In Arabidopsis, NO regulates the photoperiod pathway (He et al., 2004). However, the molecular insights into how light regulation of NO levels is happening are still obscure. The blue-photoreceptor LreA seems to be pivotal in the arginine-dependent regulation by light of agaA and fhbB, thus promoting sexual development and repressing conidiation. But this does not fully explain the increment of NO levels after illumination of the mycelia. Indeed, increased expression of agaA would argue against an increase in the levels of NO. But the upregulation of fhbB under light conditions could be a response to the increment in NO levels. FhbB seems to be critical for the regulation by light of conidiation. In our experimental set up the mycelial mat is grown on the surface. fhbB is developmentally regulated: immediately after induction of conidiation, fhbB is repressed, and later, it is induced. Therefore, it can be presumed that light provides further induction, as it happens with some of the conidiation regulators (Mooney and Yager, 1990; Ruger-Herreros et al., 2011). What appears to be very interesting is the fact that deletion of fhbA has a strong effect on conidiation in the light, which corresponds to the condition of fhbB induction, but also to an increase in NO levels. Flavohemoglobins mediate the transformation of NO into nitrate, which in turns induces the expression of fhbA. On nitrate media the NiaD-mediated pathway for the synthesis of NO is active. One possibility is that in the absence of the nitrate-regulated fhbA or the developmentally regulated fhbB the NO levels increase moderately in the dark to promote conidiation. This is further supported by the fact that deletion of both flavohemoglobins produced an additive effect on conidiation in the dark. However, in the light when an alternative pathway for the synthesis of NO is operative, it could be presumed that the NO levels elevate too high and inhibit conidiation in the absence of fhbA. On arginine media, the nitrate pathway plays a minor role, and it is only active through the recycling of the NO converted into nitrate. Consequently, the effects of the deletion of fhbA in the dark are lost in arginine media. This hypothesis is partially
Fig. 4. The intracellular arginine pools are regulated during development. A. nidulans FGSC A4 was grown vegetatively in liquid minimal media containing ammonium as sole carbon source (VEG). Development was induced by transfer to solid media. The fungus was grown under conditions promoting either conidiation (unsealed plates in light) or sexual development (sealed plates in the dark) and samples were taken at the indicated time points. Amino acids were extracted from the cells and quantified by HPLC. Table S3 shows the concentration of all amino acids.
Fig. 5. Interaction between arginine and light during conidiation. A. nidulans wild type FGSC A4, the single mutants ΔfhbA and ΔfhbB, and the double mutant ΔfhbA ΔfhbB were grown in minimal media containing nitrate (A) or arginine (B) as a sole nitrogen source in the dark or under constant light. Conidia were counted after three days of growth. The plot shows the average and standard error of the mean of conidial number values in 4 independent experiments.
in response to nitrogen and carbon availability (Macios et al., 2012). Thus, it is rather interesting the specific regulation by light and development found for the arginine utilization gene agaA. We found four GATAA/C in the promoter of the negative regulator areB and one GATAC site in the promoter of positive regulator arcA, which opens the possibility that the LreA effects are amplified by regulation of the regulators of arginine metabolism.
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supported by the fact that the deletion of any of the flavohemoglobins produces a general loss of the regulation by light of the NO levels. However, the deletion of either one or both flavohemoglobins did not produce an increase in the levels in comparison to the wild type. We have observed the same in the past (Marcos et al., 2016). We hypothesize that this could be due to several non-excluding reasons: other NO detoxification mechanisms operating in A. nidulans; the experimental set up consists on a snapshot at a certain moment that may not be the critical point; or that the flavohemoglobins are acting at specific locations where the NO is provoking its effects. Moreover, light-dependent increase of NO must be mediated through a different mechanism, yet-to-know, which may occur through the regulation of a putative NO synthase as happens in algae. In summary, our data show that the environmental cue light is regulating the intracellular signaling molecule NO, and both coordinately control fungal development and reproduction. The question that still remains open is whether there is an alternative pathway for NO synthesis from arginine in fungi.
eukaryotes. Cell Metab. 3, 277–287. Chen, C.H., et al., 2009. Genome-wide analysis of light-inducible responses reveals hierarchical light signalling in Neurospora. EMBO J. 28, 1029–1042. Corrochano, L.M., 2019. Light in the fungal world: from photoreception to gene transcription and beyond. Annu. Rev. Genet. Corrochano, L.M., Avalos, J., 2010. Light sensing. In: Borkovich, K.A., Ebbole, D.J. (Eds.), Cellular and molecular biology of filamentous fungi. ASM Press, Washington, pp. 417–441. Cove, D.J., 1966. The induction and repression of nitrate reductase in the fungus Aspergillus nidulans. Biochim. Biophys Acta 113, 51–56. de Vries, R.P., et al., 2017. Comparative genomics reveals high biological diversity and specific adaptations in the industrially and medically important fungal genus Aspergillus. Genome Biol. 18, 28. Dyer, P.S., O'Gorman, C.M., 2012. Sexual development and cryptic sexuality in fungi: insights from Aspergillus species. FEMS Microbiol Rev. 36, 165–192. Dzikowska, A., et al., 1994. Purification of arginase from Aspergillus nidulans. Acta Biochim. Pol. 41, 467–471. Empel, J., et al., 2001. arcA, the regulatory gene for the arginine catabolic pathway in Aspergillus nidulans. Mol. Genet. Genom. 266, 591–597. Etxebeste, O., et al., 2010. Aspergillus nidulans asexual development: making the most of cellular modules. Trends Microbiol. 18, 569–576. Flores, T., et al., 2008. Arginase-negative mutants of Arabidopsis exhibit increased nitric oxide signaling in root development. Plant Physiol. 147, 1936–1946. Foresi, N., et al., 2010. Characterization of a nitric oxide synthase from the plant kingdom: NO generation from the green alga Ostreococcus tauri is light irradiance and growth phase dependent. Plant Cell. 22, 3816–3830. Gardner, P.R., et al., 2000. Nitric-oxide dioxygenase activity and function of flavohemoglobins. sensitivity to nitric oxide and carbon monoxide inhibition. J. Biol. Chem. 275, 31581–31587. Gong, X., et al., 2007. L-arginine is essential for conidiation in the filamentous fungus Coniothyrium minitans. Fung. Genet. Biol. 44, 1368–1379. Gorren, A.C., Mayer, B., 2007. Nitric-oxide synthase: a cytochrome P450 family foster child. Biochim. Biophys. Acta 1770, 432–445. Gupta, K.J., et al., 2011. On the origins of nitric oxide. Trends Plant Sci. 16, 160–168. Hallemeesch, M.M., et al., 2002. Reduced arginine availability and nitric oxide production. Clin. Nutr. 21, 273–279. He, Y., et al., 2004. Nitric oxide represses the Arabidopsis floral transition. Science 305, 1968–1971. Ignarro, L., Freeman, B.E., 2017. Nitric Oxide: Biology and Pathobiology. Academic Press. Kwon, E., et al., 2012. AtGSNOR1 function is required for multiple developmental programs in Arabidopsis. Planta 236, 887–900. Lavie, L., et al., 2003. Plasma levels of nitric oxide and L-arginine in sleep apnea patients: effects of nCPAP treatment. J. Mol. Neurosci. 