BLUE LIGHT RESPONSES IN NITRATE REDUCTASE MUTANTS OF NEUROSPORA CRASSA

Abstract— The role of nitrate reductase in the blue light responses of photosuppression and phase shifting of circadian conidiation was studied in Neurospora crassa. The photoresponses, as assayed in three nitrate reductase mutants (nit-1,nit–2 and nit-3), showed no significant differences as compared to the responses in a strain which could utilize nitrate. In addition, the responses occur on a medium which (i) represses nitrate reductase activity due to the presence of ammonium ion and arginine or (ii) results in the production of an inactive enzyme due to the presence of tungsten. Nitrate reductase appears to be of no or secondary importance as a photoreceptor in the responses studied.     

Blue light induces circadian rhythms in the bd mutant of Neurospora: double mutants bd,wc-1 and bd,wc-2 are blind

This paper describes a new blue light effect for Neurospora crassa, the photoinduction of circadian rhythms in the bd mutant. The wc-1 and wc-2 genes are necessary for this effect.     

Photoinduction of albino-3 gene expression in Neurospora crassa conidia.

The synthesis of carotenoids is induced by blue light in Neurospora crassa mycelia, while in conidia (the vegetative spores) the accumulation of carotenoids also occurs in the dark. The expression of the albino-3 (al-3) gene (coding for the carotenogenic enzyme geranyl-geranyl pyrophosphate synthetase) in isolated conidia was analysed. The level of al-3 mRNA was shown to be increased in light-induced wild type (wt) conidia. This light response was elicited by blue light and was under the control of the white collar-1 (wc-1) and white collar-2 (wc-2) gene products. This indicates that the blue-light photoreceptor and the light transduction pathway which activate al-3 gene expression in mycelia are probably the same as in conidia.     

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.     

CORRELATION BETWEEN ABSORBANCE CHANGES AND A PHYSIOLOGICAL RESPONSE INDUCED BY BLUE LIGHT IN NEUROSPORA

Abstract— Absorbance changes due to the photoreduction of a b -type cytochrome are associated with many biological blue light-controlled processes. Evidence is presented for their causal relationship with the perception of light which induced conidia formation under conditions of starvation in Neurospora crassa mutant albino band, but not with light-induced phase shifts of conidiation bands.     

PHOTOACTIVATION OF NITRATE REDUCTASE FROM NEUROSPORA CRASSA

Abstract— The photoactivation of nitrate reductase from Neurospora crassa was studied in partially purified extracts. The inactive enzyme [inactivated by reduction in the presence of potassium cyanide] could be reactivated by chemical oxidation with ferricyanide or by irradiation with blue light. The enzyme contains a short electron transfer chain consisting of flavin adenine dinucleotide, cytochrome b ₅₅₇ and molybdenum which normally transfers electrons from reduced pyridine nucleotide to nitrate. This overall activity, which was negligible in the inactive enzyme, was restored to approximately 70% of the ferricyanide control by irradiation. However, nitrate reduction using reduced methylviologen as reducing power, which was also negligible in the inactive enzyme, was photoactivated to 100%. The diaphorase activity of the enzyme mediated by the flavin adenine dinucleotide, which was fully active in the inactivated enzyme, was inhibited approximately 30% by the irradiation treatment. The action spectrum for photoactivation showed that a flavin was the photoreceptor chromophore. Photoactivation occurs only in the presence of oxygen.     

Light Effects on Cyclic Nucleotide Levels and Phase Shifting of the Circadian Clock in Neurospora crassa

