Carole H Sellem
email: carole.sellem@i2bc.paris-saclay.fr

Adeline Humbert

Annie Sainsard-Chanet

Mutations in the Phosphatase Domain of the 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase Result in the Transcriptional Activation of the Alternative Oxidase and Gluconeogenic Pathways in Podospora anserina

Keywords: glycolysis and gluconeogenesis, PFK-2/FBPase-2, alternative oxidase regulation, Podospora anserina

scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Fungi have a very flexible metabolism that enables them to live in a variety of environments and to use diverse carbon sources. They can grow on a range of carbohydrates using glycolysis but are also able to grow on two-carbon compounds, fatty acids, or other sources of tricarboxylic acid (TCA) cycle intermediates, thereby requiring gluconeogenesis. Glycolysis and gluconeogenesis are two opposite pathways for glucose metabolism (degradation or biosynthesis of glucose), and fungi have to adapt their carbon metabolism according to the 3 available carbon source. Regulation of the enzymes involved is of great importance to avoid futile cycling between the degradation (glycolysis) and biosynthesis (gluconeogenesis) of sugars. Phosphoenolpyruvate carboxykinase (PCK-1) and fructose-1,6-bisphosphatase (FBPase-1) are the key enzymes of the gluconeogenic pathway. PCK-1 catalyzes an early step converting oxaloacetate to phosphoenolpyruvate. FBPase-1 catalyzes the final step in hexose monophosphate formation by dephosphorylating fructose-1,6-biphosphate to yield fructose-6phosphate. It has been shown that the expression of these two enzymes is controlled by two zinc cluster transcription factors called RSE2 and RSE3 in P. anserina [START_REF] Bovier | Genetic and functional investigation of Zn(2)Cys(6) transcription factors RSE2 and RSE3 in Podospora anserina[END_REF][START_REF] Sellem | Mutations in two zinc-cluster proteins activate alternative respiratory and gluconeogenic pathways and restore senescence in long-lived respiratory mutants of Podospora anserina[END_REF], ACUK and ACUM in A. nidulans [START_REF] Hynes | Transcriptional control of gluconeogenesis in Aspergillus nidulans[END_REF][START_REF] Suzuki | Reprogramming of carbon metabolism by the transcriptional activators AcuK and AcuM in Aspergillus nidulans[END_REF]. In N. crassa, the orthologs of these transcription factors (AOD2 and AOD5) also control the expression of pck-1 but not of fbpase-1 [START_REF] Qi | Alternative Oxidase Transcription Factors AOD2 and AOD5 of Neurospora crassa Control the Expression of Genes Involved in Energy Production and Metabolism[END_REF]. Surprisingly, in these three species, these transcription factors that are highly conserved, are also required for the expression of the alternative oxidase, AOX (Chae et al., 2007b;[START_REF] Sellem | Mutations in two zinc-cluster proteins activate alternative respiratory and gluconeogenic pathways and restore senescence in long-lived respiratory mutants of Podospora anserina[END_REF][START_REF] Suzuki | Reprogramming of carbon metabolism by the transcriptional activators AcuK and AcuM in Aspergillus nidulans[END_REF]. AOX is a mitochondrial non-proton-pump enzyme of nuclear origin that is able to receive electrons from reduced ubiquinone and to catalyze the reduction of oxygen to water.

AOX is found in plants, many fungal species, several protists, some animal phyla and certain bacteria [START_REF] Mcdonald | Alternative oxidase: an inter-kingdom perspective on the function and regulation of this broadly distributed 'cyanide-resistant' terminal oxidase[END_REF]. In most fungi aox transcripts are undetectable, or present at very low levels, under classical growth conditions but become expressed when the cytochrome respiratory chain is restricted by inhibitors or by mutations (Chae et al., 2007a;[START_REF] Lorin | Overexpression of the alternative oxidase restores senescence and fertility in a longlived respiration-deficient mutant of Podospora anserina[END_REF][START_REF] Suzuki | Reprogramming of carbon metabolism by the transcriptional activators AcuK and AcuM in Aspergillus nidulans[END_REF]. In response to such mitochondrial electron transport chain inhibition, AOX bypasses respiratory complexes III and IV. It is believed to contribute to the maintenance of the tricarboxylic acid (TCA) cycle turnover and to act as an overflow for electron transport, preventing the deleterious oxidative stress associated with the increased generation of mitochondrial reactive oxygen species (ROS). In N. crassa and A. nidulans the orthologs of RSE2 and RSE3 have been shown to bind constitutively to DNA, as a 4 heterodimer, by their N-termini (Chae et al., 2007a;[START_REF] Qi | Alternative Oxidase Transcription Factors AOD2 and AOD5 of Neurospora crassa Control the Expression of Genes Involved in Energy Production and Metabolism[END_REF][START_REF] Suzuki | Reprogramming of carbon metabolism by the transcriptional activators AcuK and AcuM in Aspergillus nidulans[END_REF]. Thus, it appears that the activation of these transcription factors is achieved by communication of an unknown signal(s) to the nucleus when cells are exposed to specific conditions. The identity of the signal(s) is crucial to the understanding of these regulatory pathways, as is the potential biological significance of this unexpected co-regulation of AOX and gluconeogenic enzymes.

In the current study, we report the properties of P. anserina mutants carrying mutations in a gene encoding the bi-functional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2). This enzyme catalyzes two opposite reactions [START_REF] Pilkis | 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase: a metabolic signaling enzyme[END_REF][START_REF] Rider | 6phosphofructo-2-kinase/fructose-2,6-bisphosphatase: head-to-head with a bifunctional enzyme that controls glycolysis[END_REF]: synthesis of Fru-2,6-P2 (fructose 2,6-biphosphate) from Fru-6-P (fructose-6-phosphate) and ATP via its phosphofructose-2-kinase activity (PFK-2), and degradation of Fru-2,6-P2 to Fru-6-P and inorganic phosphate via its fructose-2,6bisphosphatase activity (FBPase-2). A mutant carrying a single substitution in a conserved motif of the phosphatase domain was first isolated as a suppressor of a respiratory-deficient mutant. Null mutations of the kinase and phosphatase domains were then constructed. Their analysis showed that only mutations affecting the phosphatase domain are able to suppress the respiratory mutant phenotype and that this suppression is correlated with a strong expression of the AOX due to the activation of the RSEs transcription factors. Moreover we show that the key enzymes of gluconeogenesis are also strongly overexpressed. The phenotype of the phosphatase mutants was intriguing since two other genes predicted to each also encode a PFK-2/FBPase-2 are present in all the tested Ascomycetes, one of them being probably monofunctional, lacking FBPase-2 activity.

Materials and methods

Strains and growth conditions

The knockout of the COX5 subunit of complex IV has deleterious consequences 5 [START_REF] Dufour | A causal link between respiration and senescence in Podospora anserina[END_REF]. This respiratory mutant (∆cox) carries a phleomycin resistant (Phleo R ) cassette in the cox5 gene and exhibits a typical low growth phenotype that enabled the identification of suppressors [START_REF] Bovier | Genetic and functional investigation of Zn(2)Cys(6) transcription factors RSE2 and RSE3 in Podospora anserina[END_REF][START_REF] Humbert | Deletion of the MED13 and CDK8 subunits of the Mediator improves the phenotype of a long-lived respiratory deficient mutant of Podospora anserina[END_REF][START_REF] Sellem | Mutations in two zinc-cluster proteins activate alternative respiratory and gluconeogenic pathways and restore senescence in long-lived respiratory mutants of Podospora anserina[END_REF].

