An action spectrum for photoinduced sporulation in the fungus Trichoderma viride

When a dark grown colony of Trichoderma viride is exposed towhite light for a short time, sporulation occurs only in thenarrow region of mycelia produced just prior to illumination.The action spectrum of this light effect was obtained for wavelengthsbelow 520 mµ using monochromatic irradiation of knownintensity having a band width of 10 mµ. The action spectrumshows 4 distinct peaks at 320, 380, 430 and 480 mµ. Thewavelengths at 320 and 380 mµ are most effective in photoinducedsporulation. The longer wavelengths, 430 and 480 mµ, areconsiderably less effective. The possibility that a carotenoid or a flavin compound may bea photoreceptor in this photoinduced sporulation is discussed. (Received January 4, 1969; )     

LIGHT-INDUCED REACTIONS OF UBIQUINONE IN PHOTOSYNTHETIC BACTERIUM, CHROMATIUM D

Light-induced changes in ultraviolet absorption were investigatedin a photosynthetic bacterium, Chromatium strain D. The difference spectra (light-minus-dark) of the changes insteady state level of absorption under various experimentalconditions have common maxima (peaks or troughs) at 275 mµ,315 mµ and 340 mµ; The sign of change, however,varied according to the conditions of illumination (see below).From the shape of the difference spectra and for other reasonsdiscussed in the present paper, the changes at 275 mµwere ascribed to the oxidation-reduction changes of ubiquinone. Illumination under aerobic condition caused a reduction of ubiquinonsamounting to about 6–7 percent of total ubiquinone inthe cell. The addition of Na₂S₂O₃ enhanced the amplitude ofthe photoinduced change to about 25% of the total ubiquinonein the cell. The sign of the photoinduced absorption change at 275 mµwas reverted by the presence of malate or succinate as substrate.On illumination under anaerobic condition, there was a photoinducedoxidation of ubiquinone amounting to about 50% of the total.Under aerobic condition the amount was about 6–7 percentof the total. Transitional changes of ubiquinone in the bacterial cell werealso investigated under various experimental conditions. (Received July 24, 1967; )     

Sporophore Development in Sphaerobolus: Effect of Blue and Red Light

Experiments are described which indicate that, although lightof short wavelength (400–500 mµ) is necessary forover-all sporophore development, during the last days of theprocess light of longer wavelength (640–720 mµ)accelerates development more than blue light (400–500mµ) does. It is shown that at 20° C this effect ofthe longer rays of light begins to be exerted 8 to 10 days aftersporophore initiation and only a very few days before maturity.     

AN f-TYPE CYTOCHROME FROM CHLORELLA VULGARIS

A method for isolating an f-type cytochrome (Chlorella cytochrome554) from Chlorella vulgaris var. viridis (CHODAT) utilizingN, N-diethylaminoethylcellulose is described. The spectrum ofreduced Chlorella cyt. 554 has absorption maxima at 554 mµ(-band), 524 mµ (ß-band), 417 mµ (SORETband), 352 mµ, 319 mµ and 277 mµ (proteinband). The oxidized form has absorption maxima at 554mµ530 mµ, (ß-band), 412 mµ (SORET band),360mµ 322 mµ and 275 mµ (protein band). Thespectral characteristics resembled other f-type cytochromes,e. g. in the high SORET to -extinction ratio (6.8) and an asymmetric-absorption band (especially at liquid N₂ temperature) ; butcharacteristic differences were present. Mitochondria from whitelupine seedlings and sweet potato roots reduced Chlorella cyt.554. From the effects of antimycin A and 2-heptyl-4-hydroxyquinolineN-oxide it appears that Chlorella cyt. 554 was reduced sequentiallybefore cytochrome a+a₃ and near the level of the cytochromesof the b type. Oxidation was slow using lupine mitochondriaand nil with sweet potato mitochondria. The oxidation-reductionpotential at pH 7.2 and 30? was 0.35 V. Ascorbate, cysteine,glutathione and Na₂S₂O₄ readily reduced Chlorella cyt. 554.The cytochrome was not autoxidizable and was slowly oxidizedby excess potassium ferricyanide. The reduced form did not reactwith CO and was not adsorbed by IRC-50 or Cellex-P cation exchangers. ¹ Temporary address until September 1961: Department of HorticulturalScience, University of California, Los Angeles 24, California,U. S. A. ² Present address: Plant Industry Station, Pioneering ResearchLaboratory, Marketing Quality Research Division, AgriculturalMarketing Service, Beltsville, Maryland, U. S. A. (Received January 16, 1961; )     