Abstract— Mycelia of Neurospora crassa (band [bd] mutant) were exposed to white light (blue light intensity 3.5 μ.mol s^?1 m^?2) of different durations during constant darkness. The concentrations of different second messenger molecules and the phase shifting of the circadian rhythm were determined during light exposures at circadian time (ct) 12 and thereafter. These light exposures elicited 8-12 ct units delay phase shifts but did not change the amount of inositol 1,4,5-triphosphate (InsP₃). In contrast, significant effects of light were observed on cyclic adenosine 3′,5′-monophosphate (cAMP) levels, which increased transiently about 30-90 s after the onset of light. The same kinetics was observed under continued exposure to light as well as after 10 s and 2 s of light followed by darkness. The relative amount of the cAMP-dependent protein kinase A (PKA) in the form of its catalytic sub-units was determined in isolated nuclei of the bd strain in relation to total nuclear proteins by means of western blot analysis, using a heterologous antibody. The nuclear PKA content changed parallel to the cAMP changes. The transient increase of cAMP did not occur in the “blind”white collar mutant (wc-2). Long-term kinetics of cAMP changes after different light pulses in bd showed the initial increase and a return to control levels about 10 min after the onset of the light pulse and a subsequent longer lasting decrease. Light-induced cAMP changes and light-induced phase shifts showed different duration dependencies, thus indicating that cAMP may not play a role in the signal transduction pathway to the clock. Light exposures, furthermore, led to a significant decrease of the cyclic guanosine 3′,5′-monophosphate (cGMP) level. Long-term kinetics of the cGMP content again showed the immediate decrease after 2 min and a slow recovery to (or above) control values after several hours. Various calcium channel blockers (nickel, cobalt, nifedipine, dantrolene, lanthanum) and the calcium calmodulin inhibitor chlorpromazine did not affect the phase shifting by light nor did they significantly phase shift the circadian rhythm in the dark themselves (again determined at ct 12). The data showed that InsP₃ did not change after exposure to light, whereas cAMP increased and cGMP decreased. There was no evidence, however, that these changes play a role in light signal transmission to the clock.     

Light-induced dephosphorylation of a 33 kDa protein in the wild-type strain of Neurospora crassa: the regulatory mutants wc-1 and wc-2 are abnormal

Light induces the dephosphorylation of a 33 kdalton protein within 8 min in the wild-type strain of Neurospora crassa. The regulatory mutants, wc-1 and wc-2, have an altered pattern of phosphoproteins in darkness and also after irradiation. Because the wc genes have previously been implicated in photodifferentiation (F. Degli Innocenti and V. E. A. Russo, Genetic analysis of blue light-induced responses in Neurospora crassa, in H. Senger (ed.), Blue Light Effects in Biological Systems, Springer-Verlag, Berlin, Heidelberg, 1984, pp. 213-219), we suggest that protein dephosphorylation may constitute a necessary step in the light-transduction chain of Neurospora crassa.     

Oxidative cleavage of cellulose by fungal copper-dependent polysaccharide monooxygenases

Fungal-derived, copper-dependent polysaccharide monooxygenases (PMOs), formerly known as GH61 proteins, have recently been shown to catalyze the O(2)-dependent oxidative cleavage of recalcitrant polysaccharides. Different PMOs isolated from Neurospora crassa were found to generate oxidized cellodextrins modified at the reducing or nonreducing ends upon incubation with cellulose and cellobiose dehydrogenase. Here we show that the nonreducing end product formed by an N. crassa PMO is a 4-ketoaldose. Together with isotope labeling experiments, further support is provided for a mechanism involving oxygen insertion and subsequent elimination to break glycosidic bonds in crystalline cellulose.     

PHOTOREDUCTION OF CYTOCHROME b551 OF PARTIALLY PURIFIED NEUROSPORA NITRATE REDUCTASE VIA ITS INTERNAL FLAVIN

Abstract— Nitrate reductases (NR) from NR-normal Neurospora crassa mutant albino band and from NR-defective mutantsnit–1 andnit–3 were isolated and partially purified in order to test the photo-reducibility of their cytochrome b ₅₅₇ via the NR-internal FAD. Photoreducibility with blue light of the isolated enzyme was observed as absorbance increase at 423, 524 and553–557 nm. It was independent of NADPH-nitrate reductase activity and could be induced if the dissociable FAD was not lost in the isolation procedure. The photoreduction of cytochrome b ₅₅₇ was readily reversible due the high autoxi-dation rate of this cytochrome. Therefore, anaerobic conditions are required for photoreduction with low light intensities. If aerobic conditions are applied, high intensities become necessary to overcome the simultaneous cytochrome h₅₅₇ oxidation.     

INHIBITION OF RESPIRATION AND LOSS OF MEMBRANE INTEGRITY BY SINGLET OXYGEN GENERATED BY A PHOTOSENSITIZED REACTION IN NEUROSPORA CRASSA CONIDIA

Abstract— It was found that killing of Neurospora crassa conidia by singlet molecular oxygen generated by a photosensitized reaction in the presence of toluidine blue O, accompanied inhibition of respiration and uptake of the photosensitizing dye by conidia. Both the inactivation of respiration and the uptake of toluidine blue O were stimulated by deuterium oxide and suppressed by azide. Mitochondria of irradiated conidia were swollen and showed matrix of low density under an electron microscope.
These results suggest that singlet molecular oxygen induces the inhibition of respiration and the loss of membrane integrity of Neurospora conidia.     

