Fungal Genetics and Biology 47 (2010) 1055–1069

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Fungal Genetics and Biology

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The roles played by Aspergillus nidulans apoptosis-inducing factor (AIF)-likemitochondrial oxidoreductase (AifA) and NADH-ubiquinone oxidoreductases(NdeA-B and NdiA) in farnesol resistance

Taísa Magnani Dinamarco a,1, Bárbara de Castro Figueiredo Pimentel a,1, Marcela Savoldi a, Iran Malavazi b,Frederico Marianetti Soriani a, Sérgio Akira Uyemura a, Paula Ludovico c, Maria Helena S. Goldman d,Gustavo Henrique Goldman a,e,*

a Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, São Paulo, Brazilb Departamento de Genética e Evolução, Centro de Ciências Biológicas e da Saúde (CCBS), Universidade Federal de São Carlos, Brazilc Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugald Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, São Paulo, Brazile Laboratório Nacional de Ciência e Tecnologia do Bioetanol, Universidade de São Paulo, São Paulo, Brazil

a r t i c l e i n f o

Article history:Received 12 May 2010Accepted 9 July 2010Available online 21 July 2010

Keywords:Aspergillus nidulansApopotosis-inducing factorNADH-ubiquinone oxidoreductasesFarnesol

1087-1845/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.fgb.2010.07.006

* Corresponding author. Address: DepartamentoFaculdade de Ciências Farmacêuticas de Ribeirão PretAv. do Café S/N, CEP 14040-903, Ribeirão Preto, Sã36024281.

E-mail address: ggoldman@usp.br (G.H. Goldman)1 These authors contributed equally to this work.

a b s t r a c t

Farnesol (FOH) is a nonsterol isoprenoid produced by dephosphorylation of farnesyl pyrophosphate, acatabolite of the cholesterol biosynthetic pathway. These isoprenoids inhibit proliferation and induceapoptosis. Here, we show that Aspergillus nidulans AifA encoding the apoptosis-inducing factor (AIF)-likemitochondrial oxidoreductase plays a role in the function of the mitochondrial Complex I. Additionally,we demonstrated that ndeA-B and ndiA encode external and internal alternative NADH dehydrogenases,respectively, that have a function in FOH resistance. When exposed to FOH, the DaifA and DndeA strainshave increased ROS production while DndeB, DndeA DndeB, and DndiA mutant strains showed the sameROS accumulation than in the absence of FOH. We observed several compensatory mechanisms affectingthe differential survival of these mutants to FOH.

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1. Introduction

Isoprenoids are fundamental in the regulation of lipid biosyn-thesis, cell proliferation, apoptosis, and differentiation (Edwardsand Ericsson, 1999; Bifulco, 2005; McTaggart, 2006; Joo and Jetten,2010). Farnesol (FOH) and its related isoprenoids, such as perillylalcohol, geranylgeraniol, and geraniol have been found in fruitsand vegetables (Burke et al., 1997; Crowell, 1999). They have beenshown to inhibit proliferation and induce apoptosis in neoplasticcell lines and also to be effective in chemoprevention andchemotherapy in several in vivo cancer models (Adany et al.,1994; Ohizumi et al., 1995; Miquel et al., 1996; He et al., 1997;Burke et al., 2002; Hudes et al., 2000; Wiseman et al., 2007; Jooand Jetten, 2010). FOH is a catabolite of the cholesterol biosyn-thetic pathway, a nonsterol isoprenoid produced by dephosphoryl-ation of farnesyl pyrophosphate (FPP), which is crucial in the

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control of cell growth, differentiation, proliferation and survival(Goldstein and Brown, 1990; Edwards and Ericsson, 1999). FOHalso plays a role as an extracellular quorum-sensing molecule inthe dimorphic fungus Candida albicans (Nickerson et al., 2006;Langford et al., 2009). FOH inhibits the yeast to mycelium dimor-phic transition and induces C. albicans to grow as actively buddingyeasts (Hornby et al., 2001). In Saccharomyces cerevisiae, FOHblocks growth by raising the concentration of mitochondrial reac-tive oxygen species (ROS) (Machida et al., 1998, 1999). FOH trig-gers hyperpolarization of the mitochondrial transmembranepotential in budding yeast through regulation of F0F1-ATPaseand a corresponding increase in its ATP-hydrolyzing activity(Machida and Tanaka, 1999). S. cerevisiae FOH-chemogenomic pro-filing revealed 48 genes whose inactivation increased sensitivity toFOH (Fairn et al., 2007). These genes indicated a role for the gener-ation of oxygen radicals by the Rieske iron-sulfur component ofcomplex III of the electron transport chain as a major mediator offarnesol-induced cell death. Recently, it was shown that duringFOH-mediated cell death in C. albicans there is ROS accumulation,mitochondrial degradation and increased expression of antioxi-dant-encoding genes (Shirtliff et al., 2009). Taken together, thesedata indicate that the generation of ROS by the electron transportchain is a primary mechanism by which FOH kills cells.

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Mitochondria are considered to be a major source of ROS andtheir generation is closely associated with the primary functionof these organelles, i.e., oxidative metabolism and ATP synthesis(for a review, see Adam-Vizi and Chinopoulos (2006)). In most ani-mals mitochondrial Complex I provides the single mechanism forentry of electrons from NADH into the respiratory chain. However,in plants and fungi, alternatives to Complex I are widely distrib-uted enabling direct oxidation of external NADH, or act in parallelwith Complex I to give rotenone insensitivity of internal NADH (fora review, see Joseph-Horne et al. (2001)). The alternative NADHdehydrogenases are encoded by single nuclear genes, have a ma-ture peptide molecular weight of 50–60 kDa, and their only pros-tetic group is flavin adenine dinucleotide (FAD). Furthermore, themost remarkable functional difference between alternative NADHdehydrogenases and Complex I is that electron transport to ubiqui-none is not coupled to proton translocation (for a review, see Jo-seph-Horne et al. (2001)). In S. cerevisiae, that does not have theComplex I, there are three NADH dehydrogenases, two externalrotenone-insensitive NADH:ubiquinone oxidoreductases (NDE1and NDE2), and one internal rotenone-insensitive NADH:ubiqui-none reductase (NDI1) (Joseph-Horne et al., 2001). NDI1 is the yeastAMID [in humans, it encodes the apoptosis-inducing factor (AIF)-homologous mitochondrion-associated inducer of death] homo-logue, and its overexpression in yeast causes apoptosis-like celldeath (Li et al., 2006). The apoptotic effect of NDI1 overexpressionis associated with increased production of ROS by mitochondria.Neurospora crassa is another fungus where unlike S. cerevisiae Com-plex I is also present and alternative NADH dehydrogenases havealso been characterized in detail (Videira and Duarte, 2001,2002). In N. crassa there are three external and one internal alter-native NADH dehydrogenases (Duarte et al., 2003; Carneiro et al.,2004, 2007).

