Krystyna Nahlik

Marc Dumkow

Özgür Bayram

Kerstin Helmstaedt

Silke Busch

Oliver Valerius

Jennifer Gerke

Michael Hoppert

Elke Schwier

Lennart Opitz

Lennart Opitz

Mieke Westermann

Stephanie Grond

Kirstin Feussner

Cornelia Goebel

Alexander Kaever

Peter Meinecke

Ivo Feussner

Gerhard H Braus
email: gbraus@gwdg.de

The COP9 signalosome

Keywords: COP9 signalosome, oxidative stress, cell wall remodeling, fungal development, secondary metabolism COP9 signalosome, oxidative stress, cell wall remodeling, fungal development, secondary metabolism

The COP9 signalosome mediates transcriptional and metabolic response for hormones, oxidative stress protection and cell wall rearrangement during fungal development

Introduction

The conserved COP9 signalosome (CSN) multiprotein complex is present in higher eukaryotic cells [START_REF] Wei | Arabidopsis COP9 is a component of a novel signaling complex mediating light control of development[END_REF]. CSN controls light regulation, pathogen response, pigment production and seedling survival in plants and is involved in regulation of tumor growth, inflammation, hormone signalling, oxygen homeostasis and early development in mammals. Defects in CSN result in embryonic lethality, presumbably because CSN affects various processes at the cellular level including the ubiquitin-proteasome pathway, DNA damage response, cell cycle control and gene expression [START_REF] Wei | The COP9 signalosome: more than a protease[END_REF]. The main known molecular function of CSN is the control of the activity of cullin-RING E3 ubiquitin ligases (CRLs) conveyed by the metalloprotease-containing Csn5 subunit. CSN is able to regulate

CRLs by cleaving the ubiquitin-like peptide Nedd8 from lysine residues of cullins [START_REF] Wu | Neddylation and deneddylation regulate Cul1 and Cul3 protein accumulation[END_REF]. In addition, CSN-associated proteins mediate phosphorylation and deubiquitination of substrates, including transcription factors such as plant HY5 [START_REF] Osterlund | The role of COP1 in repression of Arabidopsis photomorphogenic development[END_REF] or cJUN [START_REF] Naumann | COP9 signalosome-directed c-Jun activation/stabilization is independent of JNK[END_REF] in animals affecting their stability and activity [START_REF] Sun | Inositol 1,3,4-trisphosphate 5/6-kinase associates with the COP9 signalosome by binding to CSN1[END_REF][START_REF] Naumann | COP9 signalosome-directed c-Jun activation/stabilization is independent of JNK[END_REF]. CSN also controls transcriptional networks as a transcriptional corepressor of thyroid hormone receptor [START_REF] Tenbaum | Alien/CSN2 gene expression is regulated by thyroid hormone in rat brain[END_REF] and as a transcriptional repressor during Drosophila development [START_REF] Oron | Genomic analysis of COP9 signalosome function in Drosophila melanogaster reveals a role in temporal regulation of gene expression[END_REF]. Fungi often possess incomplete and non-essential versions of CSN, but the mold Aspergillus nidulans carries an eight-subunit CSN holocomplex [START_REF] Busch | An eight-subunit COP9 signalosome with an intact JAMM motif is required for fungal fruit body formation[END_REF]. A. nidulans CSN is required for fruit body formation but is not essential for filamentous growth or asexual sporulation [START_REF] Busch | The COP9 signalosome is an essential regulator of development in the filamentous fungus Aspergillus nidulans[END_REF].

In contrast to simple modular vegetative hyphae, the development of sexual fruit bodies results in a complex three-dimensional structure and requires formation of specialized cells and tissues. A. nidulans fruit body formation can be induced in the dark by oxygen limitation and is inhibited by light, which causes primarily airborne asexual spores to be formed [START_REF] Adams | Asexual sporulation in Aspergillus nidulans[END_REF][START_REF] Purschwitz | Functional and physical interaction of blue-and red-light sensors in Aspergillus nidulans[END_REF], Bayram et al., 2008a). CSN is essential for this from primordia emerging from nests (Fig. 1A). Within these fruit bodies, meiotic cells are enclosed by maternal envelope tissue and surrounded by nursing Hülle cells. CSN is required for the light-dependent inhibition of fruit body formation, but also for its maturation, because defective CSN results in a block in fruit body formation at the state of primordia [START_REF] Busch | The COP9 signalosome is an essential regulator of development in the filamentous fungus Aspergillus nidulans[END_REF].

In filamentous fungi, reactive oxygen species produced by NADPH oxidases provide a specific trigger for sexual development [START_REF] Lara-Ortiz | Reactive oxygen species generated by microbial NADPH oxidase NoxA regulate sexual development in Aspergillus nidulans[END_REF][START_REF] Aguirre | Reactive oxygen species and development in microbial eukaryotes[END_REF].

Endogenous lipogenic signal molecules called psi (precocious sexual inducer) factors, derived from linoleic acid, also play a role in regulation of spore development; the proportion of different psi factors controls the ratio of asexual to sexual spore production (Tsitsigiannis et al., 2004a). The reorganization of vegetative hyphae to form the specialized cells and tissues and ultimately the structure of the fruit body is accompanied by massive cell proliferation. This process requires mobilization of energy and building materials, as well as protection of the fruit body, coordinated by internal signaling. The energy accumulated during growth in cell wall glucans has to be mobilized by degrading enzymes during development, mostly α-1,3-glucanases such as MutA, expressed during sexual development in Hülle cells [START_REF] Wei | Aspergillus nidulans alpha-1,3 glucanase (mutanase), mutA, is expressed during sexual development and mobilizes mutan[END_REF]. Protecting the fruit body involves developmental synchronisation of the synthesis of specific secondary metabolites including bioactive molecules with the potential to repel fungivores (Bayram et al., 2008b[START_REF] Rohlfs | Secondary chemicals protect mould from fungivory[END_REF].

We took advantage of an organism possessing a complete non-essential CSN to investigate the molecular mechanisms of CSN and its role in A. nidulans development by a first combined transcriptome, proteome and metabolome analysis of this fungus. We found that vegetative CSN functions necessary to handle oxidative stress are linked to development.