21, 57–63. Liu, L., et al., 2000. Protection from nitrosative stress by yeast flavohemoglobin. Proc. Natl. Acad. Sci. USA 97, 4672–4676. Lorenz, M.C., Fink, G.R., 2001. The glyoxylate cycle is required for fungal virulence. Nature 412, 83–86. Macios, M., et al., 2012. The GATA factors AREA and AREB together with the co-repressor NMRA, negatively regulate arginine catabolism in Aspergillus nidulans in response to nitrogen and carbon source. Fung. Genet. Biol. 49, 189–198. Maier, J., et al., 2001. Role of nitric oxide synthase in the light-induced development of sporangiophores in Phycomyces blakesleeanus. Plant Physiol. 126, 1323–1330. Marcos, A.T., et al., 2016. Nitric oxide synthesis by nitrate reductase is regulated during development in Aspergillus. Mol. Microbiol. 99, 15–33. Mooney, J.L., Yager, L.N., 1990. Light is required for conidiation in Aspergillus nidulans. Genes Dev. 4, 1473–1482. Ninnemann, H., Maier, J., 1996. Indications for the occurrence of nitric oxide synthases in fungi and plants and the involvement in photoconidiation of Neurospora crassa. Photochem. Photobiol. 64, 393–398. Niu, S., et al., 2011. The circadian deadenylase Nocturnin is necessary for stabilization of the iNOS mRNA in mice. PLoS One 6, e26954. Ojeda-López, M., et al., 2018. Evolution of asexual and sexual reproduction in the aspergilli. Stud. Mycol. 91. Paoletti, M., et al., 2007. Mating type and the genetic basis of self-fertility in the model fungus Aspergillus nidulans. Curr. Biol. 17, 1384–1389. Park, H.S., Yu, J.H., 2012. Genetic control of asexual sporulation in filamentous fungi. Curr. Opin. Microbiol. 15, 669–677. Pengkit, A., et al., 2016. Identification and functional analysis of endogenous nitric oxide in a filamentous fungus. Sci. Rep. 6, 30037. Prochazkova, D., et al., 2014. Nitric oxide biosynthesis in plants - the short overview. Plant, Soil Environ. 60, 129–134. Purschwitz, J., et al., 2008. Functional and physical interaction of blue- and red-light sensors in Aspergillus nidulans. Curr. Biol. 18, 255–259. Rodriguez-Romero, J., et al., 2010. Fungi, hidden in soil or up in the air: light makes a difference. Annu. Rev. Microbiol. 64, 585–610. Ruger-Herreros, C., et al., 2011. Regulation of conidiation by light in Aspergillus nidulans. Genetics 188, 809–822. Samalova, M., et al., 2013. Nitric oxide generated by the rice blast fungus Magnaporthe oryzae drives plant infection. New Phytol. 197, 207–222. Sarikaya Bayram, O., et al., 2010. LaeA control of velvet family regulatory proteins for light-dependent development and fungal cell-type specificity. PLoS Genet. 6, e1001226. Schinko, T., et al., 2010. Transcriptome analysis of nitrate assimilation in Aspergillus nidulans reveals connections to nitric oxide metabolism. Mol. Microbiol. 78, 720–738. Serlupi-Crescenzi, O., et al., 1983. Developmental defects resulting from arginine auxotrophy in Aspergillus nidulans. J. Gen. Microbiol. 129, 3535–3544.
Acknowledgments We thank the Fungal Genetics Stock Center (FGSC), Reinhard Fischer and Gerhard Braus for strains. D.C. would also like to thank Luis Corrochano and Jose Marcos for helpful discussions. M.C. RugerHerreros is acknowledged for help with the photoinducible experiments, Thomas Dalik for initial amino acids quantifications, and Antonio Franco and Myrsini Charikleous for help with the conidial counts. We would also like to thank Modesto Carballo for help with the fluorometric assays (Servicio de Biología, Centro de Investigación Tecnología e Innovación, Universidad de Sevilla). Work in Vienna was supported by the Lower Austria Science Fund grant “Bioactive Microbial Metabolites” Nr. K3-G-2/26-2013 to JS and by grant M01693-B22 from the FWF to DC. Work in Sevilla was supported by grant RTI2018-098636-B-I00 from the MICINN to DC. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fgb.2020.103337. References Adams, T.H., et al., 1998. Asexual sporulation in Aspergillus nidulans. Microbiol. Mol. Biol. Rev. 62, 35–54. Andrianopoulos, A., Timberlake, W.E., 1994. The Aspergillus nidulans abaA gene encodes a transcriptional activator that acts as a genetic switch to control development. Mol. Cell Biol. 14, 2503–2515. Bayram, O., et al., 2008a. More than a repair enzyme: Aspergillus nidulans photolyase-like CryA is a regulator of sexual development. Mol. Biol. Cell. 19, 3254–3262. Bayram, O., Braus, G.H., 2012. Coordination of secondary metabolism and development in fungi: the velvet family of regulatory proteins. FEMS Microbiol. Rev. 36, 1–24. Bayram, O., et al., 2010. Spotlight on Aspergillus nidulans photosensory systems. Fung. Genet. Biol. 47, 900–908. Bayram, O., et al., 2008b. VelB/VeA/LaeA complex coordinates light signal with fungal development and secondary metabolism. Science 320, 1504–1506. Berger, H., et al., 2008. Dissecting individual steps of nitrogen transcription factor cooperation in the Aspergillus nidulans nitrate cluster. Mol. Microbiol. 69, 1385–1398. Blumenstein, A., et al., 2005. The Aspergillus nidulans phytochrome FphA represses sexual development in red light. Curr. Biol. 15, 1833–1838. Borsuk, P., et al., 1999. Structure of the arginase coding gene and its transcript in Aspergillus nidulans. Acta Biochim. Pol. 46, 391–403. Borsuk, P., et al., 2007. L-arginine influences the structure and function of arginase mRNA in Aspergillus nidulans. Biol. Chem. 388, 135–144. Canovas, D., Andrianopoulos, A., 2006. Developmental regulation of the glyoxylate cycle in the human pathogen Penicillium marneffei. Mol. Microbiol. 62, 1725–1738. Canovas, D., et al., 2014. The histone acetyltransferase GcnE (GCN5) plays a central role in the regulation of Aspergillus asexual development. Genetics 197, 1175–1189. Canovas, D., et al., 2016. Nitric oxide in fungi: is there NO light at the end of the tunnel? Curr. Genet. 62, 513–518. Canovas, D., et al., 2017. High-throughput format for the phenotyping of fungi on solid substrates. Sci. Rep. 7, 4289. Castello, P.R., et al., 2006. Mitochondrial cytochrome oxidase produces nitric oxide under hypoxic conditions: implications for oxygen sensing and hypoxic signaling in
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A.T. Marcos, et al. Seth, D., et al., 2012. Endogenous protein S-Nitrosylation in E. coli: regulation by OxyR. Science 336, 470–473. Seth, D., et al., 2018. A multiplex enzymatic machinery for cellular protein S-nitrosylation. Mol. Cell. 69 (451–464), e6. te Biesebeke, R., et al., 2010. Phylogeny of fungal hemoglobins and expression analysis of the Aspergillus oryzae flavohemoglobin gene fhbA during hyphal growth. Fung. Biol. 114, 135–143. Yamasaki, H., Sakihama, Y., 2000. Simultaneous production of nitric oxide and
peroxynitrite by plant nitrate reductase: in vitro evidence for the NR-dependent formation of active nitrogen species. FEBS Lett. 468, 89–92. Zhang, Y., et al., 2015. MoARG1, MoARG5,6 and MoARG7 involved in arginine biosynthesis are essential for growth, conidiogenesis, sexual reproduction, and pathogenicity in Magnaporthe oryzae. Microbiol. Res. 180, 11–22. Zhou, S., et al., 2011. Functional analysis and subcellular location of two flavohemoglobins from Aspergillus oryzae. Fung. Genet. Biol. 48, 200–207.