The apoptosis-inducing factor (AIF) is a NADH oxidase with a lo-cal redox function that is essential for optimal oxidative phosphor-ylation and for an efficient antioxidant defense (for reviews, seeModjtahedi et al. (2006), Boujrad et al. (2007), Krantic et al.(2007), and Hangen et al. (2010)). The AIF is a mammalian flavo-protein that uses FAD as a cofactor and is synthesized as a�67 kDa precursor, imported into mitochondria via its N-terminalprodomain containing two mitochondrial localization sequences(MLS) and then processed to a �62 kDa mature protein (for re-views, see Modjtahedi et al. (2006), Boujrad et al. (2007), andKrantic et al. (2007)). In healthy cells, AIF is confined to the mito-chondria where it performs functions in bioenergetic and redoxmetabolism (for reviews, see Modjtahedi et al. (2006) and Kranticet al. (2007)). Upon mitochondrial outer membrane permeabiliza-tion mature AIF is further processed to a �57 kDa that is translo-cated to the nucleus via its C-terminal domain nuclearlocalization signal, leading to large-scale DNA fragmentation, ahallmark of caspase-independent apoptosis (Susin et al., 1999;Otera et al., 2005; Wissing et al., 2004).

Previously, we investigated which pathways are influencedthrough FOH by examining the transcriptional profile of Aspergillusnidulans exposed to this isoprenoid (Savoldi et al., 2008). We ob-served decreased mRNA abundance of several genes involved inRNA processing and modification, transcription, translation, ribo-somal structure and biogenesis, amino acid transport and metabo-lism, and ergosterol biosynthesis. We also observed increasedmRNA expression of genes encoding a number of mitochondrialproteins such as the AifA, encoding the AIF homologue, and NdeA,encoding a NADH-ubiquinone oxidoreductase (NdeA). The DaifAmutant was more sensitive to FOH than the wild type and it is pos-sibly important for decreasing the effects of FOH and ROS. Further-more, we showed an involvement of autophagy and protein kinaseC in A. nidulans FOH-induced apoptosis. Here, we extended thesestudies by showing that A. nidulans AifA plays a role in the function

of the mitochondrial Complex I. Additionally, we demonstratedthat ndeA encodes an external alternative NADH dehydrogenasethat has a function in FOH resistance. We also identified and char-acterized an additional external and a single internal alternativeNADH dehydrogenases that also play a role in FOH-tolerance.

2. Materials and methods

2.1. Strains, media methods, and determination of viability

Aspergilli strains used in this study are described in Supplemen-tary Table 1S. Media were of two basic types. A complete mediumwith three variants: YAG (2% glucose, 0.5% yeast extract, 2% agar,trace elements), YUU (YAG supplemented with 1.2 g/l each of ura-cil and uridine) and liquid YG or YG + UU medium of the samecompositions (but without agar). A modified minimal medium(MM: 1% glucose, original high nitrate salts, trace elements, 2%agar, pH 6.5) was also used. Trace elements, vitamins, and nitratesalts are described by Kafer (1977).

Five-hours-old germlings were incubated in FOH (10 lM) at37 �C (Noventa-Jordão et al., 1999). In all cases appropriate dilu-tions were made and 100 ll aliquots spread on YAG plates. Viabil-ity was determined as the percentage of colonies on treated platescompared to untreated controls. The results were expressed by theaverage of four independent experiments and means ± standarddeviation are shown (�, p < 0.001). Statistical differences weredetermined by One-Way analysis of variance (ANOVA) followed,when significant, by Newman–Keuls Multiple Comparison Test,using GraphPad Prism statistical software (GraphPad Software,Inc., version 3, 2003).

2.2. RNA isolation and real-time PCR reactions

For total RNA isolation, the germlings were disrupted by grind-ing in liquid nitrogen with pestle and mortar and total RNA was ex-tracted with Trizol reagent (Invitrogen, USA). Ten micrograms ofRNA from each treatment were then fractionated in 2.2 M formal-dehyde, 1.2% w/v agarose gel, stained with ethidium bromide, andthen visualized with UV-light. The presence of intact 25S and 17Sribosomal RNA bands was used as a criterion to assess the integrityof the RNA. RNAse-free DNAse I treatment for the real-time PCRexperiments was carried out as previously described (Semighiniet al., 2002).

All the PCR reactions were performed using an ABI 7500 FastReal-Time PCR System (Applied Biosystems, USA) and Taq-Man™Universal PCR Master Mix kit (Applied Biosystems, USA). The reac-tions and calculations were performed according to Semighini et al.(2002). The primers and Lux™ fluorescent probes (Invitrogen, USA)used in this work are described in Supplementary Tables 2S and 3S.

2.3. Staining and microscopy

For nuclear staining of the germlings, conidia were inoculatedon coverslips. After incubation at the appropriate conditions foreach experiment, coverslips with adherent germlings were trans-ferred to fixative solution (3.7% v/v formaldehyde, 50 mM sodiumphosphate buffer pH 7.0, 0.2% v/v Triton X-100) for 30 min at roomtemperature. Then, they were briefly rinsed with PBS buffer(140 mM NaCl, 2 mM KCl, 10 mM NaHPO4, 1.8 mM KH2PO4, pH7.4) and incubated for five min in a solution with 100 ng/ml ofDAPI (40,6-diamino-2-phenylindole, Sigma). After 5 min of incuba-tion with the dye, they were washed with PBS buffer for 5–10 minat room temperature and then rinsed in distilled water, mountedand visualized in epifluorescence microscope. Slides were viewedwith a Carl Zeiss (Jena, Germany) microscope using 100�

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magnification oil immersion objective lens (EC Plan-Neofluar, NA1.3) equipped with a 100 W HBO mercury lamp epifluorescencemodule. Phase contrast for the brightfield images and fluorescentimages were captured with a AxioCam camera (Carl Zeiss), pro-cessed using the AxioVision software version 3.1 and saved as TIFFfiles. Further processing was performed using Adobe Photoshop 7.0(Adobe Systems Incorporated, CA).

2.4. Measurement of intracellular ROS

Intracellular ROS levels were measured in conidia by 5-(and-6)-chloromethyl-2070-dichlorodihydrofluorescein diacetate (H2DCFDA).1 � 109 conidia of which strain were incubated for 5 h in a YUUmedium at 37 �C with shaking (150 rpm). After that cultures werecentrifuged and resuspended in 1 ml of the same medium. An ali-quot was incubated with 5 lM CM-H2DCFDA (Molecular Probes,Eugene, OR, USA) for 30 min at 37 �C, followed by FOH incubation.The fluorescence intensity was detected using a F4500 HitachiFluorescence Spectrophotometer (excitation 503 nm and emission,529 nm).

2.5. Detection of protein oxidation

Oxidative modification of proteins by oxygen free radicals wasmonitored by Western blot analysis of carbonyl groups using theOxyBlot™ Protein Oxidation Detection Kit (Chemicon� Interna-tional, Inc.). Briefly, the cultures of mutant and wild type strainswere grown for 12 h at 37 �C in an orbital shaker (150 rpm). Afterthat, the cultures were exposed to 100 lM FOH for 120 min. Bothtreated and control cultures (no FOH added) had the total proteinsextracted by homogenizing the mycelia using liquid nitrogen in abuffer containing 50 mM Tris–HCl, pH 7.4, 1 mM EGTA, 0.2% TritonX-100, 1 mM benzamidine and 10 mg ml-1 each of leupeptin, pep-statin and aprotinine. The homogenates were clarified by centrifu-gation at 20,800g for 60 min at 4 �C. Total protein concentrationswere determined by the Bradford method (Bradford, 1976) and20 lg were submitted to derivatization with dinitrophenylhydra-zine (DNPH). About 10 lg of proteins were loaded into a 12% (w/v) SDS–PAGE gel and electroblotted to a nitrocellulose membrane.The membrane was incubated with the antibody anti-DNP moietyof the proteins and the immunoblot was detected by chemilumi-nescent reaction. Densitometric analysis was made by using theImage J program (available at http://rsbweb.nih.gov/ij/download.html).

