The biphasic fluence response of carotenogenesis in Neurospora crassa: Temporary insensitivity of the photoreceptor system

Whether or not illumination is continuous or interrupted during the span in which increasing illumination time periods (i.e., increasing fluences) have no different effect on carotenogenesis is optional, in regard to the amount of carotenoids produced (revealing the plateau of the biphasic fluence response curve). This indicates temporary insensitivity. When the time delay between the onsets of the initial and second illumination is extended beyond the expanse of the plateau, the amount of carotenoids induced by the second illumination depends on the time elapsed following the first exposure; after ca. 2 h, maximum competence for a second induction is completely restored. On the other hand, the amount of carotenoids induced by a second illumination also depends on the duration of this second illumination, but, unlike the dependence in a single illumination, results in a different fluence response curve for the second exposure. When UV-A is used for induction, the refractory period which follows the first exposure seems to be the same as for blue light, suggesting vision of UV-A and blue light by the same photoreceptor system.     

Relationship of photocarotenogenesis to other behavioural and regulatory responses inPhycomyces

The effect of light on carotene accumulation was studied by analyzing the -carotene content of 4--old mycelia continuously exposed to illumination of different intensities. The wild-type, mutants defective in phototropism, mutants defective in carotene regulation, and newpic mutants specifically defective for photocarotenogenesis were examined. The results indicate that photocarotenogenesis depends on a single sensory pathway which shares its earlier steps (governed by genesmadA andmadB) with the sensory pathway for phototropism. It shares its later steps (probably governed by genescarA andpicB) with one of the pathways for carotene regulation, and includes at least one specific step (governed by genepicA) not known to be involved in other responses.     

Effects of light-dark cycles on the circadian conidiation rhythm inNeurospora crassa

The effects of 24 hr light-dark cycles on the circadian conidiation rhythm inNeurospora crassa were compared among will-typefrq ⁺ and clock mutantsfrq ⁺,frq ³,frq ⁷,frq ⁹ andfrq ¹¹. The minimum length of the light period necessary for complete entrainment to the light-dark cycles was almost 2 hr infrq ⁺,frq ³ andfrq ⁷ strains. The minimum duration of the dark period necessary for the appearance of circadian conidiation was almost 4 hr in all of the strains except thefrq ¹¹ strain. The phase of the conidiation rhythm was dependent on the light to dark transition in thefrq ¹ strain in all light-dark cycles examined and in thefrq ⁺ andfrq ³ strains when the light period was shorter than 16 hr. In contrast, the phase of thefrq ⁷ strain was dependent on the light to dark transition when the light period was shorter than 10 hr.     

Transport properties of N-acyl derivatives of the coprogen and ferrichrysin classes of siderophores inNeurospora crassa

Several derivatives of the coprogen and ferrichrysin classes of siderophores were synthesized as potential affinity labels of the iron uptake system inNeurospora crassa. While only one of these compounds has proved useful as an affinity label, all were recognized and transported byNeurospora crassa. One derivative, chloroacetyl-ferrichrysin, proved to be an unexpectedly potent reversible inhibitor (K ₁=0.4 M) of both ferrichrysin and coprogen uptake, similar to the natural siderophore, ferrirubin. The reported results provide further understanding of the steric and electronic requirements of siderophores for the iron uptake system inNeurospora crassa.Abbreviations amu atomic mass units - DMF dimethylformamide - FAB tast atom bombardment - NMR nuclear magnetic resonance - ppm parts per million - tlc thin layer chromatography     

Are carotenoids the blue-light photoreceptor in the photoinduction of protoperithecia in Neurospora crassa?