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Nitric oxide homeostasis is required for light-dependent regulation of conidiation in Aspergillus Ana T. Marcosa,1, María S. Ramosa,2, Thorsten Schinkob,3, Joseph Straussb, David Cánovasa,b,
T ⁎
a
Department of Genetics, Faculty of Biology, University of Seville, Spain Department of Applied Genetics and Cell Biology, BOKU University of Natural Resources and Life Science, University and Research Center – Campus Tulln, Tulln - Donau, Austria
b
ARTICLE INFO
ABSTRACT
Keywords: Aspergillus Nitric oxide Light Conidiation Flavohemoglobin Arginase
Nitric oxide (NO) can be biologically synthesized from nitrite or from arginine. Although NO is involved as a signal in many biological processes in bacteria, plants, and mammals, still little is known about the role of NO in fungi. Here we show that NO levels are regulated by light as an environmental signal in Aspergillus nidulans. The flavohaemoglobin-encoding fhbB gene involved in NO oxidation to nitrate, and the arginine-regulated arginase encoded by agaA, which controls the intracellular concentration of arginine, are both up-regulated by light. The phytochrome fphA is required for the light-dependent induction of fhbB and agaA, while the white-collar gene lreA acts as a repressor when arginine is present in the media. The intracellular arginine pools increase upon induction of both developmental programs (conidiation and sexual development), and the increase is higher under conditions promoting sexual development. The presence of low concentrations of arginine does not affect the light-dependent regulation of conidiation, but high concentrations of arginine overrun the light signal. Deletion of fhbB results in the partial loss of the light regulation of conidiation on arginine and on nitrate media, while deletion of fhbA only affects the light regulation of conidiation on nitrate media. Our working model considers a cross-talk between environmental cues and intracellular signals to regulate fungal reproduction.
1. Introduction Reproduction is an essential part of the biological cycle of fungi and their dispersion in the environment. As a consequence, during asexual development infectious propagules are formed and are disseminated in the air. Among fungi, Aspergillus nidulans has been used as a model organism to study reproduction for decades. A. nidulans displays two modes of reproduction: sexual and asexual (Adams et al., 1998). The asexual program (conidiation) is initiated when aerial hyphae are exposed to oxygen and light. The asexual reproductive structure of A. nidulans is the conidiophore, which forms green-pigmented conidiospores approximately 24 h after induction (Adams et al., 1998). Conidiation is controlled by a central regulatory pathway encompassing BrlA, AbaA and WetA (see reviews by (Adams et al., 1998; Etxebeste et al., 2010; Park and Yu, 2012) and references therein) that is well conserved in Eurotiomycetes (de Vries et al., 2017; Ojeda-López et al., 2018). The sexual program is promoted by signals such as hypoxia and
dark conditions. A. nidulans is homothallic, i.e. each strain harbors both mating type genes, MAT1 and MAT2 (Paoletti et al., 2007). During this program A. nidulans develops spherical fruiting bodies called cleistothecia, which contain meiospores called ascospores (Dyer and O'Gorman, 2012). Light is one of the signals that regulates the balance between asexual and sexual development (Bayram et al., 2010; Mooney and Yager, 1990; Rodriguez-Romero et al., 2010), and it has been shown that light increases the accumulation of brlA mRNA (Mooney and Yager, 1990; Ruger-Herreros et al., 2011). Several photoreceptors have been discovered in A. nidulans (Corrochano, 2019; Rodriguez-Romero et al., 2010): a phytochrome, FphA, for red light detection (Blumenstein et al., 2005; Corrochano, 2019; Purschwitz et al., 2008), a homolog of WC-1, LreA, and a homolog of WC-2, LreB, for blue light detection (Purschwitz et al., 2008), and a cryptochrome, CryA, for blue/UV light detection (Bayram et al., 2008a). The phytochrome forms a large complex with the A. nidulans homologs of WC-1 and WC-2, and the velvet A (VeA)
Corresponding author at: Department of Genetics, Faculty of Biology, University of Seville, Spain. E-mail address: [email protected] (D. Cánovas). 1 Present address: Instituto para el Estudio de la Reproducción Humana (Inebir), Avda de la Cruz Roja 1, 41009 Sevilla, Spain. 2 Present address: Faculty of Psychology, University of Sevilla, Spain. 3 Present address: Novartis Pharma Stein AG, Switzerland. ⁎
https://doi.org/10.1016/j.fgb.2020.103337 Received 8 January 2020; Accepted 15 January 2020 Available online 25 January 2020 1087-1845/ © 2020 Elsevier Inc. All rights reserved.
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protein, a repressor of light-regulated conidiation and an activator of sexual development (Purschwitz et al., 2008). VeA interacts with additional proteins to regulate sexual development and the synthesis of secondary metabolites (Bayram et al., 2008b; Sarikaya Bayram et al., 2010). In addition, the UV/blue light sensing cryptochrome is involved in the regulation of sexual development in A. nidulans by light (Bayram et al., 2008a). Nitric oxide (NO) is a signaling molecule in living organisms from bacteria to mammals. For example, NO is involved in the response to hypoxic conditions in bacteria (Seth et al., 2012, 2018), or regulates growth, development, photoperiod and flowering in plants (He et al., 2004; Kwon et al., 2012). In mammals it was shown to control a vast number of physiological processes such as smooth muscle tone, platelet aggregation and adhesion, cell growth, apoptosis, neurotransmission, vasoconstriction, the reproductive system and sexual behavior (Ignarro and Freeman, 2017). However, little is known about the role of NO in the biology of fungi. Biologically NO can be synthesized through different pathways. An oxidative pathway is mediated by the nitric oxide synthase and converts L-arginine and O2 into citrulline and NO (Gorren and Mayer, 2007). This pathway has been extensively studied in mammals, where several isoforms are expressed in different tissues (Ignarro and Freeman, 2017). The synthesis of NO from arginine has also been demonstrated in plants, although the responsible gene has not been unequivocally identified yet (Gupta et al., 2011; Prochazkova et al., 2014). The reductive pathway employs nitrite as a substrate, which is reduced to NO in two different pathways either using mitochondrial cytochrome c oxidase or cytosolic nitrate reductase. Both pathways have been demonstrated in plants (Prochazkova et al., 2014; Yamasaki and Sakihama, 2000) and also in fungi (Castello et al., 2006; Marcos et al., 2016). Since NO is a radical, organisms have developed mechanisms to metabolize NO for detoxification purposes (Canovas et al., 2016). One of these enzymes is the flavhohemoglobin, which converts NO to harmless nitrate that can be stored (in plant vacuoles), secreted (in animals) or assimilated (in bacteria, fungi and plants) (Gardner et al., 2000; Liu et al., 2000). A. nidulans contains two flavohaemoglobin genes, fhbA and fhbB. FhbA is located in the cytoplasm and induced when nitrate is present in the medium, while FhbB is mitochondrial and regulated during development (Marcos et al., 2016; Schinko et al., 2010; te Biesebeke et al., 2010; Zhou et al., 2011). Early work by Ninneman and Maier found indications that nitric oxide was involved in the regulation of photoconidiation in Neurospora crassa (Ninnemann and Maier, 1996). Here we present data showing that the genes known to be involved in the regulation of NO levels are regulated by light, and how these genes interact to affect the balance between conidiation and sexual development.