Results

Fungal CSNE deneddylase is expressed during vegetative growth and early development

A. nidulans CSN expression was estimated by monitoring the appearance of the CsnE/CSN5 deneddylase subunit during different developmental stages (Fig. 1B). A functional Nterminal fusion of csnE to TAP tag (TAP-CsnE) was highly expressed in vegetative mycelia and expression ceased by 48 h in developmentally induced tissues. Reduction of CsnE protein levels coincided with the time point of the developmental block in ∆csn strains.

Developmental markers such as the catalase-peroxidase (CpeA-sGFP), expressed in Hülle cells [START_REF] Scherer | Aspergillus nidulans catalase-peroxidase gene (cpeA) is transcriptionally induced during sexual development through the transcription factor StuA[END_REF], only appeared at this time point (Fig. 1B), confirming that CSN expression ceases after development is initiated. The pattern of CsnE expression implies that CSN activity is restricted to vegetative mycelia and early development, prior to the appearance of the ∆csn phenotypes.

CsnE is able to assemble the CSN holocomplex only when all other subunits are present [START_REF] Busch | An eight-subunit COP9 signalosome with an intact JAMM motif is required for fungal fruit body formation[END_REF], but the levels of CsnE do not depend on the presence of other subunits.

Expression of CsnE was monitored in strains lacking CsnA/CSN1 or CsnD/CSN4, which showed the same developmental block as ∆csnE (Fig. 1C). We observed the same pattern of TAP-CsnE expression independent on the presence of the complex. Whereas TAP-CsnE ran as a single band in the presence of all other csn genes, additional lower molecular weight bands appeared in strains lacking CsnA or CsnE. This suggests that an intact CSN complex might prevent degradation of the CsnE subunit.

CSN affects transcription of 15 % of the fungal genome during development

Microarrays of csn mutants in D. melanogaster revealed an achronic gene expression pattern suggesting that CSN acts as a transcriptional co-repressor during development [START_REF] Oron | Genomic analysis of COP9 signalosome function in Drosophila melanogaster reveals a role in temporal regulation of gene expression[END_REF]. We compared fungal transcriptomes of wild type and ∆csnE mutant strains during Four time points were compared pairwise between both strains (Fig. S1): after 14 and 20 hours of vegetative growth in liquid culture (V14 and V20 respectively, before and after achieving developmental competence) and after 48 h of growth on solid medium, induced to develop either asexually in light (A48) or sexually in darkness (S48). At these timepoints mutant and wild type morphologies were similar (Fig. 2A,B).

The microarray data derived from two biological and four technical replicates revealed that the expression levels of 15.4 % of 10,546 annotated A. nidulans genes were strongly (log 2 ratio ≥ 3.0 and adjusted p value ≤ 0.01) or moderately (log 2 ratio ≥ 2 and adjusted p value ≤ 0.01) altered in the mutant throughout development (Table S1). The expression of 1,256 genes, representing 11.9 % of the genome, was moderately altered, and expression of 369 genes, representing 3.5 % of the genes on the array, was strongly altered at least at one developmental stage. The transcript levels of the remaining seven CSN subunits were similar in wild type and ∆csnE (data not shown). Only the genes showing strongly altered expression were analyzed further.

Differentially expressed genes were assigned to 11 functional categories based on available annotation or similarities to characterized proteins from other organisms (Fig. 2C). During vegetative growth, the most changes in the ∆csnE mutant were observed among genes involved in secondary metabolism and redox reactions. In developing cultures, CSN repressed many genes playing a role in protein degradation, stress response and defense, cell wall biosynthesis as well as redox reactions, which were mainly upregulated in the mutant.

At the same time, CSN activated numerous genes involved in secondary metabolism and cell wall degradation, accordingly downregulated in the mutant.

During vegetative growth CSN acts mainly as a transcriptional activator, with two to three times (at V20 and V14, respectively) as many genes downregulated than upregulated in the S1). More than half of the genes differentially expressed during vegetative growth showed parallel regulation at both time points; notably, twice as many genes were downregulated in ∆csnE at V14 than at V20. Thus, the transcriptome underwent significant changes in the absence of the COP9 signalosome even before achieving developmental competence by the fungus. This data does not support an exclusive transcriptional corepressor function of CSN, but suggests its involvement in control of both transcriptional activation and repression. Although CSN is expressed only during the early stages of development (until 24 h), the largest changes in the transcriptome were observed later in development (at 48 h), indicating delayed downstream effects on gene expression.

csnE deletion leads to changes in protein procuction reflecting impaired redox regulation

CSN-mediated deneddylation regulates the activity of cullin-based ubiquitin ligases, which should be reflected by changes in the stability of proteins directed for degradation in the proteasome. To examine this posttranscriptional role of CSN, we examined the changes in the proteome of the ∆csnE strain during vegetative growth in the time window when developmental competence is acquired (Fig. 3). After 2D gel electrophoresis of soluble protein extracts, ten spots representing eight unique proteins with significant differences in their intensities between the two strains were identified by mass spectrometry (Table S3).

Deletion of csnE significantly increased the levels of five proteins (Fig 3). They include four metabolic enzymes involved in carbon and amino acid metabolism. The branched-chain amino acid biosynthetic enzyme ketol-acid reductoisomerase and carbon metabolism enzyme pyruvate decarboxylase were described as oxidative stress targets in fungi [START_REF] Cabiscol | Oxidative stress promotes specific protein damage in Saccharomyces cerevisiae[END_REF][START_REF] Reverter-Branchat | Oxidative damage to specific proteins in replicative and chronological-aged Saccharomyces cerevisiae: common targets and prevention by calorie restriction[END_REF][START_REF] Yoo | Proteomic analysis of carbonylated proteins in twodimensional gel electrophoresis using avidin-fluorescein affinity staining[END_REF]. S-adenosylmethionine (SAM) synthetase is required for biosynthesis of cysteine, which is a precursor of the antioxidant glutathione. HMG-CoA synthase is a key enzyme in the mevalonate pathway required for synthesis of many primary and secondary metabolites. The putative monooxygenase is thought to be involved in detoxification reactions or complex metabolite modifications [START_REF] Harayama | Functional and evolutionary relationships among diverse oxygenases[END_REF].