The new suppressor mutation described in this work (momentarily named su) was obtained in the ∆cox ∆med13 strain. Inactivation of the med13 gene by insertion of a nourseothricin resistant (Nat R ) cassette [START_REF] Humbert | Deletion of the MED13 and CDK8 subunits of the Mediator improves the phenotype of a long-lived respiratory deficient mutant of Podospora anserina[END_REF] was already shown to be a suppressor of the ∆cox phenotype and its weak suppressor effect allowed us to select a sector of more aerial and faster growing mycelium from a culture. The fast growing sector, containing three mutant alleles ∆cox (Phleo R ), ∆med13 (Nat R ) and a new one (su), was crossed with the wild-type strain. The presence of an extragenic suppressor was indicated by the recovery in the progeny of two types of ∆cox ∆med13 ascospores (Phleo R , Nat R ): those giving rise to a mycelium identical to that of ∆cox ∆med13 strain, and those giving rise to an improved fast-growing mycelium, ∆cox ∆med13 su. Two types of ∆cox ascospores (Phleo R , Nat S ) were also identified in the progeny: again, those giving rise to a mycelium identical to the ∆cox strain, and those giving rise to an improved fast-growing mycelium, ∆cox su. There is no additive effect of the ∆med13 and su mutations, since the ∆cox ∆med13 su and ∆cox su strains showed the same phenotype. Construction of the strains with ∆pfkfbase-1, pfkfbase-1 L24A, D103A and pfkfbase-1 H233A was done by a gene replacement strategy [START_REF] El-Khoury | Gene deletion and allelic replacement in the filamentous fungus Podospora anserina[END_REF] with several PCR amplification steps, and a final integration with a Nat R cassette in the Podospora ∆Ku70 strain (Details of the constructions are given in supplemental figure S1A et S1B for constructions, supplemental Table S1A for list of primers). The M2 medium is a minimal medium in which carbon is supplied as dextrin (0.5%) and nitrogen as urea (0.05%). Acetate medium is a minimal medium in which dextrin is replaced by sodium acetate (1.4%). When necessary, 50µg/ml nourseothricin, 10µg/ml phleomycin, 20µg/ml antimycin A and 150µg/ml salicylhdroxamic acid (SHAM) were added to the medium. The germination medium (G) 6 contains ground corn meal (5%) and ammonium acetate (0.5%).

Fertility tests

As each strain produces spermatia (male cells) and proto-perithecia (female cells) regardless of its mating type, the male and female fertility of a strain was determined by growing this strain with the wild-type strain of opposite mating type on the same plate of M2 medium. When the two cultures were confluent, sterile water was poured and dispersed on the surface of the mycelium previously grown for 3 days in the light to allow formation of gametes. Reciprocal fertilization of the two strains took place and revealed whether the strain was fertile as a male (donor) and/or as a female (receptor).

Deep sequencing

A pool of five DNA preparations extracted from five ascospores carrying the su allele (wild-type phenotype with pigmented mycelium) and a pool of five DNA preparations extracted from five wild-type progenies were sequenced on Illumina genome analyzer (platform IMAGIG, CNRS Gif-sur-Yvette). The sequence reads were mapped together and the differences recorded.

Database searches and phylogenetic analysis

The Podospora anserina genome, EST and protein databases are available at http://podospora.i2bc.paris-saclay.fr/. Sequences in different species of Ascomycetes of the homologues of the three presumed PFK-2/FBPase-2 proteins of P. anserina were obtained from the JGI fungal genomics resource (http///genome.jgi.doe.gov/). Sequences were aligned using MUSCLE (https://www.ebi.ac.uk/Tools/msa/muscle/) and the resulting alignments manipulated using JALVIEW. Trees were constructed with PhyML 3.0. We used SH-like for approximate like hood-ratio test for statistical tests for branch support. The tree was 7 visualized with the iTOL server (http://itol.embl.de/)

Real-time RT-PCR experiments

For RNA extraction, the different strains were grown on cellophane sheets (Bio-Rad) overlaid on Petri dishes containing M2 medium for 48 h at 27 °C. Mycelia were harvested, flash frozen in liquid nitrogen, and ground in a Fastprep apparatus as described [START_REF] Bovier | Genetic and functional investigation of Zn(2)Cys(6) transcription factors RSE2 and RSE3 in Podospora anserina[END_REF]. Total RNA was extracted using the RNAeasy plant kit (Qiagen). For quantitative reverse transcription-PCR (qRT-PCR) analysis, 2 or 4µg of freshly extracted RNA were reverse transcribed using Superscript II reverse transcriptase (Invitrogen) and oligo (dT) 15 as a primer. The qRT-PCR amplification mixture of cDNAs was performed with the Light Cycler SYBR green I master kit (Roche). Reactions were run on and read in a Light Cycler 480 realtime PCR system (Roche) as described. Amplification efficiencies were measured for each pair of primers based on standard curves established using four serial dilution points. The couples of gene-specific primers used are listed in supplemental Table S1A. For all genes, a non-reverse-transcribed control was performed on each biological replicate to control the absence of genomic DNA contamination. The level of expression of each gene was estimated relative to that of two normalization genes used for qRT-PCR in P. anserina: Pa_3_5110 (gene gpd, high level of expression, encoding the glyceraldehyde phosphate dehydrogenase) and Pa_4_8980 (low level of expression, encoding a transcription factor). For a given strain and a given gene, the abundance of transcripts is expressed relative to the level of expression of this gene in the wild-type strain. It is a mean value obtained from two or three biological and two or three technical replicates for each strain.

Results

A mutation in a gene encoding PFK-2/FBPase-2 partially corrects the phenotype of the respiratory mutant ∆cox

The great majority of the suppressors of the respiratory mutant phenotype cox5::ble (∆cox) obtained in a previous screen fell in the regulatory genes rse2 and rse3 [START_REF] Bovier | Genetic and functional investigation of Zn(2)Cys(6) transcription factors RSE2 and RSE3 in Podospora anserina[END_REF] encoding two zinc cluster proteins, RSE2 and RSE3 [START_REF] Sellem | Mutations in two zinc-cluster proteins activate alternative respiratory and gluconeogenic pathways and restore senescence in long-lived respiratory mutants of Podospora anserina[END_REF], required for the expression of the alternative oxidase (AOX) and of the gluconeogenic enzymes, fructose-1,6 bi-phosphatase (FBPase-1) and phosphoenolpyruvate carboxykinase (PCK-1). All of them are responsible for a strong constitutive expression of the AOX, shown previously to suppress the detrimental effects (slow growth, thinness of the mycelium) due to the loss of function of the cytochrome pathway of respiration in P. anserina [START_REF] Lorin | Overexpression of the alternative oxidase restores senescence and fertility in a longlived respiration-deficient mutant of Podospora anserina[END_REF][START_REF] Sellem | Mutations in two zinc-cluster proteins activate alternative respiratory and gluconeogenic pathways and restore senescence in long-lived respiratory mutants of Podospora anserina[END_REF].