2.3. RNA isolation and real time RT-PCR Isolation of RNA and quantification of mRNA was performed as previously described (Ruger-Herreros et al., 2011). The primers employed for real time RT-PCR are detailed in Table S2. Real time RT-PCR experiments were performed in a LightCycler 480 II (Roche) by using the One Step SYBR® PrimeScript™ RT-PCR Kit (Takara Bio Inc.). The fluorescent signal obtained for each gene was normalized to the corresponding fluorescent signal obtained with benA to correct for sampling errors. Expression data are the average of at least three independent experiments. 2.4. Physiological experiments Strains were grown in liquid medium for 18 h at 37 °C and then transferred to solid media. At the indicated time intervals, plugs were cut from the plate and resuspended in Tween 0,1% buffer. Conidia were counted. Data shown are the average of at least three independent experiments. 2.5. Amino acid quantification Amino acids were extracted by adding boiling 75% ethanol to frozen mycelia and then breaking up the cells in a cell homogeneizer with glass beads. Extraction was repeated three times and the supernatants were pooled after spinning down the cell debris (Berger et al., 2008). Samples were derivatized with 6-aminoquinolyl-N-hydrosysuccinimidyl carbamate following the manufacturer’s directions (Waters AccQ tag kit). Amino acids were analyzed by HPLC following the Waters AccQ-Tag Instruction Manual (Millipore Corporation, Milford, Mass) in a chromatographer equipped with a pre-column WAT044380, Waters AccQ-Tag column 3.9 × 150 mm WAT052885, an automatic injector (Waters Alliance 2695), and a scanning fluorescence detector (Waters 474). Five dilutions (7.8 mM, 15.61 mM, 31.25 mM, 62.5 mM, 100 mM) of an equimolar solution of amino acids in HCl (Amino Acid Standard H, Pierce) were used as standards. Integration and processing of data were performed with the Waters Millennium 32 software. 2.6. NO quantification Nitric oxide was quantified in samples by using the NO indicator DAF-FM (Invitrogen) as previously reported (Marcos et al., 2016). Briefly, A. nidulans wild type strain FGSC4 was grown in solid media with the indicated nitrogen source in the dark or under constant light for 18 h as reported (Canovas et al., 2017) and 2.5 µM DAF-FM was added. Fluorescence was detected and quantified in a Synergy HT Multi-mode Microplate Reader (Biotek) with the appropriate filter sets. Data was analyzed with Gen5TM Data Analysis Software.
2. Materials and methods 2.1. Strains, media and culture conditions
3. Results
Strains used in this study are listed in Table S1. Strains were grown in complete or minimal media containing the appropriate supplements (Cove, 1966). 1% glucose was used as carbon source. Ammonium tartrate, sodium nitrate and L-arginine were used as sole nitrogen sources at 5 mM, 10 mM or 3 mM respectively, unless otherwise indicated.
3.1. NO levels are regulated by light Our previous work in A. nidulans showed that nitrate reductase was responsible for part of the NO synthesis (Marcos et al., 2016). Here A. nidulans FGSC A4 (wild type veA+) conidia were inoculated on the surface of solid media containing nitrate as sole N-source and incubated in the dark or under constant light for 18 h. Then, NO was quantified by addition of the NO-sensitive fluorescent dye DAF-FM, and fluorescence was recorded during 60 min. Under these conditions fluorescence was higher in mycelia grown under constant light than in the dark (Fig. 1A), indicating that the levels of NO were higher when A. nidulans grows on nitrate media in the light than in the dark, and it suggests that nitrate-dependent NO levels are either induced by light or repressed in the dark. In order to get further insight into the light-dependent regulation of NO production, the expression of genes known to be involved in NO
2.2. Light induction experiments The experiments involving induction by light were performed as previously described (Ruger-Herreros et al., 2011). Briefly, cultures were grown for 18 h at 37 °C in the dark and then, mycelial mats were exposed to light (11 W/m2) for the indicated times. After the exposure to light mycelia were collected in the dark and immediately frozen in liquid nitrogen. Samples were stored at −80 °C. Control samples were harvested in complete darkness. 2
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Fig. 1. Modulation of NO levels by light. (A) A. nidulans wild type FGSC A4 (veA+) was grown on minimal media containing nitrate as sole nitrogen sources for 18 h in the dark or under constant light, and then 2.5 µM DAF-FM was added to the cultures. The emitted fluorescence was detected by fluorometry. Fluorescence is shown as relative units (r.u.). (B-D) A. nidulans FGSC A4 was grown on minimal media containing nitrite or arginine as sole nitrogen source in the dark. The mycelial mat was illuminated for 5, 30 or 60 min and then immediately frozen in liquid nitrogen. Gene expression was quantified by real time RT-qPCR. Data shown are the average of at least three biological replicates and the standard error of the mean, and were normalized against the expression of the ßtubulin gene (benA) (rel. expression). The nitrate reductase niaD (B) and the flavohaemoglobin fhbA (C) expression was induced by nitrate, but not by light. The flavohaemoglobin fhbB (D) expression was induced by light.
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synthesis and metabolism was examined. niaD and the flavohaemoglobin fhbA were both highly expressed in the presence of nitrate, as expected, but light did not change this expression pattern (Fig. 1B-C). In contrast, the expression of fhbB, which was formerly considered to be constitutive (Schinko et al., 2010) but later found to be developmentally regulated (Marcos et al., 2016), appeared to be photoinducible (Fig. 1D). Already after 5 min of illumination, fhbB was detectable and steadily increased over 50 fold until the end of the light incubation period in both nitrogen sources.
