Evidence from studies with acifluorfen for participation of a flavin-cytochrome complex in blue light photoreception for phototropism of oat coleoptiles

The diphenyl ether acifluorfen enhances the blue light-induced absorbance change in Triton X100-solubilized crude membrane preparations from etiolated oat (Avena sativa L. cv. Lodi) coleoptiles. Enhancement of the spectral change is correlated with a change in rate of dark reoxidation of a b-type cytochrome. Similar, although smaller, enhancement was obtained with oxyfluorfen, nitrofen, and bifenox. Light-minus-dark difference spectra in the presence and absence of acifluorfen, and the dithionite-reduced-minus oxidized difference spectrum indicate that acifluorfen is acting specifically at a blue light-sensitive cytochrome-flavin complex. Sodium azide, a flavin inhibitor, decreases the light-induced absorbance change significantly, but does not affect the dark reoxidation of the cytochrome. Hence, it is acting on the light reaction, suggesting that the photoreceptor itself is a flavin. Acifluorfen sensitizes phototropism in dark-grown oat seedlings such that the first positive response occurs with blue light fluences as little as one-third of those required to elicit the same response in seedlings grown in the absence of the herbicide. Both this increase in sensitivity to light and the enhancement of the light-induced cytochrome reduction vary with the applied acifluorfen concentration in a similar manner. The herbicide is without effect either on elongation or on the geotropic response of dark-grown oat seedlings, indicating that acifluorfen is acting specifically close to, or at the photoreceptor end of, the stimulus-response chain. It seems likely that the flavin-cytochrome complex serves to transduce the light signal into curvature in phototropism in oats, with the flavin moiety itself serving as the photoreceptor.     

Effects of acifluorfen on auxin translocation in bean seedlings

The effect of 5-(2-chloro-4-(trifluoromethyl)phenoxy)-2-nitrobenzoic acid (acifluorfen) on the translocation of the¹⁴C-labeled auxins 2,4,5-trichlorophenoxyacetic acid (2,4,5-T-1-¹⁴C) and indole-3-acetic acid (IAA-1-¹⁴C) was determined. The auxins and acifluorfen were injected into the stem at the cotyledonary node of 9-day-old bean (Phaseolus vulgaris L. cv Tenderpod) seedlings. The plants were harvested 4 h after treatment and analyses of¹⁴C were made of various plant parts. Acifluorfen increased 2,4,5-T,-1-¹⁴C translocation out of the treated area and especially into the large primary leaves. This translocation pattern is indicative of apoplastic translocation and suggests that acifluorfen inhibited vein loading of the auxins. Acifluorfen affected auxin translocation in the dark as effectively as in the light even though the herbicidal effects of acifluorfen are known to be expressed only after light treatment.Journal article no. 4403 of the Agric. Exp. Stn., Oklahoma State Univ.     

Chloroplast-Diphenyl Ether Interactions II

Acifluorfen, a p-nitrodiphenyl ether herbicide, is inhibitory to those photosynthetic functions that require a functioning chloroplast envelope. Functions involving the stroma are also affected. Acifluorfen does not lyse intact spinach chloroplasts, yet does increase the sensitivity of CO₂-dependent O₂ evolution to exogenous inorganic phosphate without directly affecting the function of the phosphate translocator. Acifluorfen penetrates into the chloroplast stroma in a light-independent fashion. Once inside, it causes the inactivation of light and dithiothreitol-activated fructose 1,6-bisphosphatase. Light-activated glyceraldehyde-3-phosphate dehydrogenase (NADP) is also inactivated by acifluorfen.

These data suggest that acifluorfen stimulates a pathway for inactivation of fructose 1,6-bisphosphatase and glyceraldehyde 3-phosphate dehydrogenase (NADP) which uses oxygen as a terminal oxidant and which involves thioredoxin and ferredoxin-thioredoxin reductase.

     

Effects of Acifluorfen on Endogenous Antioxidants and Protective Enzymes in Cucumber (Cucumis sativus L.) Cotyledons

The herbicide acifluorfen (2-chloro-4-(trifluoromethyl)phenoxy-2-nitrobenzoate) causes strong photooxidative destruction of pigments and lipids in sensitive plant species. Antioxidants and oxygen radical scavengers slow the bleaching action of the herbicide. The effect of acifluorfen on glutathione and ascorbate levels in cucumber (Cucumis sativus L.) cotyledon discs was investigated to assess the relationship between herbicide activity and endogenous antioxidants. Acifluorfen decreased the levels of glutathione and ascorbate over 50% in discs exposed to less than 1.5 hours of white light (450 microeinsteins per square meter per second). Coincident increases in dehydroascorbate and glutathione disulfide were not observed. Acifluorfen also caused the rapid depletion of ascorbate in far-red light grown plants which were photosynthetically incompetent.