The levels of three proteins are decreased in the csnE deletion mutant. The first two, actin and elongation factor 2 (EF-2) have also been described as targets of oxidative stress [START_REF] Cabiscol | Oxidative stress promotes specific protein damage in Saccharomyces cerevisiae[END_REF][START_REF] Reverter-Branchat | Oxidative damage to specific proteins in replicative and chronological-aged Saccharomyces cerevisiae: common targets and prevention by calorie restriction[END_REF][START_REF] Yoo | Proteomic analysis of carbonylated proteins in twodimensional gel electrophoresis using avidin-fluorescein affinity staining[END_REF]. The third protein is a peroxiredoxin, mediating protection against oxidative stress; peroxiredoxins are important cellular antioxidants identified in a wide range of organisms [START_REF] Rhee | Peroxiredoxins: a historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling[END_REF].

This proteome comparison reveals an effect of CSN on the levels of cytoplasmatic proteins sharing a connection to oxidative stress. Although putative stress-related genes were also affected by CSN at the transcriptional level, there was almost no correlation between differentially expressed genes and proteins during vegetative growth. This is not surprising as the changes in the proteome are likely due to a direct impact of CSN on protein stability, whereas the transcriptional regulation probably reflects a role of CSN in regulating the corresponding transcription factors. A notable exception is the putative monooxygenase, which is less abundant at both transcript and protein level in the ∆csnE strain and might be worth investigating further.

CSN controls stress and defense genes making the ∆ ∆ ∆ ∆csnE strain sensitive to redox imbalance

Comparative analysis of the vegetative transcriptome and proteome suggested a significant impact of CSN on stress and defense-related processes. The largest group of genes affected in the ∆csnE strain encode putative oxidoreductases of primary or secondary metabolism (Fig. S4). Genes encoding proteins of known enzymatic function were mostly assigned to the primary or secondary metabolism functional categories. Therefore the 'redox enzymes' category comprises mostly oxidoreductases where the molecular function remains to be determined. The transcriptome data suggests that CSN affects some of these genes during vegetative growth and others primarily during development.

Northern hybridization of four oxidoreductase transcripts verified this phase-specific transcriptional regulation (Fig. 4). CSN induces developmental transcription of catD gene, encoding catalase D required for oxidative stress response and expressed in Hülle cells [START_REF] Aguirre | Reactive oxygen species and development in microbial eukaryotes[END_REF]. it also activates transcription of a putative defensin gene primarily during vegetative growth. On the other hand, CSN represses a tyrosinase gene involved in protective melanin production during vegetative growth and a gene encoding a putative secondary metabolism oxygenase throughout the life cycle (Fig. 4, Table S4).

These changes in gene expression translate to marked differences in response to oxidative stress between wild type and ∆csnE strains (Fig. 5A). ∆csnE is more sensitive to menadione (which increases cellular levels of ROS) and to the thiol-oxidizing drug diamide causing redox imbalance. Diamide shifts the cellular ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG). Accordingly, GSSG had a greater limiting impact on growth of the ∆csnE strain than GSH. Treatment with either GSH or GSSG resulted in concentrationdependent appearance of red colouring in the growth medium of the mutant, and to a lesser extent in the wild type. It had been described earlier that aging csn mutant colonies also accumulate a red pigment even in the absence of any redox stress [START_REF] Busch | The COP9 signalosome is an essential regulator of development in the filamentous fungus Aspergillus nidulans[END_REF]. Under conditions inducing asexual development, low GSH concentrations led to increased number of sexual structures in all strains, whereas higher concentrations increased asexual conidiation (data not shown). The growth of the ∆csnE strain was also impaired on medium Furthermore, ∆csnE was sensitive to increased temperature (Fig. S2).

A functional CSN deneddylase is required for efficient redox and temperature stress response of A. nidulans. Both transcriptome and proteome analysis support a strong connection between oxidative stress and fungal development and point towards CSN-mediated regulation of catalase D as well as many still uncharacterized oxidoreductases in this process, ensuring resistance to a wide variety of oxidising and other stress factors.

Loss of CSN leads to significant changes of the fungal metabolome, inducing accumulation of orsellinic acid-derived metabolites and sterigmatocystin intermediates

We investigated the chemical composition of compounds producing the red colour released to the growth medium in the mutant to explore the link between oxidative stress and the mutant phenotype. The yield of the compounds increased by cultivating the wild type and ∆csnE strains on the surface of liquid medium for ten days. Whereas several high polymeric compounds could not be identified, extraction of culture filtrates with ethylacetate and thinlayer chromatography revealed many more unique red-coloured substances in the ∆csnE mutant than in the wild type (Fig. 5B). Strains grown on ammonium instead of nitrate as a nitrogen source produced highest yields. Pure compounds were isolated and five orsellinic acid-related metabolites could be identified: orcinol, diorcinol, cordyol C, violaceol I, and violaceol II (Fig. 5C, S3, S4). Four colourless substances were obtained, probably undergoing polymerization to produce yellow, orange or red coloured substances when dissolved in methanol.

The archetypal polyketide orsenillic acid is produced by enzymes encoded by a recently discovered A. nidulans gene cluster (Schroeckh et al., 2009). Indeed, several orsellinic acid synthesis genes are differentially expressed in the ∆csnE mutant (Table S4). orsB, encoding asexual conditions and orsB also under sexual conditions. In contrast, the orsA alcohol dehydrogenase gene is repressed during late vegetative growth in the mutant (V20). This suggests that the production of these secondary metabolites by the mutant is mainly a result of transcriptional derepression.

A more comprehensive untargeted metabolite fingerprinting analysis was then performed at different time points to investigate intracellular compounds. Overall, the analysis revealed more than 100 metabolite markers differing in their occurrence at least at one developmental stage between wild type and ∆csnE mutant. Intensity-based clustering by one-dimensional self-organizing maps (1D-SOMs) [START_REF] Meinicke | Metabolite-based clustering and visualization of mass spectrometry data using one-dimensional self-organizing maps[END_REF] was used to get an global overview,

shown here for marker candidates of the non-polar extracts (negative ionization mode). The intensity profiles of the marker candidates are represented by 20 prototypes separated in blocks of different intensity patterns (Fig. 6A). A first dominant block represents ∆csnEspecific marker candidates, which accumulate mainly under asexual (prototypes 6-8) or sexual conditions (prototype 13) in the mutant. A second block (prototypes 16-20) contains developmental markers specific for the wild type and downregulated in ∆csnE strain.

Prototypes 10-12 and 14-15 can be combined to a block representing mutation-independent markers specific for asexual and/or sexual development.