(Ruger-Herreros et al., 2011). In this dataset we searched for light-induced genes with possible enzymatic functions in NO biosynthesis. Interestingly, this search revealed only one gene that meets such criteria: the arginase gene agaA was among the light-induced genes, and showed a 22-fold up-regulation under illumination. Arginase catalyzes the conversion of arginine into ornithine in the urea cycle in A. nidulans (Dzikowska et al., 1994), and also regulates arginine levels in plants (Flores et al., 2008). As arginine might serve as substrate for an unknown NO synthase, levels of this amino acid may also influence NO production as it happens in plants. The other genes in the urea cycle, the ornithine transaminase otaA and the zinc-finger transcription factor regulating arginine catabolism arcA were not found to be regulated by light in this transcriptomics dataset (Ruger-Herreros et al., 2011). When we tested the expression of agaA in our illumination conditions and on media containing arginine or nitrate as sole nitrogen sources, we found that agaA was induced in the presence of arginine, as previously reported (Borsuk et al., 2007; Empel et al., 2001), but this level was further increased by exposure to light (Fig. 2B). As already observed for fhbB, also the regulation of agaA by light appeared to be independent of the nitrogen source tested. agaA was also induced 40 fold after 60 min of illumination in the presence of nitrate. However, the accumulation of
3.2. Effect of light and arginine on NO production When arginine was employed as sole nitrogen source in the culture media, the effect of light on NO production was similar to the effect on nitrate media with an increase in fluorescence in the mycelia grown under constant light (Fig. 2A), suggesting that light regulates the production of NO in A. nidulans, regardless whether the nitrogen source was nitrate or arginine. In order to identify genes putatively involved in NO generation during light exposure, we examined transcriptomics data previously obtained from mycelia grown on the surface of complete media in the dark, and subjected to 30 min of illumination
Fig. 2. Light-dependent modulation of NO levels on arginine. (A) A. nidulans wild type FGSC A4 (veA+) was grown in minimal media containing arginine as sole nitrogen sources for 18 h in the dark or under constant light, and then 2.5 µM DAF-FM was added to the cultures. The emitted fluorescence was detected by fluorometry. Fluorescence is shown as relative units (r.u.). (B) Expression of the arginase, agaA, under the same conditions as in Fig. 1. agaA was induced by arginine and by light independently. Gene expression was quantified by real time RTqPCR. Data shown are the average of at least three biological replicates and the standard error of the mean, and were normalized against the expression of the ß-tubulin gene (benA) (rel. expression).
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the agaA transcript was higher when both arginine and light signals were applied at the same time, suggesting that independent and additive inducing mechanisms are regulating the expression of the arginase. Whether the arginase is involved in the regulation of the arginine levels available for NO synthesis that are concomitantly increased upon light exposure is still obscure.
media. Plates were further incubated under conditions promoting sexual development in the dark or conidiation under illumination at the indicated times, and the intracellular amino acids were quantified. The arginine pool increased 4 h after the fungal mycelia were transferred to solid media to induce either of the developmental programs (3–4 fold), and decreased continuously afterwards to reach the basal levels found during vegetative growth (Fig. 4 and Table S3). The pool of ornithine also increased slightly after induction of development (1.4 fold after 4 h), and steadily decreased during the remaining incubation period (10–48 h). The intracellular pool of these two amino acids were basically the same after placing pre-grown mycelia onto solid media in the dark promoting sexual development. These data suggest that arginine pools are responding to the change of the cellular environment from liquid to solid medium rather than to light conditions.
3.3. Role of the photoreceptors in light- and N-dependent regulation of agaA and fhbB expression The next question was whether the photoreceptors were responsible for the light regulation of agaA and fhbB. For that, single deletion mutants of lreA, lreB and fphA and the triple deletion mutant were used. A cryA mutant was also analyzed. In these experiments strains were grown on the surface of minimal media containing nitrate or arginine as sole nitrogen source, and under continuous light or dark conditions. Deletion of the blue photoreceptor lreA led to the derepression of agaA and fhbB in the dark, and increased expression under illumination (12–25 fold compared to the wild type), when cells were grown in the presence of arginine (Fig. 3). Interestingly, no differences between the wild type and the ΔlreA mutant were found on nitrate. Hence, LreA seems to be acting as a repressor of agaA and fhbB expression, but this effect is dependent on the presence of arginine. It could be reasoned that LreA may play a general role in arginine utilization. Indeed, while the wild type strain grew faster on arginine than on nitrate, the ΔlreA deletion strain grew at the same rate on nitrate and on arginine (Fig. S1). Combination of arginine and nitrate did not increase the growth rate above the growth rate on nitrate in the ΔlreA mutant, as occurred in the wild type strain. This suggests a general role of the lreA photoreceptor in the metabolism and physiology of arginine. LreB does not contain a photoreceptor LOV domain but it also forms part of a large photoreceptor complex in A. nidulans (Purschwitz et al., 2008). At difference to the deletion of lreA, the deletion of lreB did not affect the regulation of agaA and fhbB by light. In the phytochrome ΔfphA mutant strain both agaA and fhbB were not induced by light (Fig. 3), suggesting that FphA acts as a light-dependent positive regulator of agaA and fhbB. Differently from LreA, the effects observed in the ΔfphA strain were independent of the nitrogen source. When the three photoreceptors (lreA, lreB, fphA) were deleted, the light-induced overexpression observed in the ΔlreA mutant was lost indicating that FphA and LreA have opposite functions in the same pathway. Deletion of the cryptochrome cryA did not have any significant effects on the expression of either gene. Taken altogether, the photoreceptor complex is required for the correct light-dependent regulation of agaA and fhbB transcription, where FphA seems to function as an activator, and LreA is an arginine-specific repressor.
3.6. Combined effects of arginine or nitrate, and light to regulate conidiation A direct relationship between arginine auxotrophic mutants and reduced sporulation in fungi was postulated in A. nidulans (SerlupiCrescenzi et al., 1983) and in Coniothyrium minitans (Gong et al., 2007). In view of the above results we investigated the combined effects of arginine or nitrate, and light on conidiation. In both nitrogen sources there were a significant reduction in the conidiation levels in the dark, consistent with the veA+ genotype of the strains. Serlupi-Crescenzi et al. observed that increasing the arginine concentration in the media up to 3 mM increased conidiation of an argB12 auxotrophic mutant. These authors also suggested using a fluffy mutant that arginine could be required for the formation of aerial hyphae (Serlupi-Crescenzi et al., 1983). One possibility is that there is a fine tuning of arginine concentrations to regulate conidiation. Therefore, we tested the effect of higher concentrations of arginine on conidiation. 10 mM arginine inhibited conidiation and resulted in a nearly fluffy aconidial phenotype (Fig. S3). Notably formation of cleistothecia did not appear to be inhibited by 10 mM arginine. Deletion of fhbA resulted in lower conidiation levels in both nitrogen sources in the light (Fig. 5A-B). Deletion of the flavohemoglobin B (fhbB) provoked a partial loss of the light regulation of conidiation, showing an increase in conidiation levels in the dark both on nitrate and on arginine. The double mutant fhbA fhbB behaved similar to the single fhbA deletion mutant and showed lower conidiation levels in the light than the wild type. Two additional interesting patterns were observed. First, on both nitrogen sources there was a loss of light regulation of conidiation when fhbB was deleted either in the single or in the double mutant with increase levels of conidiation in the dark. Second, on nitrate an increase in conidiation levels was observed in the dark, when either one or both flavohemoglobins were deleted. Furthermore, deletion of both flavohemoglobins had an additive effect on the increase in the conidiation levels on nitrate in the dark, which it is probably a reflection of the combined effects of the deletion of the nitrate-regulated fhbA and the light-regulated fhbB. Taken altogether, flavohemoglobins are required for the correct light regulation of conidiation.