2.6. Oxygen uptake assays

Germlings were obtained by growing 1 � 108 conidia of wildtype, DaifA and DndeA mutant strains in 50 ml of YUU or YG med-ium for 15 h at 37 �C (250 rpm). They were harvested by centrifu-gation and incubated for 5 h (90 rpm) in a standard solution usedfor A. nidulans protoplasting (Osmani et al., 1987) at 30 �C aimingpartially disrupting the cell wall. After incubation, germlings werewashed three times with buffer containing 0.7 mM sorbitol, 10 mMHepes-KOH, pH 7.2 and kept in this buffer on ice during the mea-surements. The effects of FOH in mitochondria were determinatedby the incubation of spheroplasts with 10, 50 or 100 lM of FOH for10 min. Oxygen uptake was measured in spheroplasts with a Clark-type electrode fitted to a Gilson oxygraph (Gilson Medical Elec-tronics Inc., Middleton, WI) in 1.8 ml of medium containing10 mM Hepes–KOH, 0.7 mM Sorbitol pH 7.2, 5 mM MgCl2,2.5 mM KH2PO4, 0.5 mM EGTA, 0.5% (w/v) BSA, 5 lmol Digitoninand an appropriated substrate (Tudella et al., 2004). The initial sol-ubility of oxygen in the reaction buffer was considered to be445 ng atoms of O/ml (Helmerhorst et al., 2002). Further additionsare indicated in the figure legends.

2.7. DNA manipulations and construction of the A. nidulans DndeA,DndeB, DndiA and alcA::ndiA mutants

Standard genetic techniques for A. nidulans were used for allstrain constructions and transformations (Kafer, 1977). DNAmanipulations were according to Sambrook and Russell (2001).All PCR reactions were performed using Platinum Taq DNA Polim-erase High Fidelity (Invitrogen).

For the DNA-mediated transformation, the deletion cassetteswere constructed by ‘‘in vivo” recombination in S. cerevisiae as pre-viously described by Colot et al. (2006). Briefly, about 1.5 kb re-gions on either side of the ORFs were selected for primer design.For every construction, the primers were named as 5F and 5R inthat de 5F primer contains a short homologue sequence to theMCS of the plasmid pRS426. Both primers were used to amplifythe 50-UTR flanking region of the targeted ORF. Likewise, the prim-ers 3F and 3R were used to amplify the 30-UTR ORF flanking regionand the 3R primer also contains a short homologue sequence to theMCS of the plasmid pRS426 (small letters indicated in Supplemen-tary Table 1S). Both fragments 5- and 3-UTR were PCR-amplifiedfrom genomic DNA using as templates the A4 strain for A. nidulanscassettes. The pyrG used in the A. nidulans cassettes for generatingthe strains DndiA and DndeB was used as marker for auxotrophyand was amplified from pCDA21 plasmid (Chaveroche et al.,2000), and pyroA for DndeA was amplified from A. fumigatusAf293 (for details see Supplementary Fig. 1S).

Cassettes generation was achieved by transforming each frag-ment for each construction along with the plasmid pRS426 Bam-HI/EcoRI cut in the in S. cerevisiae strain SC94721 by the lithiumacetate method (Schiestl and Gietz, 1989). The DNA of the yeasttransformants was extracted by the method described by Goldmanet al. (2003), dialyzed and transformed by electroporation in Esch-erichia coli strain DH10B to rescue the pRS426 plasmid harboringthe cassettes. The cassettes were PCR-amplified from these plas-mids and used for transformation of Aspergilli according to the pro-cedure of Osmani et al. (1987). Transformants were scored for theirability to grow on minimal medium. PCR or Southern blot analyseswere used throughout of the manuscript to demonstrate that thetransformation cassettes had integrated homologously at the tar-geted A. nidulans loci (Supplementary Fig. 1S).

3. Results

3.1. Depletion of AifA affects oxygen consumption by Complex I

We have previously shown that the DaifA mutant strain wasmore sensitive to FOH and that AifA is important for decreasingthe effects of oxidative stress caused by FOH and menadione(Savoldi et al., 2008). A striking observation from our work is thefact that when germlings were exposed to FOH, AifA::GFP did nottranslocate to the nucleus but instead remained in the cytoplasm.To verify if the DaifA mutant is more sensitive to cell death causedby other non-primarily oxidative stressing agents, we exposedconidia of this strain to several concentrations/dosages of ampho-tericin, itraconazole, caspofungin, camptothecin, hydroxyurea,4-nitroquinoline oxide, UV-light, sirolimus, propolis, phytosphin-gosine, calcium, amiodarone, etoposide, and stausporine. The wildtype and the DaifA mutant strains showed comparable levels ofsusceptibility to all these agents (data not shown). We also con-structed an AifA::mRFP strain that has the same degree of FOH-sensitivity than the wild strain and exposed it to FOH. As it waspreviously shown for an AifA::GFP fusion, the AifA::mRFP accumu-lation was detected along the cytoplasm but not in the nuclei (datanot shown). Taken together, these results strongly indicate that inthe absence of AifA cells are more susceptible to cell death induced

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by primary oxidative stress inducing agents such as FOH. Notice-ably, and in contrast to previous reports on mammalian and otherfungi, A. nidulans AifA is definitively not translocated to the nucleusduring cell death. Instead, A. nidulans AifA is released from mito-chondria and detected in cytoplasm which seems to be an eventstrong enough to avoid its ROS protecting effects on mitochondria.

As a first step to understand the role played by AifA on mito-chondrial activities, we measured the rates of oxygen consumptionin A. nidulans wild type and DaifA strains in the presence of a sub-strate cocktail containing glutamate, malate, pyruvate, and succi-nate (Fig. 1). These experiments were performed after theincubation in the presence of different FOH concentrations(Fig. 1). In the absence of FOH (control), the wild type strainshowed higher oxygen consumption than the DaifA mutant strain(100.00 versus 44.18 ng atom O min�1 mg protein�1; Fig. 1A andB), suggesting an inefficient electron transport chain in the DaifAmutant strain. The effects of FOH on the mitochondria activitywere determined by the incubation of both strains in the presenceof either 10 lM, 50 lM, or 100 lM FOH for 10 min. After this per-iod, the measurement of oxygen uptake in the wild type strainshowed a decrease of 10.0%, 20.4%, and 46.5%, respectively, whilein the DaifA mutant strain, caused a reduction of 28.9%, 35.2%and 57.3% (Fig. 1A and B). These results suggest that the cellular

Fig. 1. FOH affects more the oxygen consumption of the A. nidulans aifA inactivationthan the wild type strain. Oxygen uptake by A. nidulans wild type (A) and DaifAmutant (B) strains in the absence (Control) and in the presence of different FOHconcentrations. Graphs are representative of at least three independent assaysperformed by measuring polarographically the oxygen uptake in cells from bothstrains grown for 15 h.