Light and Spore Discharge in Ascobolus

Spore discharge in Ascobolus crenulatus occurs both in the lightand in the dark. In a 12 h light: 12 h dark daily regime discharge-ratehas peaks in the dark periods, due apparently to light stimulationwith about half a day's interval between stimulus and response. Using a ‘spore clock’ the course of discharge hasbeen followed for a single apothecium on changing from darknessto light. Exposure to light (500 lux) of wave-lengths around400, 440 and 460 mµ immediately causes ’puffing‘,whilst light of longer wave-length (504 and 580 mµ) hasno effect. Change from darkness to white light has no immediateeffect, but there is a delayed stimulation with a marked increasein discharge-rate 10–14 h later. Simultaneous illuminationof an apothecium, which has been in darkness, by blue light(420 mµ, or 440 mµ, 500lux) and yellow light (580mµ, 500 lux) does not result in puffing. The yellow appearsto prevent the blue light from exerting its effects.     

Fruiting in Sphaerobolus with Special Reference to Light

Sphaerobolus grows and, provided there is sufficient illumination,fruits readily on oatmeal agar or on malt agar. No effect oflight on vegetative growth can be demonstrated. On the maltmedium, increased fruiting occurs with increase of nutrientup to 4 per cent, malt, but at higher concentrations fruitingis not increased and may be retarded. A chemically defined mediumwith starch as the carbon source allows fruiting, but at a lowlevel. Temperature has a profound effect on basidiocarp development;above 25 C. no fruit-bodies are normally formed although vegetativegrowth is approximately optimal at that temperature. For overallfruit-body production at 20 C, light above 100 lux is necessaryand light remains a limiting factor up to about 1, 000 lux.Under continuous light of suitable intensity, fruit-bodies continueto develop and discharge glebal-masses for many weeks. Thereis a distinct periodicity of discharge with (at 20 C.) about12 days between peaks of activity. This corresponds with thetime taken for basidiocarp initiation and development. A number of developmental stages of the basidiocarp are recognized.The final stage, glebal-mass discharge from stellately openedfruit-bodies, is indifferent to light, but all other stagesare stimulated by light. The light intensity for effective stimulationfalls during development and for the penultimate stage an intensityas low as 1 lux is effective. Only light of wave-length below500 mµ is active in overall basidiocarp development. Inthe sensitive region between 400 mµ and 500 mµ,there appear to be peaks of sensitivity around 440 mµand 480 mµ. In alternating light and darkness, simulating natural conditions,glebal-mass discharge occurs in the light periods. With a regimenof 24 hours light and 24 hours of darkness discharge is mainlyin the dark periods.     