3.4. Role of the flavohemoglobins in the light regulation of the NO levels Since the flavohemoglobins are involved in the detoxification of NO (Gardner et al., 2000; Liu et al., 2000), the next question was to study whether they play any role in the regulation of the NO levels by light. For this, first we obtained the deletion mutants in a veA+ background and then, the NO levels were quantified in the dark and in the light (Fig. S2). A general loss of light regulation of the NO levels by light was observed in all the flavohemoglobin mutants, which seems to be slightly more pronounced in the case of the double mutant. Furthermore, it was observed that the NO levels were not increased in any of the mutants compared to the wild-type levels. Although it might seem surprising, these data are in agreement with previous data obtained in our lab (Marcos et al., 2016).
4. Discussion NO is one of the earliest signals accumulating immediately after the induction of development in fungi (Marcos et al., 2016). In this work, we report that NO production is regulated by light, an environmental signal involved in the regulation of different biological processes, such as development and reproduction, secondary metabolism and circadian rhythms (Bayram and Braus, 2012; Bayram et al., 2010; Corrochano, 2019; Corrochano and Avalos, 2010; Rodriguez-Romero et al., 2010). There are several pathways operating in nature for the synthesis of NO, involving either nitrite or arginine as a precursor (Canovas et al., 2016). A nitrate route has been identified in fungi (Marcos et al., 2016), but not the arginine one (Canovas et al., 2016; Pengkit et al., 2016; Samalova et al., 2013).
3.5. Developmental control of the intracellular pool of arginine The expression data of agaA suggest that the intracellular pool of arginine is regulated by light. For that, the wild-type strain was grown in liquid minimal media for 18 h and then transferred to solid minimal 4
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Fig. 3. Light-dependent inducion of agaA and fhbB is regulated by LreA and FphA, but not LreB or CryA. Mycelia of the wild type, ΔlreA, ΔlreB, ΔfphA, ΔlreAΔlreBΔfphA or ΔcryA strains were grown in minimal media containing nitrate or arginine as sole nitrogen source, and then exposed to white light for 60 min (L), or kept in the dark (D) prior to RNA extraction. The expression of the agaA and fhbB genes was quantified by real time RT-qPCR. Data were normalized against the expression of the ß-tubulin gene (rel. expression). Expression of agaA and fhbB was deregulated in the ΔlreA strain only in arginine but not in nitrate. Light induction of both genes was dependent on fphA.
Here we have found that the nitrate reductase gene (niaD) and the flavohemoglobin A (fhbA), which are involved in the nitrate route, are not regulated by light. On the other hand, we provide evidences for a role of light in arginine metabolism and an interaction between arginine and light to control the conidiation levels. Therefore, it could happen that arginine is playing a pivotal role in the regulation of the balance between asexual and sexual reproducion. An early work by Serlupi-Crescenzi and coworkers showed that the arginine-auxotrophic mutant in the arginine pathway argB12 was aconidial and sexual sterile (Serlupi-Crescenzi et al., 1983). In green algae, plants and mammals it was found that the concentration of arginine affects NO production (Flores et al., 2008; Foresi et al., 2010; Lavie et al., 2003). However, deletion of some of the genes in the arginine biosynthetic pathway did not result in any loss of NO synthesis in Magnaporthe (Zhang et al., 2015). So the question of whether arginine is a substrate for the
synthesis of NO still remains open. How are the arginine levels regulated? The intracellular concentrations of arginine can be regulated in several ways, one of which is through the activity of the arginase (Flores et al., 2008; Hallemeesch et al., 2002). Our data are in agreement with these previous reports (Borsuk et al., 2007), showing a clear induction of agaA on arginine. Frequently, genes in the same pathway are coordinately regulated (Canovas and Andrianopoulos, 2006; Lorenz and Fink, 2001). Remarkably, other genes in the urea cycle were not regulated by light or development in microarray experiments (Canovas et al., 2014; Ruger-Herreros et al., 2011). agaA was previously reported to be regulated specifically by arginine to the point that it contains an arginine aptamer in the 5′ UTR region, which stabilizes the mRNA (Borsuk et al., 1999; Borsuk et al., 2007). agaA is positively regulated by the Zn2Cys6 transcripition factor ArcA in response to arginine (Empel et al., 2001), and negatively regulated by the GATA factors AreA and AreB
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The regulation of the NO levels by light and development is further supported by the expression profile of genes and the pattern of arginine accumulation during both developmental programs. agaA and fhbB contain both light and developmental response elements in their promoter sequences. Both genes contain one AbaA-binding site (Andrianopoulos and Timberlake, 1994). AbaA is an ATTS transcriptional factor located downstream of BrlA in the central regulatory pathway of conidiation. It specifically regulates conidiation genes (Andrianopoulos and Timberlake, 1994). Genes regulated by light can be classified according to the temporal expression into Early Light Response Genes (ELRGs) and Late Light Response Genes (LLRGs). The expression of ELRGs peaks between 15 and 45 min after onset of light, whereas LLRGs peak later between 45 and 90 min (Chen et al., 2009). Early Light Response Elements (ELREs) and Late Light Response Elements (LLREs) have been identified in N. crassa in the promoters of the corresponding genes (Chen et al., 2009). 4 ELREs and 2 LLREs were found in the promoter of agaA and 1 ELRE in the promoter of fhbB, in addition to a number of other GATA and GATC sequences. The whitecollar homologe LreA is a GATA factor which seems to repress the expression of agaA and fhbB in the presence of arginine, but not nitrate. This arginine-specific and interesting phenomenon is further supported by the lack of growth rate increase in the ΔlreA mutant in arginine compared to nitrate. This suggests that LreA is playing some general role in arginine utilization. Protein binding to the promoter of agaA was shown by gel retardation assays using cell extracts but the proteins responsible have not been identified yet (Borsuk et al., 2007). LreA arises as a possible candidate for binding the agaA promoter. The regulation of NO synthesis by light seems to be a general mechanism conserved from fungi to humans: in the green alga Ostreococcus tauri and in the zygomycete P. blakesleeanus the production of NO increases in light (Foresi et al., 2010; Maier et al., 2001). In mammals, NO has a role in the synchronization of the circadian rhythms. The circadian deadenylase nocturnin stabilizes iNOS mRNA in mice (Niu et al., 2011). In Arabidopsis, NO regulates the photoperiod pathway (He et al., 2004). However, the molecular insights into how light regulation of NO levels is happening are still obscure. The blue-photoreceptor LreA seems to be pivotal in the arginine-dependent regulation by light of agaA and fhbB, thus promoting sexual development and repressing conidiation. But this does not fully explain the increment of NO levels after illumination of the mycelia. Indeed, increased expression of agaA would argue against an increase in the levels of NO. But the upregulation of fhbB under light conditions could be a response to the increment in NO levels. FhbB seems to be critical for the regulation by light of conidiation. In our experimental set up the mycelial mat is grown on the surface. fhbB is developmentally regulated: immediately after induction of conidiation, fhbB is repressed, and later, it is induced. Therefore, it can be presumed that light provides further induction, as it happens with some of the conidiation regulators (Mooney and Yager, 1990; Ruger-Herreros et al., 2011). What appears to be very interesting is the fact that deletion of fhbA has a strong effect on conidiation in the light, which corresponds to the condition of fhbB induction, but also to an increase in NO levels. Flavohemoglobins mediate the transformation of NO into nitrate, which in turns induces the expression of fhbA. On nitrate media the NiaD-mediated pathway for the synthesis of NO is active. One possibility is that in the absence of the nitrate-regulated fhbA or the developmentally regulated fhbB the NO levels increase moderately in the dark to promote conidiation. This is further supported by the fact that deletion of both flavohemoglobins produced an additive effect on conidiation in the dark. However, in the light when an alternative pathway for the synthesis of NO is operative, it could be presumed that the NO levels elevate too high and inhibit conidiation in the absence of fhbA. On arginine media, the nitrate pathway plays a minor role, and it is only active through the recycling of the NO converted into nitrate. Consequently, the effects of the deletion of fhbA in the dark are lost in arginine media. This hypothesis is partially
Fig. 4. The intracellular arginine pools are regulated during development. A. nidulans FGSC A4 was grown vegetatively in liquid minimal media containing ammonium as sole carbon source (VEG). Development was induced by transfer to solid media. The fungus was grown under conditions promoting either conidiation (unsealed plates in light) or sexual development (sealed plates in the dark) and samples were taken at the indicated time points. Amino acids were extracted from the cells and quantified by HPLC. Table S3 shows the concentration of all amino acids.
