Role of nitric oxide synthase in the light-induced development of sporangiophores in Phycomyces blakesleeanus.

Blue light controls the development of sporangiophores in the zygomycete Phycomyces blakesleeanus Burgeff. Light represses the production of microsporangiophores and enhances the development of macrosporangiophores. Inhibition of the biosynthesis of tetrahydrobiopterin, a cofactor of NO synthase, inhibits this photomorphogenesis. Light induces production of citrulline from arginine in the mycelium and in sporangiophores. The citrulline-forming activity is dependent on NADPH, independent of calcium, and inhibited by NO synthase inhibitors. It is reduced in tetrahydrobiopterin-depleted mycelium. Light induces emission of NO from the developing fungus in the same order of magnitude as citrulline formation from arginine. The NO donor sodium nitroprusside can replace the light effect on sporangiophore development, and inhibitors of NO synthase repress it. We suggest that a fungal NO synthase is involved in sporangiophore development and propose its participation in light signaling.

Indications for the occurrence of nitric oxide synthases in fungi and plants and the involvement in photoconidiation of Neurospora crassa.

Indications for the occurrence of nitric oxide synthases in Dictyostelium, Neurospora, Phycomyces and the leguminous plant Mucuna hassjoo as well as a physiological role of nitric oxide in Neurospora crassa are demonstrated. An exogenous nitic oxide donor, sodium nitroprusside, inhibited light-stimulated conidiation in N. crassa. Specific inhibitors of nitric oxide synthase, like the arginine derivatives NG -nitro-L-arginine (L-NA) and NG-nitro-L-arginine-methyl ester (L-NAME), enhanced conidiation in darkness nad in the light, whereas the stereoisomer D-NAME was inactive. This communication reports to our knowledge the first time the presence of enzymatic activity of nitric oxide synthase in fungi and a higher plant and an effect of nitric oxide in fungal photo-physiology.

Somatic hybrids between the cultivated potato Solanum tuberosum L. and the 1EBN wild species Solanum pinnatisectum Dun.: morphological and molecular characterization.

Interspecific somatic hybrids between the 1EBN-wild species Solanum pinnatisectum (S. pnt) and four different diploid breeding lines of Solanum tuberosum (S. tbr) were produced by electrofusion. S. pnt exhibits resistance to Phytophthora infestans and Erwinia blackleg. Somatic hybrids were identified by RFLP analysis using the oligonucleotide (GATA)4 as a probe. In three of four combinations all regenerates obtained were somatic hybrids. All 86 somatic hybrids between the breeding line H256/1 and S. pnt were analyzed in detail with respect to morphological and molecular characters; 50% of the somatic hybrids showed normal intermediate leaf morphology. Tubers of somatic hybrid plants grown in the greenhouse as well as in the field were evenly shaped and remarkably similar to those of the S. tbr breeding line. Analysis of relative DNA content by flow cytometry revealed that 75% of the somatic hybrids were tetraploid, some were hypotetraploid and others polyploid or mixoploid. Slotblot and RFLP analyses were carried out using repetitive and some single-copy DNA probes. The genome portion of the S. tbr breeding line was determined by slot-blot analysis using the species-specific repetitive probe pSA287. Obviously, most somatic hybrids contain the complete genomes of both fusion partners. In some of the somatic hybrids, a significantly lower intensity of the S. pnt-specific hybridization signal indicated a certain degree of asymmetry.

Homology cloning of GTP-cyclohydrolase I from various unrelated eukaryotes by reverse-transcription polymerase chain reaction using a general set of degenerate primers.

GTP-cyclohydrolase I is the primary enzyme of tetrahydrobiopterin and folic acid biosynthesis. cDNA fragments of GTP-cyclohydrolase I were obtained from rainbow trout, chicken, the fungi Neurospora crassa, Phycomyces blakesleeanus and Saccharomyces cerevisiae, the cellular slime mold Dictyostelium discoideum, the phytoflagellate Euglena gracilis and the higher plant Mucuna hassjo using primers specific for conserved regions of the open reading frame and the reverse transcription polymerase chain reaction (RT-PCR) technique. A number of regions were found to be strictly conserved between unrelated eukaryotes. These regions may be essential for the function of GTP-cyclohydrolase I and are discussed with respect to the recently resolved crystal structure of the Escherichia coli enzyme.

Biosynthesis of pteridines in Neurospora crassa, Phycomyces blakesleeanus and Euglena gracilis: detection and characterization of biosynthetic enzymes.

Occurrence, biosynthesis and some functions of tetrahydrobiopterin (H4biopterin) in animals are well known. The biochemistry of H4biopterin in other organisms than animals was hitherto not widely investigated. Recently H4biopterin was found in the phytoflagellate Euglena gracilis, in the zygomycete Phycomyces blakesleeanus and in the ascomycete Neurospora crassa. In Euglena, Neurospora and Phycomyces the enzymatic activities of GTP cyclohydrolase I, 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase are detectable and the biosynthesis follows the same steps as were shown for animals. The biosynthetic enzymes, however, show a much lower sensitivity to those inhibitors that act on vertebrate enzymes. 2,4-Diamino-6-hydroxypyrimidine as inhibitor of GTP cyclohydrolase I and N-acetylserotonin or N-methoxyacetylserotonin as inhibitors of sepiapterin reductase can decrease pteridine biosynthesis significantly, in vitro and in vivo. The apparent Km values are in general higher when compared with the respective animal enzymes. In Neurospora, the conversion of GTP to dihydroneopterin triphosphate was closely associated with subsequent production of 6-hydroxymethyl-7,8-dihydropterin due to the high activity of dihydroneopterin aldolase, different from all other tested organisms. Investigations involving inhibition of pteridine synthesis might be a useful tool for evaluating the hypothesis that pteridines in fungi and plants are co-chromophores of various blue light-dependent, flavin-containing photoreceptors.