Two other loss of functions mutations are able to partially suppress the ∆cox phenotype, one in the kinase module of the Mediator (∆med13 or ∆cdk8) with a weak suppressor effect proceeding via an AOX-independent pathway [START_REF] Humbert | Deletion of the MED13 and CDK8 subunits of the Mediator improves the phenotype of a long-lived respiratory deficient mutant of Podospora anserina[END_REF] and one in the gene described in this work (see Materials and Methods for selection). Transiently called su, this suppressor has a stronger effect than that of ∆med13 but less strong than that of the rse mutations. As shown in Figure 1 it improves the aspect of the growing mycelium that appears aerial and colored and increases the growth rate (150% compared to that of ∆cox strain).

Conversely it does not restore female fertility and, in contrast to the rse mutations, it does not improve the germination rate to the ∆cox ascospores. It does not either restore a wild type longevity: the ∆cox pfkfb-1 R284C strain is still alive after two months whereas the longevity of the wild type strain is about three weeks and that of the ∆cox strain is more than two years [START_REF] Dufour | A causal link between respiration and senescence in Podospora anserina[END_REF]. Sequencing of the rse2 and rse3 genes revealed that the su strain does not harbor mutation in any of these two genes. Crosses between the ∆cox su and the wildtype (wt) strains led to the recovery of the cox + su + (wt) and cox + su progenies that can be distinguished thanks to the more pigmented aspect of the mycelium of the su strain compared to the wild type (see Table 1). Deep sequencing of the two strains revealed a C-to-T substitution in gene Pa_3_11390 (pfkfb-1) located on chromosome III in the mutant compared 9 to the wild-type strain. The gene pfkfb-1 encodes a protein of 443 amino acids (PFK2/FBPase-2) and the substitution is predicted to change an arginine into a cysteine codon at position 284 (pfkfb-1 R284C ). PFK-2/FBPase-2, the 6-phosphofructose-2-kinase/fructose-2,6bisphosphatase is a bi-functional enzyme [START_REF] Pilkis | 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase: a metabolic signaling enzyme[END_REF][START_REF] Rider | 6phosphofructo-2-kinase/fructose-2,6-bisphosphatase: head-to-head with a bifunctional enzyme that controls glycolysis[END_REF] whose kinase and phosphatase activities determine the steady-state level of fructose-2,6-biphosphate (Fru-2, 6-P2). The former is a potent positive allosteric activator of the glycolytic enzyme PFK-1 (6phosphofructo-1-kinase) and the latter a negative effector of the gluconeogenic enzyme FBPase-1 (fructose-1-6, bisphosphatase), and thus a key regulator metabolite of glucose homeostasis (supplemental Fig. S2). PFK-2/FBPase-2 isoenzymes have been identified in animals, plants, fungi and Trypanosomatidae with an N-terminal PFK-2 domain and a Cterminal FBPase-2 domain, the functional form of the enzyme being a dimer ( [START_REF] Rider | 6phosphofructo-2-kinase/fructose-2,6-bisphosphatase: head-to-head with a bifunctional enzyme that controls glycolysis[END_REF] for a review).

Mutations in the phosphatase but not in the kinase domain of PFK-2/FBPase-2 alter sexual development and lead to suppression of the ∆cox phenotype

Mutagenesis experiments and the resolution of crystal structures of the rat testis [START_REF] Hasemann | The crystal structure of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase reveals distinct domain homologies[END_REF] and the human liver [START_REF] Lee | Tissue-specific structure/function differentiation of the liver isoform of 6-phosphofructo-2-kinase/fructose-2,6bisphosphatase[END_REF] PFK-2/FBPase-2 enzymes have led to the characterization of key catalytic residues for each PFK-2 and FBPase-2 activities and a better understanding of the catalytic mechanisms of this enzyme. The R284 amino acid is conserved in all known FBPase-2 sequences and is part of a large conserved structural motif that defines the catalytic center of all the FBPase-2 enzymes (Fig. 2). This suggests that the pfkfb-1 R284C mutant could be a null mutant for the FBPase-2 activity that has conserved the PFK-2 activity. This conclusion prompted us to construct a null mutant for the two activities by deletion of the complete gene and two mutants carrying non-conservative substitutions in key catalytic residues of the PFK-2 or FBPase-2 domains (other than R284).

10

The FBPase-2 domain presents structural homology with the cofactor-dependent phosphoglycerate mutases and acid phosphatases. These enzymes share a common catalytic mechanism and use the histidine of a conserved RHG motif as a crucial component of the catalytic machinery [START_REF] Okar | PFK-2/FBPase-2: maker and breaker of the essential biofactor fructose-2,6bisphosphate[END_REF][START_REF] Pilkis | 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase: a metabolic signaling enzyme[END_REF][START_REF] Rigden | The histidine phosphatase superfamily: structure and function[END_REF]. Mutation of this histidine in the rat liver enzyme [START_REF] Tauler | Hepatic 6-phosphofructo-2-kinase/fructose-2,6bisphosphatase. Use of site-directed mutagenesis to evaluate the roles of His-258 and His-392 in catalysis[END_REF][START_REF] Yuen | Crystal structure of the H256A mutant of rat testis fructose-6-phosphate,2kinase/fructose-2,6-bisphosphatase. Fructose 6-phosphate in the active site leads to mechanisms for both mutant and wild type bisphosphatase activities[END_REF] or in the yeast PFK26 enzyme (Kretschmer et al., 1991) abolishes any FBPase-2 activity. We therefore changed the corresponding histidine codon of the RHG motif (H233) in the phosphatase domain of pfkfb-1 into an alanine codon to obtain a pfkfb-1 H233A mutant allele. The PFK-2 domain is related to a superfamily of mononucleotide binding proteins with motifs that present some similarity with the "Walker A" (GLPXXGKS/T) and "Walker B" motifs [START_REF] Rider | 6phosphofructo-2-kinase/fructose-2,6-bisphosphatase: head-to-head with a bifunctional enzyme that controls glycolysis[END_REF] for review).

Mutagenesis in the Walker A motif (G, K or T residues) of the rat liver enzyme [START_REF] Li | Site-directed mutagenesis in rat liver 6-phosphofructo-2-kinase. Mutation at the fructose 6-phosphate binding site 24 affects phosphate activation[END_REF][START_REF] Vertommen | The ATP-binding site in the 2-kinase domain of liver 6phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Study of the role of Lys-54 and Thr-55 by site-directed mutagenesis[END_REF] drastically decreased the PFK-2 activity. Also, mutagenesis of a conserved D residue in the Walker B motif of the rat muscle enzyme abolishes the PFK-2 activity [START_REF] Rider | Site-directed mutagenesis of rat muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: role of Asp-130 in the 2-kinase domain[END_REF]. In view of these data, we introduced two mutations into the kinase domain of pfkfb-1, one in the GLP conserved "Walker A" motif (L24A), the other one in the "Walker B" motif (D103A) of the enzyme. We also constructed a pfkfb-1null allele by gene replacement (∆pfkfb-1) with a cassette conferring resistance to nourseothricin (Nat R ) (see Materials and Methods and supplemental information).