oxygen consumption was inhibited in a FOH-dose dependent man-ner and that the DaifA mutant strain was more sensitive to thisinhibition probably due to its innate inefficient electron transportchain. As a second step, we investigate in more detail the mito-chondrial activities in both strains by measuring the rates of oxy-gen consumption in the presence of the same respiratorysubstrates. The initial oxygen consumption in the DaifA mutantwas reduced when compared with the wild type strain (45.20 ver-sus 100.01 ng atom O min�1 mg protein�1; Fig. 2). After the addi-tion of malonate, which inhibits the Complex II activity andflavone, an inhibitor of alternative NADH dehydrogenases, theremaining mitochondrial activity is due to Complex I, which corre-sponded to 62% and 34% of the initial respiratory activity in thewild type and DaifA mutant strains, respectively (Fig. 2A). Theaddition of rotenone (an inhibitor of Complex I) induced an inhibi-tion of 32% in the oxygen consumption while in the DaifA mutantstrain this inhibition was 37% (Supplementary Fig. 2S). Again, thisdata indicates lower activity of the Complex I in the DaifA mutantstrain, as it was shown in Fig. 2A. This strongly suggests a defect inComplex I activity in the DaifA mutant. The inhibition of oxygenconsumption by flavone was higher in DaifA mutant correspondingto 22% of oxygen uptake while in the wild type strain the respira-tory activity was inhibited about 10% (Fig. 2A), indicating the pres-ence of alternative NADH dehydrogenase activities. Thesubsequent addition of ADP (which can be phosphorylated toATP) induced a transition in respiration from resting state (i.e.,state 4) to phosphorylating state (state 3). This transition was re-versed by the addition of oligomycin. The oxygen consumptionwas partly inhibited by 1 mM KCN (an inhibitor of Complex IV),showing the presence of an alternative oxidase, which is inhibitedby the addition of SHAM (Fig. 2A). In addition, we observed forboth strains an increase in cyanide-resistant respiration mediatedby alternative oxidase, but in the DaifA mutant this activity washigher than in wild type strain (Fig. 2B).

When the same experiment was repeated after the incubationwith 50 lM FOH, we observed a reduction in the initial oxygen up-take (45.20 versus 30.01 ng atom O min�1 mg protein�1; Fig. 2B) inDaifA mutant, whereas in the wild type strain the oxygen uptakewas about 20% decreased when compared with the control(100.0 versus 80.6). We have not observed significant differencescompared with its respectively control, after the addition of malon-ate, flavone, ADP and oligomycin. On the other hand, a minoruncoupling in proton gradient was observed after the addition ofFCCP. In wild type strain, the FCCP increased about 2.9% the oxygenconsumption, while in DaifA mutant this addition showed no ef-fects (Fig. 2B). These results are likely due to a reduction of themitochondrial transmembrane potential by FOH due to its inhibi-tory effect on mitochondrial electron transport, such as reflectedby an impairment of cellular oxygen consumption.

These results suggest that DaifA mutant strain has a deficientmitochondrial Complex I, and together with the great sensitivityof this strain to oxidative stressing agents, this could be the maincause for the increased sensitivity to FOH in this mutant.

3.2. The external alternative NADH dehydrogenase NdeA is involved inresistance to FOH

We have previously shown that when A. nidulans was exposedto FOH, a gene (AN7500.3; named ndeA) encoding a NADH-ubiqui-none oxidoreductase had increased mRNA expression (Savoldiet al., 2008). NdeA shows a pyridine nucleotide-disulfide oxidore-ductase domain (a small NADH binding domain within a largerFAD binding domain; pfam00070) at amino acid positions 226–334, and an EF-hand calcium-binding domain (PROSITE entryPS50222) at amino acid positions 433–468. NdeA displays highsimilarity with the S. cerevisiae NADH alternative dehydrogenases

Fig. 2. A. nidulans aifA inactivation affects oxygen consumption by Complex I. Oxygen uptake by the wild type and DaifA mutant strains in the absence (A) and in the presenceof FOH (B). At the times indicated by arrows, malonate (1 mM), flavone (0.5 mM), ADP (2 lM), oligomycin (4 lg/ml), FCCP (25 lM), KCN (1 mM), and salicylhydroxamic acid(SHAM; 2 mM), were added together with a substrate cocktail (10 mM glutamate; 10 mM malate; 10 mM pyruvate; and 10 mM succinate) at 30 �C. The values in parenthesesrepresent the rate of oxygen uptake and were expressed as ng atoms O/min/mg of proteins. Graphs are representative of at least three independent assays performed bymeasuring polarographically the oxygen uptake in cells from both strains grown for 15 h.

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(NDE2, 44% identity, 63% similarity, 8.4e�80; NDE1, 36% identity,52% similarity, 1.5e�78; and NDI1, 43% identity, 61% similarity,2.e�69). BLAST analysis of NdeA against the A. nidulans databasedisplayed similarity with AN1094.3 (35% identity, 54% similarity,6.5e�72) and AN10660.3 (35% identity, 55% similarity, 1.4e�51).These two proteins also showed pyridine nucleotide-disulfide oxi-doreductase domains. These three genes probably encode the twoexternal and a single internal NADH alternative dehydrogenases inA. nidulans. As it was previously shown, the ndeA gene shows

increased mRNA accumulation in the presence of FOH (Table 1).We also verified the mRNA accumulation of ndeA during oxidativestress (Fig. 3). First, we determined if the oxidative stress condi-tions for induction were appropriate by checking mRNA accumula-tion of an A. nidulans gene known as involved in oxidative stress,catB (AN9339.3; Kawasaki and Aguirre, 2001). The catB mRNAaccumulation was increased in the presence of the oxidativestressing agents, hydrogen peroxide, menadione, and paraquat(Fig. 3A), thus validating our induction conditions for oxidative

Table 1Real-time RT-PCR for the wild type strain exposed to different concentrations of FOH for 120 min.

Genesa 0 10 lM FOHb 50 lM FOH 100 lM FOH

ndeA (AN7500.3) 25.74 ± 2.13 108.45 ± 18.35 (4.20 x) 963.3 ± 0.26 (37.40 x) 186.11 ± 14.17 (7.26 x)ndeB (AN1094.3) 1.48 ± 0.09 3.32 ± 0.08 (2.24 x) 11.57 ± 1.93 (9.08 x) 13.87 ± 2.35 (10.92)ndiA (AN10660.3) 0.03 ± 0.02 2.13 ± 0.16 (168.9 x) 14.6 ± 0.40 (429.0 x) 3.73 ± 0.87 (109.0 x)catB (AN9339.3) 4.49 ± 0.64 4.33 ± 0.10 (0.96 x) 33.18 ± 5.26 (7.38 x) 16.43 ± 0.20 (3.65 x)aoxA (AN2099.3) 35.43 ± 6.24 115.21 ± 17.15 (3.25 x) 226.68 ± 19.26 (6.39 x) 311.36 ± 52.16 (8.78 x)

a The measured mRNA quantity of each gene was normalized using the CT values obtained for the b-tubulin (tubC) mRNA amplifications run in the same plate. The relativequantitation of all the genes and tubulin gene expression was determined by a standard curve (i.e., CT-values plotted against logarithm of the DNA copy number). The resultsare the means ± standard deviation of four sets of experiments. The values represent the concentration of the cDNA of a specific gene divided by the cDNA concentration oftubC.

b The numbers in bold letters represent the number of times the genes are expressed compared to the corresponding control strain grown before adding FOH.