Fig. 5. Interaction between arginine and light during conidiation. A. nidulans wild type FGSC A4, the single mutants ΔfhbA and ΔfhbB, and the double mutant ΔfhbA ΔfhbB were grown in minimal media containing nitrate (A) or arginine (B) as a sole nitrogen source in the dark or under constant light. Conidia were counted after three days of growth. The plot shows the average and standard error of the mean of conidial number values in 4 independent experiments.
in response to nitrogen and carbon availability (Macios et al., 2012). Thus, it is rather interesting the specific regulation by light and development found for the arginine utilization gene agaA. We found four GATAA/C in the promoter of the negative regulator areB and one GATAC site in the promoter of positive regulator arcA, which opens the possibility that the LreA effects are amplified by regulation of the regulators of arginine metabolism.
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supported by the fact that the deletion of any of the flavohemoglobins produces a general loss of the regulation by light of the NO levels. However, the deletion of either one or both flavohemoglobins did not produce an increase in the levels in comparison to the wild type. We have observed the same in the past (Marcos et al., 2016). We hypothesize that this could be due to several non-excluding reasons: other NO detoxification mechanisms operating in A. nidulans; the experimental set up consists on a snapshot at a certain moment that may not be the critical point; or that the flavohemoglobins are acting at specific locations where the NO is provoking its effects. Moreover, light-dependent increase of NO must be mediated through a different mechanism, yet-to-know, which may occur through the regulation of a putative NO synthase as happens in algae. In summary, our data show that the environmental cue light is regulating the intracellular signaling molecule NO, and both coordinately control fungal development and reproduction. The question that still remains open is whether there is an alternative pathway for NO synthesis from arginine in fungi.
eukaryotes. Cell Metab. 3, 277–287. Chen, C.H., et al., 2009. Genome-wide analysis of light-inducible responses reveals hierarchical light signalling in Neurospora. EMBO J. 28, 1029–1042. Corrochano, L.M., 2019. Light in the fungal world: from photoreception to gene transcription and beyond. Annu. Rev. Genet. Corrochano, L.M., Avalos, J., 2010. Light sensing. In: Borkovich, K.A., Ebbole, D.J. (Eds.), Cellular and molecular biology of filamentous fungi. ASM Press, Washington, pp. 417–441. Cove, D.J., 1966. The induction and repression of nitrate reductase in the fungus Aspergillus nidulans. Biochim. Biophys Acta 113, 51–56. de Vries, R.P., et al., 2017. Comparative genomics reveals high biological diversity and specific adaptations in the industrially and medically important fungal genus Aspergillus. Genome Biol. 18, 28. Dyer, P.S., O'Gorman, C.M., 2012. Sexual development and cryptic sexuality in fungi: insights from Aspergillus species. FEMS Microbiol Rev. 36, 165–192. Dzikowska, A., et al., 1994. Purification of arginase from Aspergillus nidulans. Acta Biochim. Pol. 41, 467–471. Empel, J., et al., 2001. arcA, the regulatory gene for the arginine catabolic pathway in Aspergillus nidulans. Mol. Genet. Genom. 266, 591–597. Etxebeste, O., et al., 2010. Aspergillus nidulans asexual development: making the most of cellular modules. Trends Microbiol. 18, 569–576. Flores, T., et al., 2008. Arginase-negative mutants of Arabidopsis exhibit increased nitric oxide signaling in root development. Plant Physiol. 147, 1936–1946. Foresi, N., et al., 2010. Characterization of a nitric oxide synthase from the plant kingdom: NO generation from the green alga Ostreococcus tauri is light irradiance and growth phase dependent. Plant Cell. 22, 3816–3830. Gardner, P.R., et al., 2000. Nitric-oxide dioxygenase activity and function of flavohemoglobins. sensitivity to nitric oxide and carbon monoxide inhibition. J. Biol. Chem. 275, 31581–31587. Gong, X., et al., 2007. L-arginine is essential for conidiation in the filamentous fungus Coniothyrium minitans. Fung. Genet. Biol. 44, 1368–1379. Gorren, A.C., Mayer, B., 2007. Nitric-oxide synthase: a cytochrome P450 family foster child. Biochim. Biophys. Acta 1770, 432–445. Gupta, K.J., et al., 2011. On the origins of nitric oxide. Trends Plant Sci. 16, 160–168. Hallemeesch, M.M., et al., 2002. Reduced arginine availability and nitric oxide production. Clin. Nutr. 21, 273–279. He, Y., et al., 2004. Nitric oxide represses the Arabidopsis floral transition. Science 305, 1968–1971. Ignarro, L., Freeman, B.E., 2017. Nitric Oxide: Biology and Pathobiology. Academic Press. Kwon, E., et al., 2012. AtGSNOR1 function is required for multiple developmental programs in Arabidopsis. Planta 236, 887–900. Lavie, L., et al., 2003. Plasma levels of nitric oxide and L-arginine in sleep apnea patients: effects of nCPAP treatment. J. Mol. Neurosci. 21, 57–63. Liu, L., et al., 2000. Protection from nitrosative stress by yeast flavohemoglobin. Proc. Natl. Acad. Sci. USA 97, 4672–4676. Lorenz, M.C., Fink, G.R., 2001. The glyoxylate cycle is required for fungal virulence. Nature 412, 83–86. Macios, M., et al., 2012. The GATA factors AREA and AREB together with the co-repressor NMRA, negatively regulate arginine catabolism in Aspergillus nidulans in response to nitrogen and carbon source. Fung. Genet. Biol. 49, 189–198. Maier, J., et al., 2001. Role of nitric oxide synthase in the light-induced development of sporangiophores in Phycomyces blakesleeanus. Plant Physiol. 126, 1323–1330. Marcos, A.T., et al., 2016. Nitric oxide synthesis by nitrate reductase is regulated during development in Aspergillus. Mol. Microbiol. 99, 15–33. Mooney, J.L., Yager, L.N., 1990. Light is required for conidiation in Aspergillus nidulans. Genes Dev. 4, 1473–1482. Ninnemann, H., Maier, J., 1996. Indications for the occurrence of nitric oxide synthases in fungi and plants and the involvement in photoconidiation of Neurospora crassa. Photochem. Photobiol. 