Prototype 7 revealed several markers, identified as intermediates of the sterigmatocystin (ST) biosynthetic pathway and strongly accumulated in the ∆csnE strain at A48 time point (Fig. 6B, Table S6). Accordingly, transcription of genes of ST and ST-like pathways, presumably involved in the biosynthesis of polyketide secondary metabolites [START_REF] Brodhun | Twenty-five coregulated transcripts define a sterigmatocystin gene cluster in Aspergillus nidulans[END_REF], was upregulated in the mutant at this time (Table S4). , 1996), stcW encodes a monooxygenase synthesizing 1´-hydroxyversicolorone [START_REF] Keller | Requirement of monooxygenase-mediated steps for sterigmatocystin biosynthesis by Aspergillus nidulans[END_REF], the stcU-encoded versicolorin reductase catalyses demethyl-ST production, and stcV encodes another norsolorinic acid reductase with a predicted role in reduction of averufanin. The function of stcC is still unknown.

We next analysed secretion of sterigmatocystin to the growth medium and found that the ∆csnE mutant produced significantly less ST than wild type (Fig. S5). It seems that transcriptional stc gene upregulation in the mutant (Table S4) resulted in an accumulation of ST intermediates (Fig. 6B), but also in a defect in production and secretion of the end product, which normally protects developmental structures against threatening organisms in the soil.

CSN controls the ratio of psi factors acting as A. nidulans sexual hormones

Light normally stimulates formation of asexual spores and blocks sexual development in wild type A. nidulans, but not in ∆csnE. Two genes, ppoA and ppoC, encode antagonistic dioxygenases involved in biosynthesis of psi factors, lipogenic developmental hormones. A subtle balance between different psi factors is required to shift between developmental programs (Tsitsigiannis et al., 2004b). ppoA is necessary for formation of sexual and ppoC of asexual spores. Both overexpression of ppoA and deletion of ppoC shifs the developmental balance towards sexual sporulation (Tsitsigiannis et al., 2004a). ppoC expression is necessary already during vegetative growth to enable normal development, but it is present at much higher levels present in light (at A48) than in dark (at S48), which is necessary to induce asexual development in the wild type (Fig. S6). The ∆csnE mutant showed a strong upregulation of the ppoA gene in light (at A48), accompanied by a reduction in ppoC expression (Table S4) not only during development but also in early vegetative growth phase, prior to any visible morphological change (Fig. 4, Table S4). We went on to investigate whether these differences in ppoA and ppoC expression are affecting the actual psi hormone factor levels, which would confirm an early role of CSN in regulation of psi factor biosynthesis (Figs. S6 andS7). Three oxylipins, the PpoA products 8hydroxy-9-octadecanoic acid (8-HOE) and 8-hydroxy-9,12-octadecadienoic acid (8-HOD) (Brodhun et al., 2009a) as well as the PpoC product 10-hydroxy-9,12-octadecadienoic acid (10-HOD) (Brodhun et al., 2009b) could be detected in cultures. Concentration of PpoCdependent 10-HOD required for asexual sporulation is reduced at all time points in the absence of CsnE, which is the most likely cause of the increased initiation of sexual structures by the mutant (Fig. 7A). Concentration of PpoA-derived 8-HOD is similar in wild type and mutant strains during vegetative growth, but reduced in the mutant during development (Fig. 7B), whereas levels of 8-HOE are reduced in the mutant during late vegetative growth and asexual development (Fig. 7C). The ratio of both PpoA-derived psi factors, 8-HOE and 8-HOD, is important for the balance between sexual and asexual spore development (Tsitsigiannis et al., 2004b). Sexual and asexual development are characterized by different 8-HOE to 8-HOD ratios; 1: 8.3 under sexual conditions as compared to 1: 2.7

under asexual conditions (Table 1). High ppoA expression in the ∆csnE mutant perturbs this balance resulting in a constantly high 8-HOD proportion both in light (1:18.7) and in darkness (1:15.1), which is likely the driving factor behind uncontrolled initiation of sexual structures.

Our data indicates that CSN is required prior to initiation of development during vegetative growth, as well as at early asexual developmental stages, to ensure the right balance of sexual hormones. This is critical for the expression of the master transcriptional regulator of asexual sporulation brlA, which was indeed downregulated in the mutant in light (Table S4). The brlA-dependent wA encoding a polyketide synthase producing the conidial yellow pigment was also downregulated (Table S4).

CSN controls the activation of cell wall degrading enzymes including β β β β-glucanases

The main metabolic transcriptional networks involved in carbon metabolism were not significantly affected in the ∆csnE mutant. However, expression of a large group of genes associated with cell wall degradation was very strongly dependent on a functional CSN. They were mainly downregulated in the mutant after the initiation of development, comprising 21% and 39% of all downregulated genes at A48 and S48, respectively (Table S2, Table S5).

This group of genes encode chitinases, glucanases and enzymes involved in degradation of plant cell wall material, such as cellobiose hydrolases (Table S5). Strikingly, 32 of the 37 glycosyl hydrolases present in the A. nidulans genome were differentially expressed at least at one developmental stage and 14 were downregulated during development. Northern hybridization confirmed that ∆csnE was unable to express the cell wall degrading αglucanase gene mutA during sexual development (Fig. 4B). Surprisingly, only one more αglucanase gene was affected, while CSN had most impact on genes encoding putative βglucanases with a potential to hydrolyse both β-1,3and β-1,4-glucans (Table S5).

Laminarinase (β-1,3-glucanase) activity was reported to increase in developing cultures [START_REF] Zonneveld | Sexual differentiation in Aspergillus nidulans: the requirement for manganese and its effect on alpha-1,3 glucan synthesis and degradation[END_REF][START_REF] Bagga | Developmentally related changes in the production and expression of endo-beta-1,4-glucanases in Aspergillus nidulans[END_REF], but there was no indication up to date that it is required for sexual development.

We measured cytoplasmic laminarinase activity and confirmed that it was first induced after 48 h of development. The presence of CsnE was necessary for this induction during both asexual and sexual development (Fig. S8). During metabolite fingerprinting we detected accumulation of various disaccharides, including maltose, galactose, galactosamine and Nacetylgalactosamine during sexual and asexual development in the wild type strain, most likely as a result of high glucanase and chitinase gene expression (Table S6). Developmental downregulation of αand β-glucanases in the ∆csnE mutant, concomitant with upregulation of a chitin synthase gene (Table S5), resulted in a reduced accumulation of disaccharides.