Fig. 3. The ndeA gene has increased mRNA accumulation in the presence of oxidative stressing conditions. Fold increase in mRNA levels of catB (AN9339.3) (A) and ndeA(AN7500.3) (B). Real-time RT-PCR was the method used to quantify the mRNA. The measured quantity of the mRNA in each of the treated samples was normalized using theCT values obtained for the b-tubulin mRNA amplifications run in the same plate. The results are the means ± standard deviation of four sets of experiments. The valuesrepresent the number of times the genes are expressed during oxidative stressing conditions divided by the corresponding control wild type strain.

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stress. The catB gene mRNA accumulation was also increased whenA. nidulans was exposed to different FOH concentrations (Table 1).Next step, we verified the ndeA mRNA accumulation during oxida-tive stressing conditions: ndeA accumulated about 10 times moremRNA in the presence of hydrogen peroxide (5, 25, and 100 mM

for 60 min), about 75 and 5 times more mRNA in the presence ofmenadione (0.1 and 0.5 mM for 60 min), and about 2 and 5 timesmore mRNA accumulation in the presence of paraquat (5 and10 mM for 60 min) (Fig. 3B). We deleted the ndeA gene as a firststep to understand its biological function (Supplementary

Fig. 4. Oxygen uptake by the wild type and DndeA mutant strains. At the times indicated by arrows, rotenone (40 lM), malonate (1 mM), NADH (2 mM), KCN (1 mM), andsalicylhydroxamic acid (SHAM; 2.5 mM), were added together with a substrate cocktail (10 mM pyruvate, 10 mM a-ketoglutarate, 10 mM glutamate, and 10 mM malate;10 mM succinate) at 30 �C. The values in parentheses represent the rate of oxygen uptake and were expressed as ng atoms O/min/mg of proteins. Graphs are representative ofat least three independent assays performed by measuring polarographically the oxygen uptake in cells from both strains grown for 15 h.

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Fig. 1S). The deletion strain showed to be more sensitive to FOH(Fig. 6D; compare at 10 lM FOH the DndeA and the wild typestrains, they show 8.0% and 92.0% viability, respectively).

Finally, we used the TUNEL assay to determine if DNA fragmen-tation occurs in the ndeA mutant strain after treatment with FOH.This assay uses terminal deoxynucleotidyltransferase to label 30-OH DNA termini with TMR-conjugated dUTP, which can be directlyvisualized by fluorescence microscopy. As previously shown bySemighini et al. (2006), when germlings from the wild type strainwere treated with 100 lM FOH for 2 h, about 50% showed TUNELpositive staining, whereas untreated control hyphae showed nostaining (Supplementary Fig. 3S). Interestingly, germlings of theDndeA mutant strain have shown about 50% and 100% TUNEL po-sitive staining in the absence and presence of FOH, respectively(Supplementary Fig. 3S). Taken together, these results stronglyindicate that NdeA has an important role as a survival factor fordetoxification of oxidative stress and FOH in A. nidulans.

In order to functionally investigate if NdeA is involved in theinternal or external alternative NADH dehydrogenase activity, oxy-gen consumption from the wild type and DndeA strains were mea-sured polarographically with an oxygen electrode in mediumcontaining NADH-linked substrates (glutamate, malate, and a-ketoglutarate) which provides NADH and succinate, a Complex IIsubstrate (Fig. 4). After the addition of rotenone and malonate, aComplex I and Complex II inhibitor, respectively, the rotenone-insensitive oxidation rates for the cocktail substrates were propor-tionally the same in both strains (Fig. 4). In addition, when bothstrains were assayed with exogenous NADH, the DndeA mutant

mitochondria showed much reduced oxidation activity relative towild type strain (Fig. 4). The oxygen consumption increased about100% in wild type strain, whereas in DndeA mutant strain, this in-crease was about 52% (37.10–78.08 versus 26.87–40.84 ng atomO min�1 mg protein�1, respectively; Fig. 4). In both cases, the oxy-gen consumption was inhibited by KCN and by the addition ofSHAM (Fig. 4). Thus, our results strongly indicate that NdeA isone of the two putative external alternative NADH dehydrogenasesin A. nidulans.

After the incubation with 50 lM FOH, the oxygen consumptionwas measured in these strains and the initial oxygen uptake wasnot affected. Upon NADH addition, we observed an increase of138% and 50% in oxygen consumption in the wild type and inDndeA mutant, respectively (data not shown). These results sug-gest that in the wild type strain the increase in oxygen consump-tion after NADH addition is due to the induction of NdeA activityby FOH (as it can be seen in Table 1). On the other hand, in DndeAmutant FOH causes no induction after the addition of NADH (datanot shown), once more indicating that ndeA encodes one of theexternal alternative NADH dehydrogenases in A. nidulans.

3.3. A. nidulans has two external alternative NADH dehydrogenases

Since A. nidulans has two other genes (AN1094.3 andAN10660.3) that could potentially encode alternative NADH dehy-drogenases, we decided to investigate whether AN1094.3 wouldencode an external or internal alternative NADH dehydrogenase.First, we verified their expression in the presence of FOH (Table 1)

Fig. 5. The ndiA and ndeB genes have increased mRNA accumulation in the presence of oxidative stressing conditions. Fold increase in mRNA levels of ndiA (AN1094.3) (A) andndeB (AN10660.3) (B). Real-time RT-PCR was the method used to quantify the mRNA. The measured quantity of the mRNA in each of the treated samples was normalizedusing the CT values obtained for the b-tubulin mRNA amplifications run in the same plate. The results are the means ± standard deviation of four sets of experiments. Thevalues represent the number of times the genes are expressed during oxidative stressing conditions divided by the corresponding control wild type strain.

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and oxidative stressing conditions (Fig. 5). Although both geneshad increased mRNA accumulation in the presence of FOH,AN10660.3 showed very high mRNA levels, reaching up to 430times at 50 lM FOH (Table 1). Both genes also had increased mRNAlevels during oxidative stressing conditions, but again AN10660.3showed more robust levels of mRNA accumulation thanAN1094.3 (Fig. 5A and B). Interestingly, the alternative oxidasegene (aoxA) also has increased mRNA accumulation upon FOHincubation (3.25, 6.3, and 8.78 times at 10, 50, and 100 lM, respec-tively, Table 1). To identify if AN1094.3 gene encodes either anexternal or internal NADH alternative dehydrogenase, we per-formed similar oxygen consumption experiments as previously de-scribed for the DndeA mutant. Thus, oxygen consumption from theAN1094.3 gene deletion mutant strain was measured polarograph-ically with an oxygen electrode in medium containing NADH-linked substrates and succinate (Fig. 6A). When the AN1094.3 genedeletion mutant strain was assayed with exogenous NADH, themitochondria showed much reduced oxidation activity relative towild type strain (compare Figs. 4–6A). The oxygen consumption in-creased about 100% in wild type strain (Fig. 4), whereas inAN1094.3 gene deletion mutant strain, this increase was about

20% (37.10–78.08 versus 31.87–38.37 ng atom O min�1 mg pro-tein�1, respectively; Figs. 4A and 6A). Thus, our results stronglyindicate that AN1094.3 gene encodes another A. nidulans externalalternative NADH dehydrogenase, and accordingly this gene wasnamed ndeB. We also constructed by sexual crossings a doubledeletion mutant DndeA DndeB and once more investigated the im-pact of these double deletion on the external supply of NADH tothe mitochondria by observing oxygen consumption from the dou-ble mutant in medium containing NADH-linked substrates(Fig. 6B). There is no increase in the oxygen consumption uponexogenous NADH addition (Fig. 6B), once more demonstrating thatndeA-B encode the two external alternative NADH dehydrogenasesin A. nidulans.