As shown in Table 1, the null ∆pfkfb-1 allele and the two pfkfb-1 H233A and pfkfb-1 R284C mutations in the phosphatase domain, associated with a functional cytochrome pathway, confer slight vegetative defects: the growth rate is reduced by 12%, compared to the wild-type and, as previously noticed, the pigmentation of the mycelium is increased. Furthermore, germinating thalli derived from ascospores carrying these alleles appeared less dense than those of the wild-type strain. These mutant strains also show a delay in the development of the fruiting body (perithecium) when used as female and the expulsion of ascospores takes 11 place from the 7 th day after fertilization instead of the 4 th day for the wild-type strain. In contrast, the pfkfb-1 L24A, D103A mutant strain in the kinase domain does not lead to any visible vegetative or sexual development defects. Associated with the cox+ allele, none of these different mutant alleles significantly modify the longevity of the strain, a parameter that is often addressed when the metabolism of this fungus is perturbed [START_REF] Lorin | Mitochondrial metabolism and aging in the filamentous fungus Podospora anserina[END_REF][START_REF] Scheckhuber | Podospora anserina: a model organism to study 25 mechanisms of healthy ageing[END_REF]. To determine whether, as pfkfb-1 R284C , the other generated alleles are able to suppress the ∆cox phenotype they were associated by genetic crosses with the ∆cox allele. The phenotype of the strains, presented in Figure 3, revealed that the null allele ∆pfkfb-1and the mutation pfkfb-1 H333A located into the phosphatase domain showed the same suppressor effect as the mutation pfkfb-1 R284C when associated with ∆cox. As pfkfb-1 R284C , these two alleles lead to a wild-type-like aspect of the mycelium and improve the growth rate (150-175%). By contrast, the pfkfb-1 L24A,D103A mutant allele located into the kinase domain does not confer any improvement to the ∆cox phenotype. All these data (Fig. 3 and Table 1) indicate therefore that only the mutations expected to inactivate the phosphatase activity, and not those expected to inactivate the kinase activity, are able to partially suppress the phenotype of the ∆cox respiratory mutant strain and lead to vegetative and sexual defects.

The inactivation of the phosphatase domain of pfkfb-1 causes the transcriptional activation of genes positively regulated by the transcription factors RSE2 and RSE3

In P. anserina, the major way to improve the phenotype of cytochrome deficient mutants is to constitutively overexpress the alternative oxidase (AOX). This has been performed by the introduction of an aox gene under the control of a strong constitutive promoter [START_REF] Lorin | Overexpression of the alternative oxidase restores senescence and fertility in a longlived respiration-deficient mutant of Podospora anserina[END_REF] or gain-of-function mutations in the rse2 or rse3 genes [START_REF] Bovier | Genetic and functional investigation of Zn(2)Cys(6) transcription factors RSE2 and RSE3 in Podospora anserina[END_REF][START_REF] Sellem | Mutations in two zinc-cluster proteins activate alternative respiratory and gluconeogenic pathways and restore senescence in long-lived respiratory mutants of Podospora anserina[END_REF]. Genes rse2 and rse3 encode transcription factors highly conserved in the filamentous Ascomycetes P. anserina, N. crassa and A. nidulans and 12 required for the expression of the aox gene in these three species [START_REF] Bovier | Genetic and functional investigation of Zn(2)Cys(6) transcription factors RSE2 and RSE3 in Podospora anserina[END_REF]Chae et al., 2007b;[START_REF] Van Schaftingen | Fructose 2,6-bisphosphate[END_REF]. They have also been shown to be responsible for the activation of the gluconeogenic genes encoding fructose-1,6 bisphosphatase (FBPase-1) and phosphoenolpyruvate carboxykinase (PCK-1) in P. anserina [START_REF] Bovier | Genetic and functional investigation of Zn(2)Cys(6) transcription factors RSE2 and RSE3 in Podospora anserina[END_REF][START_REF] Sellem | Mutations in two zinc-cluster proteins activate alternative respiratory and gluconeogenic pathways and restore senescence in long-lived respiratory mutants of Podospora anserina[END_REF] andA . nidulans (Van Schaftingen, 1987). We thus addressed the question of the level of expression of the aox, fbpase-1 and pck-1 genes in the strains carrying the different pfkfb-1 mutations in a functional respiratory context. As shown on Figure 4A real-time quantitative RT-qPCR experiments reveal that the expression of the three genes is strongly activated in the null (∆pfkfb-1) and in the mutants of the phosphatase domain (pfkfb-1 R284C and pfkfb-1 H333A ) alleles. The strongest induction was observed for pck-1 (about 40-100X). For fbpase-1, it was about 10-20X and for aox about 10X. By contrast, in the mutant of the kinase domain (pfkfb-1 L24A,D103A ) no change in the expression levels of the three genes was observed.

We therefore propose that the suppressor effect of the pfkfb-1 R284C and pfkfb-1 H333A mutant alleles in the ∆cox strain results from the high expression level of the alternative oxidase (AOXp) in presence of these alleles. The amount of AOXp was estimated by Western blot and also by the growth resistance to salicylhydroxamic acid (SHAM), a specific inhibitor of the alternative respiratory pathway. These 2 parameters examined in the ∆cox pfkfb-1 R284C and ∆cox strains (Fig. S4 and Fig. 3, respectively) reveal a higher amount of AOXp in the ∆cox pfkfb-1 R284C strain. Our data also suggest that 1) the strong constitutive expression of AOXp depends on the RSEs since there is co-activation of the three RSE-regulated genes tested, 2) inactivation of the phosphatase activity of PFK-2/FBPase-2 results in the activation of the RSEs and consequently in the transcriptional activation of the RSE-target genes. Genes other than aox, fbpase-1 and pck-1 were also recently shown to be regulated by the RSEs in P.

anserina: Pa_7_2610 encoding a Zn 2 Cys6 transcription factor, Pa_5_1700 encoding a flavohemoglobin and various hydrolases specially Pa_4_3860 [START_REF] Bovier | Genetic and functional investigation of Zn(2)Cys(6) transcription factors RSE2 and RSE3 in Podospora anserina[END_REF]. So, to 13 further investigate the role of the RSEs in the pfkfb-1 mutant strains, the expression level of these genes was determined in the pfkfb-1 R284C mutant and compared to the wild-type strain.

As a control, we also investigated the level of expression of 2 glycolytic enzymes, hexokinase (Pa_1_6760) and 6-phosphofructo-1-kinase (Pa_1_14910). Figure 4B shows that the expression of two of the three RSE-regulated genes are induced (about 3-10X) in this mutant, supporting the hypothesis that the RSEs are under an active form in the pfkfb-1 phosphatasedead mutants. No modification of expression of the glycolytic genes was detected.