CHANGES IN EMISSION SPECTRA OF PHOTOSYNTHETIC PIGMENTS IN VIVO

1. The effects of 3-(4'-chlorophenyl)-1, 1-dimethylurea (CMU)onthe fluorescence of photosynthetic pigments in vivo wereinvestigatedin blue-green, red and brown algae and in isolatedspinach chloroplasts.CMU caused an increase in steady statelevel of fluorescenceof chlorophyll a, but did not influencethe fluorescence ofphycobilins. The spectrum of the fluorescenceincrement hada peak at 685 m/µ and a shoulder at 730–740mµ.These two bands probably arise from chlorophyll a(C_f684) belongingto pigment system II.
2. On excitation of chlorophylla in a red alga, Porphyra yezoensis,a fluorescence band witha peak at 720 mµ was observedbesides a shoulder at 685mµ. The 720 m band is inferredto arise from chlorophylla (probably, C_f-1) in pigment systemI.
3. On addition of CMUto the algal cells, the induction of fluorescencewas modifiedto take a simple time course. The induction wasobserved onlywith respect to the fluorescence of chlorophylla, but not inthe fluorescence of phycobilins. The spectrumof the "transient"fluorescence showed two emission bands ofchlorophyll a at 685mµ and 740 mµ, and was quitesimilar in form tothe spectrum of the CMU-caused increase insteady state fluorescence.
4. These facts were interpreted in terms of the correlation offluorescence of chlorophyll a and the photochemical reactionsof photosynthesis

(Received July 20, 1967; )     

Effects of Monochromatic Radiations on Growth of Pelargonium Callus Tissue

Pith callus tissues were grown under continuous blue (450 mµ),green (545 mµ), red (650 mµ), and ‘white’(full-spectrum) light, and in the dark for 22 days at 27±2°C at energy levels of 15,000 ergs cm^–2 sec^–1. Mean increases in fresh weight of tissues grown under ‘white’and blue light were significantly greater than those of tissuesgrown in green and red light and in the dark. Tissues grownin the dark yielded mean fresh weight increases significantlylower than tissues grown under blue, red, and ‘white’light. No significant differences were shown between blue and‘white’, red and green, and green and dark treatmentsrespectively. Cell differentiation occurred in all treatmentsonly to the extent of vessel element formation. There were nodifferences in degree of differentiation between treatments. It was proposed that the high-energy reaction of photomorphogenesiswas in operation in the Pelargonium callus tissue. The resultsindicated the presence in the tissue of high-energy photoreceptor(s).The use of high-intensity, incandescent illumination for experimentalprocedures approximating natural conditions of irradiation wasindicated as desirable for pith callus tissues of Pelargoniumzonale var. Enchantress Fiat.     

Nature of light-induced absorbance changes at 682 m{micro} and 702 m{micro} in photosynthesis of Anacystis nidulans

Light-induced absorbance changes in the region around the redabsorption band of chlorophyll a were measured in cells andlamella fragments of Anacystis nidulans. In both materials,absorbance decreases were observed at 702 mµ and 682 mµ.(The pigments are designated as P₇₀₀ and P₆₈₀.) The nature ofP₆₈₀ was investigated with special reference to its relationshipto P₇₀₀. In the cells, light absorbed by chlorophyll a causedan absorbance decrease at 682 mµ; Simultaneous illuminationwith light absorbed by phycocyanin caused a partial recoveryof the absorbance decrease. Similar results were observed withthe light-induced absorbance change at 702 mµ. This indicatesthat P₆₈₀ is also an electron carrier in the electron transportchain and occupies a place between the two photoreactions. Inlamella fragments, both the light-induced reversible absorbancechanges of P₆₈₀ and P₇₀₀ appeared in the presence of an electrondonor system; i.e., ascorbate and 2,6-dichlorophenolindophenolor N,N,N',N'-tetramethyl-l,4-phenylenediamine. The experimentsin which the oxidation-reduction potential of the reaction mediumwas changed showed that both P₆₈₀ and P₇₀₀ are one-electroncarriers, having a normal oxidation-reduction potential of 0.44v (assuming that the normal oxidation-reduction potential ofthe ferricyanide-ferrocyanide system is 0.409 v). A possibilitywas suggested that the absorbance change observed at 682 mµis another expression of the oxidation-reduction reaction ofP₇₀₀). (Received October 30, 1968; )     