Discussion

The underlying cause of embryonic lethality in higher eukaryotes deficient in CSN is a crucial question in the field of COP9 signalosome research. We used a filamentous fungus which can survive without CSN, but is impaired in sexual development as a model to investigate the role of COP9 signalosome in development. This work represents the first comprehensive genome-wide transcriptome, proteome and metabolome analysis of both the COP9 signalosome function and of fungal development. As a result we were able to identify multiple processes depending on an intact CSN during the differentiation of the complex multicellular structure of the fruit body (Fig. 8). It is feasible that the role in some of these processes, which are conserved on a molecular level in multicellular eukaryotes, is making CSN indispensable for plant and animal development. The presence of CSN is required to ensure resistance to oxidative stress, the right concentration of sexual hormones, protection of the fruit body (including the production of toxic secondary metabolites) and mobilization of cell wall glucans during development.

A connection between CSN and oxidative stress response was observed in higher eukaryotes, where CSN5 binding partners accumulate under hypoxic conditions. CSN5 associates with several redox-sensitive transcription factors such as AP-1 [START_REF] Abate | Redox regulation of fos and jun DNA-binding activity in vitro[END_REF], NF-κB [START_REF] Galter | Distinct effects of glutathione disulphide on the nuclear transcription factor kappa B and the activator protein-1[END_REF]), p53 (Bech-Otschir et al., 2001) and with the antioxidative enzyme thioredoxin [START_REF] Hwang | Thioredoxin modulates activator protein 1 (AP-1) activity and p27Kip1 degradation through direct interaction with Jab1[END_REF]. Plant seedlings of CSN mutants accumulate the antioxidant anthocyanin which is upregulated by high levels of light-induced ROS [START_REF] Dohmann | Loss of the CONSTITUTIVE PHOTOMORPHOGENIC9 signalosome subunit 5 is sufficient to cause the cop/det/fus mutant phenotype in Arabidopsis[END_REF]. Protection against oxidative stress is required for fungal development, because a transient increase in ROS levels acts as an internal signal for developmental progression [START_REF] Aguirre | Reactive oxygen species and development in microbial eukaryotes[END_REF]. This resistance, involving the expression of catD catalase gene (Fig. 4), is dependent on the COP9 signalosome, and while not essential for normal growth it seems to set the stage for development by allowing the cell to quench the ROS signal and endogenous antioxidants is emerging as a shared feature in the regulation of multicellular development [START_REF] Covarrubias | Function of reactive oxygen species during animal development: passive or active?[END_REF][START_REF] Matsuzawa | Redox control of cell fate by MAP kinase: physiological roles of ASK1-MAP kinase pathway in stress signaling[END_REF].

The A. nidulans csnE mutant strain develops Hülle cells, but cannot develop sexual structures beyond the stage of primordia. Hülle cells have a nursing function and supply the developing fruit bodies with nutrients. Developmental progression is also accompanied by specific expression of catalase peroxidase and NADPH oxidase (NoxA) in Hülle cells [START_REF] Scherer | Aspergillus nidulans catalase-peroxidase gene (cpeA) is transcriptionally induced during sexual development through the transcription factor StuA[END_REF]. These enzymes lead to the production of superoxide and H 2 O 2 . Genes for Nox enzymes are restricted to fungi with the potential for fruit body formation, but are common in higher eukaryotes. Fungal ∆noxA strains are blocked at the same primordial state [START_REF] Lara-Ortiz | Reactive oxygen species generated by microbial NADPH oxidase NoxA regulate sexual development in Aspergillus nidulans[END_REF] as csn deletion strains. Thus, the most apparent effect of CSN is involved in the only ROS-dependent developmental transition known so far in fungi. In mammalian development, the first ROS-induced programmed cell death occurs in the blastocyst, causing apoptosis of inner cells destined to develop into trophectoderm [START_REF] Parchment | The implications of a unified theory of programmed cell death, polyamines, oxyradicals and histogenesis in the embryo[END_REF].

Strikingly, mouse embryos lacking Csn5 survive to the blastocyst state but die soon after implantation without undergoing gastrulation [START_REF] Tomoda | Multiple functions of Jab1 are required for early embryonic development and growth potential in mice[END_REF] suggesting a corresponding involvement of COP9 in ROS-mediated signaling. During the acquisition of developmental competence in A. nidulans a protective peroxiredoxin PrxB (AN10223) is expressed in emerging Hülle cells of wild type, but not of the ∆csnE strain (data not shown).

The PrxB murine homologue Prdx6 plays an important role in antioxidant defense in the lungs and protects the lens epithelium from UV-induced cell death [START_REF] Manevich | Peroxiredoxin 6, a 1-Cys peroxiredoxin, functions in antioxidant defense and lung phospholipid metabolism[END_REF][START_REF] Kubo | Protein expression profiling of lens epithelial cells from Prdx6-depleted mice and their vulnerability to UV radiation exposure[END_REF]. It would be interesting to see if mammalian Prdx6 is also targeted by CSN.

Impaired oxidative stress response resulting in the accumulation of unusual secondary metabolites becomes apparent in the ∆csnE mutant already during vegetative growth. Oxidative stress responses require catalase, superoxide dismutase and peroxiredoxin enzyme activities as well as thioredoxin and glutathione as redox buffering systems [START_REF] Aguirre | Reactive oxygen species and development in microbial eukaryotes[END_REF]. Increased levels of S-adenosylmethionine synthetase (required for glutathione biosynthesis) and of HMG-CoA synthase levels in the mutant suggest increased isoprenoid biosynthesis presumably to produce protective carotenoids [START_REF] Iigusa | Oxygen and hydrogen peroxide enhance light-induced carotenoid synthesis in Neurospora crassa[END_REF], providing an additional indication of elevated oxidative stress (Fig. 3).

The red-coloured compounds conspicously released into the medium by the csn mutant are more complex than we had anticipated (Fig. 5, S3, S4). They include several orsellinol-and orsellinic acid-derived compounds that have only recently been assigned to A. nidulans when cocultivated with a competitor organism, Streptomyces hygroscopicus (Schroeckh et al., 2009). Production of these metabolites depends on a physical interaction of A. nidulans and S. hygroscopicus and comprises a putative defense mechanism induced by the exchange of reactive oxygen species. Secretion of these phenolic compounds by the hypersensitive mutant could also be interpreted as a visible constititutive reaction to oxidative stress. Moreover, gene expression of several oxidoreductases and a polyketide synthase in several yet unexplored gene clusters involved in the production of unknown secondary metabolites is upregulated in the ∆csnE strain (data not shown), suggesting an even broader fungal metabolic repertoire in response to stress.