The nature of the signal(s) responsible for the activation of the RSEs and the mechanism(s) of this activation are not currently understood. As previously shown [START_REF] Bovier | Genetic and functional investigation of Zn(2)Cys(6) transcription factors RSE2 and RSE3 in Podospora anserina[END_REF] in the wild type strain, some genes whose expression is controlled by the RSEs are differently induced according to the growth culture conditions: in the presence of antimycin A (a strong inhibitor of complex III) the aox, Pa_5_1700, Pa_7_2610 and Pa_4_3860 genes are induced more strongly than when acetate is used as the only source of carbon. The opposite is observed for genes fbpase-1 and pck-1. These observations suggest that the inducer-mediated activation of RSEs is different on acetate and antimycin A. The expression level of these different genes was estimated in the pfkfb-1 R284C mutant and compared to the wild-type grown on non-inducer or various inducer conditions (Fig. 5). Consistent with our previous results, the tested genes can be classified in two categories, differing by their induction level according to the inducer medium. Interestingly, the expression level of these two categories of genes in the pfkfb-1 R284C mutant is close to that observed in the wild-type strain grown on acetate. These data suggest that in this mutant, the RSEs are under an active form similar or close to that they adopt in the wild-type strain grown on acetate. In agreement with this hypothesis is the fact that acetate has only a very poor, or no inducer effect in the pfkfb-1 R284C mutant (Fig. 6A). In contrast to acetate, antimycin A still acts as an inducer on the genes that it strongly induced in the wild-type strain (Fig. 6B). However the induction factor on 14 antimycin A is decreased in the pfkfb-1 R284C mutant compared to the wild-type strain.

Three genes are predicted to each encode PFK-2/FBPase-2 in all the tested Ascomycetes

The lack of distinguishable phenotype of strains with a mutant kinase domain, and the relatively small phenotypic defects of strains with a mutant phosphatase domain and with loss-of-function mutations (Table 1) is a slightly surprising. A search for pfkfb-1 (Pa_3_11390) homologous genes in the Podospora anserina protein database revealed two other possible homologs: Pa_2_10590 and Pa_3_6610 (supplemental Fig. S3). However, the RHG motif is not conserved in Pa_2_10590 due to the change of H274 to A. In view of the importance of this motif (Kretschmer et al., 1991;[START_REF] Tauler | Hepatic 6-phosphofructo-2-kinase/fructose-2,6bisphosphatase. Use of site-directed mutagenesis to evaluate the roles of His-258 and His-392 in catalysis[END_REF][START_REF] Yuen | Crystal structure of the H256A mutant of rat testis fructose-6-phosphate,2kinase/fructose-2,6-bisphosphatase. Fructose 6-phosphate in the active site leads to mechanisms for both mutant and wild type bisphosphatase activities[END_REF], it is highly likely that the Pa_2_10590 protein lacks FBPase-2 activity. Deep sequencing of cDNA libraries constructed from various stages of the life cycle of the fungus was undertaken to help the genome annotation [START_REF] Espagne | The genome sequence of the model ascomycete fungus Podospora anserina[END_REF]. A search for transcripts corresponding to each of the three possible enzymes in various Podospora ESTs database revealed that only Pa_3_11390 (pfkfb-1) is expressed all along the life cycle of the fungus (supplemental Table S2). Transcripts for Pa_2_10590 and Pa_3_6610 are only found during the sexual cycle (in the fruiting bodies where meiosis take place). Using the JGI fungal genomics resource, we carried out searches for orthologs in the genomes of at least one species of each of the ten classes of Ascomycetes. Four sequences were found in S. pombe but were omitted due to their low level of conservation and long evolutionary distance. For the same reason, we also omitted the S. cerevisiae mono-functional PFK27 protein that only contains a PFK-2 domain and a highly aberrant C-terminal domain [START_REF] Boles | Cloning of a second gene encoding 5phosphofructo-2-kinase in yeast, and characterization of mutant strains without fructose-2,6-bisphosphate[END_REF]. For all the other tested Ascomycetes, we found one ortholog of each of the three Podospora proteins. We attributed number 1 to Pa_3_11390 orthologs, (subfamily 1) the number 2 to Pa_2_10590 orthologs, (subfamily 2) and number 3 to Pa_3_6610 orthologs, (subfamily 3). To explore the 15 phylogenetic relationships among these different proteins, we generated a maximum likelihood phylogenetic tree shown in Figure 7. Inside each of the 3 subfamilies, the topological relationship of these proteins is highly consistent with the evolutionary relationships between the different species considered. Furthermore it can be seen that the three subfamilies are well supported in the tree, as indicated by high statistical values for tree branch supports. As said previously, the RHG motif is important for the catalytic function of the FBPase-2 domain but is not conserved in Pa_2_10590. Interestingly, like Pa_2_10590, all the proteins of the subfamily 2 lack this motif that is replaced by RAG, except in A. oligospora (RSG). This suggests that no protein of subfamily 2 acts as fructose-2,6bisphosphatase. In contrast, all the proteins of subfamilies 1 and 3 possess the RHG motif except S. cerevisiae PFK26 (subfamily 3) where it is replaced by RSG.

Discussion

Abolishing the phosphatase activity of PFK-2/FBPase-2 partially suppresses the growth defect of the respiratory deficient ∆cox mutant

The null mutation in the COX5 subunit of complex IV of P. anserina [START_REF] Dufour | A causal link between respiration and senescence in Podospora anserina[END_REF] leads to a slow growth rate and a thin mycelium in the corresponding ∆cox mutant strain and offers a positive selection for suppressor screening. We previously reported a largescale screening of suppressors of this mutation and found only two ways to improve the growth rate of this respiratory mutant: The strong constitutive expression of the alternative oxidase (more than 20 gain-of-function mutations in rse2 and rse3 genes that control the expression of the AOX) and, to a lesser extent, the loss-of-function of the kinase module of the Mediator [START_REF] Humbert | Deletion of the MED13 and CDK8 subunits of the Mediator improves the phenotype of a long-lived respiratory deficient mutant of Podospora anserina[END_REF]. The new suppressor described in this paper encodes a mutated PFK-2/FBPase-2 enzyme. In mammals, plants and fungi, PFK-2/FBPase-2 is a bifunctional enzyme that catalyzes both the synthesis of Fru-2,6-P2 via its PFK-2 activity and 16 its degradation via its FBPase-2 activity. The two activities of this enzyme therefore determine the steady-state level of Fru-2,6-P2 that is a cellular signaling molecule found in all eukaryotes [START_REF] Rousseau | Mammalian 6-phosphofructo-2-kinase/fructose-2,6bisphosphatase: a bifunctional enzyme that controls glycolysis[END_REF]. The crystal structure of the rat testis [START_REF] Hasemann | The crystal structure of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase reveals distinct domain homologies[END_REF], of the rat liver [START_REF] Lee | Crystal structure of the rat liver fructose-2,6-bisphosphatase based on selenomethionine multiwavelength anomalous dispersion phases[END_REF] and of the human liver isozyme [START_REF] Lee | Tissue-specific structure/function differentiation of the liver isoform of 6-phosphofructo-2-kinase/fructose-2,6bisphosphatase[END_REF] indicated that the FBPase-2 domain is related to the phosphoglycerate mutases and to the histidine phosphatases. These enzymes possess a common catalytic center involving a covalent phosphohistidine intermediate (H233 in P. anserina) located in a RHG motif whose importance for the FBPase-2 activity was confirmed when histidine was mutated in the rat liver [START_REF] Tauler | Hepatic 6-phosphofructo-2-kinase/fructose-2,6bisphosphatase. Use of site-directed mutagenesis to evaluate the roles of His-258 and His-392 in catalysis[END_REF] and testis [START_REF] Yuen | Crystal structure of the H256A mutant of rat testis fructose-6-phosphate,2kinase/fructose-2,6-bisphosphatase. Fructose 6-phosphate in the active site leads to mechanisms for both mutant and wild type bisphosphatase activities[END_REF] isoenzyme or in the yeast PFK26 enzyme (Kretschmer et al., 1991). The RHG motif is itself located in a large conserved structural region that includes an arginine corresponding to R284 in P. anserina. This residue was shown to be important for substrate binding in the rat liver enzyme [START_REF] Lee | Crystal structure of the rat liver fructose-2,6-bisphosphatase based on selenomethionine multiwavelength anomalous dispersion phases[END_REF], and this is reinforced by the fact that this residue is conserved in other members of the histidine phosphatase family [START_REF] Bazan | Evolution of a bifunctional enzyme: 6phosphofructo-2-kinase/fructose-2,6-bisphosphatase[END_REF][START_REF] Fothergill-Gilmore | The phosphoglycerate mutases[END_REF]. Furthermore, mutagenesis studies in the liver isozyme have confirmed the functional importance of this R residue in the catalysis reaction [START_REF] Lin | Arg-257 and Arg-307 of 6-phosphofructo-2kinase/fructose-2,6-bisphosphatase bind the C-2 phospho group of fructose-2,6bisphosphate in the fructose-2,6-bisphosphatase domain[END_REF]. These data suggested that the R284C suppressor is a loss of function mutation of the FBPase-2 part of the protein. To confirm this hypothesis, we mutated the other critical position, H233 of the RHG motif into alanine. We also constructed a strain inactivated for the two activities (kinase and phosphatase) by replacement of the gene with a resistance cassette.