BIOTIN LIBERATION BY THE LICHEN ALGA COCCOMYXA SP. AND BY CHLORELLA PYRENOIDOSA

The unicellular green alga Coccomyxa, a component of the lichenPeltigera aphthosa, liberated about 7.2mµg biotin permg dry weight of cells into the culture medium during a growthperiod of 15–20 days. The corresponding figure for thefree-living alga Chlorella pyrenoidosa was 0.45mµg ofbiotin. Chromatographic analysis indicated that this was freebiotin and not a bound form of the vitamin. The biotin concentrationof rinsed Coccomyxa cells was 1.88mµg per mg dry weightof cells, of which less than 0.01mµg was extractable byhot water. Cells of Chlorella contained 0.16mµg of biotinper mg dry weight, of which 0.11mµg was extractable byhot water. The biotin content of Coccomyxa, which was about12 times that of Chlorella, is thus almost entirely in the boundform. The importance of biotin in the symbiotic interactionsbetween the alga and the fungus in Peltigera is discussed. ¹Present address: University Department of Agriculture, Oxford,England. ²Present address: Institute of Marine Resources, Universityof California, La Jolla, California, U.S.A.     

LOCALIZATION OF LIGHT-INDUCED STRONTIUM ACCUMULATION IN SPINACH CHLOROPLASTS

Electron-microscopic evidence showing the location of strontiumand calcium deposits on the lamellae of spinach chloroplastsis presented. The chloroplast limiting membrane did not actas a barrier for this ion accumulation, since the chloroplastswere ruptured during the isolation and incubation, especiallyin the light. The grana stacks also were extensively swollen.The number of electrondense deposits attached on the lamellaewas greatly increased by light under conditions favoring thehydrolysis of ATP. The average diameter of the deposits aftera 6 minute illumination with 1 mM SrCl₂ was 14 mµ, after15 minutes with 1 mM SrCl₂ was 21 mµ, and after 15 minuteswith 20 µM SrCl₂ was 13 mµ. The findings are inharmony with the results of ion-uptake experiments using radioactivestrontium and calcium. ¹ United States National Science Foundation Postdoctoral Fellow.     

Effects of Growth Regulators on Polyamine Content and Peroxidase Activity in Hevea brasiliensis Callus

3, 4-dichlorophenoxyacetic acid (3,4-D) and benzylaminopurine(BAP) at 9 µM (control medium) was compared with 4.5,2.25, and 0.45 µM for ability to induce callogenesis andembryogenesis from seed explants of Hevea brasiliensis. Supplyingthese growth regulators at 4.5 µM for 20 d improved embryogenicpotential compared with the control medium (El Hadrami, Carronand d'Auzac, 1991, Annals of Botany 67, 511–515), sustainedputrescine, spermidine and spermine at a higher level throughoutof much of the culture period (40–70 d), and maintainedlow levels of peroxidase activity. In the control medium, poorcallus embryogenesis is considered a consequence of rapid ageingof tissues characterized by (i) acceleration of an early buttransient production of polyamines, which promoted embryogeniccapacity, and (ii) an early peak in peroxidase activity thatwas positively correlated with callus browning, one of the factorslimiting embryogenesis. Somatic embryogenesis, polyamines, peroxidase, Hevea brasiliensis, rubber-tree     

Effect of blue light on the photonastic reaction of rice leaves

Effect of blue light on the epinastic reaction of rice plantleaves (Oryza saliva L.) was studied. The inclination angleof intact leaves, when the plants were grown either in the lightor in the dark, was greatly increased by blue light irradiationbut not much increased by green, yellow, orange, red or whitelight. The effect of blue light on the epinastic reaction was,however, obscure in excised leaves, especially when they wereplaced on the horizontal. The photonastic reaction requireda prolonged irradiation of high intensity. The photosensitivesite localized at the leaf-nodal part. Response spectra forthe photonastic reaction were characterized by 2 distinct peaksat 420–440 mµ and 460–480 mµ and a fairlystrong response in the near ultra-violet region. (Received April 17, 1969; )     