The presence of CSN during vegetative growth is prerequisite for developmental processes accompanied by rearrangement of the cell wall. This requires the activity of numerous cell wall degrading enzymes and a major adjustment of the membrane transport systems to shuttle metabolites. ∆csnE strain showed a marked decrease in expression of genes involved in cell wall and membrane-associated functions (Table S5), indicating that a functional CSN is required for cell wall remodelling. In particular, it is necessary for induction of β-glucanase activity targeting β-1,3-glucans and possibly also β-1,4-glucans (Fig. S8), which appear to be degradation of cell wall α-glucans is necessary for fruit body formation in A. nidulans [START_REF] Zonneveld | Morphogenesis in Aspergillus nidulans. The significance of a alpha-1, 3-glucan of the cell wall and alpha-1, 3-glucanase for cleistothecium development[END_REF][START_REF] Zonneveld | Alpha-1,3 glucan synthesis correlated with alpha-1,3 glucanase synthesis, conidiation and fructification in morphogenetic mutants of Aspergillus nidulans[END_REF]. β-glucans belong to an alkali-insoluble fraction of the Aspergillus cell wall and were thought to be responsible mostly for its structural framework.

Production of endo-β-1,4-glucanases is induced during cleistothecial development [START_REF] Bagga | Developmentally related changes in the production and expression of endo-beta-1,4-glucanases in Aspergillus nidulans[END_REF], but a requirement for β-glucanase activity for fruit body formation had not been described. The extent of cell wall remodelling necessary for fruit body formation might therefore be much greater than previously anticipated.

Other genes involved in cell wall remodelling are also affected in the ∆csnE mutant, which is consequently unable to accumulate decomposition products of αand β-glucans as well as of chitins during development. Concomitant upregulation of putative genes responsible for synthesis of α-glucan and chitin suggests that CSN is either directly or indirectly required for cell wall recycling during development at many levels. Accumulation of mono-and disaccharides which is impaired in the mutant (Table S6) might be an early trigger for the initiation of later steps of sexual development that are blocked in the ∆csnE mutant.

Synthesis of secondary metabolites by fungi is intimately related to development; toxins such as ST have been proposed to protect against fungivores [START_REF] Rohlfs | Secondary chemicals protect mould from fungivory[END_REF]. CSN appears to be required for production of secondary metabolites coinciding with development. The ∆csnE mutant produces almost no ST, which is accompanied by a misregulation of genes in the ST biosynthesis pathway and an accumulation of various ST intermediates during development.

CSN was also necessary for the expression of genes encoding cell wall proteases (Table S5)

and protective structural components of the spore wall, mainly hydrophobins (Table S7).

Taken together, these data suggest that CsnE is crucial in coordination of multiple pathways A. nidulans ∆csnE strain is unable to complete and regulate sexual development, and constitutively develops sexual Hülle cells and primordia. CSN-dependent secondary metabolites also include psi factors, hormone-like oxylipins, which have direct sporogenic effects and regulate the balance of asexual and sexual spore formation in A. nidulans. Control of psi synthesis depends on at least two ppo genes encoding psi factor-producing fatty acid oxygenases [START_REF] Tsitsigiannis | Three putative oxylipin biosynthetic genes integrate sexual and asexual development in Aspergillus nidulans[END_REF]. PpoA and PpoC have antagonistic functions, whereby PpoC ultimately promotes asexual and PpoA sexual sporulation (Brodhun et al., 2009a). CSN is required for the expression of ppoC in early vegetative stage, which probably contributes to the mutant's inability to inhibit sexual development under asexually inducing conditions such as light. Indeed, misregulation of ppoA and ppoC results in an imbalance of psi factor biosynthesis. Constitutive ppoC downregulation is reflected by permanently low levels of the PpoC-derived 10-HOD during vegetative growth (Fig. S7). Decreased 10-HOD concentation at an early vegetative stage, when developmental competence is not yet achieved, seems to be the underlying cause of the light insensitive phenotype of the mutant. Moreover, the ratio of the PpoA-derived psi factors 8-HOE to 8-HOD is essential for the timing and balance of asexual and sexual differentiation (Tsitsigiannis et al., 2004a). The lower the ratio, the more the developmental balance shifts towards asexual differentiation. Constitutively increased ppoA expression in the mutant results in the distortion of the 8-HOE:8-HOD ratio (Table 1), further enhancing the shift of developmental balance towards sexual differentiation.

The effect of CSN on hormone signals could account for the lack of transcriptional activation of the asexual master regulatory gene brlA by light in the ∆csnE mutant. The BrlA transcription factor initiates a cascade of additional regulatory genes including the AbaA transcription factor which further activates brlA and numerous downstream genes 22 [START_REF] Andrianopoulos | The Aspergillus nidulans abaA gene encodes a transcriptional activator that acts as a genetic switch to control development[END_REF]. CSN is known as a negative regulator of stability of transcription factors in other systems, such as JY5 in plants and p53 in mammals. The stability of these proteins is modulated by CSN-regulated degradation by the ubiquitin system [START_REF] Bech-Otschir | COP9 signalosome-specific phosphorylation targets p53 to degradation by the ubiquitin system[END_REF][START_REF] Schwechheimer | Multiple ubiquitin ligase-mediated processes require COP9 signalosome and AXR1 function[END_REF]. It is currently unknown whether CSN regulates the stability or activity of developmental transcription factors in A. nidulans.

However, genes encoding components of the ubiquitin system were upregulated in the mutant during development and include several putative F-box proteins (Table S7, Fig. 4A), which convey target specificity in SCF ubiquitin ligases, a prototypical group of cullin-RING E3 ubiquitin ligases (CRLs). Analysis of promoter regions of genes downregulated during development in the ∆csnE mutant revealed statistically overrepresented binding sites for AbaA transcription factor [START_REF] Andrianopoulos | The Aspergillus nidulans abaA gene encodes a transcriptional activator that acts as a genetic switch to control development[END_REF]; McmA, a homologue of yeast transcription factor Mcm1involved in sexual development [START_REF] Passmore | A protein involved in minichromosome maintenance in yeast binds a transcriptional enhancer conserved in eukaryotes[END_REF]; and stress response elements (STREs) interacting proteins [START_REF] Marchler | A Saccharomyces cerevisiae UAS element controlled by protein kinase A activates transcription in response to a variety of stress conditions[END_REF] (Fig. S6). These factors do not appear to be regulated by CSN on a transcript level, suggesting that it might be acting instead by regulating their protein stability, which remains to be investigated experimentally.