A triple albino mutant of Neurospora crassa with a measured content of carotenoids absorbing at 470 nm less than 0.5% of that of the wild type (calculated value less than 8·10^-4%) had the same threshold for photoinduction of protoperithecia as the wild type when illuminated with monochromatic light at 471 nm. This is strong evidence against the hypothesis that the bulk of carotenoids are the blue-light photoreceptor for this phenomenon. However, it is impossible to exclude traces of carotenoids acting as the photoreceptor at less than 3·10^-12 M in a very efficient sensory transduction chain.Abbreviations A absorbance - al albino mutant - WT wild type     

Effect of light intensity and ammonium-N on carotenogenesis ofTrentepohlia odorata andDunaliella bardawil

AxenicTrentepohlia odorata was cultured at three different NH₄Cl levels (3.5 × 10^–2, 3.5 × 10^–3, 3.5 × 10^–4 M) and three different light intensities (48, 76, 122 µmol m^–2 s^–1). Chloride had no effect on growth over this range of concentration. High light intensity and high NH₄Cl concentration enhanced the specific growth rate. The carotenoid content increased under a combination of high light intensity and low N concentration. WhenD. bardawil was exposed to the same combination of growth conditions, there was an increase in its carotenoid content. The light saturation and the light inhibition constants (K _s andK _i, respectively) for growth, and the saturation constant (K _m) for NH₄Cl were determined. TheK _s andK _i values were higher inT. odorata (66.7 and> 122 mol m^–2 s^–1, respectively) than inD. bardawil (5.1 and 14.7 µmol m^–2 s^–1, respectively). TheK _m value determined at 122 µmol m^–2 s^–1, however, was lower inT. odorata (0.048 µM) than inD. bardawil (0.062 µM).Author for correspondence     

Calcium and tip growth inNeurospora crassa

Summary We examined the ionic regulation of tip growth inNeurospora crassa by a combination of electrophysiology and confocal microscopy. To determine if transmembrane ionic fluxes are required for tip growth, we voltage clamped the membrane from –200 to +50 mV. In this voltage range, transmembrane ionic fluxes would either reverse (e.g., K⁺) or change dramatically (e.g., Ca²⁺ influx) but had no effect on hyphal growth rates. Therefore, ionic fluxes (including Ca²⁺ influx) may not be required for tip growth. However, intracellular Ca²⁺ may still play an obligatory role in tip growth. To assess this possibility, we first increased cytosolic Ca²⁺ directly by ionophoresis. Elevated Ca²⁺ induced subapical branch initiation, often multiple tips. At hyphal tips, fluorescence ratio imaging using fluo-3 and fura-red revealed a pronounced tip-high Ca²⁺ gradient within 10 m of the tip in growing hyphae which was not observed in nongrowing hyphae. Injection of the Ca²⁺ chelator 1,2-bis(ortho-aminophenoxy)ethane-N,N,N,N-tetrapotassium acetate consistently inhibited growth concomitantly with a depletion of intracellular Ca²⁺ and dissipation of the tip-high gradient. We conclude that Ca²⁺ plays a regulatory role in tip initiation and the maintenance of tip growth. Because plasma membrane ionic fluxes do not play a role in tip growth, we suggest that the tip-high Ca²⁺ gradient is generated from intracellular Ca²⁺ stores in the ascomyceteN. crassa.Abbreviations BAPTA 1,2-bis(ortho-aminophenoxy)ethane-N,N,N,N-tetrapotassium acetate - [Ca²⁺]_i intracellular Ca²⁺ concentration - fluo-3 2,7-dichloro-6-hydroxy-3-oxo-9-xanthenyl-4-methyl-2,2-(ethylenedioxy)dianiline-N,N,N,N-tetraacetic acid     

Role of nitrogen in the photoinduction of protoperithecia and carotenoids in Neurospora crassa

Nitrogen, as KNO₃ or NH₄NO₃, can inhibit the photoinduction of protoperithecia in Neurospora crassa when present in the medium at a high concentration but does not inhibit the photoinduction of carotenoids. The point at which the presence of high nitrogen levels is no longer inhibitory is 5 h after illumination.Abbreviations al albino mutant - wc white-collar mutant - WM Westergaard and Mitchell (1947) medium Dedicated to Professor Wilhelm Nultsch in honour of his 60th birthday     

Kinetics of photoaccumulation of β-carotene in Phycomyces blakesleeanus

Upon carbon starvation the -carotene content of Phycomyces mycelium grown on minimal agar medium disappears with a time lag of about 90 min and a T_1/2 of 68–75 min. If continuous light is given 2 h after starvation, there is an increase in -carotene content with respect to the dark control. This increase has a time lag of 20–25 min. The fluence rate-response curve of wt is biphasic and two mutants in the gene madA (madA7, madA35) and in the gene madB (madB101, madB104) have higher thresholds than wt; madB mutants are blinder than madA mutants. Only blue light is effective and we suggest that it has an effect solely on the catabolism of -carotene.Abbreviations D dark - L light - wt wild type     