LACTATE OXIDATION SYSTEM IN ACETOBACTER SUBOXYDANS, WITH SPECIAL REFERENCE TO CARBON MONOXIDE-BINDING PIGMENT

1. Lactate oxidation system was investigated in Acetobacter suboxydans,which was found to have cytochromes a and b, but no cytochromec. Haemoprotein 558 was also found to exist.
2. Carbon monoxideinhibited the lactate oxidation in the darkbut not in the light.WARBURG'S partition constant was estimatedto be 7.
3. Additionof haemoprotein 558 noticeably enhanced the oxygenuptake bythe LDH preparation, which was obtained from bacterialcellsand partially purified.
4. Haemoprotein 558 has protohaem asits prosthetic group. Notonly its absorption spectrum is reminiscentof that of peroxidase,but it also shows peroxidase-like reactivitywith some ligandswith a few exceptions.
5. Ferrohaemoprotein558 reacts with oxygen, forming, at first,a complex, whichhas its SORET absorption peak at 423 mµ.The absorptionmaximum then shifts to 417 mµ, indicatinga transformationto another compound. One of these two productsis likely tobe the oxygenated ferro-haemoprotein 558.
6. The mutual transformationbetween cytochrome B and haemoprotein558 is discussed.

(Received October 8, 1965; )     

Copper Nutrition of Sugarbeet

Sugarbeet (Beta vulgaris L.) plants were grown in refined sandat graded levels of copper ranging from acute deficiency (0.000325µg Cu cm^–3) to excess (65 µg Cu cm^–3).Visible effects of copper deficiency appeared up to 0-00065µg Cu cm^–3and depression in growth up to 00065µCucm^–3. Copper deficiency decreased the concentrations ofDNA and RNA and the activities of polyphenol oxidase, cytochrome-coxidase, catalase and aldolase; and it increased the activitiesof peroxidase, ribonuclease and acid phosphatase in leaves.The maximum sucrose concentration in roots was obtained at 0-65µCucm^–3 Twenty four h after infiltration of a solution of 65µCucm^–3into copper deficient leaves, the activities of cytochrome-coxidase and peroxidase had increased even in the presence ofcycloheximide but that of polyphenol oxidase increased onlyin the absence of this inhibitor. Key words: Beta vulgaris, Cu deficiency: Enzymes     

Isolation and characterization of the 337 m{micro} UV-absorbing substance in red alga, Porphyra yezoensis UEDA

Using a variety of Sephadex gel filtrations, starch block zoneelectrophoresis, Avicel SF preparative TLC and DEAE cellulosecolumn chromatography, the characteristic 337 mµ UV-absorbingsubstance from marine red alga, Porphyra yezoensis UEDA wasisolated. The molecular weight of this substance was about 1,000,and its chemical composition was 43.11% C, 5.85% H, 7.23% N,34.73% O and 9.08% Na. In vivo it is located in the chloroplast.On irradiation at 378 mµ, it has a fluorescence actionspectrum peak at 470 mµ. Structural studies on this substanceare still underway, but it could be a kind of aminosugar judgingfrom NMR, IR spectra and other chemical properties. ¹This work was partially supported by the grant of the Ministryof Education in 1969 ²Present address: Department of Biochemistry and Biophysics,University of Hawaii, Honolulu, Hawaii 96822, U.S.A. (Received December 21, 1969; )     

PHOSFON-INHIBITION OF GERMINATION AS AFFECTED BY TEMPERATURE AND RADIATION

Inhibition of radiation-induced germination in Verbascum thapsusL. by low concentrations of the growth retardant Phosfon isincreased by maintenance temperature of 30 or higher. The germinationprocess is most sensitive to combined supraoptimal temperatureand Phosfon treatments during the time when root protrusionor processes preparatory to it occur in control seeds. At 25,inhibition of induced germination by Phosfon is relieved bycontinuous irradiation, with effective wavebands centered onwave-lengths of 430, 535, 600 and 650 mµ. The bands at485 and 735 mµ reduce germination to or below the levelof briefly red-irradiated seeds. (Received November 22, 1965; )     