CIRCADIAN RHYTHM OF Robinia pseudoacacia LEAFLET MOVEMENTS: ROLE OF CALCIUM AND PHYTOCHROME

Abstract— The effect of external calcium level, calcium ionophore A23187 and red light on the circadian rhythm of Robinia pseudoacacia leaflet movements has been studied. Fifteen minute red light pulses shifted the phase of leaflet rhythmic movement with a phase-response curve type 0. Maximum advances and delays (about 10 h and 8 h, respectively) were obtained between circadian time (CT) 10 and CT 12 at the end of a subjective day. An almost null effect was obtained at the end of a subjective night. Phytochrome is the photoreceptor involved in phase shifting since this effect of red light is reversed by 5 min of far red light. Two hour pulses of external calcium, applied as CaCl₂ (10 m M ), and 2 h pulses of calcium ionophore A23187 (10–50 μM) also shifted the phase of leaflet circadian movement and caused the same type of phase-response curve, with maximum advances and delays at the same time as those produced by red light. Two hour pulses of an external calcium chelator, EGTA (5 m M ), and a calcium channel blocker, LaCl₃ (10–50 m M ), damped the circadian rhythm or did not change the phase when they were applied at lower concentration. These results indicate that phytochrome could control the circadian oscillator, which drives Robinia leaflet movements by increasing the intracellular calcium concentration.     

PHOTOINDUCTION OF PROTOPERITHECIA IN NEUROSPORA CRASSA BY BLUE LIGHT

Blue light induces the formation of Neurospora crassa protoperithecia.This photoinduction is completed in less than 24 h. Its threshold is about 4.2 J/m². Red light is ineffective. The Bunsen-Roscoe law is obeyed at the fluence of 12.6 J/m² for fluence rates from 5.25 × 10 ² to 1.05 W/m².     

The opposed effect of 5-azacytidine and light on the development of reproductive structures in Neurospora crassa

Blue light inhibits the formation of asexual cycle spores (conidia) and stimulates the development of the sexual (female) reproductive structures (protoperithecia) in the nitrogen-starved mycelium of Neurospora crassa. The DNA methylation inhibitor, 5-azacytidine (3-300 microM), opposed the effect of light by suppressing the protoperithecia formation and stimulating a conidiation. The addition of 300 microM 5-azacytidine inhibited protoperithecia formation in the dark-cultivated mycelium by about two orders of magnitude and activated conidiation in the light-exposed mycelium by almost three orders of magnitude. Both in the dark-cultivated and the irradiated mycelium treated with various 5-azacytidine concentrations, the yield of conidia and protoperithecia demonstrated an inverse relationship. We suggest that DNA methylation and blue light are involved in the organism's selection of sexual or asexual reproductive cycle.     

TEMPERATURE-SENSITIVITY OF LIGHT-INDUCED PHASE SHIFTING OF THE CIRCADIAN CLOCK OF NEUROSPORA

Two new mutants of Neurospora craasa , designated hth-1 and hth-2 , have been isolated which allow clear expression of the circadian conidiation rhythm at high temperature (36°C). Both strains showed single-gene segregation and produced similar phenotypes but mapped to different genetic loci. These mutants allowed an analysis of the effect of temperature on (1) light-induced phase-shifting of the circadian rhythm, (2) period length of rhythm, and (3) growth rate. The amplitude of the phase response curve to light was drastically reduced as the temperature was increased from 25°C to 34°C. Phase advances were decreased more than phase delays. As previously reported (Sargent et al. , 1966), the period length of the rhythm is temperature-compensated below 30°C ( Q ₁₀˜ 1) but not well-compensated above 30°C ( Q ₁₀ 1.3–1.7). The decrease in amplitude of the light phase response curve occurred in both temperature ranges. Furthermore, the Q ₁₀ value was lowered by addition of yeast extract in the high temperature range but not in the low range. Q ₁₀ values for growth rate also differed in these strains both in the low temperature range (25–30°C) and the high temperature range (30–34°C).     