In agreement with our hypothesis, these two alleles (pfkfb-1 H233A and ∆pfkfb-1), that a priori confer a loss of phosphatase activity, act as suppressors of the ∆cox phenotype similarly to the R284C allele. In contrast, mutations in the kinase domain do not behave as suppressors. The kinase domain consists of a central six-stranded ß sheet surrounded by seven α-helices. A Walker A motif or nucleotide-binding fold (G48LPARG53KT in rat liver) is located at the C-17 terminus of the first ß-sheet [START_REF] Hasemann | The crystal structure of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase reveals distinct domain homologies[END_REF][START_REF] Rider | 6phosphofructo-2-kinase/fructose-2,6-bisphosphatase: head-to-head with a bifunctional enzyme that controls glycolysis[END_REF]. This motif is conserved in the Podospora gene (G23LPARG28KS). Mutagenesis of G48 [START_REF] Li | Site-directed mutagenesis in rat liver 6-phosphofructo-2-kinase. Mutation at the fructose 6-phosphate binding site 24 affects phosphate activation[END_REF][START_REF] Vertommen | The ATP-binding site in the 2-kinase domain of liver 6phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Study of the role of Lys-54 and Thr-55 by site-directed mutagenesis[END_REF] in the rat liver isozyme abolished PFK-2 activity without affecting bisphosphatase activity [START_REF] Li | Site-directed mutagenesis in rat liver 6-phosphofructo-2-kinase. Mutation at the fructose 6-phosphate binding site 24 affects phosphate activation[END_REF] and a comparable result was obtained when D130 (D103 in Podospora) of the Walker B motif (Z-Z-Z-Z-D where Z is any hydrophobic residue) was mutated into alanine [START_REF] Rider | Site-directed mutagenesis of rat muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: role of Asp-130 in the 2-kinase domain[END_REF]. We therefore constructed a strain whose pfkfb-1 gene carried both a mutated Walker A (L24A) and a mutated Walker B (D103A) motif. These two mutations were expected to drastically reduce the kinase activity without affecting the phosphatase activity of the corresponding protein. We show that this mutated allele does not suppress the ∆cox phenotype.

How do null alleles in the phosphatase domain of pfkfb-1 partially suppress the ∆cox phenotype?

To our knowledge, the R284C mutation of gene pfkfb-1 encoding a PFK-2/FBPase-2 in Podospora anserina is the first mutation affecting this enzyme that has been obtained in a genetic screen. All the mutations described in the mammalian isozymes have been obtained in site-directed mutagenesis studies and their effects analyzed in vitro. In S. cerevisiae, the strains PFK26 and PFK27, devoid of FBPase-2 activity and the strain FBP26 devoid of PFK-2 activity are supposed to result from crucial substitutions or major deletions that occurred during evolution in one of the two domains [START_REF] Chevalier | 6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase in Trypanosomatidae. Molecular characterization, database searches, modelling studies and evolutionary analysis[END_REF][START_REF] Rider | 6phosphofructo-2-kinase/fructose-2,6-bisphosphatase: head-to-head with a bifunctional enzyme that controls glycolysis[END_REF]. The selection of R284C as a genetic suppressor of the ∆cox respiratory mutant strain is interesting. Identification of genetic suppressors of a given mutant is a powerful way to discover unexpected interactions between functions affected in the mutant on one hand and the suppressor on the other hand.