Effect of Gibberellic Acid and Ethylene on Peroxidase in Pea Internodes

Ethylene at 5–80 µl l^–1 inhibited elongationand induced swelling in internodes of light-grown normal anddwarf pea plants; GA₃ did not prevent swelling in response toethylene. GA₃ neither inhibited nor enhanced the activity of isoperoxidasesin the internodes, regardless of its effect on their elongation.Ethylene at 80 µl l^–1 enhanced peroxidase in GA₃-untreatedand treated normal and dwarf plants. At 5 µl l^–1,ethylene had only a weak effect on peroxidase activity or none.The enzyme enhancement by ethylene was not related to its effecton cell expansion and seems do be due, at least in part, tochemical injury. Electron microscopy revealed peroxidase activity in the roughER and cell walls, including intercellular spaces. Stainingof walls in ethylene-treated tissues was more pronounced thanin untreated ones. Golgi vesicles did not seem to be involvedin the assembly of the enzyme carbohydrate moiety in ethylene-treatedcells. The peroxidase fraction extracted with 20 mM phosphate buffer,pH 6, and that extracted from wall debris with 1 M NaCl accountedfor 98% of total enzyme activity. Both fractions contained thesame six cathodic isoforms which comprised 85–90% of theiractivity. Electrophoresis did not reveal differences in thequalitative isoenzyme patterns in relation to variety, age,GA₃, or ethylene. The only observed quantitative differenceswere age-dependent. Procedural artefacts during separation of protoplast and wallionically bound peroxidase fractions are discussed.     

A PHENOLIC PIGMENT REACTIVE TO L-LACTATE CYTOCHROME C REDUCTASE OF YEAST

1. A phenolic pigment was extracted from baker's yeast. The pigmentis slowly autooxidizable, and rapidly oxidized with Rhus-laccaseor polyphenol oxidase and reduced by dithionite.
2. The pigmentdissolved in ethylether had an absorption peak at258 mµ,shoulders at 289 and 382 mµ and a plateauat 450–500mµ. The difference spectrum between oxidizedand reducedforms of the pigment showed a wide plateau around500 mµ.
3. The pigment supported the oxygen uptake by reconstructed enzymesystem: L-lactate, L-lactate cytochhrome c reductase and Rhuslaccaseor polyphenol oxidase. In its absence, no oxygen uptake tookplace. The pigment was replaced successfully with p-quinone,catechol and menadione, but not with ubiquinone. The sequenceof hydrogen transport can be represented: L-lactate L-lactatecytochrome c reductase "phenolic pigment" oxidase oxygen.

(Received August 11, 1967; )     

OCCURRENCE OF A FAR-RED-ABSORBING PIGMENT (P 715) IN LEAF EXTRACT OF GYNKGO BILOBA

The occasional occurrence of far-red-absorption band in thegreen extract of Gynkgo biloba leaves was noticed. It resultedfrom the conversion of the ordinary red-absorbing form of thechlorophyllous pigments with absorption maximum at 675 mµ(P 675), to the far-red-absorbing form with absorption maximumat 710–715 mµ (P 715). The conversion seems to takeplace in two steps, i.e., first, decrease in thered-absorptionband, and secondly, appearance of the far-red-absorption band.Under some experimental conditions, the latter step occurs onlyafter a certain time lag. The conversion is accompanied by thechangeof chlorophylls a and b, contained in the initial formof the pigment, P 675, to the pheophytins and pheophorbides.The properties of the far-red-absorbing pigment are differentfrom those of other chlorophyllous pigments discovered in otherplant materials. ( Accepted January 29, 1965)