Carbon starvation mediated changes in carbohydrate metabolism inNeurospora crassa

Carbon starvation conditions were found to increase the activities of gluconeogenic enzymes such as malic enzyme, cytosolic malate dehydrogenase and isocitrate lyase along with proteases and inhibition in glucose catabolic enzymes such as G6P dehydrogenase and FDP aldolase inNeurospora crassa     

Inversion of gravitropism by symmetric blue light on the clinostat

Gravitropic stimulation of maize (Zea mays L.) seedlings resulted in a continuous curvature of the coleoptiles in a direction opposing the vector of gravity when the seedlings were rotated on a horizontal clinostat. The orientation of this response, however, was reversed when the gravitropic stimulation was preceeded by symmetric preirradiation with blue light (12.7 mol photons·m^–2). The fluence-response curve of this blue light exhibited a lower threshold at 0.5 mol·m^–2, and could be separated into two parts: fluences exceeding 5 mol·m^–2 reversed the direction of the gravitropic response, whereas for a range between the threshold and 4 mol·m^–2 a split population was obtained. In all cases a very strong curvature resulted either in the direction of gravity or in the opposite orientation. A minor fraction of seedlings, however, curved towards the caryopsis. Furthermore, the capacity of blue light to reverse the direction of the gravitropic response disappeared with the duration of gravitropic stimulation and it depended on the delay time between both stimulations. Thistonic blue-light influence appears to be transient, which is in contrast to the stability observed fortropistic blue-light effects.This work was supported by the Deutsche Forschungsgemeinschaft.     

Strong-light response of Micrasterias thomasiana Archer

Prostrate cells of Micrasterias thomasiana Archer were irradiated from above with intensive blue light. Many of the cells reacted by rising to a profile position. During a period of 15 to 90 min the response is linearly dependent on the duration of irradiation, inferred from the number of rising cells. In a range from 10 to 30 mol m^-2 s^-1 (equalling 2.7–8.0 W m^-2 at a wavelength of 450 nm) and for an irradiation time of 30 min, the rising reaction was linearly dependent on the quantum flux density. Choosing 30 min irradiation time and a quantum flux density of 30 mol m^-2 s^-1, the reactive number of rising cells was employed in establishing an action spectrum. As a result of this, a flavin is postulated as the light-percepting pigment in the reaction, whereas chlorophylls do not appear to be involved. The rising reaction can be distinguished from other light-induced movements as a strong-light response, resembling in this respect the movement of chloroplasts within cells. The different sensitivity of individual cells and the importance of this strong-light response for the algal cells is discussed.     

The effect of copper on histidine biosynthesis inNeurospora crassa

The inhibition of growth of a wild strain ofNeurospora crassa by Cu²⁺ is counteracted by histidine, histidine methyl ester, histidinol and Mn²⁺. In the presence of Cu²⁺, the total free amino acid content decreased by 30%. The decreased free amino acid pools of arginine, histidine and tyrosine were restored on the addition of Mn²⁺. Histidinol phosphate phosphatase showed a decrease in activity in the presence of Cu²⁺. This inhibition was reversed on the addition of excess Mn²⁺. The data suggest that copper toxicity in the mould is due to suppression of histidine biosynthesis.     

Photomovement in Dunaliella salina: Fluence rate-response curves and action spectra