Glutathione reductase, dehydroascorbate reductase, superoxide dismutase, ascorbate oxidase, ascorbate free radical reductase, peroxidase, and catalase activities rapidly decreased in acifluorfen-treated tissue exposed to white light. None of the enzymes were inhibited in vitro by the herbicide. Acifluorfen causes irreversible photooxidative destruction of plant tissue, in part, by depleting endogenous antioxidants and inhibiting the activities of protective enzymes.

     

Stimulation of conidiation by derivatives of cAMP inTrichoderma viride

Derivatives of cyclic-3′,5′-adenosine monophosphate, N⁶,2′-O-dibutyryl-cAMP, 8-(chlorophenylthio)-cAMP and 8-bromoadenosine-cAMP added at 0.1–10 μmol/L concentrations into the growth medium have markedly stimulated conidiation inTrichoderma viride. Their stimulatory effect on conidiation was best observed in colonies that were constantly kept in the dark but was also marked in colonies illuminated by sub-saturating doses of light. The relative stimulation of conidiation depended not only on the concentration of exogenous cyclic nucleotides but also on the concentration of glucose in the medium. It was more pronounced in glucose-rich media than in media where the concentration of the sugar was low. Concentrations of cAMP analogues equal to 50 μmol/L and higher inhibited the conidiation.     

Growth and conidiation ofTrichoderma viride are affected by non-steroidal antiinflammatory agents

Nonsteroid antiinflammatory agents (NSAIA's) (inhibitors of cyclooxygenase and lipoxygenase) of several structural series inhibited growth ofTrichoderma viride. The most potent growth inhibitors were indomethacin and its derivative repanidal which inhibited in the range of 0.1 mmol/L. The weakest inhibitors were acetylosalicylic acid and lysine salicylate which exerted only a weak effect at concentrations above 1 mmol/L. The inhibition of growth was accompanied by a stimulation of conidiation in the dark. A light pulse increased the efficiency of these drugs to stimulate the conidiation.Saccharomyces cerevisiae was as sensitive to NSAIA's asT. viride whileBotrytis cinerea was less sensitive. The results indicate that arachidonate metabolism may play a role in the growth of fungi and may participate also in the process of conidiation.     

Vegetative growth, aging- and light-induced conidiation ofTrichoderma viride cultivated on different carbon sources

The growth and conidiation of the agedTrichoderma viride culture grown in the dark, and after an induction by a light pulse. was examined in the presence of selected mono-, di(tri)saccharides, amino acids and alcohols as sole carbon sources. Hexoses and disaccharides, but not pentoses and amino acids, promoted proportionally both growth and conidiation induced by aging or light. All compounds but pentoses promoted the conidiation in aged cultures and photoconidiation in a close correlation. Ethanol, glycerol and ethylene glycol supported both growth and conidiation but these processes were not supported equally. Conidia formation with hexoses and amino acids as sole carbon sources seems to be a function of growth promotion, rather than of growth restriction (starvation, stress, aging). With glucose as sole carbon source the conidiation was not triggered by nutrient limitation, nor by the accumulation of waste metabolites. The aging-induced conidiation can be considered to be triggered by the genetic program of the microorganism rather than by its nutrient status.     

Physiological basis for differential sensitivities of plant species to protoporphyrinogen oxidase-inhibiting herbicides