We assume that most of the effects observed in the fungal ∆csnE mutant result from the lack of deneddylase activity, removing Nedd8 ubiquitin-like peptide from CRLs. Deneddylation regulates the activity of CRLs, which are disassembled and can be reassembled only after another round of neddylation [START_REF] Cope | Role of predicted metalloprotease motif of Jab1/Csn5 in cleavage of Nedd8 from Cul1[END_REF]. We hypothesise that the wide scope of vegetative and developmental CSN functions in A. nidulans reflects the regulation of various CRLs fulfilling regulatory roles in different regulatory networks and time points. As additional function, it has been shown in N. crassa that CSN is also involved in the circadian clock [START_REF] He | The COP9 signalosome regulates the Neurospora circadian clock by controlling the stability of the SCFFWD-1 complex[END_REF]. The identity of neddylated proteins involved in the control of A. of substrates are also involved in these processes [START_REF] Sun | Inositol 1,3,4-trisphosphate 5/6-kinase associates with the COP9 signalosome by binding to CSN1[END_REF][START_REF] Naumann | COP9 signalosome-directed c-Jun activation/stabilization is independent of JNK[END_REF].

In

Microarray RNA extraction, labelling and hybridisation

RNA was extracted from 2 ml of ground fungal mycelium with Trizol (Invitrogen), followed by a double phenol/chlorophorm extraction [START_REF] Chomczynski | A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples[END_REF]. The RNA was then treated with DNAse I for 20 min at 37 °C in the presence of RNAseOUT™ (Invitrogen). RNA was extracted with phenol/chlorophorm/isoamyl alcohol and additionally purified using RNeasy columns (Qiagen, Hilden, Germany). For each array hybridisation, 1 µg RNA was amplified using the Message Amp II kit (Ambion, Darmstadt, Germany) and 2 µg resulting aRNA was labeled with NHS-activated Cy-3 or Cy-5 dyes (Amersham Bioscience). RNAs from two independent biological replicates were analysed and four technical replicates were performed.

Data analysis

Microarray intensity data were extracted using the 'Automatic Image Processing for Microarrays' software [START_REF] Katzer | Methods for Automatic Microarray Image Segmentation[END_REF]. Normalization of the raw intensity data was performed with a non-linear loess regression method. Differentially expressed genes were identified by an ANOVA fixed effects model [START_REF] Landgrebe | Efficient two-sample designs for microarray experiments with biological replications[END_REF]. Normalization and statistical computation was performed for two independent data sets derived from a high gain and a low gain scan. 'Contrasts' refer to log 2 normalized intensity ratios between wild type and ∆csnE samples. Genes with log 2 ratios ≥ 3.0 and adjusted p values ≤ 0.01 were regarded as significantly regulated and genes with log 2 ratios ≥ 2 and adjusted p values ≤ 0.01 were regarded as moderately regulated.

Two-dimensional electrophoresis, protein visualization and image analysis

70 µg of protein were treated with chloroform and methanol, pelleted and resolved in rehydration buffer to a final volume of 350 µl. The mixture was used for the hydration of IPG strips, followed by the multistep focusing protocol using the IPGphor (GE Healthcare Life Sciences, Munich, Germany).

After the first dimension, the individual strips were equilibrated for 60 min. The second dimension was performed on homogeneous polyacrylamide gels, which were silver stained and analyzed with PDQuest (Bio-Rad Industries, München, Germany). Extracts of three independent cultures were analyzed with at least two replicate gels for each.

In gel digestion and nano-ESI-MS

Tryptic peptides were separated by water-acetonitrile gradients on Dionex-NAN75-15-03-C18-PM columns on an ultimate-nano-HPLC system (Dionex, Sunnyvale, CA, USA). Online ESI-MS/MS2 spectra were generated on an LCQ-DecaXP plus mass spectrometer (Thermo Finnegan, San Jose CA, USA).

Summary of metabolite analysis

Untargeted metabolic fingerprinting was performed by Ultra Performance Liquid Chromatography (UPLC) coupled with time-of-flight mass spectrometry (TOF-MS).

Sterigmatocystin extracts were analysed by thin layer chromatography. Psi factor composition was analysed by gas chromatography mass spectrometry (GC MS). Isolated

Supplementary information

A.nidulans strains (Table S8), plasmids (Table S9) primers (Table S10

  control and its defects result in 'blind' organisms constitutively forming initial sexual tissues. Mature A. nidulans fruit bodies are closed cleistothecia, developing

  in setting up the stage for the light-mediated decision between asexual and sexual development by ensuring the appropriate ratio of psi factor hormone-like molecules.CSN also appears to protect the fungus during the internal reactive oxygen species (ROS) signalling triggering development. Furthermore, in absence of CSN we found dramatic changes in secondary metabolism and redcued expression of glucanases necessary to mobilize sugars stored in cell wall during development.

  the effects of CSN on gene expression at different developmental stages.

  development-inducing conditions, the number of up-and downregulated genes was similar (Table

  ethanol as an inducer of oxidative stress (data not shown).

  orsC, encoding a tyrosinase are upregulated in the ∆csnE strain under

  Fig 6C summarises the correlation between ∆csnE metabolome data and the corresponding upregulated genes of the A. nidulans ST gene cluster: stcE encodes a putative norsolorinic acid reductase producing averantin (Brown et

  the importance of CsnE in regulating accumulation of cell wallderived sugars acting both as a reserve for energy-consuming developmental processes and materials for cell wall remodeling.CSN-dependent cellular remodelling also involves membrane transport, which is apparent by major changes in expression of membrane transport related genes induced by csnE deletion (Fig.2C). The second largest group of genes differentially expressed at all developmental stages (up to 8 %) fell into this 'transport' category. They encoded proteins facilitating membrane transport of different metabolites, predominantly carbohydrates, amino acids and toxins. This might be an additional reason why only the wild type accumulated β-glucan and chitin decomposition products, which provide building blocks for restructuring cell wall during development.