The DndeA and DndeB deletion mutant strains (see Supplemen-tary Fig. 1S) showed lower viability than the wild type when ex-posed to FOH 10 lM (about 8% and 65% in the DndeA and DndeBmutant strains compared to 92% in the wild type strain, Fig. 6D).Surprisingly, the double mutant displays 60% viability when ex-posed to FOH 10 lM (Fig. 6D). These results suggest that thereare other mitochondrial pathways (for instance the internal NADHalternative dehydrogenase and/or the alternative oxidase) that

Fig. 6. Viability and oxygen uptake by DndeB (A), DndeA DndeB (B), and wild type and DndiA (C) mutant strains. In (A) and (B), the initial oxygen uptake was measured with aSubstrate Cocktail (SC; 10 mM pyruvate, 10 mM a-ketoglutarate, 10 mM glutamate, and 10 mM malate; 10 mM succinate), and at the times indicated by arrows, rotenone(40 lM), malonate (1 mM), NADH (2 mM), KCN (1 mM), and salicylhydroxamic acid (SHAM; 2.5 mM), at 30 �C. In (C), rotenone (40 lM), substrate cocktail (CS) (10 mMmalate, 10 mM a-ketoglutarate, and 10 mM pyruvate), NADH (2 mM) and KCN (1 mM) were added as indicated. The values in parentheses represent the rate of oxygenuptake and were expressed as ng atoms O/min/mg of proteins. Graphs are representative of at least three independent assays performed by measuring polarographically theoxygen uptake in germlings from both strains grown for 15 h. (D) Viability of 5-h-old germlings exposed to FOH (10 lM) at 37 �C for 2 h. (E) The aoxA gene has increasedmRNA accumulation when the DndiA mutant strain is exposed to FOH. Fold increase in mRNA levels of aoxA (AN2099.3). The measured quantity of the mRNA in each of thetreated samples was normalized using the CT values obtained for the b-tubulin mRNA amplifications run in the same plate. The results are the means ± standard deviation offour sets of experiments. The values represent the number of times the gene is expressed during FOH stressing conditions divided by the corresponding control wild typestrain.

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could compensate for the depletion of the external NADH alterna-tive dehydrogenases. In fact, there is an increase of 34% of the alter-native oxidase activity in the double mutant DndeA DndeB whencompared to the wild type strain (compare oxygen consumptionfrom KCN to SHAM inhibition and Fig. 6A with Fig. 4).

3.4. Overexpression of ndiA increases FOH resistance

To corroborate ndiA (AN10660.3) encodes the A. nidulans inter-nal alternative NADH dehydrogenase, we deleted this gene (seeSupplementary Fig. 1S) and performed similar oxygen consump-tion experiments as previously described. Thus, once more wemeasured polarographically oxygen consumption from the wildtype and DndiA gene deletion mutant strains with an oxygenelectrode in medium containing Rotenone aiming to inhibit theComplex I. Then, a Substrate Cocktail (SC; 10 mM malate, 10 mMa-ketoglutarate, and 10 mM pyruvate), that generate NADH, wasadded to the cell suspension. Since Complex I was inhibited, thesingle activity is due to the presence of NdiA (Fig. 6C). Thus, inthe wild type strain there was an increase in the oxygen consump-tion (21.52 versus 25.64 ng atom O min�1 108 conidia�1) that is notobserved in the DndiA mutant (Fig. 6C). Interestingly, the additionof exogenous NADH activates the external NADH dehydrogenasesabout three times more in the DndiA mutant than in the wild type(about 52% and 100% in the wild type and DndiA mutant, respec-tively; Fig. 6C). Further the addition of KCN showed an inhibition

of 77% and 42% in the wild type and DndiA respectively, suggestingan increase in the alternative oxidase in the DndiA mutant. Theseresults strongly indicate that ndiA encodes the single A. nidulansinternal NADH dehydrogenase. In addition, compared to the wildtype strain, we also observed an increase of the aoxA mRNA accu-mulation when the DndiA mutant was exposed to increasing con-centrations of FOH (compare Fig. 6E with Table 1). However, ndeAand ndeB mRNA levels were comparable to the wild type strain(data not shown).

The DndiA deletion strain showed about 80% viability when ex-posed to FOH 10 lM (compared to 92% in the wild type strain,Fig. 6D). Again, as it was previously observed, this increased sur-vival could be due to increased compensatory pathways that willhelp the cells to survive in the presence of FOH, such as the in-creased external NADH dehydrogenase activity above shown forthe DndiA mutant strain (Fig. 6C).

It was previously shown that overexpression of S. cerevisiaehomologue internal NADH dehydrogenase (NDI1) can cause apop-tosis-like cell death (Li et al., 2006). The apoptotic effect of NDI1overexpression is associated with increased production of ROS inmitochondria. We investigate the effects of overexpressing ndiAin A. nidulans by constructing a conditional alcA mutant of the ndiAgene. The alcA promoter is repressed by glucose, derepressed byglycerol and induced to high levels by ethanol or L-threonine(Flipphi et al., 2002). We were able to select two transformantswith different levels of ndiA mRNA accumulation (Fig. 7). When

Fig. 6 (continued)

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they were grown in the presence of glycerol 2% as single carbonsource for 16 h and transferred to glycerol 2% + ethanol 2% for 6 hat 37 �C, the ndiA mRNA accumulates about 3 and 5 times, respec-tively (Fig. 7A). However, when transferred to 2% glucose as singlecarbon source, the ndiA mRNA accumulation was repressed(Fig. 7A). The ndiA overexpression in both transformants did notcause any observable phenotypic difference in terms of growth inliquid (data not show) and solid media (Fig. 7B). However, interest-ingly the ndiA overexpression increased the viability in the pres-ence of FOH when compared to the wild type (Fig. 7C). Thisincrease was proportional to the ndiA mRNA levels since trans-formant alcA::ndiA10 has increased viability than transformant al-cA::ndiA13 (Fig. 7C). Furthermore, we observed that in contrast towhat was seen during growth on YG medium (Fig. 6D), the DndiAmutant showed the same FOH-tolerance than the wild type strainat 10 and 50 lM when grown on MM + 2% glycerol + 100 mM

threonine (Fig. 7C); however, when DndiA mutant was grown at100 lM FOH, it showed higher FOH-tolerance than the wild type(30% versus 3% viability, Fig. 7C).