As mentioned above, the only mutations having a robust suppressor effect on the ∆cox phenotype identified so far were those leading to the constitutive overexpression of the alternative oxidase [START_REF] Lorin | Overexpression of the alternative oxidase restores senescence and fertility in a longlived respiration-deficient mutant of Podospora anserina[END_REF][START_REF] Sellem | Mutations in two zinc-cluster proteins activate alternative respiratory and gluconeogenic pathways and restore senescence in long-lived respiratory mutants of Podospora anserina[END_REF][START_REF] Bovier | Genetic and functional investigation of Zn(2)Cys(6) transcription factors RSE2 and RSE3 in Podospora anserina[END_REF]. We show here that both mutations in the phosphatase domain (R284C and H233A) of the PFK-2/FBPase-2, lead to a constitutive expression of the aox gene and to a suppressor effect when associated with ∆cox mutation. The double mutation in the kinase domain has no effect on the aox gene expression and no suppressor effect on the ∆cox phenotype. The correlation between a strong constitutive induction of the alternative pathway and the suppressor effect was also attested at the protein level showing an increased resistance to SHAM, an inhibitor of the AOXp, and a higher steady state level of AOXp of the ∆cox strains when they carry the PFK-2/FBPase-2 mutated in the phosphatase domain. The aox gene expression is nevertheless lower in pfkfb-1 R284C and ∆pfkfb-1 (7-12 x more than in wild type, Fig. 4) than in the rse mutant strains (25-50x more than in wild type, [START_REF] Sellem | Mutations in two zinc-cluster proteins activate alternative respiratory and gluconeogenic pathways and restore senescence in long-lived respiratory mutants of Podospora anserina[END_REF]) and correlates with a lower suppression effect. Previous results strongly suggest that RSE2 and RSE3 are the main, if not the only, transcriptional regulators of the aox gene [START_REF] Bovier | Genetic and functional investigation of Zn(2)Cys(6) transcription factors RSE2 and RSE3 in Podospora anserina[END_REF]. They are highly conserved in the filamentous ascomycetes P. anserina, N. crassa and A. nidulans. In addition to being required for the expression of the aox gene in the three species [START_REF] Bovier | Genetic and functional investigation of Zn(2)Cys(6) transcription factors RSE2 and RSE3 in Podospora anserina[END_REF]Chae et al., 2007b;[START_REF] Suzuki | Reprogramming of carbon metabolism by the transcriptional activators AcuK and AcuM in Aspergillus nidulans[END_REF], they have been shown to control the expression of the gluconeogenic genes encoding fbpase-1 and pck-1 in P. anserina [START_REF] Bovier | Genetic and functional investigation of Zn(2)Cys(6) transcription factors RSE2 and RSE3 in Podospora anserina[END_REF][START_REF] Sellem | Mutations in two zinc-cluster proteins activate alternative respiratory and gluconeogenic pathways and restore senescence in long-lived respiratory mutants of Podospora anserina[END_REF] and A. nidulans [START_REF] Suzuki | Reprogramming of carbon metabolism by the transcriptional activators AcuK and AcuM in Aspergillus nidulans[END_REF] and of a number of other genes that contribute to various adaptative strategies and defense mechanisms in P. anserina [START_REF] Bovier | Genetic and functional investigation of Zn(2)Cys(6) transcription factors RSE2 and RSE3 in Podospora anserina[END_REF] or involved in energetic metabolism in N. crassa [START_REF] Qi | Alternative Oxidase Transcription Factors AOD2 and AOD5 of Neurospora crassa Control the Expression of Genes Involved in Energy Production and Metabolism[END_REF]. We demonstrate here that the mutations in the phosphatase domain of pfkfb-1 not only confer higher expression of the aox gene but also of fbpase-1 and pck-1 and of two other RSE-target genes supporting the hypothesis that these mutations lead to the activation of the RSEs transcription factors. Various mechanisms that might control the activation of transcription factors are conceivable: nuclear localization, ligand binding, post-translational modification…. The RSEs orthologs in N. crassa (Chae et al., 2007b;[START_REF] Qi | Alternative Oxidase Transcription Factors AOD2 and AOD5 of Neurospora crassa Control the Expression of Genes Involved in Energy Production and Metabolism[END_REF] and A. nidulans [START_REF] Suzuki | Reprogramming of carbon metabolism by the transcriptional activators AcuK and AcuM in Aspergillus nidulans[END_REF] have been shown to be constitutively localized in the nucleus and a large body of data suggest that heterodimerization of the two proteins is essential for activation. These data support the idea that the activation of these transcription factors is determined by signal(s) to the heterodimer when there are modifications of the respiratory function or availability of carbon sources. The nature of the signal(s) is not currently known though malate has been suggested as an inducing molecule for the RSEs orthologs in A. nidulans [START_REF] Suzuki | Reprogramming of carbon metabolism by the transcriptional activators AcuK and AcuM in Aspergillus nidulans[END_REF].

We have proposed that the activation of the RSEs is different according to inducer (acetate or antimycin A containing medium). In the presence of antimycin A, induction of genes such as aox is strong whereas that of genes such as fbpase-1 and pck-1 is weak. On acetate as the sole carbon source, induction of fbpase-1 and pck-1 is strong and that of aox much less ( [START_REF] Bovier | Genetic and functional investigation of Zn(2)Cys(6) transcription factors RSE2 and RSE3 in Podospora anserina[END_REF] and this work). These data strongly suggest that different signals arising from different growth conditions can differentially affect the transcription of the RSE-target genes.

We show here that in the mutants of the phosphatase domain of pfkfb-1, the level of expression of the RSE-target genes is comparable to that in the wild-type grown on acetate. In addition, we show that in these mutants, acetate has only a slight, or no inducer effect on the expression of genes of the second category, whereas antimycin A is still able to induce the expression of genes of the first category. Altogether, these observations support the hypothesis that in the phosphatase mutants of pfkfb-1, the RSEs are under an activated form similar or close to which they adopt in the wild-type strain grown on acetate with the transcriptional activation of the gluconeogenic enzymes.

In mammalian tissues, the balance between the kinase and the bisphosphatase reactions of PFK-2/FBPase-2 determines the cellular content of Fru-2,6-P2 that is a potent allosteric positive effector of PFK-1 and inhibitor of FBPase-1, and thus stimulates glycolysis 20 20 [START_REF] Rousseau | Mammalian 6-phosphofructo-2-kinase/fructose-2,6bisphosphatase: a bifunctional enzyme that controls glycolysis[END_REF][START_REF] Van Schaftingen | Fructose 2,6-bisphosphate[END_REF]. Bisphosphatase-dead mutants are therefore expected to increase the level of Fru-2,6-P2 and stimulate glycolysis (Fig. 2). This effect could, in addition of the overexpression of the AOX, participate to the suppressor effect of the phosphatase mutants, by compensating partially the ATP deficit of the ∆cox mutant.

However, if glycolysis is in fact, activated in the phosphatase-dead mutants, there is then an apparent discrepancy between this stimulation and the activation of the RSEs that, under their active form, increase the expression of the gluconeogenic gene fbpase-1. In contrast to mammals, where the level of Fru-2,6-P2 was shown to be the main control element in carbohydrate metabolism [START_REF] Rousseau | Mammalian 6-phosphofructo-2-kinase/fructose-2,6bisphosphatase: a bifunctional enzyme that controls glycolysis[END_REF][START_REF] Van Schaftingen | Fructose 2,6-bisphosphate[END_REF], the physiological importance of Fru-2,6-P2 in yeast is not clear and contradictory results have been presented for the regulation of glycolytic flux rates by Fru-2, 6-P2 [START_REF] Boles | Cloning of a second gene encoding 5phosphofructo-2-kinase in yeast, and characterization of mutant strains without fructose-2,6-bisphosphate[END_REF][START_REF] Müller | Mutant studies of phosphofructo-2-kinases do not reveal an essential role of fructose-2,6-bisphosphate in the regulation of carbon fluxes in yeast cells[END_REF][START_REF] Navas | Futile cycles in Saccharomyces cerevisiae strains expressing the gluconeogenic enzymes during growth on glucose[END_REF]. Mutants affected in the amount of Fru-2,6-P2 or in the control of FBPase-1 are able to grow normally on glucose indicating that in this organism, the concentration of Fru-2,6-P2 does not regulate glycolytic flux rates during growth on glucose and that Fru-2,6-P2 does not play a major role in the prevention of futile cycling of the FBPase-1/PFK-1 pair [START_REF] Boles | Cloning of a second gene encoding 5phosphofructo-2-kinase in yeast, and characterization of mutant strains without fructose-2,6-bisphosphate[END_REF][START_REF] Müller | Mutant studies of phosphofructo-2-kinases do not reveal an essential role of fructose-2,6-bisphosphate in the regulation of carbon fluxes in yeast cells[END_REF]. Unfortunately, data in fungi other than S. cerevisiae is missing. In P. anserina, consistently with a decrease in intracellular ATP, the phosphatase-dead mutants present a reduction of the growth rate, a delay in the development of the fruiting bodies (Table 1) and alterations of the germinating mycelium. It is tempting to attribute these phenotypes to futile cycling, what focus on the unsolved and exciting question: why does abolishing the phosphatase activity of PFK-2/FBPase-2 lead to the activation of the RSE and the transcriptional activation the FBPase-1 gluconeogenic enzyme while we rather expected an increase of Fru-2,6-P2 level and thus a negative effect on gluconeogenesis? Although experimental data for this functional link are missing so far, we are convinced that the elucidation of this intriguing metabolic network will allow gaining new 21 21 insights into the control of carbohydrate metabolism in fungi.