  . ROS-mediated signaling balanced by protection conferred by

  α-glucans during development. The accumulation and subsequent

  protection of newly produced sexual structures against various external threats.

  nidulans development and secondary metabolism remains to be explored. Another open CSN-associated proteins mediating phosphorylation and deubiquitination

  The TIGR A. nidulans microarrays were provided by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA. The polymer-coated glass arrays contained 11,481 70-mer oligonucleotide probes spotted in duplicate, providing 98% genome ,701 predicted genes. The slides were prehybridised in 5× SSC, 0.1 % SDS, 1 % BSA for 1 h at 65 °C. After washing in distilled water and isopropanol the slides were dried in nitrogen stream. 1.5 µg (ca. 130 pmol) of each labeled RNA was hybridised to the slides using the Agilent '60-mer Oligo Microarray Processing' protocol. The slides were washed and dried in a nitrogen stream. Following hybridisation, spot intensities were determined using an Agilent G2505B microarray scanner.

  were analysed by HPLC. Details are described in supplementary material.

Fig. 1 .

 1 Fig. 1. Fungal CsnE/CSN5 protein expression during development.

Fig. 2 .Fig. 3 .

 23 Fig. 2. CSN-dependent transcriptome during fungal development.

Fig. 4 .

 4 Fig. 4. Comparison of CSN effect on gene expression by Northern and microarray analysis.

Fig. 5 .

 5 Fig. 5. ∆csnE strain has impaired resistance to oxidative stress and produces an unusual

  metabolite production of wild type and ∆csnE under grown in NH 4 -medium (NH 4 ) or NO 3 -medium (NO 3 ) in dark (d) or light (l). 1-5 indicate the identified orcellinic acid derivates. C. Structures of the identified substances: 1: orcinol; 2: violaceol II; 3: violaceol I; 4: cordyol C; 5: diorcinol.

Fig. 6 .

 6 Fig. 6. CSN-dependent metabolite fingerprinting during fungal development.

Fig. 7 .Fig. 8 .

 78 Fig. 7. Amounts of the PpoA and PpoC-derived psi factors 8-HOE, 8-HOD and 10-HOD

F

  Figure 1 160x99mm (300 x 300 DPI)

  summary, we demonstrate that CSN exerts a broad role, both during vegetative growth when it is crucial for determining later developmental fate and during development itself. Our data suggest a much greater extent of cell wall remodelling prior to fruit body formation than previously recognized in fungi. The broad metabolic repertoire of CSN-deficient strain confirms the fungal potential for producing novel secondary metabolites under stress revealed by recent research. Our data also provide evidence for a crucial biological role of CSN in regulating redox homeostasis of eukaryotic cells, especially during processes dependent on ROS signaling. Combined deficiencies in these functions, although not essential for vegetative growth of a single cell or modular organism under laboratory conditions, might be the reason why the development of multicellular plant or mammalian embryo, or a fungal fruit body cannot be completed. Our comprehensive analysis opens further avenues into research on the molecular functions of the COP9 signalosome in filamentous fungi, both as a way to explore novel natural products and as a model of mammalian development.

	Materials and methods
	A. nidulans culture
	A. nidulans strains were culitvated at 37 °C or 30 °C in minimal medium and fungal
	development was induced as described (Clutterbuck, 1974). Briefly, asexual sporulation was
	induced by incubation in continuous white light and cleistothecia formation by oxygen-
	limiting conditions on plates sealed with cellophane film grown in the dark. Oxidative stress F r o assays were performed with 4% ethanol, menadione (0.04, 0.06, 0.08, 0.10 mM), diamide
	(0.8, 1.2, 1.4, 1.6 mM), GSH or GSSG (5, 10, 15, 20 mM), added to the medium. Growth, P conidiation and cleistothecia development of A. nidulans was observed by differential e r e interference contrast microscopy (DIC).
	R
	e
	v i e
	w

  The data are deposited in NCBI's Gene Expression

	rabbit anti-calmodulin binding protein epitope tag (Upstate/Milliore, Schwalbach, Germany),
	mouse anti-GFP (Clontech-Takara Bio Europe, Saint-Germain-en-Laye, France) or rabbit
	anti-actin C11 peptide (Sigma-Aldrich, Steinheim, Germany) antibodies.
	F o r
	P
	e
	e r
	R
	e
	v i e
	Omnibus; http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=pluhlwwaocgqmpa&acc=GSE22442 w
	and were generated conforming to the MIAME guidelines. Gene probes were annotated and
	assigned to functional categories manually by homology searches via translated nucleotide
	BLAST algorithm (blastx). Each gene probe was assigned to one functional category only.
	Immunoblotting
	15-20 µg of crude extracts were separated by SDS-PAGE, electroblotted and hybridized with
	26

  ), complete molecular biology, protein extraction, electron microscopy, chemical extraction and analysis procedures as well as full characteristics of the metabolites produced by the A. nidulans ∆csnE strain are listed in the supplementary information.

	F o r F o r F o r F o r F o r F o r F o r F o r
	P P P P P P P P
	e e e e e e e e
	e r e r e r e r e r e r e r e r
	R R R R R R R R
	e e e e e e e e
	v i e v i e v i e v i e v i e v i e v i e v i e
	w w w w w w w w

Table 1 .

 1 Ratios of oleic to linoleic acid-derived psi factors in the A. nidulans ∆csnE mutant and the wild type at different developmental timepoints

	Time point	strain	8-HOE:8-HOD 1
	V14	∆csnE	1 : 6,2
		wild type	1 : 4,8
	V20	∆csnE	1 : 8,2
		wild type	1 : 19,6
	A48	∆csnE	1 : 18,7
		wild type	1 : 2,7
	S48	∆csnE	1 : 15,1
	r o	wild type	1 : 8,3
	P
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		R
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			39

1 8-HOE: 8-hydroxy oleic acid; 8-HOD: 8-hydroxy linoleic acid

Molecular Microbiology

Acknowledgements

We thank B. Könnecke, P. Meyer and M. Landesfeind for experimental or bioinformatic support, S. Freitag for technical support and S. Krappmann for plasmid pSK341. Our work has been supported by grants from the Deutsche Forschungsgemeinschaft, the Volkswagen-Stiftung and the Fonds der Chemischen Industrie. K. Nahlik was supported by a Lichtenberg scholarship.