3.5. aifA and ndeA mutants have increased accumulation of ROS in thepresence of FOH

The increased/decreased sensitivity of these mutant strains toFOH could be due to a decreased/increased ROS accumulation inthese mutants. Thus, their decreased viability in the presence ofFOH could be due to their inability to cope with such high ROS con-centrations. We verified ROS accumulation upon FOH expositionwithin A. nidulans wild type and mutant strains by using 5-(and-6)-chloromethyl-20,70-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), a cell-permeable ROS indicator that is non-fluorescentuntil acetate groups are removed (H2DCF) by intracellular esterases

Gly 2 % + etOH 2%

6

5

4

3

2

1Fold

incr

ease

0Wild type alcA::ndiA 10 alcA::ndiA 13

Glu 4 %

Glu 4 % Gly 2 % Gly 2 % +Threo 100 mM

alcA::ndiA 13

alcA::ndiA 10

Wild type

Control10 μM FOH50 μM FOH

100 μM FOH

100

80

60

40

20

0Wild type alcA::ndiA 10 alcA::ndiA 13ΔndiA

Surv

ivva

l (%

)

A

B

C

Fig. 7. Overexpression of ndiA increases the resistance to FOH. (A) Fold increase in mRNA levels of ndiA (AN1094.3). Real-time RT-PCR was the method used to quantify themRNA. The measured quantity of the mRNA in each of the treated samples was normalized using the CT values obtained for the b-tubulin mRNA amplifications run in thesame plate. The relative quantitation of all the genes and tubulin gene expression was determined by a standard curve (i.e., CT-values plotted against logarithm of the DNAcopy number). The results are the means ± standard deviation of four sets of experiments. The values represent the number of times the gene is expressed during oxidativestressing conditions divided by the corresponding control wild type strain. (B) Growth phenotypes of the wild type and alcA::ndiA in MM + glucose 4% (Gluc), MM + glycerol2% (Gly), and MM + glycerol 2% + threonine 100 mM (Gly + Threo) grown for 72 h at 4 �C. (C) Viability of 5-h-old germlings grown in MM + glycerol 2% + threonine 100 mMafter exposed to FOH (10 lM) at 37 �C for 2 h.

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and oxidation occurs within the cell (H2DCF can be oxidized by dif-ferent ROS). By using this method, germlings of the wild type andmutant strains were incubated with CM-H2DCFDA, and subse-quently with FOH and their fluorescence was examined. Uponexposure to FOH, the wild type, DaifA, DndeA, and DndeB strains

showed about 1.5, 10.0, 1.8, and 1.3 times more fluorescence thanwithout FOH (Fig. 8A). In contrast, the DndeA DndeB and DndiAmutant strains showed comparable levels of fluorescence to thewild type strain (Fig. 8A). Another method used to evaluate ROSaccumulation, protein carbonylation, shows the increased

Fig. 8. AifA and NdeA mutants have increased accumulation of ROS in the presence of FOH. (A) The wild type, DaifA, DndeA, DndeB, and DndeA DndeB mutants were grown for6 h at 37 �C in YG medium, and germlings were stained with CM-H2DCFDA for 30 min, and incubated for further 10 min in the presence of 50 lM FOH. The fluorescenceintensity was detected using a F4500 Hitachi Fluorescence Spectrophotometer. Results are the average of three repetitions with standard deviation. (B) Pattern of oxidativelydamaged proteins in the wild type, DndeA, and DaifA mutant strains exposed or not to 50 lM FOH for 10 min. In each strain, the left and right panels show the Coomassiestaining of a polyacrylamide gel and the same proteins blotted onto a nitrocellulose membrane and probed by the antibody anti-DNP moiety of the proteins (the immunoblotwas detected by chemiluminescent reaction), respectively. (C) Western blot densitometry.

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concentration of carbonyl groups introduced into proteins by oxi-dative reactions with FOH, reflecting the inability to efficiently re-pair proteins damaged by oxidative stress. Thus, total proteinsfrom these strains previously exposed to FOH were derivatizedwith dinitrophenylhydrazine, electroblotted to a nitrocellulosemembrane, and monitored by Western blot analysis of carbonylgroups using the OxyBlot™ Protein Oxidation Detection Kit. Themembrane was incubated with the antibody anti-DNP moiety ofthe proteins and the immunoblot was detected by chemilumines-cent reaction. The intensity of the bands in the wild type exposedto FOH was about 7.5 times higher than in the wild type strain notexposed to FOH. The DaifA, DndeA, DndeB, and DndeA DndeB mu-tant strains had about 17.8, 10, 1.3, and 1.1 times more intensity,respectively, when exposed to FOH than the same strains not ex-posed to FOH (Fig. 8B). The DndiA mutant strain showed no in-crease in the intensity of the band levels when exposed to FOH(Fig. 8B). The NdiA overexpression showed comparable levels ofROS and carbonylated protein when exposed or not to FOH (datanot shown).

Although the results obtained with the two different methodsdisplayed a relative discrepancy, such as differences in fold in-crease for both assays, they clearly demonstrate that FOH induceshigh ROS levels and AifA and NdeA are important for monitoringand/or repairing the oxidative stress caused by ROS. In contrast,the ROS levels seem to be diminished in the DndeB, DndeA DndeB,and DndiA mutant strains, what could help to explain their in-creased survival levels in the presence of FOH.

4. Discussion

Besides producing most of the cellular energy in eukaryotes,mitochondria have also been involved in programmed cell death(PCD), through the generation of ROS and the release of mitochon-drial proteins (Pradelli et al., 2010; Circu and Aw, 2010). We previ-ously showed that FOH induces at transcriptional level a series ofgenes that have a mitochondrial function in A. nidulans (Savoldiet al., 2008). Here, we characterized in more detail the functionof four of these genes: AifA oxidoreductase (aifA), and the externaland internal NADH-ubiquinone oxidoreductases (ndeA-B and ndiA,respectively). AIF is a flavoprotein usually confined to mitochon-dria but translocates to the nucleus when apoptosis is induced,causing chromatin condensation and large-scale fragmentation ofDNA (Modjtahedi et al., 2006; Porter and Urbano, 2006; Boujradet al., 2007; Krantic et al., 2007). Porter and Urbano (2006) haveproposed that the main function of AIF is to support energy pro-duction in both normal and transformed cells, whereas nuclear-translocated AIF might contribute to stress-induced or pathologicalcell death in some circumstances. However, it is well establishedthat AIF is not a universal cell death effector but its function isdependent on the cell type, the apoptotic insult, and its intrinsicDNA binding capacity (Hangen et al., 2010). AIF is indispensablefor the optimal function of the mitochondrial respiratory chainand is involved in redox metabolism (Klein et al., 2002; Vahsenet al., 2004). Although it is still controversial, AIF has also been pro-posed to act as a putative ROS scavenger (Klein et al., 2002). Thereare several reports that the AIF depletion causes increased sensitiv-ity to oxidative stress and compromises oxidative phosphorylation(Wissing et al., 2004; Vahsen et al., 2004; Apostolova et al., 2006;Chinta et al., 2009). Perturbations of oxidative phosphorylationcan enhance ROS generation, and most intracellular ROS are gener-ated at the Complexes I and III of the mitochondrial respiratorychain, particularly under mitochondrial malfunction (Circu andAw, 2010). The major biochemical defect caused by the globalreduction or time-specific ablation of AIF is a defective respiratorychain Complex I (Hangen et al., 2010). AIF deficiency causes Com-

plex I defects in vitro in embryonic stem cells, and in vivo in skeletalmuscle, heart, liver, and brain tissues from which AIF has been re-moved by conditional knockout (Vahsen et al., 2004; Joza et al.,2005; Cheung et al., 2006; Pospisilik et al., 2007). Knockout ofAIF also reduces Complex I formation in Drosophila melanogaster(Joza et al., 2008) and negatively affects respiration in yeast, whichlacks a Complex I (Wissing et al., 2004). We have exposed the A.nidulans AifA inactivation mutant to several apoptotic stimuli,but we have never observed any susceptibility of this strain tothese stimuli, except for oxidative stressing agents. Actually, theDaifA mutant strain has very high levels of constitutive ROS asshown by DCFDA and protein carbonylation. An interesting featureof the A. nidulans AifA again observed here in this study was thefact that AifA is present along the cytoplasm upon oxidative stressbut never translocates to the nuclei.