With a leaf disc assay, 11 species were tested for effects of the herbicide acifluorfen on porphyrin accumulation in darkness and subsequent electrolyte leakage and photobleaching of chlorophyll after exposure to light. Protoporphyrin IX (Proto IX) was the only porphyrin that was substantially increased by the herbicide in any of the species. However, there was a wide range in the amount of Proto IX accumulation caused by 0.1 millimolar acifluorfen between species. Within species, there was a reduced effect of the herbicide in older tissues. Therefore, direct quantitative comparisons between species are difficult. Nevertheless, when data from different species and from tissues of different age within a species were plotted, there was a curvilinear relationship between the amount of Proto IX caused to accumulate during 20 hours of darkness and the amount of electrolyte leakage or chlorophyll photobleaching caused after 6 and 24 hours of light, respectively, following the dark period. Herbicidal damage plateaued at about 10 nanomoles of Proto IX per gram of fresh weight. Little difference was found between in vitro acifluorfen inhibition of protoporphyrinogen oxidase (Protox) of plastid preparations of mustard, cucumber, and morning glory, three species with large differences in their susceptibility at the tissue level. Mustard, a highly tolerant species, produced little Proto IX in response to the herbicide, despite having a highly susceptible Protox. Acifluorfen blocked carbon flow from δ-aminolevulinic acid to protochlorophyllide in mustard, indicating that it inhibits Protox in vivo. Increasing δ-aminolevulinic acid concentrations (33-333 micromolar) supplied to mustard with 0.1 millimolar acifluorfen increased Proto IX accumulation and herbicidal activity, demonstrating that mustard sensitivity to Proto IX was similar to other species. Differential susceptibility to acifluorfen of the species examined in this study appears to be due in large part to differences in Proto IX accumulation in response to the herbicide. In some cases, differences in Proto IX accumulation appear to be due to differences in activity of the porphyrin pathway.     

Metarhizium robertsii illuminated during mycelial growth produces conidia with increased germination speed and virulence

Light conditions during fungal growth are well known to cause several physiological adaptations in the conidia produced. In this study, conidia of the entomopathogenic fungi Metarhizium robertsii were produced on: 1) potato dextrose agar (PDA) medium in the dark; 2) PDA medium under white light (4.98 W m^?2); 3) PDA medium under blue light (4.8 W m^?2); 4) PDA medium under red light (2.8 W m^?2); and 5) minimum medium (Czapek medium without sucrose) supplemented with 3 % lactose (MML) in the dark. The conidial production, the speed of conidial germination, and the virulence to the insect Tenebrio molitor (Coleoptera: Tenebrionidae) were evaluated. Conidia produced on MML or PDA medium under white or blue light germinated faster than conidia produced on PDA medium in the dark. Conidia produced under red light germinated slower than conidia produced on PDA medium in the dark. Conidia produced on MML were the most virulent, followed by conidia produced on PDA medium under white light. The fungus grown under blue light produced more conidia than the fungus grown in the dark. The quantity of conidia produced for the fungus grown in the dark, under white, and red light was similar. The MML afforded the least conidial production. In conclusion, white light produced conidia that germinated faster and killed the insects faster; in addition, blue light afforded the highest conidial production.     

Photoinduced conidiation inTrichoderma viride: A study with inhibitors

The influence of a number of inhibitors affecting respiration, oxidative phosphorylation, cAMP-phosphodiesterase and of the antioxidant 1,4-dithiothreitol on growth and photoinduced conidiation ofTrichoderma viride were investigated. In all cases, growth and conidiation were influenced to a different extent. Among the first group of compounds, antimycin A was the most potent inhibitor of conidiation while it influenced growth much less. A similar effect was obtained with 2,4-dinitrophenol and 1,4-dithiothreitol. On the other hand, 3-isobutyl-1-methylxanthine (inhibitor of phosphodiesterase) greatly stimulated the conidiation induced by light without affecting growth. It is concluded that the redox reactions represent a vital component of the differentiation pathway and that cAMP may play a regulatory role in this process.     

Isolate-specific conidiation in Trichoderma in response to different nitrogen sources

A characteristic feature of Trichoderma is the production of concentric rings of conidia in response to alternating light/dark conditions and a single ring of conidia in response to a single burst of light. In this study, conidiation was investigated in four biocontrol isolates (T. hamatum, T. atroviride, T. asperellum, T. virens) and one isolate from the mushroom pathogen species, T. pleuroticola. All five isolates produced concentric conidial rings under alternating light/dark conditions on potato-dextrose agar (PDA), however, in response to a 15min burst of blue light, only T. asperellum and T. virens produced a clearly defined conidial ring. Both T. pleuroticola and T. hamatum photoconidiated in a disk-like fashion and T.?atroviride produced a broken ring with a partially filled in appearance. In the presence of primary nitrogen, T. asperellum and T. pleuroticola conidiated in a disk, whereas, when grown in the presence of secondary nitrogen, a ring of conidia was produced. Primary nitrogen promoted photoconidiation and competency to conidiate in response to light appeared dependent on the nitrogen catabolite repression state of the cell. Mycelial injury was also investigated in the same five isolates of Trichoderma on PDA and under different nitrogen statuses. For the first time, we report that conidiation in response to injury is differentially regulated in different isolates/species of Trichoderma.     