Molybdenum cofactor biosynthesis in Neurospora crassa: biochemical characterization of pleiotropic molybdoenzyme mutants nit-7, nit-8, nit-9A, B and C.

Available mutants of molybdenum cofactor (MoCo) biosynthesis of Neurospora crassa were studied for converting factor activity and for in vitro molybdate repair of nitrate reductase (NR) activity. Mutant nit-7 was found to contain an activity that fits the functional definition of converting factor activity in Escherichia coli. Its high molecular weight fraction converts a low molecular weight compound from nit-1 and nit-8 into biologically active molybdopterin (MPT). Like nit-1, mutant nit-8 is devoid of this activity. Mutants nit-9 A, B and C contain a protein-bound precursor form of MoCo, which is presumed to be MPT bound to apo-NR. It is converted into active MoCo as part of NR in the presence of reduced glutathione and high exogenous molybdate concentrations. The NR apoenzyme of nit-1 is needed to detect the total amount of MoCo after molybdate repair, because mutants nit-9 A, B and C build no detectable content of functional NR apoenzyme. Evidence is presented for the transfer of MPT from demolybdo-NR to free NR apoenzyme.

A specific oligonucleotide of the 5S rDNA spacer and species-specific elements identify symmetric somatic hybrids between Solanum tuberosum and S. pinnatisectum.

The nucleotide sequences of the 5S rRNA genes (5S rDNA) of two Solanum tuberosum breeding lines (R1 and B15) and of the Mexican wild species S. pinnatisectum were determined and compared with each other and to the 5S rDNA of other Solanaceae species (Lycopersicon esculentum, Nicotiana rustica and Petunia hybrida). The 5S rDNA repeats of the Solanum species are 324-329 bp in length, and they exhibit 91-95% sequence identity. Sequence variability is mainly located in a short region of the spacer separating the 5S rRNA coding regions. A synthetic 28-mer oligonucleotide constructed according to this region can be used as a specific hybridization probe to distinguish symmetric somatic hybrids between S. tubersosum breeding line B15 and S. pinnatisectum produced by protoplast fusion. Interestingly, the two Solanum breeding lines R1 and B15 differ also in this spacer region.

Photostimulation of conidiation in mutants of Neurospora crassa.

Various mutants of Neurospora crassa were screened for light-stimulated conidiation which is a blue light effect and, at least in strain albino-band, is mediated by the flavoprotein nitrate reductase (NR). NR- mutants showed practically no photoconidiation under standard conditions. However, in fusion products of nit-1 (diaphorase activity present, terminal activity missing) plus nit-3 (terminal activity present, diaphorase activity missing), NR activities and photoconidiation were partially restored. Mutants with altered light sensitivities, such as white collar WC-1 and light-insensitive lis-2 and lis-3, had normal NR activities and their conidiation was promoted by light, whereas WC-2 and lis-1 responded only slightly. These two mutants showed low NR activities especially when grown on solid medium which might be the cause of their blindness. Experiments with NR- mutants indicated that nitrite reductase might also act as a blue light photoreceptor.

Method for identification of intracellular free flavin species in the photosensitive fungus Neurospora crassa.

Establishing the relative intracellular proportions of flavins in Neurospora crassa (and in other organisms) in vivo may be hampered by degradation of flavins after homogenization of the cells. The system described here allows separation and identification of intracellular free and bound flavins under conditions restrictive for the FAD-degrading enzyme(s). A "protective buffer" containing 0.1 M citrate adjusted to pH 4.0 with K2HPO4, 5 mM ATP, and 0.5 mM EDTA prevents FAD from rapid enzymatic cleavage in crude cell lysates of the Neurospora crassa mutant "slime."

Nitrate reductase activity test: phenazine methosulfate-ferricyanide stop reagent replaces postassay treatment.

Nitrate reductase activity is usually measured by colorimetric determination of the nitrite formed. Since reduced pyridine nucleotides interfere with color formation, the use of NADPH or NADH in the assay requires a specific postassay treatment to remove excess substrate. A "stop mix" containing 1.5 mM phenazine methosulfate and 4.0 mM ferricyanide (final concentrations 0.136 and 0.36 mM, respectively) can remove excess NAD(P)H and terminate the enzymatic reaction quickly in a single, time-saving step. For activity tests containing dithionite we recommend the use of a 1:1 mixture of the two color reagents to avoid incomplete color formation. This may occur during longer time intervals between addition of the color reagents due to destruction of the diazonium salt formed with the first reagent by oxidation product(s) of dithionite.