We determined the action spectra of the photophobic responses as well as the phototactic response in Dunaliella salina (Volvocales) using both single cells and populations. The action spectra of the photophobic responses have maxima at 510 nm, the spectrum for phototaxis has a maximum at 450–460 nm. These action spectra are not compatible with the hypothesis that flavoproteins are the photoreceptor pigments, and we suggest that carotenoproteins or rhodopsins act as the photoreceptor pigments. We also conclude that the phototactic response in Dunaliella is an elementary response, quite independent of the step-up and step-down photophobic responses. We also determined the action spectra of the photoaccumulation response in populations of cells adapted to two different salt conditions. Both action spectra have a peak a 490 nm. The photoaccumulation response may be a complex response composed of the phototactic and photophobic responses. Blue or blue-green light does not elicit a photokinetic response in Dunaliella.Diagrams of the optical set-ups used for measuring the responses at the single-cell level and of the plans for building the phototaxometer described in this paper are available to the interested readerWe thank Mr. M. Kubota for a tremendous amount of technical assistance and Mr. R. Nagy for building the phototaxometer. We thank T. Kondo, Professor H. Imaseki and the members of the Laboratory of Biological Regulation, NIBB, for their help and support in various aspects of this research. This research was supported, in part, from grants from the Okazaki Large Spectrograph (Project Nos. 86-535, 87-518, 88-523), the Japanese Society for the Promotion of Science, and the College of Agriculture and Life Sciences at Cornell University to R. W.     

Growth and carotenogenesis in eight strains ofDunaliella salina Teodoresco from Chile

A study was made of growth and carotenogenesis in eight strains of the green algaDunaliella salina collected from salt ponds at Salar de Atacama (23° 30′ S; 68° 15′ W) and Antofagasta (23° 39′ S; 70° 24′ W), Chile and kept in unialgal cultures at the Laboratorio Cultivo de Algas, University of Concepcion. The algae were grown in Erdschreiber medium supplemented with 12.5% w/v NaCl, under a continuous photon flux density of approximately 150 μmol m^-2 s^-1 at 25 ± 4 °C without aeration. When growth reached the stationary phase, the amount of NaCl was increased to 25%. Total carotenoid content was measured during the exponential growth phase and 20 days after the addition of salt. Strain CONC-001 (Laguna La Rinconada, Antofagasta) exhibited the highest growth rate (k = 0.32 div d^-1) and the lowest total carotenoid content (7.2 and 13.7 mg l^-1 at 12.5 and 25% NaCl, respectively). Strain CONC-007 (Salar de Atacama) had the lowest growth rate (k = 0.14 div d^-1) and yielded the highest total carotenes per volume unit (23.1 and 35.6 mg 1^-1 at 12.5 and 25% NaCl) and per cell (ca. 42 pg at 25% NaCl). Total carotenoid synthesis did not increase in strains CONC-001 and CONC-006 with the increase of salinity. These strains had the greatest increase of total carotenoid-to-chlorophyll ratio (4.5- and 9.3-fold, respectively). The seven strains from Salar de Atacama had higher total carotenoid contents than the strain from Antofagasta. Cell size also varied. Strain CONC-001 cells were smallest; strain CONC-006 had the largest cells. There was an inverse correlation between maximum cell density and mean cell size.     

A single positive phototropic response induced with pulsed light in hypocotyls of Arabidopsis thaliana seedlings

The fluence-response curves were measured for phototropic curvature in response to unilateral 450-nm light in hypocotyls of Arabidopsis thaliana (L.) Heynh. These show the classical first positive (peak curvature of 9–10°), indifferent and second positive phototropic response. Reciprocity is valid only for the first positive response; the fluence requirements for its induction are similar to those for induction of the first positive phototropic response of coleoptiles. Large angles of curvature also may be induced by multiple pulses if the individual pulses are separated by an optimum dark period of about 15 min. The curvature induced by a given fluence, whether applied in continuous irradiation or a sequence of pulses, is a linear function of the duration of continuous irradiation or the duration between first and last pulse, respectively. For a given fluence applied in a sequence of pulses, reciprocity remains valid provided the duration between first and last exposure is kept constant. When the duration between first and last pulse is sufficiently long, the fluence required for high phototropic curvature falls in the first positive fluence range. These results are interpreted to indicate the existence of a kinetic limitation in the transduction sequence, and a relatively short lifetime of an initial physiologically active photoproduct. The apparent existence of more than one positive response may have resulted from these characteristics of the transduction sequence.     