Photoinduced conidiation inTrichoderma viride

Trichoderma viride is a deuteromycete in which conidiations is photo-inducible. Conidiation results when colonies grow in the day-night regime or when colonies grown in the dark are exposed to short pulses of near UV or blue light. Conidiation was induced by light pulses at intervals of 8, 16, 24, 48 or 72 hours. Several membrane-damaging agents, DNA-intercalating drugs and inhibitors of RNA or protein synthesis prevent photo-conidiation. A hypothetical scheme of photo-induced conidiation, based on the results with metabolic inhibitors, is presented. A sudden increase of intracellular ATP was observed as an immediate photo-response. The ATP level is dose-dependent, with a maximum at 1.2 klx. Drugs interfering with various signalling pathways were tested in an attempt to analyze the signal pathways whereby light pulses induce conidiation. Nonconidiating and color mutants have been obtained and used in complementation studied by means of heterokaryosis and protoplast fusion. In a color mutant with brown conidia, conidiation is accompanied by high production and excretion of anthraquinone metabolites. Modified version of a lecture given at the7th International Congress of IUMS Mycology Division, Prague, July 3rd–8th, 1994.     

Rhythmic conidiation in the blue-light fungus Trichoderma pleuroticola

Trichoderma species conidiate in response to blue light, however, unlike in the blue-light model fungus Neurospora crassa, conidiation in Trichoderma spp. has been considered to be non-circadian. In this study we uncovered evidence for circadian conidiation in Trichoderma pleuroticola and identified orthologues of the key N. crassa clock components, wc-1 (blr-1) and frq.     

Protoporphyrin IX Content Correlates with Activity of Photobleaching Herbicides

Several laboratories have demonstrated recently that photobleaching herbicides such as acifluorfen and oxadiazon cause accumulation of protoporphyrin IX (PPIX), a photodynamic pigment capable of herbicidal activity. We investigated, in acifluorfen-treated tissues, the in vivo stability of PPIX, the kinetics of accumulation, and the correlation between concentration of PPIX and herbicidal damage. During a 20 hour dark period, PPIX levels rose from barely detectable concentrations to 1 to 2 nanomoles per 50 cucumber (Cucumis sativus L.) cotyledon discs treated with 10 micromolar acifluorfen. When placed in 500 micromoles per square meter per second PAR, PPIX levels decayed logarithmically, with an initial half-life of about 2.5 hours. PPIX levels at each time after exposure to light correlated positively with the cellular damage that occurred during the following 1 hour in both green and yellow (tentoxin-treated) cucumber cotyledon tissues. PPIX levels in discs incubated for 20 hours in darkness correlated positively with the acifluorfen concentration in which they were incubated. In cucumber, the level of herbicidal damage caused by several p-nitrodiphenyl other herbicides, a p-chlorodiphenylether herbicide, and oxadiazon correlated positively with the amount of PPIX induced to accumulate by each of the herbicide treatments. Similar results were obtained with acifluorfen-treated pigweed and velvetleaf primary leaf tissues. In cucumber, PPIX levels increased within 15 and 30 minutes after exposure of discs to 10 micromolar acifluorfen in the dark and light, respectively. These data strengthen the view that PPIX is responsible for all or a major part of the photobleaching activity of acifluorfen and related herbicides.     

Effects of light,phosphate, and oxygen on ethylene formation and conidiation in surface cultures ofpenicillium cyclopium westling

Penicillium cyclopium growing in a surface culture with 2-ketoglutarate or glutamate as a sole carbon source produced ethylene in two phases. The first peak of ethylene production (EP 1) was associated with aerial mycelium growth whereas the second peak of ethylene production (EP 2) occurred with formation and maturation of conidia. Conidiation was induced by blue light between 120 and 172 h after the culture was started and depended on the presence of a carbon source at the stage of conidiophore initiation. Exogenous phosphate content dropper rapidly before the onset of conidiation. The EP 2 was connected with conidiation via this drop. Addition of phosphate prior to the conidiophore initiation and during conidiation inhibited EP 2 without affecting conidiation, but conidia lacked a green pigment and their germination ability decreased by 905. Exogenous ethylene did not restore normal development. The EP 2 in asporogenic cultures was evoked by incubation in the dark and by phosphate removal. The EP 2 and conidiation were accompanied by an increased oxygen consumption. The EP 1 yield of ethylene depended only on biomass growth and was unaffected by any treatment mentioned above.     