64, 393–398. Niu, S., et al., 2011. The circadian deadenylase Nocturnin is necessary for stabilization of the iNOS mRNA in mice. PLoS One 6, e26954. Ojeda-López, M., et al., 2018. Evolution of asexual and sexual reproduction in the aspergilli. Stud. Mycol. 91. Paoletti, M., et al., 2007. Mating type and the genetic basis of self-fertility in the model fungus Aspergillus nidulans. Curr. Biol. 17, 1384–1389. Park, H.S., Yu, J.H., 2012. Genetic control of asexual sporulation in filamentous fungi. Curr. Opin. Microbiol. 15, 669–677. Pengkit, A., et al., 2016. Identification and functional analysis of endogenous nitric oxide in a filamentous fungus. Sci. Rep. 6, 30037. Prochazkova, D., et al., 2014. Nitric oxide biosynthesis in plants - the short overview. Plant, Soil Environ. 60, 129–134. Purschwitz, J., et al., 2008. Functional and physical interaction of blue- and red-light sensors in Aspergillus nidulans. Curr. Biol. 18, 255–259. Rodriguez-Romero, J., et al., 2010. Fungi, hidden in soil or up in the air: light makes a difference. Annu. Rev. Microbiol. 64, 585–610. Ruger-Herreros, C., et al., 2011. Regulation of conidiation by light in Aspergillus nidulans. Genetics 188, 809–822. Samalova, M., et al., 2013. Nitric oxide generated by the rice blast fungus Magnaporthe oryzae drives plant infection. New Phytol. 197, 207–222. Sarikaya Bayram, O., et al., 2010. LaeA control of velvet family regulatory proteins for light-dependent development and fungal cell-type specificity. PLoS Genet. 6, e1001226. Schinko, T., et al., 2010. Transcriptome analysis of nitrate assimilation in Aspergillus nidulans reveals connections to nitric oxide metabolism. Mol. Microbiol. 78, 720–738. Serlupi-Crescenzi, O., et al., 1983. Developmental defects resulting from arginine auxotrophy in Aspergillus nidulans. J. Gen. Microbiol. 129, 3535–3544.
Acknowledgments We thank the Fungal Genetics Stock Center (FGSC), Reinhard Fischer and Gerhard Braus for strains. D.C. would also like to thank Luis Corrochano and Jose Marcos for helpful discussions. M.C. RugerHerreros is acknowledged for help with the photoinducible experiments, Thomas Dalik for initial amino acids quantifications, and Antonio Franco and Myrsini Charikleous for help with the conidial counts. We would also like to thank Modesto Carballo for help with the fluorometric assays (Servicio de Biología, Centro de Investigación Tecnología e Innovación, Universidad de Sevilla). Work in Vienna was supported by the Lower Austria Science Fund grant “Bioactive Microbial Metabolites” Nr. K3-G-2/26-2013 to JS and by grant M01693-B22 from the FWF to DC. Work in Sevilla was supported by grant RTI2018-098636-B-I00 from the MICINN to DC. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fgb.2020.103337. References Adams, T.H., et al., 1998. Asexual sporulation in Aspergillus nidulans. Microbiol. Mol. Biol. Rev. 62, 35–54. Andrianopoulos, A., Timberlake, W.E., 1994. The Aspergillus nidulans abaA gene encodes a transcriptional activator that acts as a genetic switch to control development. Mol. Cell Biol. 14, 2503–2515. Bayram, O., et al., 2008a. More than a repair enzyme: Aspergillus nidulans photolyase-like CryA is a regulator of sexual development. Mol. Biol. Cell. 19, 3254–3262. Bayram, O., Braus, G.H., 2012. Coordination of secondary metabolism and development in fungi: the velvet family of regulatory proteins. FEMS Microbiol. Rev. 36, 1–24. Bayram, O., et al., 2010. Spotlight on Aspergillus nidulans photosensory systems. Fung. Genet. Biol. 47, 900–908. Bayram, O., et al., 2008b. VelB/VeA/LaeA complex coordinates light signal with fungal development and secondary metabolism. Science 320, 1504–1506. Berger, H., et al., 2008. Dissecting individual steps of nitrogen transcription factor cooperation in the Aspergillus nidulans nitrate cluster. Mol. Microbiol. 69, 1385–1398. Blumenstein, A., et al., 2005. The Aspergillus nidulans phytochrome FphA represses sexual development in red light. Curr. Biol. 15, 1833–1838. Borsuk, P., et al., 1999. Structure of the arginase coding gene and its transcript in Aspergillus nidulans. Acta Biochim. Pol. 46, 391–403. Borsuk, P., et al., 2007. L-arginine influences the structure and function of arginase mRNA in Aspergillus nidulans. Biol. Chem. 388, 135–144. Canovas, D., Andrianopoulos, A., 2006. Developmental regulation of the glyoxylate cycle in the human pathogen Penicillium marneffei. Mol. Microbiol. 62, 1725–1738. Canovas, D., et al., 2014. The histone acetyltransferase GcnE (GCN5) plays a central role in the regulation of Aspergillus asexual development. Genetics 197, 1175–1189. Canovas, D., et al., 2016. Nitric oxide in fungi: is there NO light at the end of the tunnel? Curr. Genet. 62, 513–518. Canovas, D., et al., 2017. High-throughput format for the phenotyping of fungi on solid substrates. Sci. Rep. 7, 4289. Castello, P.R., et al., 2006. Mitochondrial cytochrome oxidase produces nitric oxide under hypoxic conditions: implications for oxygen sensing and hypoxic signaling in
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A.T. Marcos, et al. Seth, D., et al., 2012. Endogenous protein S-Nitrosylation in E. coli: regulation by OxyR. Science 336, 470–473. Seth, D., et al., 2018. A multiplex enzymatic machinery for cellular protein S-nitrosylation. Mol. Cell. 69 (451–464), e6. te Biesebeke, R., et al., 2010. Phylogeny of fungal hemoglobins and expression analysis of the Aspergillus oryzae flavohemoglobin gene fhbA during hyphal growth. Fung. Biol. 114, 135–143. Yamasaki, H., Sakihama, Y., 2000. Simultaneous production of nitric oxide and
peroxynitrite by plant nitrate reductase: in vitro evidence for the NR-dependent formation of active nitrogen species. FEBS Lett. 468, 89–92. Zhang, Y., et al., 2015. MoARG1, MoARG5,6 and MoARG7 involved in arginine biosynthesis are essential for growth, conidiogenesis, sexual reproduction, and pathogenicity in Magnaporthe oryzae. Microbiol. Res. 180, 11–22. Zhou, S., et al., 2011. Functional analysis and subcellular location of two flavohemoglobins from Aspergillus oryzae. Fung. Genet. Biol. 48, 200–207.
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