Effect of visible light on carotenogenesis in arthroconidiating Trichophyton mentagrophytes

The effect of visible light on carotenoid content in the dermatophyte Trichophyton mentagrophytes ATCC 26323 was investigated. The fungus T. mentagrophytes accumulated several carotenoids when arthroconidiated on Sabouraud glucose agar at 37°C. When this fungus was irradiated with moderate fluence rates of white light, the resultant arthroconidia contained considerably less carotenoids in comparison with dark controls although growth and arthroconidiation of this fungus were not at all affected by visible light. The reduction of carotenoid content in arthroconidia was due primarily to blue light, although red light caused a slight decrease in pigmentation. The suppressive effect of visible light on pigmentation was fluence rate dependent. Carotenoid accumulation in arthroconidia was inversely and exponentially related to the fluence rate of light. Carotenoid formation in arthroconidiating T. mentagrophytes was neither photoinducible nor photostimulative. An analysis of isolated carotenoids revealed that visible light caused a quantitative reduction in pigmentation, and no single carotenoid was selectively decreased.Non-standard abbreviations PI pigmentation index - r coefficient of correlation     

Two distinct blue-light responses regulate the levels of transcripts of specific nuclear-coded genes in pea

Fluence-response characteristics for bluelight(BL)-mediated changes in the steady-state levels ofCab-, pEA215 and pEA207-RNA in red-light(RL) grown pea (Pisum sativum L. cv. Alaska) seedlings indicate the existence of two BL responses: a blue-lowfluence (BLF) response, causing an increase inCab- and pEA215-RNA, and a blue-high-fluence (BHF) response, causing a return to control levels forCab- and pEA215-RNA and a decrease in pEA207-RNA levels (Warpeha and Kaufman, 1989, Plant Physiol.,91, 1030–1035). We now show that under dark growth conditions, only the BLF response is apparent;Cab- and pEA215-RNA increase at all fluences tested, whereas pEA207-RNA levels are unaltered over the range of BL fluence tested. The treatment of dark-grown seedlings with RL immediately prior to BHF irradiation does not elicit the BHF response forCab-, pEA215 and pEA207-RNA, indicating that the role of growth in RL is to enable the seedling to reach a particular developmental state, rather than ensuring the presence of active phytochrome at the time of BL-irradiation. The apical bud of RL-grown seedlings has only the BLF response;Cab-RNA levels increase while pEA207-RNA exhibits no change at any of the fluences tested. The developing leaves of the fourth node show the BHF response; bothCab- and pEA207-RNA decrease following treatment with high-fluence BL. These data also indicate the necessity for reaching a specific developmental state before the BHF response can be activated. This research was supported by U.S. Department of Agriculture, Competitive Grants Office, grant No. 86CRCR12228 to L.S.K. K.M.F.W. gratefully acknowledges a University of Illinois at Chicago Laboratory for Molecular Biology Fellowship.     

Nitrogen regulation of amino acid catabolism in Neurospora crassa

Neurospora crassa can utilize numerous compounds including certain amino acids as a sole nitrogen source. Mutants of the nit-2 locus, a regulatory gene which is postulated to mediate nitrogen catabolite repression, are deficient in the ability to utilize several amino acids as well as other nitrogen sources used by wild type. Various enzymes involved in amino acid catabolism were found to be regulated in distinct ways. Arginase, ornithine transaminase, and pyrroline-5-carboxylate dehydrogenase are all inducible enzymes but are not subject to nitrogen catabolite repression. By contrast, proline oxidase and the amino acid transport system(s) are controlled by nitrogen repression and their synthesis is increased markedly when nitrogen source is limiting. Unlike wild type, the nit-2 mutant cannot derepress amino acid transport, although proline oxidase is regulated in a normal fashion.This work was supported by Grant R01 GM-23367 from the National Institutes of Health. T. J. F. was supported by an NIH Predoctoral Traineeship in Developmental Biology; G. A. M. is supported by NIH Career Development Award GM-00052.     

Investigation of electrophysiological responses of Neurospora crassa to blue light

The influence of light in a spectrum range of 350–500 nm 20 W m^-2 (20,000 erg · cm^-2 · s^-1) has been studied in the mycelial cells of Neurospora crassa. Light-induced input resistance and membrane potential changes can be measured by means of intracellular microelectrodes. The value of the input resistance reached maximum after a 2–5 min illumination. The maximum hyperpolarization of the cell membrane reaching 30–40 mV was observed after 20–25 min illumination, when the input resistance values did not differ significantly in the illuminated and non-illuminated cells.