The effect of different wavelengths of light on polyketide metabolism inAlternaria alternata

The effect of visible light on alternariol content in the moldAlternaria alternata was investigated. When the mold was irradiated with white light at moderate fluence rates the mycelia contained little or no alternariol in comparison with dark controls. This reduction of alternariol content in mycelia was due primarily to blue light, although red light also resulted in a slight decrease. The results show that red light above 700 nm also inhibits alternariol synthesis. The suppressive effect of blue light was fluence rate dependent; however, very low fluence rates also caused inhibition. Growth and conidiation were not affected by the light treatments.     

The A mating type and blue light regulate all known differentiation processes in the basidiomycete Coprinus cinereus

Monokaryons of Coprinus cinereus constitutively form small spores (oidia) in the aerial mycelium. Some strains also produce large, inflated single cells (chlamydospores) at the agar/air interface, and hyphal aggregates (hyphal knots) that can develop into sclerotia. Monokaryons show various reactions upon transformation with heterologous A mating type genes. Production of oidia in such A-activated transformants is repressed in the dark and induced by blue light. Five of six monokaryons tested following transformation with A genes showed induced production of hyphal knots and sclerotia in the dark, and at least three strains showed enhanced chlamydospore production in the dark. Continuous incubation under blue light inhibited formation of hyphal knots, sclerotia and chlamydospores in both competent monokaryons and in A-activated transformants. On artificial medium and on a 12?h light/12?h dark regime, A-activated transformants of one distinct monokaryon (218) formed fruit-body primordia that were arrested in development before karyogamy. Our studies show that A mating type genes control all major differentiation processes in Coprinus, but whether developmental processes can proceed depends on the genetic background of the strain.     

Control of the Photosynthetic Apparatus of Acetabularia mediterranea by Blue Light : Analysis by Light-Saturation Curves

During growth, Acetabularia mediterranea requires the action of blue light to maintain high rates of photosynthesis. In the present study, blue light-dependent alterations of the photosynthetic apparatus, which can be detected by analysis of light-saturation curves and by measurements of partial reactions of the photosynthetic electron transport chain, are described. Light-saturation curves of photosynthesis in vivo were measured with a new closed oxygen electrode system after culture of Acetabularia in continuous red or blue light. These curves were compared to those of 2,6-dichlorophenol-indophenol reduction by isolated chloroplast membranes. The analysis lead to the following statements: (a) only one reaction limits electron transport rates in vitro (dichlorophenol-indophenol reduction) at all light intensities irrespective of the light quality during growth, and (b) the limiting step is light driven and located in the reaction center of photosystem II. Presumably, this same reaction determines the flow of electrons under low light intensities in vivo in cells from white, blue, and red light. In addition to photosynthesis, the rates of dark respiration changed due to the action of blue light. Concomitantly, the light compensation point of apparent photosynthesis was shifted during monochromatic irradiations.     

Induction of conidiation by endogenous volatile compounds in Trichoderma spp

Light and starvation are two principal environmental stimuli inducing conidiation in the soil micromycete Trichoderma spp. We observed that volatiles produced by conidiating colonies of Trichoderma spp. elicited conidiation in colonies that had not been induced previously by exposure to light. The inducing effect of volatiles was both intra- and interspecific. Chemical profiles of the volatile organic compounds (VOCs) produced by the nonconidiated colonies grown in the dark and by the conidiating colonies were compared using solid-phase microextraction of headspace samples followed by tandem GC-MS. The conidiation was accompanied by increased production of eight-carbon compounds 1-octen-3-ol and its analogs 3-octanol and 3-octanone. When vapors of these compounds were applied individually to dark-grown colonies, they elicited their conidiation already at submicromolar concentrations. It is concluded that the eight-carbon VOCs act as signaling molecules regulating development and mediating intercolony communication in Trichoderma.