A third member of the photolyase/blue-light photoreceptor family in Drosophila: a putative circadian photoreceptor

Two photolyases, specific for cyclobutane pyrimidine dimers and (6-4) photoproducts, have been reported in Drosophila. These enzymes share extensive sequence homologies with the plant blue-light photoreceptor. We have now identified a third gene in Drosophila melanogaster with extensive sequence homology to the photolyase gene. The newly identified gene, which we named dCRY, was expressed as a recombinant protein and tested for photolyase activity. The recombinant protein exhibited photochemical properties similar to those of Drosophila pyrimidine dimer and (6-4) photolyases but lacked photolyase activity. In light of recent evidence that blue-light photoreceptors regulate the circadian clock in mammals, we propose that dCRY is the circadian photoreceptor in this organism.     

Fast induction of translatable mRNA by blue light in Neurospora crassa wt: the wc-1 and wc-2 mutants are blind

After blue-light irradiation of Neurospora crassa (wt) mycelia we observed an increase of about 13 translatable mRNA species within a period of 30 min. The induction of translatable mRNA species followed a specific temporal pattern which permitted the identification of four distinct classes. One of the translatable mRNAs was induced in less than 2 min, while the others showed lag periods of 5, 10 or 20 min from the beginning of illumination. The white collar mutants, wc-1 and wc-2, which do not display any of the blue-light-induced physiological effects tested until now were found to be defective for the photoinduction of translatable mRNAs.     

INACTIVATION OF NORMAL AND MUTANT NEUROSPORA CRASSA CONIDIA BY VISIBLE LIGHT AND NEAR-UV: ROLE OF 1O2, CAROTENOID COMPOSITION AND SENSITIZER LOCATION

Abstract— Inactivation of Neurospora crassa conidia from wild-type and mutant strains by visible and near-UV light has been investigated in the presence and absence of photosensitizing dyes. Inactivation by near-UV is virtually unchanged by the presence of deuterium oxide or azide suggesting that, contrary to the situation with visible light and photosensitizing dyes, ¹O₂ is not involved in any substantial way in the formation of lethal lesions. The finding that carotenoid deficient strains are similar to wild-type strains in sensitivity to near-UV inactivation is consistent with ¹O₂ not being involved.
Photodynamic inactivation of conidia by visible light occurs in the presence of methylene blue (MB), toluidine blue O (TB), or acridine orange (AO). Carotenoid deficient strains are more sensitive to such inactivation only when MB and TB are used. These results support the contention that MB and TB mediated damage involves the cell membrane where carotenoids are available for quenching, whereas AO mediated damage occurs in the nucleus sequestered from the protective influence of carotenoids.
A newly isolated, lemon–yellow mutant, mapping to the al -1 locus, exhibits sensitivities to photodynamic inactivation similar to other pure-white mutants at the same locus. The sensitivity of this pigmented mutant is apparently related to insufficient unsaturation (seven to nine double bonds) of the two colored carotenoids, zeta–carotene and neurosporene, produced by the mutant.     

Chitosan as a macroaffinity ligand purification of chitinases by affinity precipitation and aqueous two-phase extractions

(1) Chitosan was found to be a suitable macroaffinity ligand for affinity precipitation of chitinase from Neurospora crassa, cabbage and puffballs. (2) The activity recoveries of 85, 82 and 90% with concomitant fold purifications in terms of specific activities were 27, 15 and 30 with N. crassa, cabbage and puffballs and were obtained with affinity precipitation. These results were obtained with clarified extracts/homogenates as the starting materials. (3) The incorporation of chitosan in poly(ethylene glycol) (PEG)-salt aqueous two-phase system allowed purification of chitinases from these sources directly from unclarified extracts/homogenates. (4) The 96% (w/v) chitosan (of initially introduced into the aqueous two-phase system) partitioned into PEG-phase and this enhanced the partitioning of chitinases into PEG-phase. The chitosan, free as well as bound to chitinases, could be separated from PEG-phase by increasing the pH to 7. (5) By the process of desorption with 2.0 M MgCl2, 86, 80 and 88% activity recoveries (% expressed in terms of total units of enzyme activities in the crude extract) were obtained in the case of N. crassa, cabbage and puffballs, respectively. The corresponding fold purifications in terms of specific activities were 34, 20 and 38. (6) The purified preparations gave single bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the estimated molecular masses agreed with the reported values in the literature.