Our results also suggest that A. nidulans AifA plays an importantrole in the activity of the mitochondrial Complex I. Although wehave not addressed if AifA-deficient cells exhibit a reduced contentof Complex I and of its components, the oxygen consumption mea-surements clearly show that this mutant has defects in Complex Ieven in the absence of FOH or any other stress that are severelypronounced in the presence of FOH. Mutants impaired in the AifAactivity have the increased tendency to produce ROS and increasedsensitivity to oxidative stress. Interestingly, the activity of thealternative NADH dehydrogenases and alternative oxidase (AoxA)was much higher in the DaifA mutant than in the wild type whenexposed to FOH. In fact, ndeA, ndeB, ndiA, and aoxA have highermRNA accumulation even in the absence of FOH (about 12.0-,4.0-, 7.0-, and 13.0-times, respectively) in the DaifA mutant strainthan in the wild type strain (data not shown).

However, there are differences between A. nidulans and N. crassaDaifA mutants. Recently, transcriptional profiling and functionalanalysis of Heterokaryon Incompatibility (HI) (a nonself recogni-tion process that occurs in filamentous fungi (Glass and Demen-thon, 2006) in N. crassa revealed that ROS, but not N. crassa AIFhomologue, is required for HI or programmed cell death (PCD)(Hutchinson et al., 2009). Hyphal death during HI shows some fea-tures that are similar to apoptosis in mammalian cells, includingshrinkage of plasma membrane, membrane-bound vesicle forma-tion, DNA condensation and TUNEL-positive nuclei (Glass andDementhon, 2006). Additionally, treatment of A. nidulans and N.crassa cells with the sphingolipid phytosphingosine (PHS) inducesPCD by an unknown mechanism (Castro et al., 2008). As comparedwith the N. crassa wild type strain, Castro et al. (2008) found that astrain containing a deletion in the gene encoding an AIF (apoptosis-inducing factor)-like protein is more resistant to PHS and H2O2.Actually, PHS resistance was correlated with a lower accumulationof ROS production in Complex I mutants in the presence of PHS(Videira et al., 2009). A. nidulans DaifA mutant has decreased Com-plex I activity and increased sensitivity to FOH but presented com-parable levels of sensitivity to PHS than the wild type strain (datanot shown). These results suggest that there are differences in theAIF and mitochondrial function related to PCD in A. nidulans and N.crassa.

Complex I to Complex IV form a functional electron transportchain (ETC), referred as the cytochrome ETC. In contrast, internaland external NADH dehydrogenases as well as alternative oxidasecan form a functional ETC, referred to as the alternative ETC (vanAken et al., 2009). The importance of these pathways in terms ofthe physiology of the cell is not very well understood. It has beensuggested that these pathways are involved in the prevention ofROS formation (Moller, 2001; Fernie et al., 2004). However, someother authors have shown they may increase ROS production andcause cell death (Fang and Beattie, 2003; Li et al., 2006). The fungihave extensive redundancy within their respiratory chains andthe presence of these nonproton-pumping alternative NADH

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dehydrogenases contrasts between different fungi (Joseph-Horneet al., 2001; Melo et al., 2001; Duarte et al., 2003; Guerrero-Castilloet al., 2009; Carneiro et al., 2004, 2007; Tarrío et al., 2006). S. cere-visiae lacks Complex I and has three NADH dehydrogenases: NDE1,NDE2 (both external) and NDI (internal) (Luttik et al., 1998; Li et al.,2006). However, their specific function is not clear. During normalrespiration in animals, superoxide anion is generated and ComplexI is strongly implicated in this generation, due to the low redox po-tential required for one electron reduction of dioxygen to superox-ide (Liu, 1997; Joseph-Horne et al., 2001). It is generally believedthat the presence of internal and/or external alternative dehydro-genases should enable NADH oxidation and decrease the produc-tion of ROS. Our results suggest that A. nidulans has two externaland one internal alternative dehydrogenases and they (and proba-bly the alternative oxidase too) could play an important role in re-sponse to the great amount of ROS produced during FOH-inducedcell death. The main evidence that A. nidulans ndeA-B and ndiA en-code external and internal NADH dehydrogenases, respectively,was provided by the great reduction in oxidation activity observedin the corresponding deletion mutants when Complexes I and IIwere inhibited and either exogenous NADH or substrates that gen-erate NADH were added as substrates. This was further confirmedby the construction of a double DndeA DndeB mutant strain thatshowed no oxidation activity under the same conditions. These re-sults emphasize the importance of alternative respiratory pathwaysfor A. nidulans when the mitochondrial environment is extensivelydamaged by ROS.

Two interesting observations from our work emphasize theimportance of internal and external NADH dehydrogenases asgenes that promote the survival during conditions that need by-pass of the intense accumulation of ROS produced by Complex I.First, the increased survival in the presence of FOH of DndeB,DndeA DndeB, and DndiA mutant strains when compared to thewild type and DndeA strains correlates with the decreased accu-mulation of ROS in these former strains. It is possible that compen-satory pathways could be activated in the absence of specific genesto aid these mutants to cope with increased ROS. We were able toobserve some of these genes with increased expression, such as thealternative oxidase in the DndeA DndeB mutant and alternativeoxidase and external NADH dehydrogenase in the DndiA mutantstrain. Interestingly, a similar compensation has already been ob-served in the N. crassa nde-3 mutant (nde-3 encodes an externalNADH dehydrogenase) since there is an up-regulation of the nde-2 transcript (whose gene encodes an external NADH dehydroge-nase) from early to late exponential growth, in contrast to whathappens in the wild type where the nde-2 transcript is stronglydown-regulated (Carneiro et al., 2007). The second observationwould be the differences between the phenotypes of ndiA overex-pression in A. nidulans and NDI1 overexpression in S. cerevisiae. TheA. nidulans ndiA overexpression shows no modification in the ROSconcentration and increases the survival to FOH while S. cerevisiaeNDI1 overexpression is associated with increased production ofROS in mitochondria and can cause apoptosis-like cell death. Thesedifferences between these two species could be due to the absenceof Complex I in S. cerevisiae, but they strongly suggest that modifi-cations in the copy number of these genes can probably causeimbalances in the capacity of ROS detoxification in the mitochon-dria. Actually, this could help to explain the apparently paradoxicalincreased survival in the presence of FOH when ndiA is either over-expressed or deleted. It remains to be investigated which otherpathways are modulated in these mutants and can help to increasetheir survival in the presence of FOH.

In summary, our study shows a cooperative involvement ofalternative respiratory pathways and AifA in the FOH-induced celldeath opening exciting new avenues for research into FOH-inducedcell death and apoptosis in filamentous fungi.

Acknowledgments

This research was supported by the Fundação de Amparo à Pes-quisa do Estado de São Paulo (FAPESP), Conselho Nacional deDesenvolvimento Científico e Tecnológico (CNPq), Brazil, John Si-mon Guggenheim Memorial Foundation, USA. We also thank Dr.Mario de Barros for the critical reading of the manuscript.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.fgb.2010.07.006.

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