Evolution of PFK-2/FBPase-2 in Ascomycota

In mammals, multiple tissue-specific isoforms encoded from four genes, designated PFKFB1-4, have been identified whereas in plants, molecular data suggest that only one gene encodes the bi-functional enzyme [START_REF] Nielsen | Fructose-2,6-bisphosphate: a traffic signal in plant metabolism[END_REF][START_REF] Okar | PFK-2/FBPase-2: maker and breaker of the essential biofactor fructose-2,6bisphosphate[END_REF]. In fungi, data on PFK-2/FBPase-2 have only been reported in S. cerevisiae in which three isoforms are present, but each displays only one activity, two for kinase (PFK26, PFK27) and one for phosphatase (FBP26, [START_REF] Boles | Cloning of a second gene encoding 5phosphofructo-2-kinase in yeast, and characterization of mutant strains without fructose-2,6-bisphosphate[END_REF]Kretschmer and Fraenkel, 1991;[START_REF] Paravicini | The yeast FBP26 gene codes for a fructose-2,6bisphosphatase[END_REF]. Searching Ascomycota databases, we found that all the tested Ascomycetes, except S. pombe that is evolutionary distant, contain three homologous sequences that fall into 3 subfamilies (subfamilies 1, 2 and 3, Fig. 7). FBP26 and PFK27 of S. cerevisiae can be clustered in subfamilies 1 and 3 respectively. Noticeably, all the isoforms of subfamily 2 lack the key catalytic residue RHG that is replaced by RAG (or RSG in A. oligospora). These data strongly suggest that all these isoforms are mono-functional PFK-2s. In contrast the RHG is conserved in all the isoforms of subfamily 1 and in most of the isoforms of subfamily 3 except PFK26 of S. cerevisiae. Trypanosomatidae have also been shown to contain four isoenzymes for PFK-2/FBPase-2. Two of them are predicted to be mono-functional PFK-2s and the two others mono-functional FBPase-2 enzymes respectively [START_REF] Chevalier | 6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase in Trypanosomatidae. Molecular characterization, database searches, modelling studies and evolutionary analysis[END_REF].

As mentioned earlier, it is clear that the FBPase-2 domain is homologous to the phosphoglycerate mutases, and the PFK-2 domain to several nucleotide-binding proteins. The most plausible hypothesis for the origin of PFK-2/FBPase-2 is a gene fusion followed by duplication [START_REF] Rider | 6phosphofructo-2-kinase/fructose-2,6-bisphosphatase: head-to-head with a bifunctional enzyme that controls glycolysis[END_REF]. Why the bi-functional PFK-2/FBPase-2 evolved into several mono-functional enzymes in S. cerevisiae and Trypanosomatidae is unclear. It has been proposed that expression of multiple mono-functional enzymes offers additional flexibility of metabolic regulation in the trypanosomes whose life cycles involve dramatic environmental shifts [START_REF] Chevalier | 6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase in Trypanosomatidae. Molecular characterization, database searches, modelling studies and evolutionary analysis[END_REF]. We show here that all the Ascomycetes contain several PFK-2/FBPase-2 isoenzymes. Aside from the yeasts (Saccharomycotina), two are probably bifunctional and one a mono-functional PFK-2 enzyme. Our data do not permit to draw any conclusions as to the reason why Ascomycetes contain these different isoenzymes. However, as said in the introduction, a remarkable feature of fungi is their important flexible metabolism that enables their adaptation to the available carbon source. As proposed for the Trypanosomatidae, the presence of multiple FBK-2/FBPase-2 isoforms could participate to an as yet not understood complex system of regulation of carbohydrate metabolism in these organisms. carry either the pfkfb-1 + wild type allele or the pfkfb-1 R384C (called "su" in Fig. 1) suppressor allele. The deleted ∆pfkfb-1 and mutated pfkfb-1 H233A constructed alleles in the phosphatase domain or the double mutated (pfkfb-1 L24A, D103A ) constructed allele in the kinase domain were also associated with ∆cox. ∆cox pfkfb-1 R384C and ∆cox pfkfb-1 H233A having the same phenotype only ∆cox pfkfb-1 R384C was shown. a The growth rate is given as a mean value expressed in mm/day (± standard deviation). The resistance to salicylhdroxamic acid (SHAM R ), reflecting the level of AOX expression, is given as the percentage of growth in the presence of 150µg/ml SHAM compared to the growth in absence of SHAM. The growth rate is a mean value expressed in mm/day (± standard deviation). b ++ referred to an increased pigmentation of the mycelium compared to that of the wt (+) c Female fertility (F) is determined according to the number of fruiting bodies or ascospores produced in a cross with the wild type strain used as a male. -corresponds to sterility and + to wild type fertility. d Growth on antimycin reflects a constitutive expression of AOX e Longevity is expressed in centimeters of growth (± standard deviation), measured on M2 medium on three parallel subcultures derived from two to five independent ascospores with a given genotype as previously described [START_REF] Sellem | Mutations in two zinc-cluster proteins activate alternative respiratory and gluconeogenic pathways and restore senescence in long-lived respiratory mutants of Podospora anserina[END_REF]

LEGENDS OF FIGURES

Fig. 1 .

 1 Fig. 1. Phenotypic characteristics of the different ∆cox suppressors. For each strain

Fig. 2 .

 2 Fig. 2. Alignment of PFK-2/FBPase-2 in R. norvegicus (R), H. sapiens (H), N. crassa

Fig. 3 .

 3 Fig. 3. Characteristics of the suppressor effect of mutations in pfkfb-1. Strains ∆cox

Fig. 4 .

 4 Fig. 4. Level of transcripts in the pfkfb-1 mutants relative to the wild type strain. The

Fig. 5 .

 5 Fig. 5. Level of transcripts in the pfkfb-1 R284C mutant strain grown on non inducer

Fig. 6 .

 6 Fig. 6. Level of transcripts in the pfkfb-1 R284C mutant and wild type strains grown on

Fig. 7 .

 7 Fig. 7. Phylogenetic tree of the PFK-2/FBPase-2 proteins in Ascomycetes.

  Figure 1

  Figure 2

  Figure 3

Figure 7

 7 Figure 7

TABLE 1

 1 Phenotypic characterization of the pfkfb-1 mutant strains

		wt	∆pfkfb-1	pfkfb-1 R284C	pfkfb-1 H233A pfkfb-1 L23A D103A
	GROWTH a	6.9±0.2	6.1±0.1	6.1±0.1	6.2±0.1	7.0±0.1
	PIGMENTATION b	+	++	++	++	+
	GERMINATION	+	+/-	+/-	+/-	+
	FERTILITY c	WT	delayed	delayed	delayed	WT
	ANTIMYCIN d	+/-	++	++	++	+/-
	LONGEVITY e	11.3±0.8	9.7±0.8	9.7±1.9	11.2±1.8	13.8±1.8
	a

Acknowledgements

This work has benefited from the facilities and expertise of the high throughputsequencing platform of IMAGIF (I2BC, UMR9198). We are thankful to Cécile Fairhaed, Fabienne Malagnac and Pierre Grognet for critical reading of the manuscript.